EA046872B1 - CONFORMAL COATING OF CELLS FOR IMMUNOISOLATION - Google Patents

Abstract

Hydrodynamic methods for conformally coating cells and cell clusters of heterogeneous sizes with biomaterials for implantation are described, thereby preventing immune rejection, inflammation, or autoimmune destruction while preserving cellular functionality. Also described are reagents, a device, and methods for conformally coating cells and cell clusters with biocompatible, mechanically and chemically stable hydrogels having pore sizes corresponding to the required cutoff threshold.

Description

This application claims benefit from U.S. Provisional Application No. 63/016787, filed April 28, 2020, the contents of which are hereby incorporated by reference in their entirety.

This application is related to US Patent Application No. 15/478320, filed April 4, 2017, which is now US Patent No. 10653816, which is a divisional application claiming priority to US Patent Application No. 14/114690, filed February 12, 2014. which is now US Patent No. 10660987, which under 35 USC §371 is the national stage application of International Patent Application No. PCT/US 2012/035696, filed April 28, 2012, which claims priority to US Provisional Patent Application No. 61 /480513, filed April 29, 2011, the contents of each of these applications are hereby incorporated herein by reference in their entirety.

U.S. Federal Government Research Sponsorship Statement

This invention was supported by Grant No. R01 DK109929 from the US National Institutes of Health. The US Government has certain rights to this invention.

Technical field

This invention relates to the field of cell transplantation and provides reagents and methods for facilitating cell transplantation into a human or animal with minimal immunological reactions or minimal graft rejection. More specifically, this invention provides biomaterials, including cells for transplantation, that have been conformally coated at neutral pH with a coating mixture that minimally affects the viability, metabolism or biological activity of such cells or other biomaterials, and reagents, apparatus and methods for obtaining and using said biomaterials onto which a conformal coating is applied and, in particular, said cells. In specific embodiments of the present invention, the conformally coated cells and/or cell clusters comprise insulin-producing cells (e.g., islets of Langerhans containing beta cells) differentiated from stem cells or isolated from the pancreas of an animal, more particularly a mammal and most specifically - a person. In particular, this document provides methods of using biomaterials, in particular cells and/or cell clusters, obtained using the specified reagents and apparatus, and the specified methods.

State of the art

Cell encapsulation is a promising strategy for immunoisolating individual cells and cell clusters, and therefore preventing any immune response that might compromise the functionality of these cells after implantation. Bioencapsulation has been widely used in new therapeutic trials in the areas of diabetes, hemophilia, cancer and renal failure. However, most trials were not completely successful for a variety of reasons:

insufficient reproducibility of cell encapsulation and isolation methods;

lack of suitable encapsulation materials that must be biocompatible, mechanically and chemically stable and have pore sizes that meet the required cutoff threshold to allow nutrients and byproducts to flow into and out of the capsule, protecting the encapsulated biomaterial from the effects of the immune system;

production of capsules with non-uniform or non-conformal coating (which affects (i) the diffusion of oxygen and nutrients through the capsule, and (ii) the cells' secretion of bioactive molecules and therefore the viability of the encapsulated cells, functionality as a delivery vehicle for therapeutic molecules and the overall volume of the encapsulated graft );

failure to scale up the encapsulation process from small animal studies to preclinical studies in non-human primates; and selection of unfavorable areas of the body for transplantation.

Such problems associated with encapsulation technology can be considered in the context of work in one of the most promising therapeutic areas for cell encapsulation - the field of diabetes.

Diabetes mellitus results from the autoimmune destruction of pancreatic beta cells, one of several types of cells that make up the islets of Langerhans. Throughout their lives, patients with diabetes must frequently monitor their blood glucose levels and administer insulin when they become hyperglycemic, which has many adverse effects, including suboptimal metabolic control, a higher risk of hypoglycemia, and decreased quality of life. Islet allotransplantation is an extremely promising therapy for the treatment of patients with diabetes mellitus, but requires lifelong systemic immunosuppression to avoid allograft rejection. See Robertson, 2010, Endocrinol Metab Clin North

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Am 39, 655-667; Herring et al., 2016, Diabetes Care 39: 1230–1240; Shapiro et al., 2017, Nat. Rev. Endocrinol. 13: 268-277; Pepper et al., 2018, Curr. Opin Organ Transplant. 23: 428-439; LaBlanche et al., 2018, Lancet Diabetes Endocrinol. 6: 527-537; Foster et al., 2018, Diabetes Care 41: 1001-1008; Rickels & Robertson, 2019, Endocrin. Rev. 40: 631-668.

To avoid the administration of immunosuppressive drugs at the systemic level, islet allografts can be protected from immune reactions by coating the transplanted cells with a semi-permeable polymer capsule that allows the diffusion of oxygen, glucose and insulin, while preventing cell-cell contact and the diffusion of cytotoxic molecules that would otherwise cause immune response against a given graft and its complete rejection by the host (de Vos et al., 2010, Adv Exp Med Biol 670, 38-53; Desai & Shea, 2017, Nat Rev Drug Discov 16: 338-350; Vairhilingham et al., 2017, Rev Diabetes Stud 14: 51-78; Krosgren, 2017, Diabetes 66: 1748-1754; Smink et al., 2018, Am J. Tranplant 18: 2113-2118). The islands have a heterogeneous size that varies from about 50 to 300 μm in diameter. Most coating procedures known in the art do not allow for conformal coating of islands; the capsule diameter is generally constant and independent of islet size, and therefore typically greater than 300 µm to ensure coverage of larger islets (Teramura & Iwata, 2010, Adv Drug Deliv Rev 62, 827-840). Due to the excess of acellular coating material, the total volume of the islet implant increases significantly, so that the only suitable area for transplantation is the poorly oxygenated abdominal cavity, which contributes to hypoxia of the encapsulated cells. In addition, the thickness of the capsule increases the diffusion barrier to oxygen through this coating, which also exacerbates cellular hypoxia and delays glucose uptake and hence the insulin secretion response (de Groot et al., 2004, J Surg Res 121, 141-150; Buchwald et al. al., 2015, Biomed. Online 14: 28; Expert Opin. Biol. 15: 1321-1326; Buchwald et al., 2018, Biotechnol. Bioeng 115; Most of these encapsulation methods are based on the formation of droplets of coating material mixed with islands using an air-jet pump or electrostatic droplet generators (Rabanel et al., 2009, Biotechnol Prog 25, 946-963).

In contrast to droplet-based encapsulation methods, conformal coating of cell clusters of different diameters has been the focus of some recent studies. Most of these methods rely on either (a) layer-by-layer coating directly onto the cells (e.g., by chemical reaction or photopolymerization) or (b) a completely hydrodynamic procedure, usually involving the formation of particles by forming a water-in-oil emulsion or by jet disruption water in oil according to the hydrodynamic principle of Rayleigh-Plateau instability (Teramura et al., Id.; Chabert & Viovy, 2008, Proc Natl Acad Sci USA 105, 3191-3196). Using these methods, it is possible to generate water particles with a constant diameter, specifically depending on the characteristics of the water and oil phases, the surface tension between the two phases and the ratio of the hydrodynamic parameters of the two phases (Eggleton et al., 2001, Phys Rev Lett 87, 048302; Utada et al. al., 2008, Phys Rev Lett 100, 014502; Loscertales et al., 2020, Science 295, 1695-1698; Cohen et al., 2001, Science 292, 265-267). In the food and pharmaceutical industries, these methods have been widely used for the nanoencapsulation of water-soluble drugs and other substances (Loscertales et al., Id.), and have only recently been extended to the encapsulation of micron-sized individual cells and cell clusters, with some success, as described below.

Chabert and colleagues have developed a microfluidic high-throughput system for single cell encapsulation and self-sorting based on the principle described above (Chabert. & Viovy, Id). However, their system is designed for encapsulating and sorting single cells (those having a diameter of 40 μm or less) and cannot be applied to clusters of cells due to the microscale nature of their design, which would subject non-single cells to unacceptable shear stresses.

Garfinkel and his colleagues developed another way to encapsulate the islets by selectively withdrawing the islet-containing aqueous phase from the outer oil phase to create a thin coating on cell clusters. In this method, jet atomization of the aqueous phase into the oil phase is achieved by sucking a layer of the aqueous phase on top of the oil phase. This design creates turbulent flow in the water drainage area, ultimately resulting in incomplete coverage, requiring a second round of encapsulation, increasing the stress to which the cells are exposed and reducing the productivity of the process (Wyman et al., 2007, Small 3, 683 -690). In addition, gel polymerization is achieved through photopolymerization, which may compromise the long-term function of coated cells.

Hubbell and colleagues developed a coating approach by chemical reaction directly on the cell surface, in which a photosensitizer was adsorbed onto the islet surface and the photosensitizer-treated islets were suspended in an aqueous solution of photopolymerizable macromers (US Patent No. 6,911,227). Illumination with suspension light

- 2 046872 islets led to polymerization and cross-linking of the macromer with the formation of a conformal polymer gel associated with the surface of the islets.

Hubbell et al describe a conformal coating method in US Patent No. 10653816, US Patent No. 10660987, and International Publication WO2012/149496. In the example provided, Hubbell et al. disclose that the aqueous phase consisted of (1) multi-arm PEG (10 kDa, 8 arms, 7590% functionalized PEG-maleimide (PEG-MAL) or PEG-vinyl sulfone (PEG-WS)), an excess amount of cross-linker (dithiothreitol or 2 kDa PEG-dithiol at a molar ratio of 3-4 to 1) to achieve complete gelation of PEG and viscosity increasing agent (e.g. alginate, amphiphilic self-assembling polymers or peptides, etc.). However, the inclusion of crosslinker and viscosity increasing agent requires that the pH of the aqueous phase be maintained at 3.5-6 (depending on the specific hydrogel composition) and the process must be performed within 15 minutes to prevent premature gelation of the aqueous phase before application coating that occurs after jet atomization and decomposition of the aqueous phase in the encapsulation chamber due to the high reactivity of the Michael type reaction occurring between the multi-arm PEG and the thiolated cross-linkers. This low pH level can affect long-term cellular functionality. In addition, the rapid gelation reaction that occurs when the base polymer and cross-linker are premixed limits the overall throughput of the process and complicates scale-up efforts. Finally, the inclusion of a viscosity increasing agent can affect the immunoinsulating properties of coatings and biocompatibility. See, for example, Manzoli et al., Am. J. Transplant, 18 (3): 590-603 (2018).

In view of the above, there remains a need in the art for efficient methods of conformally coating cells and cell clusters in high yield without compromising cell functionality or biocompatibility.

Brief description of the invention

This document provides reagents and methods, as well as a conformal coating apparatus, for conformally coating biomaterials, in particular cells, and in particular insulin-secreting cells derived from stem cells and beta cells containing islets of Langerhans isolated from vertebrates. , more specifically mammals, and in particular humans. Biomaterials, particularly cells and/or cell clusters, and more particularly islet cells and/or islet cell clusters, are coated and maintained at a neutral pH (e.g., pH ~6 to ~7.4) as described herein. document. This document presents further modifications of the specified reagents, apparatus and methods relative to the reagents, apparatus and methods set forth in related applications.

Therefore, the first aspect of the present invention provides methods for conformally coating biomaterials with a coating material, comprising the following steps:

(a) injecting the aqueous phase into the coaxial oil phase in a coating device configured to provide a transition from droplet spray to jet spray and elongation of the flow of said aqueous phase into said oil phase;

(b) adding said biomaterial and said coating material to said aqueous phase, wherein the coating material from said step (b) does not contain a viscosity increasing agent; and wherein said aqueous phase has a pH of from about 6 to about 7.4;

(c) allowing the stream of said aqueous phase to disintegrate into particles; and (d) adding a component of said coating material after the jet of said aqueous phase has disintegrated into particles, said component being a gelling emulsion that promotes or catalyzes the polymerization of said coating material; As a result, a biomaterial with a conformal coating is obtained.

In some embodiments of the present invention, the method further includes the step of collecting material flowing from the coating device (ie, the conformally coated biomaterial and any coating material that does not contain the biomaterial).

In some embodiments of the present invention, said method further includes the step of purifying said conformally coated biomaterial and said biomaterial-free coating material from said oil phase.

In some embodiments of the present invention, the method further includes the step of separating said conformally coated biomaterial from said non-biomaterial coating material.

In some embodiments of the present invention, purifying the conformally coated biomaterial and any non-biomaterial coating material from the oil phase includes step (e) pouring the product from step (d) of the method above into mineral oil while stirring the resulting mixture (i.e., a conformally coated biomaterial and any coating material that does not contain a biomaterial, an oil phase, and a gelling emulsion (containing

- 3 046872 dithiothreitol (DTT) solution dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80) in mineral oil).

In some embodiments of the present invention, purification of the conformally coated biomaterial and the biomaterial-free coating material from the oil phase includes step (f) adding Hanks' Balanced Salt Solution (HBSS) to the product obtained in step (e) (i.e. Conformally coated biomaterial and any coating material that does not contain a biomaterial, an oil phase and a gelling emulsion (containing a solution of dithiothreitol (DTT) dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80) in mineral oil) exiting said device, and mineral oil).

In some embodiments of the methods described herein, the product from step (f) is centrifuged and washed in HBSS.

In some embodiments of the methods described herein, after centrifugation and washing with Hanks' Balanced Salt Solution (HBSS), the coated biomaterial and any non-biomaterial material are incubated with a PEG-dithiol solution.

In some embodiments of the present invention, the viscosity increasing agent, which is excluded from the methods described herein, is selected from polysaccharides such as alginate, decellularized tissue, PEG nanomaterial assemblies, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate , heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water-soluble cellulose derivatives, gelatin, collagen and albumin.

In some embodiments of the present invention, methods for conformally coating biomaterials with a coating material are provided, comprising the following steps: (a) injecting an aqueous phase into a coaxial oil phase in a coating device configured to provide a transition from droplet spray to jet spray and extension flow of said aqueous phase into said oil phase; (b) adding said biomaterial and said coating material to said aqueous phase, wherein polymerization of said coating material occurs after the jet of said aqueous phase disintegrates into particles, resulting in conformal coating of said biomaterial with said coating material; (c) adding a component of said coating material after the jet of said aqueous phase disintegrates into particles, wherein said component stimulates/catalyzes the polymerization of said coating material; (d) optionally collecting material flowing from said coating device; e) optionally, purifying said conformally coated biomaterial and said biomaterial-free coating material from said oil phase; and (f) optionally, separating said conformally coated biomaterial from said coating material not containing the biomaterial.

In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG), polyethylene oxide (PEO), poly(vinylpyrrolidinone) (PVP), polyethyloxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK), poly(amino acids), derivatives thereof, or their combinations. In some embodiments of the present invention, said coating material is one or more of the following: polyethylene glycol (PEG), polyethylene oxide (PEO), poly(N-vinylpyrrolidinone) (PVP), polyethyloxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK), and (or) poly(amino acids). In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG), PEG maleimide, PEG acrylate, PEG vinyl sulfone, PEGthiol, or modified derivatives thereof, or combinations of specific moieties thereof. In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG), PEG maleimide, PEG acrylate, or PEG vinyl sulfone. In some embodiments of this invention, the coating material comprises a multi-arm polyethylene glycol (PEG) minimally cross-linked (5-50%) with PEG-dithiol. In some embodiments of this invention, the coating material comprises a multi-arm polyethylene glycol (PEG) minimally cross-linked (1-30%) with PEG-dithiol. In some embodiments of this invention, said coating material contains 5-10% PEG. In some embodiments of this invention, said aqueous phase contains a serum-free medium having a pH of about 6-7.4; or Hanks' balanced salt solution (HBSS), having a pH of about 67.4; in particular, the pH of the aqueous phase is approximately pH 6-7.4. In some embodiments of this invention, said aqueous phase contains a serum-free medium having a pH of 6-7.4; or Hanks' balanced salt solution (HBSS), having a pH of 6-7.4; in particular, the pH of the aqueous phase is represented as pH 6-7.4. In some embodiments of the present invention, said aqueous phase has a pH of from about 6 to about 7.4. In some embodiments of this invention, said aqueous phase has a pH ranging from 6 to 7.4.

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In some embodiments of the present invention, the biomaterial comprises cells, clusters of cells, biomaterial-coated cells or clusters of cells, subcellular organelles, biological molecules, non-biological drugs, or a combination thereof. In some embodiments of the present invention, the biomaterial comprises one or more of the following: cells, cell clusters, biomaterial-coated cells or clusters of cells, subcellular organelles, biological molecules, and non-biological drugs. In some embodiments of the present invention, said biomaterial comprises cells or clusters of cells, more particularly islet cells or clusters thereof. In some embodiments of the present invention, said biomaterial comprises cells or clusters of cells. In some embodiments of this invention, said aqueous phase contains about 100,000,000 to about 200,000,000 cells/ml. In some embodiments of this invention, said aqueous phase contains about 200,000,000 cells/ml. In some embodiments of this invention, said aqueous phase contains about 50,000 to about 100,000 cell clusters/ml. In some embodiments of this invention, said aqueous phase contains about 100,000 cell clusters/ml. In some embodiments of this invention, said aqueous phase contains 500-750,000 cells and/or cell clusters/ml, in particular islet cells. In some embodiments of the present invention, said biomaterial contains about 100,000,000 to about 200,000,000 cells/ml and/or about 50,000 to about 100,000 cell clusters/ml. In some embodiments of the present invention, said biomaterial contains about 100,000,000 to about 200,000,000 islet cells/ml. In some embodiments of the present invention, said biomaterial contains about 50,000 to about 100,000 islet cells/ml. In some embodiments of this invention, said aqueous phase contains about 25,00250,000 islet cells and/or islet cell clusters/ml.

In some embodiments of this invention, said aqueous phase contains a thiolated reagent, a reducing reagent, a surfactant, or a combination thereof. In some embodiments of the present invention, the aqueous phase used in practicing the methods of the present invention may optionally contain one or more thiolated reagents, a reducing reagent, and/or a surfactant. In certain embodiments of this invention, said surfactant is a polyoxyethylene-polyoxypropylene block copolymer or poly(ethylene glycol-N-propylene sulfide), more specifically a 2% polyoxyethylene-polyoxypropylene block copolymer. In some embodiments of this invention, said thiolated or reducing reagent is dithiothreitol (DTT) or PEGdithiol. In some embodiments of this invention, said thiolated or reducing reagent in said aqueous phase is 0.01-0.62% dithiothreitol (DTT). In some embodiments of this invention, said thiolated or reducing reagent in said aqueous phase is 0.01-3.1% PEG-dithiol.

In some embodiments of this invention, said gelling emulsion contains dithiothreitol (DTT) dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG) with 10% v/v. sorbitan monooleate (Span80).

In some embodiments of the methods described herein, said oil phase comprises polypropylene glycol (PPG). In some embodiments of this invention, said oil phase comprises polypropylene glycol (PPG) with 10% sorbitan monooleate, wherein said oil phase optionally contains triethanolamine. In embodiments of this invention that include triethanolamine, said oil phase is 0.01-0.2% triethanolamine. In some embodiments of this invention, said oil phase contains a mineral oil with a viscosity that is at least 2.5 times the viscosity of said aqueous phase. In some embodiments of this invention, the viscosity of said oil phase is about 1300 kP.

In some embodiments of the present invention, methods are provided in which said component of the coating material is added after the stream of said aqueous phase has broken up into particles, the gelling emulsion being a solution of dithiothreitol (DTT) dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG). ) with 10% sorbitan monooleate (Span80).

In a second aspect of the present invention, a biomaterial is provided that has been conformally coated using the method described herein.

In a third aspect of the present invention, methods are provided for treating a disease or disorder in a patient, comprising the step of implanting into said patient a conformally coated biomaterial produced by the methods of the present invention. In specific embodiments of the present invention, said disease is and said conformally coated biomaterial includes cells and cell clusters. IN

- 5 046872 In some embodiments of the present invention, said disorder is diabetes mellitus.

The methods presented herein involve contacting the coated biomaterial with an emulsion of dithiothreitol (DTT) and Hanks' balanced salt solution (HBSS) in polypropylene glycol (PG) after a stream of said aqueous phase has broken up into particles. In certain embodiments of the present invention, the 3D printed device is connected to said encapsulation chamber for dispensing said gelling emulsion coaxially to said coated biomaterial. In certain embodiments of the present invention, the 3D printed device is connected to said encapsulation chamber for dispensing a DTT/HBSS/HllT gelling emulsion coaxially to said coated biomaterial.

The methods of this invention, while providing an advantage, do not necessarily provide purification of the coated biomaterials. In such embodiments of the present invention, purification of said conformally coated biomaterial and said biomaterial-free coating material from said oil phase is accomplished by infusing the product from the coating product into mineral oil with stirring. In certain embodiments of these cleaning steps in the method presented herein, Hanks' Balanced Salt Solution (HBSS) is added during the mineral oil cleaning while stirring while continuing to stir the emulsion. In additional embodiments of the present invention, after purification with mineral oil and Hanks' balanced salt solution (HBSS), the product is centrifuged and washed with HBSS. In other embodiments of the present invention, after cleaning and washing with Hanks' Balanced Salt Solution (HBSS), the coated materials are incubated with an IEG-dithiol solution.

In a fourth aspect of the present invention, there is provided a conformal coating apparatus including (1) an encapsulation chamber including (a) a housing portion connected to a catheter through a first inlet at a first end of said housing portion and connected to a first pump;

(b) a fastening portion including a first end configured to engage an inner surface of a second end of said body portion; and (c) a coating portion including a first end configured to engage an outer surface of a second end of said fastening portion, a chamber configured for coating, a second inlet port connected to the second pump, and a second end of said coating portion. coatings;

wherein said catheter coupled to said first pump is configured to inject the coating material and the biomaterial to be coated in an aqueous phase into said first inlet on the inside of said body portion, wherein said aqueous phase has a pH level of exactly or about 6-7.4; and wherein the coating material introduced does not contain a viscosity increasing agent;

wherein said second pump is configured to inject an oil phase containing a surfactant into a second inlet on the outside of said coating portion, wherein the injection of said oil phase is configured to flow coaxially to and outside of said internal aqueous phase;

(2) a capillary connected to said second end of said coating portion, said capillary located downstream of a point where a stream of said aqueous phase contacts and extends within said outer oil phase to form a two-phase fluid that is configured for coaxial flow through said capillary from said coating portion; and (3) a third pump connected to said capillary and configured to inject a gelling emulsion coaxially to said capillary, said emulsion comprising a catalyst for polymerizing said aqueous phase.

In some embodiments of this invention, said first end of said coating portion substantially surrounds said second end of said fastening portion, forming a chamber in which a stream of said aqueous phase contacts said oil phase, forming a two-phase fluid.

In some embodiments of the present invention, said encapsulation chamber includes a conical opening including a cone angle of an outlet nozzle configured to deliver a two-phase liquid into said capillary, and a second inlet port connected to said chamber at a cone angle.

In some embodiments of the present invention, said first pump is a precision flow syringe pump.

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In some embodiments of the present invention, said second pump is a first peristaltic pump.

In some embodiments of the present invention, said third pump is a second peristaltic pump.

In some embodiments, the present invention provides a conformal coating apparatus including an encapsulation chamber formed by a three-piece assembly. A catheter coupled to a precision flow syringe pump is configured to introduce the coating material and the biomaterial to which the coating is applied into a first inlet of said encapsulation chamber (internal phase), wherein said aqueous phase has a pH level of exactly or about 6- 7.4. The first peristaltic pump is configured to inject an oil phase containing a surfactant into a second inlet of said encapsulation chamber, wherein the injection of said oil phase (external phase) is configured to flow coaxially to said internal aqueous phase. A capillary connected to the end of said encapsulation chamber, wherein said capillary is located downstream of a point where the flow of said internal aqueous phase is extended within said external oil phase (two-phase liquid) such that said two-phase liquid is configured to flow into said capillary from said encapsulation chamber, and wherein said internal aqueous phase (containing the coating material and the biomaterial being coated) and said external oil phase are configured to flow coaxially through said capillary. Said device further comprises a second peristaltic pump configured to inject a gelling emulsion coaxially to said capillary, said emulsion comprising a catalyst for polymerizing said coating material, wherein said emulsion is configured to be in coaxial contact with said coating material and said biomaterial being coated. . In some embodiments of the present invention, said conformal coating device further comprises an outlet for releasing air from said device. In some embodiments of the present invention, said conformal coating device may further comprise an outlet for releasing air from said device. In some embodiments of this invention, said air outlet is located upstream of the aqueous phase inlet of said device. Said air outlet is closed during the coating process.

These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It should be noted that the scope of the claims of this invention is determined by the provisions contained therein and not by the specific discussion of features and advantages set forth herein.

Brief description of graphic materials

This patent or patent application contains at least one color image. Copies of this patent or this patent publication with color image(s) will be provided by the authorized patent organization upon request and payment of the necessary fees.

Fig. 1. Schematic illustration of conformal coating modification to eliminate viscosity enhancers (alginate, Matrigel, or amphiphilic self-assembling peptides) to improve the biocompatibility of conformal coating (CC) gels and enable encapsulation at physiological pH to maximize the functionality of non-human primate (NHP) islets. As presented in prior art applications (hereinafter referred to as the low pH method), the aqueous phase consists of a multi-arm PEG, a cross-linker (PEG-dithiol or dithiothreitol, DTT) and viscosity enhancers at pH less than 6 to prevent cross-linking of the PEG and the formation of a gel before the formation of capsules (downstream after the disintegration of the aqueous phase jet). The methods presented herein are performed at physiological pH, with the aqueous phase consisting only of multi-arm PEG that is cross-linked to 130% PEG-SH to achieve optimal viscosity at physiological pH (7.4). 100% PEG cross-linking and gelation are achieved after capsule formation, with an emulsion of DTT in Hanks' balanced salt solution (DTT/HBSS) and polypropylene glycol (PPG) being fed downstream after the breakdown of the aqueous phase jet and coaxially with the output stream of the device. Purification is achieved by dilution with light mineral oil and HBSS with stirring and centrifugation, rather than by extraction with organic solvents such as hexane as in prior art applications. Therefore, the innovation is based on (i) the composition of the aqueous phase containing only islets and pure PEG without any additives, (ii) the introduction of the PEG cross-linking agent after the formation of the capsule by dosing the gelling emulsion using a device, (iii) the method of cleaning the capsule, which does not include organic solvents.

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Fig. 1A. A schematic illustration of a conformal coating apparatus presented herein, including a conformal coating apparatus and a device for dispensing a gelling emulsion. This conformal coater is obtained by assembling three different parts: Panels (ii), (iii) and (iv) are illustrations of the three main components of one version of the conformal coater used with a 16G IV catheter to coat clusters cells 50-250 microns in size. These three parts are assembled and screwed together with a glass capillary to form a complete device. Panel (v) identifies the critical design components of the conformal coating device's internal chamber and the relative performance required to achieve flow conditions that allow the formation of coatings that are conformal around cell clusters. The table shows these relative rates and the specific rates used with a 16G IV catheter to coat cell clusters ranging in size from 50-250 µm.

Fig. 1B. Photograph of the conformal coating device presented in this document.

Fig. 1C. An illustrative image of a 3D printed part presented in this document for dispensing a gelling emulsion.

Fig. 2. Evaluation of conformally coated (CC) islets from non-human primates (NHPs) in vitro and in vivo. NNP islands from cynomolgus monkeys were conformally coated (CP) with PEG/PEG-dithiol (PEG-SH)/peptide hydrogels according to a low-pH method. Panel A shows phase contrast microscopy images of CP NPP islands, showing that the large islands had very thin and potentially incomplete coatings. Panel B: Functionality of small (<200 μm) and large (>200 μm) islets from uncoated (BC) and conformally coated (CP) NCPs based on static glucose-stimulated insulin secretion (SGSI), based on sequential stimulation with glucose concentrations 2.2 mM (1 h, L1), 16.7 mM (1 h, H), 2.2 mM (1 h, L2), and 25 mM KCl (1 h); demonstrated higher functionality of small islets (arrows), but reduced insulin secretion by PC NHP islets compared with uncoated islets. Functionality was assessed by secreted insulin (left), GISI index (ratio of insulin secretion when stimulated with glucose at a concentration of 16.7 mM and at a concentration of 2.2 mM: H divided by L1) and GISI difference (difference between insulin secretion when stimulated with glucose in concentration of 16.7 mM and at a concentration of 2.2 mM: H minus L1). Panel C: Islet Functionality of PC NHPs Based on Dynamic Glucose-Stimulated Insulin Secretion (Perifusion); demonstrated decreased insulin secretion for CP NSP islets compared to uncoated islets, but the stimulation index was 2.6, indicating functionality. Panels D-G: Functionality of PC NSP islets in vivo compared with uncoated islets after transplantation of 2000 or 4000 islet cell equivalents (IECs)/mouse into the epididymal adipose tissue (EAT) or renal capsule (RP) of diabetic NSG mice or NODscid mice based on blood glucose (panels D, F, G) and random human C-peptide in serum 7 days after islet transplantation (panel E). Shown are data from three independent NHP islands: batch #1 (panels D, E); batch #2 (panel F); batch #3 (panel G).

Fig. 3. Effect of coating solution pH on the viability of insulin-secreting pancreatic islets (nuclei, cytoplasm of live cells and nuclei of dead cells were stained according to the live/dead cell assay); demonstrated a decrease in viability with decreasing pH.

Fig. 4. Evaluation of PC islets of NHPs (low pH method, PEG/PEG^I/peptide hydrogels) in the omental bursa of NHPs with diabetes mellitus. Panel A shows the feasibility of a laparoscopic procedure for immobilizing the islets of the CP of the CP on the omental surface of the cynomolgus monkey using biological scaffolds. Panel B shows exogenous insulin requirement (PEI, dotted line), blood glucose level (dots), and C-peptide of the PC NHP islet recipient, demonstrating minimal function of the NHP KP islets. Panels C and D show histological evaluation of explanted OCP omental grafts analyzed for islet survival (insulin) and capsule immunoisolation from T cells (CD3, green label) (Panel D; arrows indicate staining for insulin (red label)) and biocompatibility (panel C, arrows indicate multinucleated gigantocytes). The inflammatory reaction to the capsule in the omental area of the ulcer was extensive, chronic and active.

Fig. 5. In vitro and in vivo evaluation of the conformal coating method presented herein (pure PEG and physiological pH) with human islets. The quality of the coating was assessed using phase contrast microscopy (panel A); viability of coated islets by live/dead cell staining and confocal microscopy (panel B); function in vitro - based on static (panels C, E) and dynamic (perifusion, panels D, F) glucose-stimulated insulin secretion by islets from three different donors and two batches

- 8 046872 encapsulation (batch No. 1: C, D; batch No. 2: E, F); function in vivo - by monitoring blood glucose (panel G), assessing glucose tolerance by intraperitoneal glucose testing (panel H) and stimulated human C-peptide (panel I) after transplantation of 4000 EOC/mouse human islets without or with CP into fat pad of immunodeficient NSG (NOD scid gamma) mice with diabetes mellitus.

Fig. 6. Transplantation of human PC islets using the physiological pH method presented herein into the omental bursa of diabetic NCP islets with costimulatory blockade.

(Panel A) Feasibility of a laparotomy procedure for implantation of CP islets into the bursa omentum of a diabetic cynomolgus monkey. (Panel B, Fig. 6C) Exogenous insulin requirement (PEI, solid line), blood glucose level (dots), C-peptide, and summary table (Panel C) for KP NHP islet recipient demonstrating minimal function of transplanted KP islets person when blood glucose levels decrease. (Panel D, Panel E, Panel F) Histological evaluation of explanted KP NCP grafts analyzed by hematoxylin-eosin staining for biocompatibility (panels D, E) and graft revascularization (panel E, CD31: blood vessels; anti-SMA staining: fibrosis and/or maturity of blood vessels). The inflammatory reaction to the capsule in the omental bursa of the NCP was chronic and active, but moderate.

Fig. 7. Transplantation of syngeneic islets from a Lewis rat into the omentum of an immunocompetent Lewis rat with diabetes mellitus. Panel A: Blood glucose from Lewis rat islet recipients without coating (solid line, dotted markers) and with conformal coating (dashed line, square markers). (Panel B) Postoperative day (POD) 30: intraperitoneal glucose tolerance test (IPGT) in Lewis rat islet recipients without coating (solid line, dotted markers) and conformally coated (dashed line, square markers), and (Panel C ) areas under the VBTTG curves. (Panel D) Fasting and post-stimulation C-peptide levels during VBTHT in uncoated (solid line, dotted markers) and conformally coated (dashed line, square markers) Lewis rat islet recipients. Statistical analysis confirms the in vivo functionality of syngeneic rat CP islet grafts comparable to that of uncoated grafts in the rat omentum.

Fig. 8. Transplantation of allogeneic clusters of MIN6 insulinoma cells into NOD mice with spontaneous diabetes mellitus. (Panel A) Phase contrast microscopy images of uncoated (top row) and conformally coated (middle and bottom rows) MIN6 clusters. (Panel B) Confocal microscopy images of conformally coated MIN6 clusters, anti-PEG stained, and adjacent orthogonal views showing capsule completeness in three dimensions. (Panel C) Mortality of recipients of uncoated (solid line, dotted markers) and conformally coated (dashed line, square markers) MIN6 clusters demonstrating improved survival of KP clusters. (Panel D) Nonfasting levels of random C-peptide in recipients of uncoated and conformally coated MIN6 clusters, demonstrating persistence of C-peptide in KP clusters but not in uncoated clusters. (Panel E) Histological evaluation of explanted MIN6 KP cluster grafts analyzed by hematoxylin-eosin staining for survival of cell clusters within KP capsules and biocompatibility assessment demonstrating that KP capsules provide immunoisolation of allogeneic cell clusters in the presence of both alloresection and autoimmunity.

Detailed description of the invention

Definitions and general techniques

Unless otherwise defined herein, scientific and technical terms used in this application have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, biochemistry, immunology, microbiology, genetics and related fields as described herein, and their methods are within the scope of the prior art. In the event of any conflict, this patent specification, including definitions, will control.

This application makes reference to various issued patents, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the references included herein and this description, the terms of this description will apply. In addition, any specific embodiment of the present invention that is prior art may be expressly excluded from any one or more claims of the present invention. Since such embodiments are considered known to one skilled in the art, they may be excluded even if the exclusion is not expressly stated herein. Any specific embodiment of the present invention may be

- 9 046872 is excluded from any claim of this invention for any reason, regardless of whether it is related to the existence of prior art or not.

The term as used herein means the entirety of this application.

It should be understood that any of the embodiments of this invention described herein, including those described in various aspects of this specification and various parts of this document (including embodiments of this invention described in the examples only), can be combined with one or a large number of other embodiments of the present invention, unless expressly stated otherwise or otherwise prohibited. The combinations of the embodiments of the present invention are not limited to those specific combinations that are claimed in the numerous dependent claims of the present invention.

In addition, this invention covers all variations, combinations and permutations in which one or more limitations, elements, disclaimers and descriptive terms from one or more of the listed claims of this invention are introduced into another claim of this invention. For example, any claim of this invention that is dependent on another claim of this invention may be modified to include one or more of the limitations found in any other claim of this invention that is dependent on the same basic claim of this invention. . When elements are presented as lists, such as the Markush group format, each subgroup of elements is also expanded, and any element(s) may be removed from that group.

Throughout this specification and embodiments of this invention, the phrase include or variations thereof, such as include(s) or including (including, including, including) should be understood to imply the inclusion of the specified integer (or components) or a group of integers (or components), but not to the exclusion of any other integer (or components) or group of integers (or components).

Throughout this document, when compositions or devices are described as having, containing, including, or including (or variations of these statements) specific components, it is intended that such compositions may also consist essentially of, or consist of, the specified components . Likewise, when methods or processes are described as having, containing, including, or involving specific process steps, such processes may also consist essentially of, or consist of, the enumerated process steps. In addition, it should be understood that the order of steps or the order in which certain actions are performed does not matter as long as the compositions, devices and methods described herein remain operational. Moreover, two or more steps or actions may be performed simultaneously. For the sake of simplicity, such embodiments of the present invention have not been specifically set forth verbatim herein.

The term including is used to mean including, but not limited to. Including and including but not limited to are used interchangeably. Therefore, these terms should be understood to mean the inclusion of the specified integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

When used in the context of this document, about or about means within the acceptable range of errors for a particular value as determined by those of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. from the limitations of the measuring system.

When used in the context of this document, the singular form (especially in the context of the claims of this invention set forth below) includes reference to both the singular and the plural, unless the context clearly indicates otherwise or the context clearly contradicts it.

When used in the context of this document, the term or should be understood to mean and/or unless the context clearly indicates otherwise.

Any example(s) following the term for example or by way of example is not intended to be exhaustive or limiting.

The ranges of values given herein are intended only to serve as a shorthand way of individually referring to each individual value falling within that range unless otherwise stated herein, and each individual value is included herein as if stated individually in this document. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use in the context of this document of any and all examples or illustrative language (for example, such as) is intended only to better illuminate embodiments of this invention and does not impose a limitation on the scope of the claims of this invention, unless otherwise indicated. None of them

- 10 046872 language in this document should not be construed as indicating any non-stated element as material.

As used herein, the term conformal coating, or variations thereof such as conformally coated and conformally coated, refers to a uniform layer of coating material (e.g., typically up to about 100 μm thick) that surrounds the outer surface of the biomaterial and conforms to its shape and size. The conformal coating is relatively monodisperse and does not depend on the size of the biomaterial. The conformal coating has a fixed and constant thickness. This is unlike traditional microencapsulation performed by droplet generation method, in which the size of the capsules is fixed and the thickness depends on the size of the encapsulated biomaterials.

The terms aqueous phase and aqueous phase are used interchangeably herein and refer to a liquid phase that contains water and may further contain hydrophilic cosolvents and water-soluble substances. Examples of the aqueous phase include, but are not limited to, water itself, aqueous buffers, cell culture media, solutions of peptides, proteins and carbohydrates in water, and organ preservation solutions.

When used in the context of this document, the term oil phase means a liquid phase that is immiscible with the aqueous phase or aqueous phase. Examples of oil phase include, but are not limited to, polypropylene glycol (PG), mineral oils (e.g., mineral oil having a viscosity that is at least 2.5 times that of the aqueous phase), any high viscosity (e.g., 1300 kN ) a liquid that does not mix with water.

As used herein, drip atomization, when applied to an aqueous phase, refers to the process by which the aqueous phase is allowed to pass through an opening, opening or aperture so that droplets are formed. The term also includes a process by which a continuous stream of aqueous phase is converted into droplets by mechanical, ultrasonic or other comparable means.

As used herein, the term aqueous phase jetting refers to the process by which a stream of liquid (eg, aqueous phase) is expelled at high speed from an opening, opening, or aperture. High speed neutralizes differences in surface tension. Jet atomization refers to the extension of the flow such that the diameter of the flow of the aqueous phase decreases as it exits the injection port by at least one order of magnitude.

As used herein, the term elongation, as applied to the aqueous phase, refers to the process by which the length of a fluid stream (eg, aqueous) is increased or lengthened while its cross-sectional diameter is decreased. The extension allows you to switch from drip spray to jet spray.

The present invention provides reagents, apparatus, and methods for immunoisolating biomaterials, such as cells and cell clusters, to prevent immune rejection, inflammation, and/or autoimmune destruction while maintaining cell functionality when said biomaterials are implanted in a subject.

The conformal coating technology presented in this document allows cell clusters of variable sizes (diameter 50-350 μm, including insulin-producing cells and primary islets) to be coated with a hydrogel ranging from a few microns to tens of microns in thickness, with a thickness of coating was relatively monodisperse and independent of the diameter of cell clusters (Tomei et al., 2014, Proc. Nat. Acad. Sci. USA 111: 10514-19). Significant advances in this technology include (i) minimizing capsule thickness, which maximizes nutrient/glucose/insulin transport to coated cells, maximizing cell viability, and minimizing delays in glucose-stimulated insulin secretion (Buchwald et al., 2018, Biotechnol . Bioeng. 115: 232-245), and (ii) minimizing the volume of encapsulated cell graft, which allows transplantation in limited areas of the body that can only accommodate limited volumes, including prevascularized sites and devices for local immunomodulation. See Tomei et al., 2015, Expert. Opin. Biol. Ther. 15: 1321-1326.

Conformal coating is achieved by injecting an aqueous phase containing the coating solution and cell clusters coaxially into a larger chamber (~10 times larger in cross-sectional diameter) containing a flowable, immiscible oil mixture (made from, among other things, high-viscosity polypropylene glycol ( PPG) and Span80 - a commercially available (Sigma Aldrich) nonionic surfactant containing oleic acid (C18:1) <60%; a balance of predominantly linoleic (C18:2), linolenic (C18:3) and palmitic (C16:0). ) acids) in an encapsulation device having a flow focusing geometry after introducing the aqueous phase into the oil phase. This geometry allows the two-phase flow to be extended and, if the viscosity of the aqueous phase is high enough (Tomei et al., 2014, Proc. Nat. Acad. Sci. USA.111: 10514-10519), a jet atomization of the aqueous phase occurs within the oil phase, as indicated V

- 11 046872 US Patent No. 10653816, US Patent No. 10660987 and International Patent Application No. PCT/US 2012/035696, the contents of each of these patent documents are hereby incorporated herein by reference in their entirety (hereinafter - prior art applications).

As used herein, the term jetting refers to the extension of the flow such that the diameter of the aqueous phase flow decreases as it exits the injection port by at least one order of magnitude. After jet atomization of the aqueous phase, the phenomenon of Plateau-Rayleigh instability results in the disintegration of said aqueous phase into nanoliter-sized droplets and hence the coating of clusters of cells contained in said aqueous phase. In this device, the encapsulation chamber is connected to a 10 cm long glass capillary with a 1 mm diameter hole through which two-phase liquids continue to flow; A collection vessel is located under the tip of the glass tube to collect the output flow.

As indicated in the examples from the prior art applications, the aqueous phase consisted of: multi-arm PEG (10 kDa, 8 arms, 75-90% functionalized PEG-maleimide (PEG-MAL) or PEG-vinyl sulfone (PEG-VS)), excess crosslinking agent (dithiothreitol and 2 kDa PEG-dithiol at a molar ratio of 3-4 to 1) 1, 8-arm PEG-MAL or PEG-BC) to achieve complete gelation of PEG; this aqueous phase also included additives to achieve a sufficiently high viscosity of this aqueous phase (including, but not limited to, alginate, amphiphilic self-assembling polymers or peptides, etc.). As presented in the prior art applications, it is necessary that the pH of the aqueous phase in the given example be maintained at a level of 3.5-6 (depending on the specific hydrogel composition) to prevent premature gelation of the aqueous phase before coating, which occurs after jet spraying and degradation of the aqueous phase in the encapsulation chamber due to the high reactivity of the Michael-type reaction occurring between the multi-arm PEG and the thiolated cross-linkers. To collect the effluent, a 50 mL conical tube was placed under a glass tube and filled with PPG and base (triethanolamine) to increase the pH of the aqueous phase and accelerate the gelation of the aqueous phase after collection and before purification. Purification was performed by extracting the gelled aqueous phase (PEG hydrogel-coated cell clusters and empty PEG gel) using hexane. Using this method, as presented in prior art applications, the in vitro and in vivo functionality of conformally coated primary islets, conformally coated stem cell-derived insulin secreting cells, and conformally coated human kidney epithelial cells has been demonstrated to presented in this document. These conformally coated cells were found to be able to reverse diabetes in mice without the need for immunosuppression. See Tomei et al., 2014, Ibid.; Manzoli et al., 2018, Ibid; Stock et al., 2020, Stem Cell Reports 14: 91-104.

However, low pH of the aqueous phase during encapsulation has been found to reduce the functionality of primary coated islets (eg, their viability), especially for non-human primate islets (as shown in the examples presented herein). Also, the aqueous phase used in low pH processes must contain both a PEG cross-linker and a viscosity enhancing additive (alginate, amphiphilic self-assembling polymers or peptides, etc.) to achieve the required viscosity for jetting of the aqueous phase and its collapse. Some of these additives reduce the biocompatibility (as demonstrated herein) and immunoisolation of hydrogel coatings in animal models, as demonstrated by Manzoli et al., 2018, Am. J. Transplant. 18: 590-603.

The modified conformal coating procedure presented herein allows the uncoupling of the base polymer (10 kDa 8-arm 75% functionalized PEG-maleimide or PEG-vinyl sulfone) and the total amount of cross-linker (dithiothreitol and 2 kDa PEG-dithiol) required to achieve gelation of PEG (molar ratio of crosslinker to multi-arm PEG is 3 to 1) into hydrogels from the aqueous phase containing cell clusters, and further eliminates any need for viscosity additives in the aqueous phase (as illustrated herein in FIG. 1 - Method at physiological pH). As described herein, deposition of the functional conformal coating was achieved using an aqueous phase consisting of multi-arm PEG minimally cross-linked by adding a 20% mol/mol required amount of cross-linker, which allowed PEG to gel into the hydrogels. Minimally cross-linked PEG (e.g., 1-30% cross-linking) has sufficient viscosity to allow the aqueous phase to jet out from the coaxial oil phase (made from polypropylene glycol (Mn ~4000), PPG with 10% Span80, or a surfactant having equivalent properties, especially in relation to a decrease in interfacial tension) and disintegrate, leading to the formation of a conformal coating around cells and/or cell clusters. Complete cross-linking of PEG is achieved after coating cells and/or cell clusters following jetting and aqueous phase disintegration in the encapsulation chamber.

- 12 046872

This is now achieved by passing a gelling emulsion of 1:15 (v:v) 25 mg/ml DTT (dithiothreitol) in HBSS-/- : PPG/10% Span80 downstream of the portion of the encapsulation chamber where jet disintegration using a device as shown in FIG. 1, 1A and 1B. As indicated in the examples from the prior art applications, the pH of the aqueous phase must be maintained at acidic values to prevent premature gelation of the aqueous phase before coating, which occurs after jetting and disintegration of the aqueous phase in the encapsulation chamber. However, the low pH aqueous phase during encapsulation reduced the functionality of the coated primary islands (as illustrated herein in the examples and in particular in Figure 2). Moreover, the aqueous phase, as indicated in the examples from the prior art applications, had to contain PEG, a cross-linking agent and an additive (i.e., viscosity increasing agent; alginate, amphiphilic self-assembling polymers or peptides, etc.) to achieve the required viscosity to ensure jet spraying of the aqueous phase and its disintegration. These additives reduced the biocompatibility and immunoisolation of the coating hydrogels in animal models, as shown in the examples presented herein and FIGS. 2 and 3. Increasing the pH of the aqueous phase to near physiological values (~6-7.4) and eliminating any viscosity enhancers improved the functionality of the encapsulated islets and the biocompatibility of the coating, as shown in the examples presented herein and FIGS. 4-8.

Therefore, in some embodiments of the present invention, a method of conformally coating biomaterials with a coating material is provided, comprising the following steps: (a) injecting an aqueous phase into a coaxial oil phase in a coating device configured to provide a transition from droplet spray to jet spray and extending the flow of said aqueous phase in said oil phase; (b) adding said biomaterial and said coating material to said aqueous phase, wherein the coating material from said step (b) does not contain a viscosity increasing agent; and wherein said aqueous phase has a pH of from about 6 to about 7.4; (c) allowing the stream of said aqueous phase to disintegrate into particles; and (d) adding a component of said coating material after the jet of said aqueous phase has disintegrated into particles, said component being a gelling emulsion that promotes or catalyzes the polymerization of said coating material; As a result, a biomaterial with a conformal coating is obtained. Examples of viscosity increasing agents include, but are not limited to, alginate, amphiphilic self-assembling polymers or peptides, etc.) In some embodiments of the present invention, a viscosity increasing agent that is excluded from the methods described herein is selected from polysaccharides such as alginate, decellularized tissues, nanomaterial assemblies based on PEG, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water-soluble cellulose derivatives, gelatin, collagen and albumin.

In some embodiments of the present invention, the method further includes the step of collecting material flowing from the coating device (ie, the conformally coated biomaterial and any coating material that does not contain the biomaterial).

In some embodiments of the present invention, said method further includes the step of purifying said conformally coated biomaterial and said biomaterial-free coating material from said oil phase.

In some embodiments of the present invention, the method further includes the step of separating said conformally coated biomaterial from said non-biomaterial coating material.

In some embodiments of the present invention, purifying the conformally coated biomaterial and any non-biomaterial coating material from the oil phase includes step (e) pouring the product from step (d) of the method above into mineral oil while stirring the resulting mixture (i.e., a conformally coated biomaterial and any coating material that does not contain a biomaterial, an oil phase, and a gelling emulsion (containing dithiothreitol (DTT) solution dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80) in mineral oil).

In some embodiments of the present invention, purification of the conformally coated biomaterial and the biomaterial-free coating material from the oil phase includes step (f) adding Hanks' Balanced Salt Solution (HBSS) to the product obtained in step (e) (i.e. a conformally coated biomaterial and any coating material that does not contain a biomaterial, an oil phase and a gelling emulsion (containing dithiothreitol (DTT) solution dissolved in Hanks' balanced salt solution (HBSS) and emulsified in

- 13 046872 polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80) in mineral oil) leaving the specified device, and mineral oil).

In some embodiments of the methods described herein, the product from step (f) is centrifuged and washed in HBSS.

In some embodiments of the methods described herein, after centrifugation and washing with Hanks' Balanced Salt Solution (HBSS), the coated biomaterial and any non-biomaterial material are incubated with a PEG-dithiol solution.

In some embodiments of the present invention, the viscosity increasing agent, which is excluded from the methods described herein, is selected from polysaccharides such as alginate, decellularized tissue, PEG nanomaterial assemblies, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate , heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water-soluble cellulose derivatives, gelatin, collagen and albumin.

In some embodiments of this invention, the size of said biomaterial is smaller than the size of said opening where said aqueous phase is injected into said oil phase.

The methods of this invention can be used to encapsulate any material that would benefit from immunoisolation when implanted into a subject. The specified material may be non-uniform in shape. In some embodiments of the present invention, said material that may benefit from immunoisolation is a biomaterial. In some embodiments of the present invention, the biomaterial comprises cells, clusters of cells, biomaterial-coated cells or clusters of cells, subcellular organelles, biological molecules, non-biological drugs, or a combination thereof. In some embodiments, the methods of this invention are used to encapsulate one or more of the following: cells, clusters of cells, subcellular organelles, biological agents such as proteins, nucleic acids and antibodies, and non-biological agents (e.g., small molecules) such as medications. In some embodiments of this invention, said biomaterial is cells. In some embodiments of this invention, said biomaterial is clusters of cells. In some embodiments of the present invention, said biomaterial is cells and clusters of cells. In some embodiments of the present invention, said biomaterial is subcellular organelles. In some embodiments of the present invention, said biomaterial is a protein. In some embodiments of the present invention, said biomaterial is nucleic acids. In some embodiments of the present invention, said biomaterial is an antibody. In some embodiments of the present invention, the methods of the present invention are used to encapsulate cells and/or clusters of cells. In some embodiments of the present invention, the methods of the present invention are used to encapsulate pancreatic islet cells and clusters of pancreatic islet cells.

In certain embodiments of the present invention, said conformally coated cells or clusters of conformally coated cells may include one or more of the following: autologous, heterologous, syngeneic, allogeneic or xenogeneic pancreatic islets, alone or in combination with other cell types ( e.g., Sertoli cells, mesenchymal and bone marrow-derived cells, endothelial progenitor cells, stem cells, Treg regulatory T cells, etc., each of which are referred to generically as implantable support cells) that provide growth factors and/or other beneficial agents to generate, maintain or increase the number of said conformally coated cells, or otherwise assist said conformally coated cells to exert a therapeutic effect when implanted into a host. In some embodiments of the present invention, said support cells are mesenchymal stem cells.

When used in the context of this document, the term host refers to the recipient of the implanted biomaterial and includes all animals. In some embodiments of the present invention, said host organism is a mammal. In an illustrative embodiment of the present invention, said host organism is a human.

The methods of this invention can be advantageously used for conformal coating in model cell therapy systems. Conformally coated cells can exert therapeutic effects, for example, by expressing a therapeutic factor in vivo after implantation. Examples of such cells include, but are not limited to, cells that produce: insulin for the treatment of diabetes; dopamine for the treatment of Parkinson's disease

- 14 046872 (Minquez-Castellanos et al., 2007, J Neurol Neurosurg Psychiatry 78: 825-831); growth hormone for the treatment of dwarfism (Chang et al., 1999, Trends Biotechnol 17: 78-83); factor VIII and factor IX (Chang et al., 1999, Trends Biotechnol 17, 78-83) for the treatment of hemophilia; and erythropoietin for the treatment of anemia (Rinsch et al., 2002, Kidney Intern 62: 1395-1401). Many more beneficial factors produced by cells or cellular/tissue functions can be imagined. In some embodiments of the present invention, conformally coated cells may express and/or deliver more than one therapeutic factor, or may include two or more cell types delivering one or more therapeutic factors. In some embodiments of the present invention, the conformally coated cells also or alternatively express and/or deliver an antagonist, agonist, analog, derivative, chimera, fusion molecule, or fragment of a therapeutic factor to provide a therapeutic effect when implanted into a host.

In some embodiments of the present invention, at least some of the conformally coated cells also or alternatively provide a therapeutic effect without releasing a diffusible factor. In certain embodiments of the present invention, the conformally coated cells provide enzymatic activity that, for example, converts a substrate into a beneficial product and/or metabolizes, binds, or absorbs a harmful substance. In certain embodiments of the present invention, said conformally coated cells exert a therapeutic effect via a factor associated with a biological material, such as a cell surface associated factor.

In some embodiments of the present invention, the conformally coated cells naturally produce a therapeutic effect, without genetic modification, when implanted into a host. In some embodiments of the present invention, said conformally coated cells are genetically engineered to provide a therapeutic effect. By way of non-limiting examples, the cells may be transfected with expression vectors or transduced with lentiviral vectors that render the cells capable of expressing one or more therapeutic and/or auxiliary cellular factors. In another embodiment of the present invention, said cells may include, consist of, or consist essentially of cells transfected with expression vectors that render said cells capable of expressing one or more therapeutic and/or auxiliary cellular factors. Such expression may be constitutive or regulated, for example, in response to biological modulators in the bloodstream or tissues to which the cells are exposed.

In some embodiments of the present invention, the cells to which the conformal coating will be applied are derived from cadaveric tissue or living tissue. In some embodiments of the present invention, the cells are mammalian or non-mammalian, human or non-human, autologous or allogeneic. These cells may be pluripotent, multipotent, totipotent or differentiated embryonic or adult stem cells; primary differentiated cells; or immortalized cells, among other cell types. In certain embodiments of the present invention, stem cells include, for example, cells derived from umbilical cord blood, amniotic fluid, menstrual blood, placenta, Wharton's jelly, cytotrophoblasts, etc. These cells may also include any combination of the types listed above cells.

Exemplary therapeutic factors that may be provided by conformally coated cells include, but are not limited to, one or more of the following: insulin, glucagon, erythropoietin; factor VIII; factor IX; hemoglobin; albumen; neurotransmitters such as dopamine, gamma-aminobutyric acid (GABA), glutamic acid, serotonin, norepinephrine, epinephrine and acetylcholine; growth factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), cholinergic differentiation factor/leukemia inhibitory factor (CDF), epidermal growth factor (EGF) , eng. EGF), insulin-like growth factor (IGF, eng. IGF), fibroblast growth factor (Eng. FGF) and platelet-derived growth factor (TGF, eng. PDGF); pain inhibitors such as substance P, catecholamines, dynorphins, endorphins or enkephalins; hormones such as parathyroid hormone or growth hormone; immunomodulators such as granulocyte-macrophage colony-stimulating factor (GM-CSF); neuromodulators; lymphokines; cytokines; cofactors; antibodies; aptamers; and enzymes. Selection of one or more therapeutic factors and the concentrations at which they are produced and

- 15 046872 released from cells is dictated by the needs of the patient undergoing treatment and can be easily determined empirically by a qualified practitioner.

In some embodiments of the present invention, the conformally coated cells produce a therapeutic factor having insulin-like or insulin-regulatory activity. In certain embodiments of the present invention, said therapeutic factor is insulin. In certain embodiments of the present invention, said therapeutic factor is a precursor form of insulin, such as preproinsulin or proinsulin. In certain embodiments of the present invention, said therapeutic factor is a chimeric or fusion protein of insulin.

In some embodiments of the present invention, the therapeutic benefit provided by the conformally coated cells includes regulating blood insulin levels. In certain embodiments of the present invention, said therapeutic effect includes regulating blood glucose levels. In other embodiments of the present invention, said therapeutic effect includes regulating the levels of one or more other biological response regulators in the patient's blood.

In some embodiments of the present invention, the therapeutic factor(s) are released from the conformally coated cells upon receipt of a stimulus or signal. For implanted cells, said stimulus or signal may be received from the host (eg, changes in blood glucose levels, hormones, metabolic signaling agents, chemical signaling molecules, etc.).

In some embodiments of the present invention, the cells and/or clusters of cells according to the present invention are generally uniform in size. In other embodiments of the present invention, the cells and/or clusters of cells according to the present invention are not uniform in size. In certain embodiments of the present invention, the diameter of said cells and/or cell clusters ranges from about 10 microns to about 10,000 microns; from about 25 μm to about 500 μm; or from about 40 μm to about 400 μm. In certain embodiments of the present invention, the diameter of said cells and/or cell clusters ranges from 10 μm to 10,000 μm; from 25 µm to 500 µm; or from 40 µm to 400 µm. In certain embodiments of the present invention, the diameter of said cells and/or cell clusters ranges from about 10 microns to about 10,000 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells varies from 10 microns to 10,000 microns. In certain embodiments of the present invention, the diameter of said cells and/or cell clusters ranges from about 25 microns to about 500 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells varies from 25 microns to 500 microns. In certain embodiments of the present invention, the diameter of said cells and/or cell clusters ranges from about 40 microns to about 400 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells varies from 40 μm to 400 μm. In specific embodiments of the present invention, the diameter of these cells and/or clusters of cells varies from about 50 to about 300 microns. In specific embodiments of the present invention, the diameter of said cells and/or clusters of cells varies from 50 to 300 microns. In certain embodiments of the present invention, the diameter of islet cells and/or islet cell clusters ranges from about 10 microns to about 10,000 microns. In certain embodiments of the present invention, the diameter of islet cells and/or islet cell clusters ranges from 10 μm to 10,000 μm. In certain embodiments of the present invention, the diameter of islet cells and/or islet cell clusters ranges from about 25 microns to about 500 microns. In certain embodiments of the present invention, the diameter of islet cells and/or islet cell clusters ranges from 25 μm to 500 μm. In certain embodiments of the present invention, the diameter of islet cells and/or islet cell clusters varies or ranges from about 40 μm to about 400 μm. In certain embodiments of the present invention, the diameter of islet cells and/or islet cell clusters varies or ranges from 40 μm to 400 μm. In a specific embodiment of the present invention, the diameter of islet cells and/or islet cell clusters ranges from about 50 to about 300 microns. In some embodiments of the present invention, islet cells and/or clusters of islet cells, the diameter of which varies from about 50 to about 300 microns, contain islet cells. In some embodiments of the present invention, cells and/or clusters of cells, the diameter of which varies from 50 to 300 microns, contain islet cells.

In some embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 , 600, 650, 700, 750, 800, 850, 900, 950 or 1000 µm. In some embodiments of the present invention, the diameter of said cells and/or cell clusters is greater than about

- 16 046872 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 50 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 60 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 70 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 80 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 90 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 100 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 150 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 200 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 250 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 300 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 350 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 400 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 450 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 500 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 550 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 600 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 650 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 700 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 750 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 800 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 850 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than about 900 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 950 microns. In some embodiments of the present invention, the diameter of said cells and/or clusters of cells is up to about 1000 microns.

In some embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 microns. In some embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 40 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 50 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 60 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 70 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 80 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 90 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 100 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 150 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 200 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 250 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 300 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 350 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 400 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 450 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 500 microns. In certain embodiments of this invention, the diameter of said cells and/or

- 17,046,872 cell clusters are more than 550 µm. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 600 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 650 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 700 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 750 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 800 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 850 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 900 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is greater than 950 microns. In some embodiments of the present invention, the diameter of these cells and/or clusters of cells is up to 1000 microns.

In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 40 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 50 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 60 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 70 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 80 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 90 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 100 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 150 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 200 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 250 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 300 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 350 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 400 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 450 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 500 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 550 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 600 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 650 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 700 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 750 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 800 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 850 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 900 microns. In certain embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 950 microns. In some embodiments of the present invention, the diameter of said cells and/or clusters of cells is about 1000 microns.

The coating materials used in the conformal coating methods of this invention are biocompatible and mechanically and chemically stable. In addition, materials suitable for conformal coating do not interfere with or significantly affect the function of the encapsulated biomaterial and reduce, minimize or eliminate the immune response when the encapsulated biomaterial is implanted into a host. In certain embodiments of this invention, said coating material may be polymerized by internal gelation. In certain embodiments of this invention, the material used in the conformal coating methods of this invention is biodegradable.

- 18 046872

In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG), polyethylene oxide (PEO), poly(N-vinylpyrrolidinone) (PVP), polyethyloxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK), poly(amino acids), derivatives thereof or combinations thereof. In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG), PEG maleimide, PEG acrylate, PEG vinyl sulfone, or combinations thereof. In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG), PEG maleimide, PEG acrylate, or PEG vinyl sulfone. In some embodiments of this invention, said coating material comprises polyethylene glycol (PEG). In some embodiments of this invention, said coating material contains PEG-maleimide. In some embodiments of this invention, said coating material comprises PEG acrylate. In some embodiments of this invention, said coating material contains PEGvinyl sulfone.

In some embodiments of the present invention, said coating material is selected from polyethylene glycol (PEG), polyethylene oxide (PEO), poly(N-vinylpyrrolidinone) (PVP), polyethyloxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK), poly(amino acids), their derivatives and their combinations. In some embodiments of the present invention, said coating material is polyethylene glycol (PEG), PEG maleimide, PEG acrylate, PEG vinyl sulfone, or combinations thereof. In some embodiments of this invention, said coating material is polyethylene glycol (PEG), PEGmaleimide, PEG acrylate, or PEG vinyl sulfone. In some embodiments of this invention, said coating material is polyethylene glycol (PEG). In some embodiments of this invention, said coating material is PEGmaleimide. In some embodiments of this invention, said coating material is PEG acrylate. In some embodiments of this invention, said coating material is PEG vinyl sulfone.

In some embodiments of this invention, said coating material comprises one or more of the following: polyethylene glycol (PEG), polyethylene oxide (PEO), poly(N-vinylpyrrolidinone) (PVP), polyethyloxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK), and (or) poly(amino acids).

In certain embodiments of this invention, said covering material is single-arm. In certain embodiments of this invention, said coating material is multi-arm. In certain embodiments of this invention, said cover material is a mixture of single-arm material and multi-arm material. In some embodiments of this invention, the coating material comprises a multi-arm polyethylene glycol (PEG) minimally cross-linked (5-50%) with PEG-dithiol. In some embodiments of this invention, said coating material comprises a multi-arm polyethylene glycol (PEG) minimally cross-linked (1-30%) with PEGdithiol. In some embodiments of this invention, said coating material contains 5-10% PEG. In some embodiments of this invention, said aqueous phase contains a serum-free medium having a pH of 6-7.4; or Hanks' balanced salt solution (HBSS), having a pH of 6-7.4; in particular, the pH of the aqueous phase is represented as pH 6-7.4. In some embodiments of the present invention, said aqueous phase has a pH of from about 6 to about 7.4. In some embodiments of this invention, said aqueous phase has a pH ranging from 6 to 7.4.

In some embodiments of this invention, said coating material comprises minimally cross-linked PEG-MAL. In some embodiments of this invention, the coating material contains PEG-MAL (10 kDa, 8 arms, 75% functionalization) mixed with PEG-SH. In some embodiments of this invention, said coating material comprises PEG-MAL (10 kDa, 8 arms, 75% functionalization) and PEG-SH. In some embodiments of this invention, said coating material comprises PEG-MAL (10 kDa, 8 arms, 75% functionalization), PEG-SH and HBSS. In some embodiments of this invention, said coating material contains 12.5% w/v. PEG-MAL (10 kDa, 8 arms, 75% functionalization), mixed with 10X PEG-SH. In some embodiments of this invention, said coating material contains 12.5% w/v. PEG-MAL (10 kDa, 8 arms, 75% functionalization) and PEG-SH. In some embodiments of this invention, said coating material contains 12.5% w/v. PEG-MAL (10 kDa, 8 arms, 75% functionalization), PEG-SH and HBSS.

In some embodiments of this invention, said aqueous phase contains a thiolated reagent, a reducing reagent, a surfactant, or a combination thereof. In some embodiments of this invention, the aqueous phase used in practicing the methods of this invention may optionally contain one or

- 19 046872 more thiolated reagents, reducing reagent and (or) surfactant. In certain embodiments of this invention, said surfactant is a polyoxyethylene-polyoxypropylene block copolymer or poly(ethylene glycol-N-propylene sulfide), more specifically a 2% polyoxyethylene-polyoxypropylene block copolymer. In some embodiments of this invention, said thiolated or reducing reagent is dithiothreitol (DTT) or PEGdithiol. In some embodiments of this invention, said thiolated or reducing reagent in said aqueous phase is 0.01-0.62% dithiothreitol (DTT).

In some embodiments of the present invention, said gelling emulsion comprises a crosslinking agent dissolved in Hanks' Balanced Salt Solution (HBSS) and emulsified in an oil having a viscosity of 1300 kP. In some embodiments of this invention, said gelling emulsion contains DTT, Hanks' balanced salt solution (HBSS) and polypropylene glycol (PPG). In some embodiments of this invention, the gelling emulsion contains dithiothreitol (DTT), Hanks' balanced salt solution (HBSS), polypropylene glycol (PPG) and sorbitan monooleate (Span80). In some embodiments of the present invention, said gelling emulsion comprises dithiothreitol (DTT) dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG) with sorbitan monooleate (Span80). In some embodiments of the present invention, said gelling emulsion comprises dithiothreitol (DTT) dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80).

In some embodiments of the present invention, the conformal coating has permeability characteristics that allow the exchange of nutrients and cellular byproducts and the release of therapeutic factors, but which can also prevent the penetration of host immune effector molecules and/or other undesirable elements into the capsules . In certain embodiments of the present invention, said conformal coating comprises pores having a cut-off size of 100, 110, 120, 130, 140, 145, 150, 155, 160, 170, 180, 190, 200, 210, 220, 230, 240 , 250 or 500 kDa. In certain embodiments of the present invention, said conformal coating comprises pores having a size cutoff of 150 kDa. In certain embodiments of this invention, said conformal coating comprises pores having a size cutoff of up to 500 kDa.

The thickness of the conformal coating is independent of the size/diameter of the coated material. In some embodiments of this invention, the thickness of the coating varies from 1 to 100 microns, from 5 to 50 microns, or from 8 to 25 microns. In some embodiments of this invention, the thickness of said coating varies from 25-50 microns. In some embodiments of this invention, the thickness of said coating varies from 10-20 microns.

In some embodiments of the present invention, the coating is visualized by tagging the coating material with a detectable marker. The marker may be, for example, a fluorescent, enzymatic, chemiluminescent or epitope tag. In certain embodiments of this invention, said coating can be visualized by entrapping high molecular weight dextran-FITC in said coating material. In a specific embodiment of the present invention, said labeled coating material is PEG-FITC.

In some embodiments of this invention, the coating material may be chemically modified to contain functional groups. In some embodiments of this invention, said functional groups help stabilize said coating. In addition, the coating material may contain therapeutic factors or other molecules that are associated with therapeutic factors such as receptors or affinity agents (see, for example, Kim et al., 2003, Biomacromolecules 4: 12141223). Therapeutic factors can be incorporated into the coating material through covalent cross-linking, emulsification, ionic interactions, specific affinity interactions, simple entrapment, or any combination thereof.

In certain embodiments of the present invention, the coating material contains anti-inflammatory molecules to reduce the host inflammatory response following implantation of conformally coated cells. Exemplary anti-inflammatory agents include corticosteroids (dexamethasone, cortisol, prednisolone, loteprednol etabonate, fluocinolone acetonide, and others), calcineurin inhibitors (cyclosporine A), interleukin-1 (IL-1), interleukin 10 (IL-10), alpha-1- antitrypsin (AAT), lysophylline, pentoxifylline, COX-2 inhibitors, interleukin-1 receptor antagonist peptide (IRAP), interleukin-10 (IL-10), alpha-1 antitrypsin (AAT), TGF-beta; antibodies against IL-1, interferon-gamma and TNF-alpha; antibodies against tissue factor and complement inhibitors; antibodies against leukocyte integrins

- 20 046872 (LFA-1 and ICAM-1). In some embodiments of the present invention, said coating material comprises extracellular matrix (ECM) molecules such as type I or type IV collagen, laminin, fibronectin, hyaluronic acid, or arginine-glycine-aspartate peptides (Beck et al., 2007, Tissue Eng 13 (3): 1-11). In some embodiments of the present invention, anti-inflammatory molecules and/or ECM molecules are attached to the surface of said coating material. In certain embodiments of this invention, these molecules are coated or encapsulated for slow release.

The application of a conformal coating to a biomaterial occurs in a coating device. As used herein, the term coating device refers to any device that is capable of applying a conformal coating to a biomaterial. In some embodiments of the present invention, said coating device is a device that provides a transition from droplet spray to jet spray and extension of the aqueous phase into an immiscible (eg, oil) phase, wherein said aqueous phase undergoes jet disintegration into particles. In some embodiments of the present invention, said coating apparatus is a flow chamber comprising one or more oil phase inlets, one or more aqueous phase inlets (which may be the same as oil phase inlets, or different from them) and one or more flow focusing areas downstream of said inlets where co-flowing oil phase jets focus the aqueous phase. Said flow chamber may further comprise one or more channels downstream of the flow focusing region(s). The diameter of the aqueous phase channel(s) may be, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4 mm in diameter . The diameter of the oil phase channel(s) can be, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19 .5 or 20 mm in diameter. In certain embodiments of this invention, the diameter of the oil phase channel(s) can be up to 100 mm. The length of the channel(s) may be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 30 mm. The channels may lead to one or more outlets from said flow chamber. In specific embodiments of the present invention, said device or installation is configured as shown in the examples and illustrated in FIGS. 1, 1A or 1B.

In some embodiments of the present invention, a coating system is provided, including a first pump 102, a conformal coating device 104 coupled to the first pump 102, a catheter 106 located within the conformal coating device 104, a second pump 108 configured to in contact with the oil phase reservoir 110 (e.g., PPG) on the inlet side and connected to the conformal coating device 104 on the output side, a third pump 112 configured to contact the emulsion reservoir 114 on the suction side and connected to the conformal coating device coating 104 on the downstream side, capillary 116 on the downstream side of conformal coater 104, and collection vessel 118, as shown in FIG. 1A.

The first pump 102 may be configured to inject the coating material and the biomaterial to be coated in an aqueous phase into said first inlet of an interior portion of said body portion. In some aspects of the present invention, the first pump 102 is selected from a glass syringe pump, a peristaltic pump, or any other pump configured to inject said material. The conformal coating apparatus 104 is coupled to the first pump 102 on the upstream side and coupled to the capillary 116 on the downstream side, and may be configured to apply a conformal coating of said coating material to said biomaterial.

The catheter 106 may be located within the conformal coating device 104 and coupled to a precision flow syringe pump 102. The catheter 106 may be configured to inject the coating material and the biomaterial to be coated into the first inlet 160 of the chamber body 152 for encapsulation (inner phase), as shown in FIG. 1A(v). The body portion 152 may be connected to a fastening portion 154 at the second end 162 of the body portion 152, which is further connected to a coating portion 156 at the second end of the fastening portion 154, as shown in FIG. 1A(v). In some aspects of the present invention, the second end 162 of the housing portion 152 is located opposite the first end 160 of the housing portion 152, as shown in FIG. 1A(v). In some aspects of the present invention, the coating portion 156 is configured to engage the outer surface of the second end 164 of the fastening portion 154, as shown in FIG. 1A(v).

The second pump 108 is connected to the PPG reservoir 110 on the inlet side and is connected on the outlet side to the second inlet 158 of the coating portion 156

- 21 046872 conformal coating device 104, and is configured to inject an oil phase containing a surfactant into the second inlet 158, as shown in FIG. 1A. Said injection of the oil phase (outer phase) may be configured to flow coaxially to the inner aqueous phase. In some embodiments of the present invention, the second pump 108 is a peristaltic pump, a roller pump, a glass syringe pump, or any other pump configured to inject said oil phase material.

The encapsulation chamber includes a body portion 152 connected to a fastening portion 154, which is connected to a coating portion 156, as shown in FIG. 1A, panel (v).

In some embodiments of the present invention, said conformal coating device further comprises an outlet for releasing air from said device. In some embodiments of the present invention, said conformal coating device may further comprise an outlet for releasing air from said device. In some embodiments of the present invention, said air outlet is located upstream of the aqueous phase inlet of said device. Said air outlet is closed during the coating process.

In some embodiments of the present invention, the device focuses the flow from channel 10d to channel d (a 1/10 reduction in diameter to allow the transition from droplet spray to jet spray). In certain embodiments of this invention, d varies from 0.5-10 mm. In certain embodiments of this invention, d varies from 1-4 mm. In a specific embodiment of the present invention, d is about 1 mm. In some embodiments of this invention, the focusing angle of the specified device varies from 100 to 5° (from greater focus to less focus). In certain embodiments of the present invention, said focusing angle varies from 90 to 10°. In certain embodiments of the present invention, said focusing angle is greater than 10, 20, 30, 40, 50, 55, 60, 65, 70, 80, or 90°. In certain embodiments of this invention, the focusing angle of the specified device is greater than 60°. In some embodiments of the present invention, the flow focusing area has a length of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mm. In certain embodiments of this invention, the flow focusing area has a length of 100 mm.

In some embodiments of this invention, the diameter of the external oil phase chamber (cylinder) is 1-20 mm. In some embodiments of this invention, the diameter of the external oil phase chamber is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mm. In a specific embodiment of the present invention, the diameter of the external oil phase chamber is 10 mm. In some embodiments of the present invention, the external oil phase chamber is filled through a side port 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm upstream (cylinder axial distance) from the coaxial water phase injection port. In certain embodiments of the present invention, said side port is located 5 mm upstream of said aqueous phase injection port. In certain embodiments of this invention, said external oil phase chamber is filled through more than one side port 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mm upstream (cylinder axial distance) from said coaxial aqueous phase injection port.

In some embodiments of the present invention, the position of the tip of the water injection needle is aligned with the base of the focusing area of the device. In some embodiments of the present invention, the end of the water injection needle is located at about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. 9, 1, 2, 3, 4 or 5 mm upstream or downstream (cylinder axial distance) from the base of the focusing area of the said device. In a specific embodiment of the present invention, the tip of the water injection needle is located about 0.5 mm upstream of said focusing area.

In a specific embodiment of the present invention, said device is characterized by a 10mm diameter external oil phase chamber which is filled through a side port 5mm upstream of a coaxial aqueous phase injection port which is 0.5mm upstream of the flow focusing region, and the focusing of the flow occurs in a channel that tapers from 10 mm to 1 mm in diameter and is 100 mm in length, with a focusing angle of 60 degrees.

In specific embodiments of the present invention, the device is configured to provide a coaxial flow of a solution containing a gelling agent, in particular a solution of dithiothreitol in Hanks' balanced salt solution (HBSS) (10, 20, 25, 30, 40, 50, 60, 70, 80 , 90 or 100 mg/ml dithiothreitol), emulsified in polypropylene glycol

- 22 046872 (ING), in a volume to volume ratio, which is 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:50 or 1:100, localized in the position after the formation of coated biomaterials.

In some embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 40 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 50 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 60 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 70 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 80 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 90 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 100 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 150 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 200 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 250 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 300 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 350 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 400 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 450 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 500 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 550 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 600 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 650 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 700 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 750 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 800 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 850 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 900 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter greater than 950 microns. In some embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of up to 1000 microns.

In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 40 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 50 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 60 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 70 microns. In certain embodiments of this invention

- 23 046872 this device is capable of coating cells and/or clusters of cells having a diameter of about 80 μm. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 90 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 100 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 150 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 200 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 250 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 300 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 350 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 400 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 450 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 500 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 550 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 600 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 650 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 700 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 750 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 800 microns. In certain embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 850 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 900 microns. In certain embodiments of this invention, the device is capable of coating cells and/or clusters of cells having a diameter of about 950 microns. In some embodiments of the present invention, the device is capable of coating cells and/or clusters of cells having a diameter of up to 1000 microns.

In some embodiments of the present invention, the aqueous phase flow, the oil phase flow, or both are supported by a peristaltic pump. In some embodiments of the present invention, the aqueous phase flow, the oil phase flow, or both are supported by a syringe pump. In some embodiments of the present invention, the aqueous phase flow is maintained by a syringe pump and the oil phase flow is maintained by a peristaltic pump. In certain embodiments of this invention, the dithiothreitol solution is applied coaxially to the conformally coated biomaterials using a syringe pump or peristaltic pump.

While this document provides performance for a specific device that uses a 16G intravenous catheter to coat 50-250 µm cell clusters, it should be understood that other devices having different configurations and sizes are also contemplated to the extent in which the relative indicators set out in FIG. 1A, panel (v).

In some embodiments of the present invention, in which the cells and/or cell clusters are the specified biomaterial to be conformally coated, the concentration of cells added to the specified aqueous phase may vary from 100-500000000 cells/ml,

100-200000000 cells/ml, 1000000-200000000 cells/ml,

10000000-200000000 cells/ml, 25000000-200000000 cells/ml, 40000000-200000000 cells/ml, 55000000-200000000 cells/ml, 70000000-200000000 cells/ml, 85 000000-200000000 cells/ml, 100000000-200000000 cells/ml,

15000000-200000000 cells/ml, 30000000-200000000 cells/ml, 45000000-200000000 cells/ml, 60000000-200000000 cells/ml, 75000000-200000000 cells/ml, 90 000000-200000000 cells/ml, 105000000-200000000 cells/ml,

5000000-200000000 cells/ml, 20000000-200000000 cells/ml, 35000000-200000000 cells/ml, 50000000-200000000 cells/ml, 65000000-200000000 cells/ml, 800 00000-200000000 cells/ml, 95000000-200000000 cells/ml, 110000000 -200000000 cells/ml,

- 24 046872

115000000-200000000 cells/ml, 120000000-200000000 cells/ml, 125000000-200000000 cells/ml,

130000000-200000000 cells/ml, 135000000-200000000 cells/ml, 140000000-200000000 cells/ml,

145000000-200000000 cells/ml, 150000000-200000000 cells/ml, 155000000-200000000 cells/ml,

160000000-200000000 cells/ml, 165000000-200000000 cells/ml, 170000000-200000000 cells/ml,

175000000-200000000 cells/ml, 180000000-200000000 cells/ml, 185000000-200000000 cells/ml,

190000000-200000000 cells/ml, 195000000-200000000 cells/ml, 100-1000000 cells/ml, 500-750000 cells/ml, 1000-500000 cells/ml or 2500-250000 cells/ml.

In some embodiments of the present invention, in which the cells and/or cell clusters are the specified biomaterial to be conformally coated, the concentration of cells/cell clusters added to the specified aqueous phase may vary from about 100 to about 500,000,000 cells/ ml, about 100 - about 200000000 cells/ml, about 10000000 - about 20000000 cells/ml, about 5000000 - about 20000000 cells/ml, about 10000000 - about 200000000 cells/ml, about 15000000 - about 2000000 00 cells/ml, about 20000000 - about 200000000 cells/ml, about 25000000 - about 200000000 cells/ml, about 30000000 - about 200000000 cells/ml, about 35000000 - about 200000000 cells/ml, about 40000000 - about 200000000 cells/ml , about 45,000,000 about 200,000,000 cells/ml, about 50000000 - about 200000000 cells/ml, about 55000000 - about 200000000 cells/ml, about 60000000 - about 200000000 cells/ml, about 65000000 - about 200000000 cells/ml, about 70000000 - about 20 0000000 cells/ml, about 75000000 - about 200000000 cells/ml, about 80000000 - about 200000000 cells/ml, about 85000000 - about 200000000 cells/ml, about 90000000 - about 200000000 cells/ml, about 95000000 - about 200000000 cells/ml, about 10000000 0 about 200,000,000 cells/ml, about 105,000,000 - about 200000000 cells/ml, about 110000000 - about 200000000 cells/ml, about 115000000 - about 200000000 cells/ml, about 120000000 - about 200000000 cells/ml, about 125000000 - about 20000000 0 cells/ml, about 130000000 - about 200000000 cells/ ml, about 135000000 - about 200000000 cells/ml, about 140000000 - about 200000000 cells/ml, about 145000000 - about 200000000 cells/ml, about 150000000 - about 200000000 cells/ml, about 1550000 00 about 200,000,000 cells/ml, about 160,000,000 - about 200000000 cells/ml, about 165000000 - about 200000000 cells/ml, about 170000000 - about 200000000 cells/ml, about 175000000 - about 200000000 cells/ml, about 180000000 - about 200000000 cells /ml, about 185,000,000 - about 200,000,000 cells/ml, about 190000000 - about 200000000 cells/ml, about 195000000 - about 200000000 cells/ml, about 100 about 1000000 cells/ml, about 500 - about 750000 cells/ml, about 1000 - about 500000 cells/ml or about 2500 - about 250,000 cells /ml.

In some embodiments of the present invention, the concentration of cells added to the aqueous phase is about 100 cells/ml, about 500 cells/ml, about 1000 cells/ml, about 2500 cells/ml, about 5000 cells/ml, about 7500 cells/ ml, about 10,000 cells/ml, about 25,000 cells/ml, about 50,000 cells/ml, about 75,000 cells/ml, about 100,000 cells/ml, about 250,000 cells/ml, about 500,000 cells/ml, about 750,000 cells/ml, about 1000000 cells/ml, about 2500000 cells/ml, about 5000000 cells/ml, about 7500000 cells/ml, about 10000000 cells/ml, about 20000000 cells/ml, about 25000000 cells/ml, about 30000000 cells/ml, about 40000 000 cells/ml, about 50000000 cells/ml, about 60000000 cells/ml, about 70000000 cells/ml, about 80000000 cells/ml, about 90000000 cells/ml, about 100000000 cells/ml, about 105000000 cells/ml, about 110000000 cells/ ml, about 115,000,000 cells/ml, about 120,000,000 cells/ml, about 125,000,000 cells/ml, about 130,000,000 cells/ml, about 135,000,000 cells/ml, about 140,000,000 cells/ml, about 145,000,000 cells/ml, about 1500,000 00 cells/ml, about 155000000 cells/ml, about 160000000 cells/ml, about 165000000 cells/ml, about 170000000 cells/ml, about 175000000 cells/ml, about 180000000 cells/ml, about 185000000 cells/ml, about 190000000 cells/ml, about 195,000,000 cells/ml, about 200,000,000 cells/ml.

In some embodiments of this invention, the concentration of cells added to said aqueous phase is 100 cells/ml, 500 cells/ml, 1000 cells/ml, 2500 cells/ml, 5000 cells/ml, 7500 cells/ml, 10000 cells/ml , 25000 cells/ml, 50000 cells/ml, 75000 cells/ml, 100000 cells/ml, 250000 cells/ml, 500000 cells/ml, 750000 cells/ml, 1000000 cells/ml, 2500000 cells/ml, 5000000 cells/ml , 7500000 cells/ml, 10000000 cells/ml, 20000000 cells/ml, 25000000 cells/ml, 30000000 cells/ml, 40000000 cells/ml, 50000000 cells/ml, 60000000 cells/ml, 70000000 cells /ml, 80000000 cells/ml , 90000000 cells/ml, 100000000 cells/ml, 105000000 cells/ml, 110000000 cells/ml, 115000000 cells/ml, 120000000 cells/ml, 125000000 cells/ml, 130000000 cells/ml, 1 35000000 cells/ml, 140000000 cells/ml , 145000000 cells/ml, 150000000 cells/ml, 155000000 cells/ml, 160000000 cells/ml, 165000000 cells/ml, 170000000 cells/ml, 175000000 cells/ml, 180000000 cells/ml, 185000000 cells/ml, 190000000 cells/ml , 195,000,000 cells/ml, 200,000,000 cells/ml.

In some embodiments of the present invention, in which the cell clusters are the specified biomaterial to be conformally coated, the concentration of the cell clusters added to the specified aqueous phase may vary from 100200000 cell clusters/ml, 100-100000 cell clusters/ml, 500-100000 cell clusters/ml, 1000-100000 cell clusters/ml, 2000-100000 cell clusters/ml, 3000-100000 cell clusters/ml, 4000- 25 046872

100000 cell clusters/ml, 5000-100000 cell clusters/ml, 6000-100000 cell clusters/ml, 7000-100000 cell clusters/ml, 8000-100000 cell clusters/ml, 9000-100000 cell clusters/ml, 10000100000 cell clusters /ml, 15000-100000 cell clusters/ml, 20000-100000 cell clusters/ml, 25000-100000 cell clusters/ml, 30000-100000 cell clusters/ml, 35000-100000 cell clusters/ml, 40000-100000 cell clusters/ml, 45 000- 100000 cell clusters/ml, 50000-100000 cell clusters/ml, 55000-100000 cell clusters/ml, 60000-100000 cell clusters/ml, 65000-100000 cell clusters/ml, 70000-100000 cell clusters/ml, 75000-10 0000 clusters cells/ml, 80000-100000 cell clusters/ml, 85000-100000 cell clusters/ml, 900000-100000 cell clusters/ml, 95000-100000 cell clusters/ml.

In some embodiments of the present invention, in which the cell clusters are the specified biomaterial to be conformally coated, the concentration of cell clusters added to the specified aqueous phase can vary from about 100 to about 200,000 cell clusters/ml, about 100 to about 100,000 cell clusters/ml, about 500 100000

100000

100000

100000

100000

100000

100000

100000

100000

100000

100000

100000

100000 clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells /ml, cells/ml, cells/ml, about about about about about about about about about about about about

1000 - approx.

3000 - approx.

5000 - approx.

7000 - approx.

9000 - approx.

15000 25000 35000 45000 55000 65000 75000 about about about about about about about

100000

100000

100000

100000

100000

100000

100000

100000

100000

100000

100000

100000 clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells /ml, cells/ml, cells/ml, about about about about about about about about about about about about

2000 4000 6000 8000 10000 20000 30000 40000 50000 60000 70000 80000 cell clusters/ml, about 85000 -100000 cell clusters/ml, about 90000 about about about about about about about about about about about about about about about

100,000 cell clusters/ml, about 95,000 - about 100,000 cell clusters/ml.

In some embodiments of this invention, the concentration of cell clusters added to said aqueous phase is about 100 cell clusters/ml, about 500 cell clusters/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml , cells/ml, cells/ml, cells/ml, about 1000 cell clusters/ml, about 4000 cell clusters/ml, about 7000 cell clusters/ml, about 2000 about 5000 about 8000 about about about about about about

10000

25000

40000

55000

70000

85000 cell clusters/ml, cell clusters/ml, cell clusters/ml, cell clusters/ml, cell clusters/ml, cell clusters/ml, about about about about about about

15000

30000

45000

60000

75000

90,000 clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, about 3000 about 6000 about 9000 about about about about about about

20000

35000

50000

65000

80000

95,000 cells/ml, about 100,000 cell clusters/ml.

In some embodiments of the present invention, clusters of clusters of clusters of clusters of clusters of clusters of clusters of clusters, the concentration of cell clusters added to said aqueous phase is 100 cell clusters/ml, 500 cell clusters/ml,

1000 cell clusters/ml, 2000 cell clusters/ml, 3000 cell clusters/ml, 4000 cells/ml, 5000 cell clusters/ml, 6000 cell clusters/ml, 7000 clusters 8000 cell clusters/ml, 9000 cell clusters/ml, 10000 cell clusters/ml, 15000 cells/ml, 20000 cell clusters/ml, 25000 cell clusters/ml, 30000 cell clusters/ml, cell clusters/ml,

35000 cell clusters/ml, 40000 cell clusters/ml, 45000 cell clusters/ml, 50000 cell clusters/ml, 55000 cell clusters/ml, 60000 cell clusters/ml, 65000 cell clusters/ml, 70000 cell clusters/ml, 75000 clusters cells/ml, 80,000 cell clusters/ml, 85,000 cell clusters/ml, 90,000 cell clusters/ml, 95,000 cell clusters/ml, 100,000 cell clusters/ml.

In some embodiments of this invention, the concentration of islet cells added to said aqueous phase may vary from 100-500000000 cells/ml, 100-200000000 cells/ml, 1000000-200000000 cells/ml, 5000000-200000000 cells/ml, -200000000 cells/ml,

15000000-200000000 30000000-200000000 45000000-200000000 60000000-200000000 75000000-200000000 90000000-200000000 10500000 0-200000000 120000000-200000000 135000000-200000000 150000000-200000000 165000000-200000000 cells/ml, cells/ml, cells/ml, cells/ml, cells /ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml,

20000000-200000000 35000000-200000000 50000000-200000000 65000000-200000000 80000000-200000000 95000000-200000000 11000000 0-200000000 125000000-200000000 140000000-200000000 155000000-200000000 170000000-200000000 cells/ml, cells/ml, cells/ml, cells/ml, cells /ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml,

25000000-200000000

40000000-200000000

55000000-200000000

70000000-200000000

85000000-200000000

100000000-200000000

115000000-200000000

130000000-200000000

145000000-200000000

160000000-200000000

175000000-200000000 cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml, cells/ml,

- 26 046872

180000000-200000000 cells/ml, 185000000-200000000 cells/ml, 190000000-200000000 cells/ml, 195000000-200000000 cells/ml, 100-1000000 cells/ml, 500-7 50000 cells/ml, 1000-500000 cells/ml or 2500 -250000 cells/ml.

In some embodiments of the present invention, the concentration of islet cells added to said aqueous phase may vary from about 100 - about 500000000 cells/ml, about 100 - about 200000000 cells/ml, about 1000000 - about 200000000 cells/ml, about 5000000 - about 200000000 cells/ml, about 10000000 - about 200000000 cells/ml, about 15000000 cells/ml, about 20000000 - about 200000000 cells/ml, about 25000000 - about 200000000 cells/ml, about 30000000 - about 200,000,000 cells/ml, approx. 35000000 - about 200000000 cells/ml, about 40000000 - about 200000000 cells/ml, about 45000000 - about 200000000 cells/ml, about 50000000 - about 200000000 cells/ml, about 55000000 - about 200 000000 cells/ml, about 60000000 about 200000000 cells/ ml, about 65000000 - about 200000000 cells/ml, about 700000000 cells/ml, about 75000000 - about 200000000 cells/ml, about 80000000 - about 200000000 cells/ml, about 85000000 - about 200000000 cells/ml, about 90000000 - about 200000000 cells/ml, about 95000000 - about 200000000 cells/ml, about 100000000 - about 200000000 cells/ml, about

105000000 - about 200000000 cells/ml, about 110000000 - about 200000000 cells/ml, about

115000000 - about 200000000 cells/ml, about 120000000 - about 200000000 cells/ml, about

125000000 - about 200000000 cells/ml, about 130000000 - about 200000000 cells/ml, about

135000000 - about 200000000 cells/ml, about 140000000 - about 200000000 cells/ml, about 145000000 about 200000000 cells/ml, about 150000000 - about 200000000 cells/ml, about 155000000 - about 200000000 cells/ml, about 160000000 - about 200000000 cells/ ml, about 165,000,000 - about

200000000 cells/ml, about 170000000 - about 200000000 cells/ml, about 175000000 - about

200000000 cells/ml, about 180000000 - about 200000000 cells/ml, about 185000000 - about

200000000 cells/ml, about 190000000 - about 200000000 cells/ml, about 195000000 - about

200000000 cells/ml, about 100 - about 1000000 cells/ml, about 500 - about 750000 cells/ml, about 1000 - about 500000 cells/ml or about 2500 - about 250000 cells/ml.

In some embodiments of the present invention, the concentration of cells added to the aqueous phase is about 100 cells/ml, about 500 cells/ml, about 1000 cells/ml, about 2500 cells/ml, about 5000 cells/ml, about 7500 cells/ ml, about 10,000 cells/ml, about 25,000 cells/ml, about 50,000 cells/ml, about 75,000 cells/ml, about 100,000 cells/ml, about 250,000 cells/ml, about 500,000 cells/ml, about 750,000 cells/ml, about 1000000 cells/ml, about 2500000 cells/ml, about 5000000 cells/ml, about 7500000 cells/ml, about 10000000 cells/ml, about 20000000 cells/ml, about 25000000 cells/ml, about 30000000 cells/ml, about 40000 000 cells/ml, about 50000000 cells/ml, about 60000000 cells/ml, about 70000000 cells/ml, about 80000000 cells/ml, about 90000000 cells/ml, about 100000000 cells/ml, about 105000000 cells/ml, about 110000000 cells/ ml, about

115000000 cells/ml, about 120000000 cells/ml, about 125000000 cells/ml, about 130000000 cells/ml, about 135000000 cells/ml, about 140000000 cells/ml, about 145000000 cells/ml, about

150000000 cells/ml, about 155000000 cells/ml, about 160000000 cells/ml, about 165000000 cells/ml, about 170000000 cells/ml, about 175000000 cells/ml, about 180000000 cells/ml, about 185000000 cells /ml, about 190,000,000 cells /ml, about 195,000,000 cells/ml, about 200,000,000 cells/ml.

In some embodiments of this invention, the concentration of islet cells added to the aqueous phase is 100 cells/ml, 500 cells/ml, 1000 cells/ml, 2500 cells/ml, 5000 cells/ml, 7500 cells/ml, 10000 cells/ ml, 25000 cells/ml, 50000 cells/ml, 75000 cells/ml, 100000 cells/ml, 250000 cells/ml, 500000 cells/ml, 750000 cells/ml, 1000000 cells/ml, 2500000 cells/ml, 5000000 cells/ ml, 7500000 cells/ml, 10000000 cells/ml, 20000000 cells/ml, 25000000 cells/ml, 30000000 cells/ml, 40000000 cells/ml, 50000000 cells/ml, 60000000 cells/ml, 70000000 cells /ml, 80000000 cells/ ml, 90000000 cells/ml, 100000000 cells/ml, 105000000 cells/ml, 110000000 cells/ml, 115000000 cells/ml, 120000000 cells/ml,

125000000 cells/ml, 130000000 cells/ml, 135000000 cells/ml, 140000000 cells/ml, 145000000 cells/ml, 150000000 cells/ml, 155000000 cells/ml, 160000000 cells/ml, 16 5000000 cells/ml, 170000000 cells/ml, 175000000 cells/ml, 180000000 cells/ml, 185000000 cells/ml,

190000000 cells/ml, 195000000 cells/ml, 200000000 cells/ml.

In some embodiments of the present invention, the concentration of islet cell clusters added to said aqueous phase may vary from 100-200,000 cell clusters/ml, 100-100,000 cell clusters/ml, 500-100,000 cell clusters/ml, 1000-100,000 cell clusters/ ml, 2000-100000 cell clusters/ml, 3000-100000 cell clusters/ml, 4000-100000 cell clusters/ml, 5000-100000 cell clusters/ml, 6000-100000 cell clusters/ml, 7000-100000 cell clusters/ml, 8000-100000 cell clusters/ml, 9000-100000 cell clusters/ml, 10000-100000 cell clusters/ml, 15000-100000 cell clusters/ml, 20000-100000 cell clusters/ml, 25000-100000 cell clusters/ml 3000, 0- 100000 cell clusters/ml, 35000-100000 cell clusters/ml, 40000-100000 cell clusters/ml, 45000-100000 cell clusters/ml, 50000-100000 cell clusters/ml, 55000-100000 clusters

- 27 046872 cells/ml, 60000-100000 cell clusters/ml, 65000-100000 cell clusters/ml, 70000-100000 cell clusters/ml, 75000-100000 cell clusters/ml, 80000-100000 cell clusters/ml, 85000- 100000 cell clusters/ml, 900000-100000 cell clusters/ml, 95000-100000 cell clusters/ml.

In some embodiments of the present invention, the concentration of islet cell clusters added to said aqueous phase may vary from about 100 to about 200,000 cell clusters/ml, about 100 to about 100,000 cell clusters/ml, about 500 to about 100,000 cell clusters/ml, about 1000 - about 100,000 cell clusters/ml, about 2000 - about 100,000 cell clusters/ml, about 3000 - about 100,000 cell clusters/ml, about 4000 - about 100,000 cell clusters/ml, about 5000 about 100,000 cell clusters/ml, about 6000 - about 100,000 cell clusters/ml, about 7000 - about 100,000 cell clusters/ml, about 8000 - about 100,000 cell clusters/ml, about 9000 - about 100,000 cell clusters/ml, about 10000 - about 100,000 cell clusters/ml, about 15000 - about 100000 cell clusters/ml, about 20000 - about 100000 cell clusters/ml, about 25000 - about 100000 cell clusters/ml, about 30000 - about 100000 cell clusters/ml, about 35000 - about 100000 cell clusters/ml, about 40,000 - about 100,000 cell clusters/ml, about 45,000 - about 100,000 cell clusters/ml, about 50,000 - about 100,000 cell clusters/ml, about 55,000 - about 100,000 cell clusters/ml, about 60,000 - about 100,000 cell clusters/ml, about 65000 - about 100000 cell clusters/ml, about 70000 - about 100000 cell clusters/ml, about 75000 - about 100000 cell clusters/ml, about 80000 - about 100000 cell clusters/ml, about 85000 -100000 cell clusters/ml, about 90000 - about 100,000 cell clusters/ml, about 95,000 - about 100,000 cell clusters/ml.

In some embodiments of the present invention, the concentration of islet cell clusters added to said aqueous phase is about 100 cell clusters/ml, about 500 cell clusters/ml, about 1000 cell clusters/ml, about 2000 cell clusters/ml, about

3000 cell clusters/ml, about 4000 cell clusters/ml, about 5000 cell clusters/ml, about

6000 cell clusters/ml, about 7000 cell clusters/ml, about 8000 cell clusters/ml, about

9000 cell clusters/ml, about 10000 cell clusters/ml, about 15000 cell clusters/ml, about

20,000 cell clusters/ml, about 25,000 cell clusters/ml, about 30,000 cell clusters/ml, about

35,000 cell clusters/ml, about 40,000 cell clusters/ml, about 45,000 cell clusters/ml, about

50,000 cell clusters/ml, about 55,000 cell clusters/ml, about 60,000 cell clusters/ml, about

65,000 cell clusters/ml, about 70,000 cell clusters/ml, about 75,000 cell clusters/ml, about

80,000 cell clusters/ml, about 85,000 cell clusters/ml, about 90,000 cell clusters/ml, about

95,000 cell clusters/ml, about 100,000 cell clusters/ml.

In some embodiments of this invention, the concentration of islet cell clusters added to said aqueous phase is 100 cell clusters/ml, 500 cell clusters/ml, 1000 cell clusters/ml, 2000 cell clusters/ml, 3000 cell clusters/ml, 4000 cell clusters cells/ml, 5000 cell clusters/ml, 6000 cell clusters/ml, 7000 cell clusters/ml, 8000 cell clusters/ml, 9000 cell clusters/ml, 10000 cell clusters/ml, 15000 cell clusters/ml, 20000 cell clusters/ ml, 25000 cell clusters/ml, 30000 cell clusters/ml, 35000 cell clusters/ml, 40000 cell clusters/ml, 45000 cell clusters/ml,

50000 cell clusters/ml, 55000 cell clusters/ml, 60000 cell clusters/ml, 65000 cell clusters/ml, 70000 cell clusters/ml, 75000 cell clusters/ml, 80000 cell clusters/ml,

85,000 cell clusters/ml, 90,000 cell clusters/ml, 95,000 cell clusters/ml, 100,000 cell clusters/ml.

In certain embodiments of this invention, the concentration of cells/clusters of cells added to said aqueous phase varies from 5,000 to 100,000 cells/ml. In a specific embodiment of the present invention, from 5,000 to 250,000 cells/ml added to the aqueous phase are pancreatic islet cells, which may optionally be enriched in insulin-secreting beta cells or clusters thereof.

In some embodiments of this invention, the pH of said aqueous phase is about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4. In some embodiments of this invention, the pH of said aqueous phase is about 6. In some embodiments of this invention, the pH of said aqueous phase is about 6.1. In some embodiments of this invention, the pH of said aqueous phase is about 6.2. In some embodiments of this invention, the pH of said aqueous phase is about 6.3. In some embodiments of this invention, the pH of said aqueous phase is about 6.4. In some embodiments of this invention, the pH of said aqueous phase is about 6.5. In some embodiments of this invention, the pH of said aqueous phase is about 6.6. In some embodiments of this invention, the pH of said aqueous phase is about 6.7. In some embodiments of this invention, the pH of said aqueous phase is about 6.8. In some embodiments of this invention, the pH of said aqueous phase is about 6.9. In some embodiments of this invention, the pH of said aqueous phase is about 7.0. In some

- 28 046872 embodiments of the present invention, the pH of said aqueous phase is about 7.1. In some embodiments of this invention, the pH of said aqueous phase is about 7.2. In some embodiments of this invention, the pH of said aqueous phase is about 7.3. In some embodiments of this invention, the pH of said aqueous phase is about 7.4. In specific embodiments of this invention, the pH of said aqueous phase is about 6-7.4.

As stated herein, the pH of said aqueous phase is 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 , 7.4, 7.5, or 8. In some embodiments of this invention, the pH of said aqueous phase is 6.0. In some embodiments of this invention, the pH of said aqueous phase is 6.1. In some embodiments of this invention, the pH of said aqueous phase is 6.2. In some embodiments of this invention, the pH of said aqueous phase is 6.3. In some embodiments of this invention, the pH of said aqueous phase is 6.4. In some embodiments of this invention, the pH of said aqueous phase is 6.5. In some embodiments of this invention, the pH of said aqueous phase is 6.6. In some embodiments of this invention, the pH of said aqueous phase is 6.7. In some embodiments of this invention, the pH of said aqueous phase is 6.8. In some embodiments of this invention, the pH of said aqueous phase is 6.9. In some embodiments of this invention, the pH of said aqueous phase is 7.0. In some embodiments of this invention, the pH of said aqueous phase is 7.1. In some embodiments of this invention, the pH of said aqueous phase is 7.2. In some embodiments of this invention, the pH of said aqueous phase is 7.3. In some embodiments of this invention, the pH of said aqueous phase is 7.4. In specific embodiments of this invention, the pH of said aqueous phase is 6-7.4.

In some embodiments of this invention, said aqueous phase contains cells and/or clusters of cells in the medium, a surfactant, and one or more thiolated or reducing reagents (which may be, for example, mono- or multifunctional agents that are linear or multi-armed). In some embodiments of the present invention, said cells/clusters of cells include islet cells, said medium is serum-free or Hanks' Balanced Salt Solution (HBSS), and/or said thiolated or reducing reagent is DTT or a linear bifunctional PEG-dithiol . In some embodiments of this invention, said aqueous phase contains 5-10% PEG (eg, 5% or 10% PEG) and 50,000-250,000 islet cells or islet cell clusters/ml at pH 7.4. In some embodiments of this invention, said aqueous phase contains 1-30% PEG (eg, 1% or 30% PEG) and 50,000-250,000 islet cells or islet cell clusters/ml at a pH of 6 to 7.4. Methods for applying conformal coating according to this invention cover any combination of these values.

In some embodiments of this invention, said oil phase comprises polypropylene glycol (PPG), a mineral oil with a viscosity that is at least 2.5 times the viscosity of said aqueous phase, polypropylene glycol (PPG) with 10% sorbitan monooleate. In some embodiments of this invention, said oil phase comprises polypropylene glycol (PPG) with 10% sorbitan monooleate, wherein said oil phase optionally contains triethanolamine. In embodiments of this invention that include triethanolamine, said oil phase is 0.01-0.2% triethanolamine. In certain embodiments of this invention, said oil phase contains one or more of, for example, polypropylene glycol (PPG) and mineral oil with a viscosity that is at least 2.5 times the viscosity of said aqueous phase. In certain embodiments of this invention, said oil phase contains polypropylene glycol (PPG). In some embodiments of this invention, said oil phase contains a mineral oil with a viscosity that is at least 2.5 times the viscosity of said aqueous phase.

Said oil phase may further contain one or more agents, for example Span80.

In some embodiments of this invention, said oil phase contains PPG. In certain embodiments of this invention, said oil phase contains PPG with 1-20%, 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10% Span80. In some embodiments of this invention, said oil phase contains PPG with 1-20% Span80. In some embodiments of this invention, said oil phase contains PPG with 515% Span80. In some embodiments of this invention, said oil phase contains PPG with 6-14% Span80. In some embodiments of this invention, said oil phase contains PPG with 7-13% Span80. In some embodiments of this

- 29 046872 of the invention, said oil phase contains PPG with 8-12% Span80. In some embodiments of this invention, said oil phase contains PPG with 9-11% Span80. In a specific embodiment of this invention, said oil phase contains PPG with 10% Span80.

In some embodiments of this invention, the flow rates of said aqueous phase (Qw) and said oil phase (Qo), respectively, are selected from: 10 μl/min and 3.5 ml/min; 15 µl/min and 3.5 ml/min; 20 µl/min and 3.5 ml/min; 1 µl/min and 3.5 ml/min; 10 µl/min and 7 ml/min; 50 µl/min and 0.5 ml/min; 50 µl/min and 2.5 ml/min; 150 µl/min and 0.5 ml/min; and 150 μl/min and 2.5 ml/min. In some embodiments of the present invention, air is introduced prior to injection of said aqueous phase to stabilize the water jet within the oil jet. In certain embodiments of the present invention, air is introduced into an injection catheter containing an aqueous phase such that an air bubble can be introduced into said oil phase prior to injecting said aqueous phase into said oil phase to facilitate visualization of the onset of said aqueous phase.

The flow rates of said aqueous phase and said oil phase must be adjusted over time. In some embodiments of this invention, said aqueous phase is reduced over time, while said oil phase is increased. In a specific embodiment of the present invention, said aqueous phase enters said oil phase initially at a rate of 50 μl/min, and then the rate is reduced to 10 μl/min. In certain embodiments of the present invention, the rate of said oil phase is gradually increased from 0.5 to 3.5 ml/min while said aqueous phase is reduced and then maintained constant throughout the encapsulation process, or the rate of said oil phase is maintained constant at 3.5 ml/min throughout the encapsulation process.

In some embodiments of this invention, the ratio of the speed of said oil phase to the speed of said aqueous phase is from 70 to 500. In some embodiments of this invention, the ratio is 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 , 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 450 or 500. In a particular embodiment of the present invention, this ratio is 350.

In some embodiments of this invention, the ratio of the viscosity of said oil phase to the viscosity of said aqueous phase is from 2.5 to 100. In some embodiments of this invention, the ratio is 2.5, 3, 3.1, 3.2, 3.3 , 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100. V In a particular embodiment of the present invention, this ratio is 3.5.

After disintegration of the water jet, the material to be encapsulated (eg, cells and/or cell clusters) is coated with a thin layer of water containing coating material proportional to the size of the jet, allowing conformal coating to be applied. Then, as described herein, the coating is converted into a polymer by coaxial contact with a gelling emulsion, which promotes polymerization. In certain embodiments of the present invention, said gelling emulsion comprises dithiothreitol and Hanks' balanced salt solution (HBSS) in polypropylene glycol (PPG).

The coated cells/clusters of coated cells are then collected from the effluent of the coater. In some embodiments of the present invention, after collection, said coated cells and/or clusters of coated cells continue to mix for, for example, 1-30, 5-20, or 8-12 minutes to avoid coalescence before polymerization of said coverings. In some embodiments of this invention, mixing occurs at a temperature of from 4 to 25°C. In specific embodiments of this invention, mixing occurs at a temperature of 25°C. In some embodiments of this invention, the mixing speed is from 50-500 rpm to 100-300 rpm. In some embodiments of the present invention, said coated cells and/or clusters of coated cells continue to mix for about 7 minutes. In some embodiments of the present invention, said coated cells and/or clusters of coated cells are collected in a vessel and allowed to settle by gravity. In some embodiments of this invention, said coated cells and/or clusters of coated cells are kept without stirring in an external bath, for example, 1-30, 5-20 or 8-12 minutes to allow polymerization of said coating to complete. In some embodiments of the present invention, said coated cells and/or clusters of coated cells are collected in a vessel containing mineral oil and 0.01-0.1% triethanolamine.

It may be desirable to separate said coated cells and/or clusters of coated cells from the biomaterial-free coating material. This separation can be achieved by any of a number of size- or density-based separation techniques well known in the art, such as gradient centrifugation. In certain embodiments of the present invention, conformally coated cells and/or

- 30 046872 conformally coated cell clusters are further purified from the coating material by gradient centrifugation, which includes the following steps:

(a) layering the solutions to form a density gradient capable of separating said conformally coated biomaterial from said coating material not containing the biomaterial; (b) introducing said conformally coated biomaterial and said coating material not containing the biomaterial into said density gradient; (c) centrifuging said density gradient to separate said conformally coated biomaterial from said coating material not containing the biomaterial; and (d) removing a portion of said gradient containing said coating material that does not contain the biomaterial.

In some embodiments of this invention, said solutions layered to form said gradient have a density of (1) 1-1.1 g/ml, eg 1.042 g/ml, and (2) media. In some embodiments of the present invention, said biomaterial-free coating material is purified to greater than 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% the specified coated biomaterial. In certain embodiments of the present invention, more than 95% of the coated biomaterial is purified from said non-biomaterial coating material.

In certain embodiments of this invention, said conformally coated biomaterials, particularly cells, are washed repeatedly in HBSS. After purification, said conformally coated cell-containing biomaterial can be cultured in vitro under appropriate culture conditions.

If said coating material contains a fluorescent label, said coated cells and/or clusters of coated cells can be visualized, for example, using fluorescence microscopy, fluorocytometry, flow cytometric cell sorting technology, or using a fluorescence plate reader. In some embodiments of the present invention, fluorescently labeled conformally coated cells can be detected and/or isolated using, for example, flow cytometry or fluorescence activated cell sorting (FACS).

In some embodiments of this invention, the methods of this invention are scaled up to apply conformal coating to at least 50,000; 100000; 150000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000; or 1,000,000 cells and/or cell clusters simultaneously. In some embodiments of the present invention, this increase in scale is achieved by performing the methods of the present invention in a series of chambers. In some embodiments of the present invention, the methods of the present invention may be scaled up by assembling a series of parallel vertical chambers, for example, in a radial configuration in which the radial flow into each chamber delivers an aqueous phase to each of the individual chambers with comparable hydrodynamic flow characteristics. In some embodiments of the present invention, the coated cells and/or clusters of coated cells and the biomaterial-free coating material from each chamber are collected into separate containers. In some embodiments of the present invention, the coated cells and/or clusters of coated cells and the biomaterial-free coating material from each chamber are collected into one container and purified simultaneously.

In some embodiments of this invention, the methods of this invention provide conformal coating to more than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the biomaterial applied to the specified coating device. In certain embodiments of the present invention, the present methods provide conformal coating to greater than 95% of the applied biomaterial.

In some embodiments of the present invention, the viability and function of coated cells and/or coated cell clusters is assessed by any of a number of methods well known in the art, e.g., MTT assay, live/dead cell staining, and/or ( for islets) based on static glucose-stimulated insulin secretion, or perifusion. In some embodiments of the present invention, said assessment is performed prior to implantation of said cells. In cases in which the coated cells are islets, immune protection for transplanted conformally coated islets can be assessed, for example, by monitoring glucose levels and/or body weight of the transplant patient, serum C-peptide levels, hemoglobin A1c and/or by histological evaluation.

The present invention further provides methods for treating a disorder in a patient, comprising the step of implanting into said patient a conformally coated biomaterial isolated using the methods provided herein. Such disorders include, but are not limited to: diabetes mellitus, hemophilia, renal failure,

- 31 046872 amyloidosis, immune system disorders, inflammation, chronic pain, arthritis, hypertension, nervous system disorders, metabolic disorders, endocrine disorders, lymphoproliferative disorders, myeloproliferative disorders, myelodysplastic syndromes, stem cell disorders, phagocyte disorders, histiocytic disorders, red blood cell abnormalities or platelets, plasma cell disorders, acute leukemia, chronic leukemia, malignancies (breast carcinoma, Ewing's sarcoma, neuroblastoma, renal cell carcinoma, etc.), hypothyroidism, hypopituitarism, hypogonadism, graft failure, graft-versus-host disease ( GVHD), veno-occlusive disease, side effects of pre-transplant chemotherapy (such as excessive bleeding, infertility and renal, pulmonary and cardiac complications) and other disorders and diseases that will be recognized by a qualified practitioner.

As used herein, the term patient refers to the recipient of therapeutic treatment and includes all animals. In some embodiments of the present invention, said patient is a mammal. In some embodiments of the present invention, said patient is a primate. In some embodiments of the present invention, said patient is a human.

The conformally coated biomaterial produced using the methods presented herein can be implanted at any suitable location within the patient. The specified biomaterial can be implanted into living or pre-vascularized areas; into physiological or transformed areas; and in or near tissues and organs. In certain embodiments of the present invention, the location of said implant may be, for example, intraomental (in the omental sac), subcutaneous, intraperitoneal, preperitoneal, intramuscular, intralymph node, or renal-subcapsular. In some embodiments of this invention, the location of said implant is subcutaneous. In some embodiments of the present invention, said implant is not located in the abdominal cavity.

In some embodiments of the present invention, a conformally coated biomaterial produced using the methods presented herein is placed into a device prior to implantation in a patient to reduce the patient's immune response and/or prolong cell survival. The device may be any device suitable for implanting biological material into a patient, for example, the device described in US Patent No. 2006/0024276 or US Patent No. 6716246, each of which is incorporated herein by reference in its entirety. In some embodiments of the present invention, said conformally coated biomaterial is implanted within or adjacent to a natural or synthetic, biodegradable or non-biodegradable scaffold substrate.

The following examples are illustrative of specific embodiments of the present invention and their various applications. They are presented for purposes of illustration only and should in no way be construed as limiting the scope or content of the present invention.

Examples

The conformal coating procedures presented herein are improvements over the procedures described in US Patent Application No. 15/478320, filed April 4, 2017, US Patent Application No. 14/114690, International Patent Application No. PCT/US2012/035696 and US Provisional Patent Application No. 61/480,513, the contents of each of which are expressly incorporated herein by reference. These improvements include, but are not limited to, not exposing biomaterials, particularly cells and particularly islet cells, to low pH conditions (i.e. less than about pH 5) and eliminating viscosity enhancers in aqueous phases containing these biomaterials, which leads to improved functioning of the encapsulated islets and biocompatibility of the coating.

Materials Used in Improved Conformal Coating Procedures Unit List_________________________________________________________________________ kDa 8-arm 75% functionalized PEG-maleimide (concentration 12.5% w/v) in HBSS-/-_______________________________________________________________________________ mg/mL dithiothreitol in HBSS-/-_________________________________________________________

10X solution of 2 kDa PEG-dithiol (PEG-SH) (42.9% w/v)_________________________________

1X PEG-SH (4.29% w/v)____________________________________________________________

Polypropylene glycol (Mn ~4000) with 10% Span80_________________________________

Light mineral oil_________________________________________________________________

Isolated islets of Langerhans__________________________________________________________

Laboratory beakers (150 ml, 1 l)_________________________________________________

Conformal coating device

- 32 046872

Syringe pump________________________________________________________________

Peristaltic pump_______________________________________________________________

Tygon tubing for peristaltic pump_____________________________________________

Rigid tubes for syringe pump________________________________________________

Hamilton's syringe____________________________________________________________

Catheter 16G (SurFlash®)__________________________________________________________________________

1N HCl solution______________________________________________________________________________

1H NaOH solution________________________________________________________________

Reagents used in improved conformal coating procedures.

Gelling emulsion: A solution of dithiothreitol in Hanks' balanced salt solution (HBSS) (25 mg/ml dithiothreitol) was slowly added by stirring to 500 ml of polypropylene glycol (PPG) until an emulsion (milky white) was formed and mixed evenly.

Minimally cross-linked PEG-MAL solution: to 400 µl of 12.5% w/v. PEG-MAL (10 kDa, 8 arms, 75% functionalization) was added with 2.6 µL of 1N HCl and then vortex mixed (which prevented flash gelation due to non-uniform mixing), then 22.22 µL of 10X PEG-SH solution was added by stirring. This provided a solution of minimally cross-linked PEG-MAL.

Separately, 1 μL of 1 N NaOH was added to 400 μL of HBSS, which was then added to 401 μL of the above minimally cross-linked PEG-MAL solution and then completely vortex mixed. This mixture was then allowed to partially gel, during which it turned into a viscous liquid, for 30 minutes. This gelation step can be performed for 5, 10, 15, 20, 25, 30, 35, 40 or 45 minutes.

Device setup

The components of the coating apparatus, as illustrated in FIG. 1 and fig. 1A, cleaned and dried, and then reassembled and sterilized (usually in an autoclave), except for the capillary, capillary screw and bubble trap. In particular, said three parts shown in FIG. 1A, panels (ii), (iii) and (iv), were screwed to each other. Before screwing over the part shown in FIG. 1A (iii), a pair of rubber valves was installed. A glass capillary was inserted into the lower hole of the part shown in FIG. 1A, panel (iv), and secured in place. For each drug, new, unused Tygon pump tubing and rigid tubing (for use with a syringe pump) were washed and sterilized. Similar washing and sterilization procedures were performed for the Hamilton syringe. All other steps of this procedure were performed in a laminar flow cell culture hood in which the apparatus was assembled as shown in FIG. 1 and 1A. The capillary tube was then mounted and secured in place using a capillary screw, and 1 waste container, typically a 50 mL conical tube, was placed under the capillary. A bubble trap was also installed on this device as shown in FIG. 1 and 1A.

A glass Hamilton syringe was then filled with mineral oil without creating gas bubbles, and a rigid tube was prepared by filling with mineral oil, conveniently using a 5 ml luer lock syringe, and then said primed tube was connected to said Hamilton syringe. Said syringe was then secured to said syringe pump as shown in FIG. 1 and 1A, ensuring that at least 2 ml of mineral oil remains in the syringe to ensure proper filling of the catheter. The distal end of said rigid tube is conveniently secured to a steel stand as shown in FIG. 1A.

The Tygon tubing was then connected to a peristaltic pump and brought into contact with a 50 ml conical tube (or other vessel) containing a solution of PPG and 10% Span 80. The distal end of said Tygon tubing was then connected to said coating device as shown in FIG. . 1 and 1A, and filled the specified pump. This pump was then adjusted to a flow rate of 3.5 ml/min by calibrating the amount supplied to, for example, 50 ml conical tubes to be 3.5 g.

A sterile catheter was then attached to the end of said rigid tube, and the flow rate of said syringe pump was set to 2 ml/ml. This catheter was filled to ensure that all air bubbles were removed and that at least 1 ml of mineral oil remained in the syringe.

Finally, two 200 μL wide bore micropipette tips were attached to said glass capillary conveniently using zippers at opposite ends of said capillary, and two peristaltic tubes attached were attached to said wide bore tips attached to said glass capillary. The assembled tube/capillary assembly was then mounted onto said peristaltic pump as shown in FIG. 1 and 1A, and the ends of said peristaltic tubes were immersed in said gelling emulsion. A photograph of the fully assembled device is shown in FIG. 1B. In addition to this,

- 33 046872 a custom made fixture by 3D-ne4amu was developed to replace the assembly described earlier in this paragraph (Fig. 1C). Briefly, this device is designed to slide over and be attached to said glass capillary from below, and has two barbed ports to which said peristaltic tube can be directly attached.

Conformal coating process.

Pancreatic islet cells were coated according to the following protocol. Approximately 10,000 islet cell equivalents (IECs) in cell culture medium were injected into sterile 1.5-mL low-binding Eppendorf tubes and centrifuged at 150 relative centrifugal force for 1 minute to pellet the cells. After removing the cell culture supernatant, islet cells were resuspended in 100 μl of minimally cross-linked PJG-MAL (at neutral pH), conveniently using a 200 μl wide-bore tip attached to a P100 pipette. Cells were then recovered into a 16G catheter (Surflash®) at a recovery rate of 100 μl per minute using a precision syringe pump. The gelling emulsion was then introduced into the capillary and allowed to flow into a 150 mL collection beaker at a flow rate of 4 mL/min. The bubble trap was opened to allow bubble removal and the PPG'/Span 80 mixture was injected into the device at a calibrated rate of 3.5 mL/min. (The bubble trap was closed as soon as the bubbles generated by the introduction of the various liquid components were removed from the device.)

A 16G catheter (Surflash®) containing islets was then inserted into the top of the device, and the islet-containing PEG-MAL suspension was injected into the capillary using a Harvard syringe pump at a rate of 15 μL per minute. The coating procedure was allowed to continue for approximately 7 min, observing the transition from drip spray to stream spray throughout the procedure to ensure a stable transition. The 2 peristaltic pumps and the Harvard syringe pump were then turned off and the conformally coated islets collected in a 150 mL collection beaker were allowed to gel for 12 min.

Cleaning process

The collected effluent from this conformal coating procedure was slowly poured into 200 mL of mineral oil, typically contained in a 1 L beaker, with a stirring speed of 240 rpm. The vessel containing the islets was then washed with HBSS-/- and the volume of the vessel containing the islets and mineral oil was adjusted to a total volume of 500 ml with HBSS-/- and stirring was continued at the same speed for two minutes. After this, this mixture was divided into two portions of 250 ml in conical tubes and subjected to centrifugation for 5 min at 1500 rpm, leaving a sediment containing cells with a volume of ~5 ml.

This cell pellet was transferred to a 50 ml conical tube, the pellet tubes were washed with HBSS-/- and the resulting wash was added to each 50 ml conical tube. The volume of these 50 ml conical tubes containing islet cells was adjusted to 50 ml with HBSS-/- and centrifuged for 5 min at 1000 rpm. The pelleted cells obtained from this centrifugation were transferred to 15 ml tubes, the 50 ml tubes in which the sedimentation was carried out were washed with HBSS-/- and the resulting wash was added to these 15 ml tubes. The volume of each of these 15 ml conical tubes containing islets was adjusted to 15 ml with HBSS-/- and centrifuged for 5 min at 1000 rpm. The supernatant was then removed and the cells were incubated with 250 µl of 1X PEG-SH for 1 minute, and after 1 minute each of these 15 ml conical tubes was brought to a volume of 15 ml with HBSS-/- and centrifuged at 1000 rpm /min for 1 minute. The cell pellet containing the conformally coated islet cells was then washed three times in 1 ml of cell culture medium and pelleted by centrifugation at 1000 rpm for 1 minute each time. The washed, conformally coated islet cells were then seeded in 10 mL of cell culture medium in a 10 cm Petri dish.

Comparisons of cell viability and functionality.

Effect of prior art methods on cell viability and functionality.

Using the conformal coating methods set forth in US Patent Application No. 15/478320 filed April 4, 2017, US Patent Application No. 14/114690, International Patent Application No. PCT/US2012/035696, and US Provisional Patent Application No. 61 /480513, the contents of each of which are expressly incorporated herein by reference, non-human primate (NHP) islets were conformally coated with PEG and a self-assembling amphiphilic peptide at low pH (shown in FIG. 2, panel A) and found that these islets have a poor response in static (SGSI) and dynamic (perifusion) glucose-stimulated insulin secretion assays in vitro (Fig. 2, panels B and C). The absolute rates of insulin secretion and stimulation ratio of PC NCP islets were lower than those of uncoated islets.

- 34 046872

Accordingly, PC NHP islets failed to reverse diabetes mellitus after transplantation into the fat pad or renal capsule of NSG mice (shown in Fig. 2, panel D), although detectable human C-peptide was detected in all recipient mice (Fig. 2, panel D). panel E). Further comparative studies showed that islet cell viability decreased at pH, as shown in Fig. 3.

Effect of prior art methods on biocompatibility in large animals.

In NHPs with diabetes mellitus, laparoscopic implantation of NHP PC islets into the omental bursa using biological scaffolds was demonstrated (Fig. 4, panel A) using a marginal dose (5 thousand EOC/kg) of fully allogeneic NHP PC islets and immunosuppression without steroids. However, only minimal PC NHP islet function was observed in the form of decreased insulin requirements and detectable C-peptide after transplantation (Fig. 4, panel B). Histological examination of grafts explanted 21 days after transplantation revealed the presence of insulin-positive PC NCP islets in the graft and the absence of T cell infiltration (Fig. 4, panel D). However, the biocompatibility of the PEG/PEGSH/peptide prepared using the low pH prior art method in the omental sac was low. Pathological examination revealed a widespread, chronic, active, severe inflammatory response characterized by a large number of multinucleated gigantocytes and fibroplasia (Fig. 4, panel C).

Testing a new conformal coating method: improved cell viability and functionality.

The conformal coating methods presented herein: (i) improve the biocompatibility of coatings by eliminating the viscosity enhancing component and producing a pure PEG coating, and (ii) allow coating at physiological pH to maximize the viability and functionality of the coated islets. coating (including islands from NChP). Variations from the methods presented in the prior art applications include increasing the viscosity of the aqueous phase, which may include, but is not limited to, the islet cells to be coated, by cross-linking a minimum of 20% of the cross-linking arms of the PEG-MAL cross-linker. PEG-SH, which provides the viscosity necessary to jet spray this aqueous phase into the oil phase to form a conformal coating. In the methods presented herein, complete gelation of PEGMAL around the islands occurs downstream of the jet disintegration when capsule formation occurs by feeding an emulsion of a solution of dithiothreitol (DTT) in polypropylene glycol and the surfactant Span80 coaxial to the aqueous phase, but downstream of the part devices where the aqueous phase jet disintegrates.

The coating method, redesigned to provide CP with pure PEG (to improve biocompatibility) and at physiological pH (to improve NHP islet functionality), was tested with both human and NHP islets and found increased signal in a glucose-stimulated secretion assay insulin in vitro for both NHP islets and human islets. The results are shown in Fig. 5, which shows that human islet cells were effectively coated with the methods presented herein (FIG. 5, panel A), were viable (FIG. 5, panel B), and demonstrated functionality in vitro (FIG. 5 , panels C-F) and in vivo (Fig. 5, panels G-I), which was comparable to that of uncoated islets and often higher than that of uncoated islets. With this method, absolute insulin secretion by KP islets was not reduced compared to uncoated islets, and glucose tolerance in animals with diabetes mellitus improved.

Transplantation of human islets, which were conformally coated at physiological pH, into NNPs.

Using the method presented in this document, human PC islets with costimulatory blockade were transplanted into the omental bursa of a diabetic patient with diabetes mellitus. Orencia® (20 mg/kg i.v.) was administered on postoperative days (POD) -1, 0, 3, 7, 14, 21 and 28, and weekly thereafter, and anti-CD154 antibody (20 mg/kg i.v.) was administered c) in POS -1, 0, 3, 7, 14, 21 and 28, and every 10 days after that. At POS 0, using a laparotomy procedure, 20,358 islet cell equivalents (IECs)/kg were injected into the animal's bursa. A routine autopsy was performed at POS 55. The feasibility of a laparotomy procedure for implantation of CP islets into the bursa of a diabetic cynomolgus monkey was assessed (Fig. 6, panel A). Analysis of exogenous insulin requirement (PEI, solid line), blood glucose levels (dots), and C-peptide in NCP islet recipients demonstrated minimal function of transplanted human KP islets with decreased blood glucose levels (Fig. 6, panels B and WITH). Histological evaluation of explanted CP NCP grafts and evaluation of graft revascularization were performed (Fig. 6, panels D and E).

Transplantation of syngeneic islets from a Lewis rat into the omentum of an immunocompetent Lewis rat with diabetes mellitus.

- 35 046872

Diabetes mellitus (blood glucose level >250 mg/dL) was induced in Lewis rats by injection of streptozotocin. 2500 uncoated or conformally coated Lewis rat islets were transplanted into the omental bursa of diabetic Lewis rats. Diabetes mellitus was monitored by measuring blood glucose levels up to postoperative day (POD) 36 and using an intraperitoneal glucose tolerance test at POS 30. Panel A: Blood glucose levels were assessed in recipients of uncoated and conformally coated Lewis islets ( Fig. 7, panel A). On postoperative day (POD) 30, an intraperitoneal glucose tolerance test (IPGT) was performed in bare and conformal coated Lewis islet recipients, and the corresponding areas under the curves were analyzed (Fig. 7, panels B and C). Fasting and post-stimulation C-peptide levels were also analyzed during VBTHT in Lewis rat islet recipients, uncoated (solid line, dotted markers) and conformally coated (dashed line, square markers) (Fig. 7, panel D).

In vivo effects of allogeneic MIN6 insulinoma cell clusters transplanted into spontaneously diabetic NOD mice.

MIN6 cells were collected into clusters via suspension culture for 4 days and then conformally coated. Non-obese diabetic mice (NOD mice) that spontaneously developed diabetes mellitus with blood glucose levels >250 mg/dL were transplanted with either uncoated or conformally coated (CO) MIN6 insulinoma cell clusters at a dose of 4000 EOC/dL. mouse. Graft functionality and survival were monitored by assessing mouse mortality and measuring C-peptide levels in the blood of mice recipients of uncoated islets or KP islets, as well as by histological evaluation of the graft. In fig. 8, panel A, shows phase contrast microscopy images of uncoated (and conformally coated) MIN6 clusters. In fig. 8, Panel B, shows confocal microscopy images of conformally coated anti-PEG stained MIN6 clusters with adjacent orthogonal views showing capsule completeness in three dimensions. Mortality of recipients of uncoated and conformally coated MIN6 clusters was assessed, showing improved survival of KP clusters (Fig. 8, panel C). (Panel D) Non-fasting levels of random C-peptide were also assessed in recipients of uncoated and conformally coated MIN6 clusters (Fig. 8, panel D). Histological evaluation of explanted MIN6 KP cluster grafts and analysis of the survival of cell clusters within KP capsules were performed (Fig. 8, panel E).

Evaluation of conformally coated islet autograft in a porcine model of diabetes mellitus.

Yorkshire-Landrace piglets, approximately 8 weeks old, are implanted with 2-4 cell pouches (8-chamber or 10-chamber) (Cell Pouch™, Sernova Corp, London, Ontario, Canada) into the deep subcutaneous space behind 4-8 weeks to 90% pancreatectomy and islet isolation (performed using standard procedures). To ensure a strict hyperglycemic state, pigs can be given intravenous streptozotocin at a dose of 150 mg/kg after pancreatectomy. The isolated islands are subjected to a conformal coating process followed by analysis against release criteria. Once conformally coated islets are confirmed to meet release criteria, said islets are transplanted into prevascularized cell bag chambers approximately five days after their release, allowing time for recovery from pancreatectomy and confirmation of diabetic status. Graft function is assessed using biweekly blood glucose and body weight measurements and monthly intravenous glucose tolerance tests (IVGTTs) as needed. Fasting and IVTG blood C-peptide values are measured and compared with controls measured before transplantation. At the end of the study, the cell sacs are explanted and dissected using standard histological techniques to evaluate islet staining for insulin, assess microvascular development in the cell sac chambers, and islet cell viability. After explantation of the cell bags, measurements of the above performance parameters are performed to confirm loss of blood glucose control. The rate of glucose disappearance can be assessed, the area under the blood glucose curve, and C-peptide levels analyzed.

Evaluation of conformally coated donor islets in an allograft porcine model of diabetes mellitus.

Female Gottingen miniature pigs, approximately one year old, are implanted with two Cell Pouch™ (Sernova Corp, London, Ontario, Canada) deep subcutaneous cells (8- or 10-chamber) six to eight weeks before administration of islet allograft. Donor islets are obtained and isolated using standard procedures from male former inseminators of Yorkshire Landrace pigs >2 years of age. The isolated islands are subjected to a conformal coating process and confirmed

- 36 046872 compliance with release criteria. One to three weeks before transplantation, diabetes mellitus is induced chemically by injecting animals intravenously with 150 mg/kg of streptozotocin. To ensure a strict hyperglycemic state, diabetic intravenous glucose tolerance tests (DIGTTs) are performed before conformal islet transplantation. Miniature pig recipients are transplanted with conformally coated allogeneic islets at a dose ranging from 2500-10,000 EOC/kg into a pre-vascularized subcutaneous Cell Pouch™ after surgically opening the cell pouch and sequentially removing the plugs. Graft function is assessed using biweekly blood glucose and body weight measurements and monthly intravenous glucose tolerance tests (IVGTTs) as needed. Fasting and IVTG blood C-peptide values are measured and compared with controls measured before transplantation. At the end of the study, the cell sacs are explanted and dissected using standard histological techniques to evaluate islet staining for insulin and other hormones important for blood glucose control (eg, glucagon, somatostatin) and to assess microvascular development within the cell sac chambers. After explantation of the cell bags, measurements of the above parameters are performed to confirm loss of blood glucose control. You can also measure the rate of glucose disappearance, analyze the area under the blood glucose curve and C-peptide levels.

Evaluation of conformally coated human stem cell-derived glucose-responsive islets in a porcine xenograft model of diabetes mellitus.

Female Göttingen miniature pigs, approximately one year old, are implanted with two Cell Pouch™ (Sernova Corp, London, Ontario, Canada) deep subcutaneous tissues (8- or 10-chamber) six to eight weeks prior to injection. conformal coating of glucose-sensitive human islets obtained by stem cell technology (iPSC or ESC). Before transplantation, insulin-producing stem cell-derived cells, or clusters thereof, are prepared and subjected to a conformal coating process, followed by confirmation that the cells meet standard release criteria. One to three weeks before transplantation of conformally coated cells obtained from stem cells, diabetes mellitus is induced chemically by intravenously injecting animals with 150 mg/kg of streptozotocin. To ensure a strict hyperglycemic state, diabetic intravenous glucose tolerance tests (DIGTTs) are performed before transplantation of conformally coated stem cell-derived cells. Miniature pig recipients are transplanted with conformally coated allogeneic islets or clusters thereof at a dose ranging from 2500-10,000 EOC/kg (or equivalent) into the chambers of a pre-vascularized subcutaneous Cell Pouch™ after surgically opening the chambers and sequentially removing the plugs. Graft function is assessed using biweekly blood glucose and body weight measurements and monthly intravenous glucose tolerance tests (IVGTTs) as needed. Fasting and IVTG blood C-peptide values are measured and compared with controls measured before transplantation. At the end of the study, the cell sacs are explanted and dissected using standard histological techniques to evaluate islet staining for insulin and other hormones important for blood glucose control (eg, glucagon, somatostatin) and to assess microvascular development within the cell sac chambers, as well as to confirm the survival of human cells derived from stem cells. After explantation of the cell bags, measurements of the above parameters are performed to confirm loss of blood glucose control. Also, this xenograft model of diabetes mellitus can measure glucose disappearance rates, analyze the area under the blood glucose curve and C-peptide levels using the conformally coated human cell xenograft model to analyze the technology of protecting conformally coated human stem cells from immune system attack .

Evaluation of conformally coated donor islets in a syngeneic mouse model of diabetes mellitus.

Four to five weeks before cell transplantation, a single-chamber mini-CM (Sernova Corp, London, Ontario, Canada) is implanted subcutaneously into the lower abdominal quadrant of male BALB/c mice weighing approximately 25 g to induce neovascularization. Briefly, CM placement is performed a small transverse incision to create a small pocket below the incision line. Once sufficient space is created, the CM is implanted into this space so that the opening is in a cephalad position. The incision is then closed with a surgical clip (Autoclip; Becton Dickinson, Sparks, MD, USA). Pancreatic islets were isolated from male BALB/c mice aged 8 to 12 weeks. Animals are kept under normal conditions with free access to food and water. Before pancreatectomy, the common bile duct is cannulated with a 27-gauge needle and 0.125 mg/mL of cold Liberase TL Research Grade in Hanks' balanced salt solution is injected into the pancreas. Islets are isolated by digesting the pancreas in a 50 ml Falcon tube placed in a water bath

- 37 046872 at 37°C for 14 minutes with gentle shaking. After the digestion phase, the islets are cleared of pancreatic digestion products by Histopaque density gradient centrifugation (1.108, 1.083 and 1.069 g/ml). Once isolated, the islets undergo a conformal coating process in preparation for transplantation into a cell bag and are confirmed to meet release criteria.

From three to 5 days before transplantation, diabetes mellitus is induced in mice that will be implanted with a cell bag (CM) by chemical induction - intraperitoneal administration of streptozotocin at a dose of 185 mg/kg in acetate-phosphate buffer, pH 4.5. Animals are considered to have diabetes mellitus if their blood glucose level exceeds 15 mmol/L on 2 consecutive daily tests.

The total islet mass (500 conformally coated islets ± 10% per diabetic recipient mouse) or border islet mass (200 conformally coated islets ± 10% per diabetic recipient) with a purity of 90% ± 5% is aspirated into a tube from polyethylene-50 using a microsyringe and centrifuged to obtain a cell pellet suitable for transplantation.

A dose level study to examine the effectiveness of a full dose of islets (500 conformally coated islets) in reversing diabetes mellitus when transplanted into the BM and a study of the mass of borderline islets (200 conformally coated islets) transplanted into the BM could be conducted.

To transplant islets into the BM, a small incision is made in the skin to gain access to the cranial portion of the device. The plug is then removed, exposing a vascularized tissue chamber into which the islet preparation is injected. The CM is closed by approximating 2 layers of the cranial portions of the CM with 4-0 vicryl suture. The skin incision is then closed with a surgical clip (Autoclip; Becton Dickinson).

Islet graft function in recipients is assessed twice weekly by measuring nonfasting blood glucose (mmol/L) using a portable glucose meter in all study groups. Graft function and diabetes reversal were defined as 2 consecutive blood glucose levels of less than 11.1 mmol/L maintained until completion of the study. In addition, glucose tolerance tests are performed in euglycemic mice up to 100 days after transplantation to further assess metabolic capacity. Recipients fast overnight before receiving an intraperitoneal bolus of glucose (3 g/kg). Blood glucose levels are measured at baseline (time 0) and 15, 30, 60, 90 and 120 minutes after injection, allowing the area under the curve (blood glucose PIC) to be calculated and analyzed between graft groups.

To confirm graft-dependent euglycemia, animals with functional grafts have their islet grafts explanted by removing the BM. BM explantation begins with a small skin incision. The ventral surface of the BM is cut off from the dermis, maintaining the integrity of the surrounding neovascularized tissue. The dorsal aspect of the engrafted island device is incised to ensure complete removal. Post-explantation BMs are placed in 10% buffered formalin for histological analysis, and animals are monitored for hyperglycemia.

Immunohistochemistry is used to determine the details of the overall structure using hematoxylin-eosin, endothelial cells to assess vascularity using antibodies against von Willebrand factor, antibodies against insulin and glucagon to detect the presence of islets within the BM. Immediately after explantation, BM tissue is fixed in 10% buffered formalin for 48 hours and then washed in phosphate-buffered saline with a final rinse in 70% ethanol. The tissue is dissected, embedded in paraffin, and sections 5 µm thick are made. Representative tissue sections are stained with hematoxylin-eosin. Additionally, tissue sections are immunofluorescently stained. Sections were deparaffinized and processed with heat antigen retrieval (Target Retrieval Solution, Dako) followed by a Tris-buffered saline (TBS)-supplemented Tween-20 (TBS-T) wash. Sections are blocked with 10% goat serum in TBS-T for 1 hour at room temperature. Sections are treated with a primary antibody, either rabbit anti-von Willebrand factor antibody (Millipore AB7356) diluted 1:100 (TBS with 1% goat serum) or guinea pig anti-insulin antibody (Dako A0564) diluted 1:1000 (TBS with 1% goat serum), or rabbit anti-glucagon antibody (AbD Serotec AHP534), diluted 1:100 (TBS with 1% goat serum) - for 15 hours at 4°C. Sections are washed in TBS-T followed by treatment with a secondary antibody—either goat anti-rabbit antibody (Molecular Probes A-11034; Alexa Fluor 488) diluted 1:1000 (TBS-T with 1% goat serum) or goat anti-guinea pig antibody (Molecular Probes A-11075; Alexa Fluor 568), diluted 1:1000 (TBS-T with 1% goat serum) - for 1 hour at room temperature. Samples are washed in TBS-T and stained with DAPI (1:1000). To reduce the autofluorescence background, sections are treated with 0.3% Sudan black solution (in 70% ethanol) for 2 minutes, washed in TBS and covered.

- 38 046872 cover glass with mounting medium to prevent fluorophores from fading. Microscopy and image analysis are performed using the Aperio-Scan Scope Console for light microscopy and the Zeiss Axio Imager Z1 for fluorescence microscopy, or similar devices.

Evaluation of conformally coated islets transplanted into a subcutaneously implanted prevascularized device (Cell Pouch™) for reversing diabetes mellitus in a syngeneic rat model.

Male Lewis rats (250 g) are used as islet donors, and female Lewis rats (up to 200 g) are used as transplant recipients. A cell bag (1-chamber or 2-chamber; (Sernova Corp, London, Ontario, Canada) is implanted subcutaneously in Lewis rats on the ventral side according to Sernova Corp. specifications and left for a minimum of 4 weeks to develop vascularized tissue chambers suitable for transplantation cells. At 4 weeks after implantation, diabetes mellitus is induced in recipient rats by administration of streptozotocin. Rats with two nonfasting blood glucose values >300 mg/dL in blood samples obtained by tail puncture (OneTouch Ultra glucose meters) are considered to have diabetes mellitus. Donor Lewis rats are isolated by enzymatic digestion followed by density gradient purification using standard protocols. Islets from Lewis rats are then conformally coated. Approximately four days after coating, isolated islets from Lewis rats are either uncoated or conformally coated. transplanted into a vascularized cell bag. For islet transplantation, the cell sac is surgically accessed and the plug(s) are removed to expose the vascularized tissue void space, and conformally coated or uncoated islets are transplanted into the cell sac chambers using a micropipette at a dose of approximately 3000-5000 EOC per recipient with diabetes mellitus. It is expected that multiple doses will be evaluated in different groups. Blood glucose levels in islet recipient rats are monitored at least three times a week by tail pricking and measurement using portable glucose meters. Graft function will be defined as fasting glucose <200 mg/dL and positive rat C-peptide measured by standard techniques. At selected time points after transplantation, islet cell recipient rats are given a glucose tolerance test to assess graft efficacy. After an overnight fast, administer a glucose bolus orally (oral glucose tolerance test [OGT]; 2.5 g/kg) or intravenously (intravenous glucose tolerance test [IGTT]; 0.5 g/kg). Monitor blood glucose values and calculate the area under the curve (AUC) of blood glucose. Blood samples from rats are collected before (t=0 min) and 30 min after the glucose bolus and fasting and stimulated C-peptide are assessed. The transplanted cell bags are isolated in different dosing groups at intervals of 60 to 300 days to confirm effectiveness in establishing glucose control. The explanted cell bags are examined by histological analysis for the presence of transplanted islets using standard histological techniques for staining against C-peptide, insulin, glucagon, etc., and to assess cell viability, as well as for other indicators. The effectiveness of uncoated and conformally coated islets within the cell sac is compared to evaluate the effect of conformal coating on protecting the islets from immune system attack. Efficacy is measured as the ability of conformally coated islets in a cell bag to reverse diabetes mellitus (blood glucose < 200 mg/dL) after graft administration and maintain graft function based on detectable C-peptide as well as positive glucose tolerance (AUC during TGT). in syngeneic recipient rats with diabetes mellitus. In addition, surviving hormone-producing grafts are identified by histological analysis to confirm the survival of conformally coated cells transplanted into the cell bag compared to uncoated cells.

Evaluation of conformally coated islets transplanted into an implanted subcutaneously prevascularized device (Cell Pouch™) for reversing diabetes mellitus in an allograft rat model of diabetes mellitus

Female Lewis rats (up to 200 g) are used as islet recipients. Male Wistar Furth, WF (RT1u) rats (250 g) were purchased as islet donors. A cell bag (1-chamber or 2-chamber; (Sernova Corp, London, Ontario, Canada) is implanted subcutaneously into female Lewis rats on the ventral side according to Sernova Corp. specifications and left for a minimum of 4 weeks to allow the formation of vascularized tissue chambers, suitable for cell transplantation. At 4 weeks after implantation, diabetes mellitus is induced in recipient rats by administration of streptozotocin. Rats with two nonfasting blood glucose values of >300 mg/dL in blood samples obtained by tail puncture (OneTouch Ultra glucometers) are considered to have diabetes mellitus. from male Wistar Firth donor rats are isolated by enzymatic digestion followed by density gradient purification using standard protocols. Then islets from Wistar Firth rats are isolated.

- 39 046872 apply a conformal coating. Approximately four days after coating, isolated uncoated or conformally coated islets from Wistar Firth rats are transplanted into a vascularized cell bag. For islet transplantation, the cell sac is surgically accessed and the plug(s) are removed to expose the vascularized tissue void space, and conformally coated or uncoated islets are transplanted into the cell sac chambers using a micropipette at a dose of approximately 3000-5000 EOC per recipient with diabetes mellitus. It is expected that multiple doses will be evaluated in different groups. Blood glucose levels in islet recipient rats are monitored at least three times a week by tail pricking and measurement using portable glucose meters. Graft function will be defined as fasting glucose <200 mg/dL and positive rat C-peptide measured by standard techniques. At selected time points after transplantation, islet cell recipient rats are given a glucose tolerance test to assess graft efficacy. After an overnight fast, administer a glucose bolus orally (oral glucose tolerance test [OGT]; 2.5 g/kg) or intravenously (intravenous glucose tolerance test [IGTT]; 0.5 g/kg). Monitor blood glucose values and calculate the area under the curve (AUC) of blood glucose. Blood samples from rats are collected before (t=0 min) and 30 min after the glucose bolus and fasting and stimulated C-peptide are assessed. The transplanted cell bags are isolated in different dosing groups at intervals of 60 to 300 days to confirm effectiveness in establishing glucose control. The explanted cell bags are examined by histological analysis for the presence of transplanted islets using standard histological techniques for staining against C-peptide, insulin, glucagon, etc., and to assess cell viability, as well as for other indicators. The effectiveness of uncoated and conformally coated islets within the cell sac is compared to evaluate the effect of conformal coating on protecting the islets from immune system attack. Efficacy is measured as the ability of conformally coated islets in a cell bag to reverse diabetes mellitus (blood glucose < 200 mg/dL) after graft administration and maintain graft function based on detectable C-peptide as well as positive glucose tolerance (AUC during TGT). in diabetic recipient rats compared with uncoated islets. For allogeneic islet transplants, graft failure is defined simply as a return to a hyperglycemic state without fasting, which is expected in the uncoated islet groups compared with the conformally coated islet groups. In addition, surviving hormone-producing grafts are identified by histological analysis to confirm the survival of conformally coated cells transplanted into the cell sac compared with uncoated cells in this experimental allograft model, which tests the ability of the conformal coating to provide protection from the immune system.

Evaluation of conformally coated human islets derived from stem cells transplanted into an implanted subcutaneously prevascularized device (Cell Pouch™) for reversing diabetes mellitus in a xenograft rat model of diabetes mellitus.

Female Lewis rats (up to 200 g) are used as islet recipients. In this xenograft model, human stem cell (iPSC or ESC)-derived islet clusters are generated using a differentiation protocol to produce insulin-producing cells for transplantation into a cell bag. A cell bag (1-chamber or 2-chamber; (Sernova Corp, London, Ontario, Canada) is implanted subcutaneously into female Lewis rats on the ventral side according to Sernova Corp. specifications and left for a minimum of 4 weeks to allow the formation of vascularized tissue chambers, 4 weeks after implantation, diabetes mellitus is induced in recipient rats by administration of streptozotocin. Rats with two nonfasting blood glucose values >300 mg/dL in tail prick blood samples (OneTouch Ultra glucometers) are considered to have diabetes mellitus. Human stem cell-derived islet clusters are prepared using standard protocols. The human stem cell-derived islet clusters are then conformally coated approximately four days after coating. coated and transplanted into a vascularized cell bag. For islet transplantation, the cell sac is surgically accessed and the plug(s) are removed to expose the vascularized tissue void space, and conformally coated or uncoated islets are transplanted into the cell sac chambers using a micropipette at a dose of approximately 3000-5000 EOC per recipient. with diabetes (or equivalent). It is expected that multiple doses of stem cell-derived islet clusters will be evaluated in different groups. Blood glucose levels in islet recipient rats are monitored at least three times a week by tail pricking and measurement using portable glucose meters. Graft function will be defined as fasting glucose <200 mg/dL and positive rat C-peptide,

- 40 046872 measured using standard methods. At selected time points after transplantation, islet cell recipient rats are given a glucose tolerance test to assess graft efficacy. After an overnight fast, administer a glucose bolus orally (oral glucose tolerance test [OGT]; 2.5 g/kg) or intravenously (intravenous glucose tolerance test [IGTT]; 0.5 g/kg). Monitor blood glucose values and calculate the area under the curve (AUC) of blood glucose. Blood samples from rats are collected before (t=0 min) and 30 min after the glucose bolus and fasting and stimulated C-peptide are assessed. The transplanted cell bags are isolated in different dosing groups at intervals of 60 to 300 days to confirm effectiveness in establishing glucose control. The explanted cell bags are examined by histological analysis for the presence of transplanted islets using standard histological techniques for staining against C-peptide, insulin, glucagon, etc., and to assess cell viability, as well as for other indicators. The effectiveness of a xenograft of uncoated islets and conformally coated islets within a cell bag is compared to evaluate the effect of conformal coating on protecting the islets from immune system attack. Efficacy is measured as the ability of conformally coated islets in a cell bag to reverse diabetes mellitus (blood glucose < 200 mg/dL) after graft administration and maintain graft function based on detectable C-peptide as well as positive glucose tolerance (AUC during TGT). in diabetic recipient rats compared with uncoated islets. For stem cell-derived islet cluster transplants, graft failure is defined simply as a return to a hyperglycemic state without fasting, which is expected in the uncoated islet groups compared with the conformally coated islet groups. In addition, surviving hormone-producing grafts are identified by histological analysis to confirm the survival of conformally coated cells transplanted into the cell sac compared with uncoated cells in this experimental xenograft model, which tests the ability of the conformal coating to provide protection against the immune system using the hormone-producing cells. insulin clusters derived from stem cells.

Analyze conformally coated cells for additional cell types and clinical indications.

Using the above examples, it should be noted that cells (donor cells, stem cell technologies) suitable for various clinical indications can be analyzed for the treatment of disease in xenograft, allograft and xenogeneic animal models. Applicable animal models can be found in the scientific literature, and efficacy metrics can be determined to test a specific cell type and clinical indication.

Numbered Embodiments

Specific embodiments of the present invention are presented in the numbered paragraphs below.

1. A method for conformally coating biomaterials with a coating material, which includes the following steps:

(a) injecting the aqueous phase into the coaxial oil phase in a coating device configured to provide a transition from droplet spray to jet spray and elongation of the flow of said aqueous phase into said oil phase;

(b) adding said biomaterial and said coating material to said aqueous phase, wherein polymerization of said coating material occurs after the jet of said aqueous phase disintegrates into particles, resulting in conformal coating of said biomaterial with said coating material;

(c) adding a component of said coating material after the jet of said aqueous phase has disintegrated into particles, said component being a gelling emulsion that promotes/catalyzes the polymerization of said coating material;

(d) optionally collecting material flowing from said coating device;

(e) optionally purifying said conformally coated biomaterial and said biomaterial-free coating material from said oil phase; and (f) optionally, separating said conformally coated biomaterial from said coating material not containing the biomaterial.

2. The method of paragraph 1, wherein said biomaterial contains one or more of the following: cells, cell clusters, biomaterial-coated cells or clusters of cells, subcellular organelles, biological molecules and non-biological drugs.

3. The method according to paragraph 2, characterized in that said biomaterial contains cells or clusters of cells.

- 41 046872

4. The method according to paragraph 3, characterized in that the specified biomaterial contains islet cells.

5. The method according to any one of paragraphs 1-3, characterized in that said coating material is selected from the group consisting of one or more of the following: polyethylene glycol (PEG), polyethylene oxide (PEO), poly(N-vinylpyrrolidinone) (PVP) , polyethyloxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK) and poly(amino acids).

6. The method of paragraph 5, wherein said coating material is one or more of the following: polyethylene glycol (PEG), PEG maleimide, PEG acrylate, PEG vinyl sulfone, or modified derivatives thereof.

7. The method according to paragraph 6, characterized in that said coating material is 5-10% PEG.

8. The method according to any of paragraphs 1-7, characterized in that said aqueous phase contains:

(a) serum-free medium with pH 6-7.4; or (b) Hanks' balanced salt solution (HBSS) pH 6-7.4.

9. The method according to paragraph 8, characterized in that said aqueous phase contains 500-750,000 islet cells or islet cell clusters/ml.

10. Method according to paragraph 9, characterized in that said aqueous phase contains about 2500250000 islet cells or islet cell clusters/ml.

11. The method according to any of paragraphs 1-10, characterized in that said aqueous phase optionally contains one or more of thiolated or reducing reagents, and/or a surfactant.

12. The method according to paragraph 11, characterized in that said surfactant is a block copolymer of polyoxyethylene-polyoxypropylene or poly(ethylene glycol-Npropylene sulfide).

13. The method according to paragraph 12, characterized in that said surfactant is a 2% polyoxyethylene-polyoxypropylene block copolymer.

14. The method according to paragraph 11, characterized in that said thiolated or reducing reagent is dithiothreitol (DTT) or PEG-dithiol.

15. The method according to paragraph 14, characterized in that said thiolated or reducing reagent is 0.01-0.62% dithiothreitol (DTT).

16. The method according to any of paragraphs 1-15, characterized in that said oil phase contains polypropylene glycol (PPG).

17. The method according to paragraph 16, characterized in that said oil phase contains polypropylene glycol (PPG) with 10% sorbitan monooleate, wherein said oil phase optionally contains triethanolamine.

18. The method according to paragraph 17, characterized in that said oil phase contains 0-0.2% triethanolamine.

19. A biomaterial that has been conformally coated in a manner described in any of paragraphs 1-18.

20. A method of treating a disorder in a patient, comprising the step of implanting into said patient the conformally coated biomaterial set forth in paragraph 19.

21. The method of paragraph 20, wherein said disorder is diabetes mellitus and said conformally coated material contains islet cells and islet cell clusters.

22. The method according to paragraph 11, characterized in that said aqueous phase is a multi-arm polyethylene glycol (PEG) minimally cross-linked (5-50%) with PEG-dithiol.

23. The method according to any of paragraphs 1-18, 21 or 22, characterized in that said gelling emulsion is added after the stream of said aqueous phase has broken up into particles, wherein said gelling emulsion is a solution of dithiothreitol (DTT) dissolved in a balanced salt solution Hanks (HBSS) and emulsified in polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80).

24. The method according to any of paragraphs 1-18, 21-23, characterized in that the purification of the specified biomaterial with a conformal coating and the specified coating material not containing biomaterial from the specified oil phase is performed by pouring the product from the coating product into mineral oil with stirring .

25. The method of paragraph 24, characterized in that during the mineral oil cleaning, Hanks' Balanced Salt Solution (HBSS) is added while stirring, while continuing to stir said gelling emulsion containing a solution of dithiothreitol (DTT) dissolved in Hanks' Balanced Salt Solution (HBSS) and emulsified in polypropylene glycol (PPG) with 10% sorbitan monooleate (Span80) in mineral oil.

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Claims (28)

26. The method of paragraph 25, wherein after purification with mineral oil and Hanks' balanced salt solution (HBSS), said product is centrifuged and washed with HBSS. 27. The method of paragraph 26, wherein after cleaning with mineral oil and washing with Hanks' Balanced Salt Solution (HBSS), said product is incubated with a PEG-dithiol solution. 28. A device for applying conformal coating, including: an encapsulation chamber formed by a three-piece assembly as illustrated in FIG. 1A, panels (ii), (iii) and (iv); a catheter connected to a precision flow syringe pump is configured to introduce the coating material and the biomaterial to which the coating is applied into a first inlet of said encapsulation chamber (internal phase), wherein said aqueous phase has a pH level of exactly or about 6- 7.4; a first peristaltic pump is configured to inject an oil phase containing a surfactant into a second inlet of said encapsulation chamber, wherein the injection of said oil phase (external phase) is configured to flow coaxially to said internal aqueous phase; a capillary connected to the end of said encapsulation chamber, wherein said capillary is located downstream of a point where the flow of said internal aqueous phase is extended within said external oil phase (two-phase liquid) such that said two-phase liquid is configured to flow into said capillary from said encapsulation chamber, and wherein said internal aqueous phase (containing the coating material and the biomaterial to be coated) and said external oil phase are configured to flow coaxially through said capillary; and a second peristaltic pump configured to inject a gelling emulsion coaxially to said capillary, wherein said emulsion comprises a catalyst for polymerizing said coating material, wherein said emulsion is configured to be in coaxial contact with said coating material and said biomaterial to which the coating is applied. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. The scope of the present embodiments described herein is not intended to be limited by the above description, but rather is as set forth in the appended claims of this invention. Those of ordinary skill in the art will appreciate that various changes and modifications may be made to this specification without departing from the spirit or scope of the present invention as defined in the following claims. CLAIM 1. A method for conformally coating biomaterials with a coating material, which includes the following steps: (a) injecting the aqueous phase into the coaxial oil phase in a coating device configured to provide a transition from droplet spray to jet spray and elongation of the flow of said aqueous phase into said oil phase; (b) adding said biomaterial and said coating material to said aqueous phase, wherein the coating material from said step (b) does not include a viscosity increasing agent; and wherein said aqueous phase has a pH of from about 6 to about 7.4; (c) allowing the stream of said aqueous phase to disintegrate into particles; and (d) adding a component of said coating material after the jet of said aqueous phase has disintegrated into particles, said component being a gelling emulsion that promotes or catalyzes the polymerization of said coating material; As a result, a biomaterial with a conformal coating is obtained. 2. The method of claim 1, further comprising the step of collecting material flowing from said coating device. 3. The method according to claims 1, 2, further comprising the step of purifying said conformally coated biomaterial and the coating material not containing biomaterial from said oil phase. 4. The method according to any one of claims 1 to 3, further comprising the step of separating said conformally coated biomaterial from said coating material not containing the biomaterial. 5. Method according to any one of claims 1 to 4, characterized in that said biomaterial includes cells, cell clusters, biomaterial-coated cells or cell clusters, subcellular organelles - 43 046872 ly, biological molecules, non-biological drugs or a combination thereof. 6. The method according to claim 5, characterized in that said biomaterial includes cells or clusters of cells. 7. The method according to claim 6, characterized in that said biomaterial includes islet cells or clusters of cells. 8. A method according to any one of claims 1 to 7, characterized in that said coating material includes an agent selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), poly(vinylpyrrolidinone) (PVP), polyethyl oxazoline, polyvinyl alcohol (PVA), polyethyloxazoline (PEOK), poly(amino acids), derivatives thereof and combinations thereof. 9. The method of claim 8, wherein said coating material includes an agent selected from the group consisting of polyethylene glycol (PEG), PEG maleimide, PEG acrylate and PEG vinyl sulfone. 10. The method according to claim 9, characterized in that said coating material includes 5-10% PEG. 11. The method of claim 8, wherein said coating material comprises multi-arm polyethylene glycol (PEG) minimally cross-linked (1-30%) with PEG-dithiol. 12. The method according to any one according to claims 1-11, characterized in that said aqueous phase includes (a) a serum-free medium with a pH of 6-7.4; or (b) Hanks' balanced salt solution (HBSS) pH 6-7.4. 13. The method according to claim 12, characterized in that said aqueous phase includes about 200,000,000 islet cells/ml. 14. The method according to claim 12, characterized in that said aqueous phase includes about 100,000 cell clusters/ml. 15. Method according to any one of claims 1 to 14, characterized in that said aqueous phase includes a thiolated reagent, a reducing reagent, a surfactant, or a combination thereof. 16. The method according to claim 15, characterized in that said surfactant is a block copolymer of polyoxyethylene-polyoxypropylene or poly(ethylene glycol-Npropylene sulfide). 17. The method according to claim 16, characterized in that said surfactant is a 2% polyoxyethylene-polyoxypropylene block copolymer. 18. The method according to claim 15, characterized in that said reducing reagent is dithiothreitol (DTT) or PEG-dithiol. 19. The method according to claim 18, characterized in that said reducing reagent is 0.01-0.62% dithiothreitol (DTT). 20. The method according to any one of claims 1 to 19, characterized in that said gelling emulsion includes dithiothreitol (DTT) dissolved in Hanks' balanced salt solution (HBSS) and emulsified in polypropylene glycol (PG) with 10% sorbitan monooleate (Span80) . 21. Method according to any one of claims 1 to 20, characterized in that said oil phase comprises polypropylene glycol (PPG). 22. Method according to any one of claims 1 to 21, characterized in that said oil phase comprises polypropylene glycol (PPG), 10% sorbitan monooleate and optionally triethanolamine. 23. The method according to claim 22, characterized in that said oil phase includes 0-0.2% triethanolamine. 24. The method according to any one of claims 3 to 23, characterized in that purification of said conformally coated biomaterial and said biomaterial-free coating material from said oil phase includes step (e) infusing the product obtained in step (d ), into mineral oil while stirring the resulting mixture. 25. The method of claim 24, wherein purification of said conformally coated biomaterial and said biomaterial-free coating material from said oil phase comprises step (f) adding Hanks' balanced salt solution (HBSS) to the product obtained in step (e). 26. Method according to claim 25, characterized in that the product of step (f) is centrifuged and washed with HBSS. 27. The method of claim 26, wherein after centrifugation and washing with Hanks' Balanced Salt Solution (HBSS), said conformally coated biomaterial is incubated with a PEG-dithiol solution. 28. A conformal coating apparatus comprising (1) an encapsulation chamber including (a) a housing portion connected to a catheter through a first inlet at a first end of said housing portion and connected to a first pump; (b) a fastening portion including a first end configured to engage an inner surface of a second end of said body portion; and (c) a coating portion including a first end configured to -