WO2025151923A1 - Apparatus for production of engineered cells - Google Patents

Abstract

An apparatus (100) for production of engineered cells, the apparatus including: at least one injection module (116) to be at least partly immersed in a fluid mixture containing cells to be engineered, the module including: an aspiration unit (118) for isolating one of the cells from the fluid mixture, the aspiration unit including: a chamber (128) to receive said one of the cells; and an inlet (122) configured to convey at least a portion of said one of the cells from the fluid mixture to the chamber upon application of suction through the aspiration unit; a porous filter (126) arranged with respect to the aspiration unit to selectively retain said one of the cells at least partially within the chamber upon the application of suction; and an injector (130) projecting through the porous filter and terminating at a tip located within the chamber for penetrating said one of the cells upon the application of suction to deliver one or more substances into said one of the cells to cause said one of the cells to express a recombinant receptor.

Description

APPARATUS FOR PRODUCTION OF ENGINEERED CELLS

Field

[0001] The present invention relates generally to instrumentation used in biochemical engineering. More particularly, the present invention relates to instrumentation for production of engineered cells. However, while some embodiments will be described herein with particular to that application, it will be appreciated that the invention is not limited to such a field of use, and is applicable in broader contexts.

Background

[0002] Therapeutic bioengineering is a pioneering discipline of medical science with the potential to provide many benefits, particularly in clinical applications to deliver targeted, personalised disease treatments.

[0003] Chimeric antigen receptor T-cell (CAR T-cell) therapy is a form of immunotherapy which involves genetically engineering T-cells to express surface chimeric antigen receptors (CARs) for binding to specific proteins, or antigens, on the surface of target cells, such as cancer cells, to induce cell death. Other immune cells, such as NK or dendritic cells, may also be engineered in a similar manner for therapeutic use.

[0004] CAR T-cell therapy may involve procedures such as chemical transfection, virus infection or electroporation of the isolated T-cells to engineer the T-cell to express CARs. Disadvantageously, such procedures may be complex, expensive and difficult to standardise. Moreover, such procedures may also suffer from:

• variability on the quantity of material delivered into the immune cell;

• intrinsic toxicity of the infection/transfection procedure;

• a requirement to select cells after the procedure to sort the infected/transduced from the group that has not received the payload;

• a requirement for purification from virus and chemicals if the engineered immune cells are utilised for therapeutic purposes; and

• an increasing level of biohazards. [0005] Extracellular vesicles (EVs) have also been proposed as possible structures for delivering therapeutics. However, improvements in efficacy are needed for engineered therapies based on the use of extracellular vesicles.

Summary of Invention

[0006] It is an object of the present invention to substantially overcome, or at least ameliorate, one or more drawbacks of present arrangements or to provide a useful alternative thereto.

[0007] In one aspect, the present invention provides an apparatus for production of engineered cells, the apparatus including: at least one injection module to be at least partly immersed in a fluid mixture containing cells to be engineered, the module including: an aspiration unit for isolating one of the cells from the fluid mixture, the aspiration unit including: a chamber to receive said one of the cells; and an inlet configured to convey at least a portion of said one of the cells from the fluid mixture to the chamber upon application of suction through the aspiration unit; a porous filter arranged with respect to the aspiration unit to selectively retain said one of the cells at least partially within the chamber upon the application of suction; and an injector projecting through the porous filter and terminating at a tip located within the chamber for penetrating said one of the cells upon the application of suction to deliver one or more substances into said one of the cells to cause said one of the cells to express a recombinant receptor.

[0008] In one or more embodiments, the injector is configured to administer a microinjection, a nanoinjection, a high pressure flux injection, or an elastic recoil injection.

[0009] In one or more embodiments, the porous filter is formed of a nanoporous membrane.

[0010] In one or more embodiments, the apparatus further includes a vacuum unit communicable with the module to provide suction through the aspiration unit.

[0011] In one or more embodiments, the apparatus further includes a sensor operatively associated with the module to sense changes in negative pressure within the aspiration unit. [0012] In one or more embodiments, the apparatus further includes: a sorting block to separate the cells to be engineered from a blood sample or other medium; a microinjection block to receive the cells from the sorting block, the microinjection block including the at least one injection module; a filtering block to receive the engineered cells from the microinjection block for purifying the engineered cells; and an incubator to receive the engineered cells from the filtering block for storing, cultivating and maintaining the engineered cells for collection.

[0013] In one or more embodiments, the at least one injection module includes a plurality of injection modules.

[0014] In one or more embodiments, the porous filter and the injector form an integrated structure within the aspiration unit.

[0015] In one or more embodiments, the inlet has a variable diameter.

[0016] In one or more embodiments, the inlet has a diameter in the range of about 4-7 pm.

[0017] In one or more embodiments, the porous filter is axially spaced from the inlet by a variable distance.

[0018] In one or more embodiments, the porous filter is axially spaced from the inlet by a distance in the range of about 4-7 pm.

[0019] In another aspect, the invention provides a microfluidic device for production of engineered cells, the device including: a first channel providing an inlet for introduction of a fluid containing cells to be engineered; a second channel fluidly communicable with the first channel, the second channel being arranged to resist flow of the fluid to modulate a flow rate thereof, enabling manipulation of each of the cells; a third channel fluidly communicable with the second channel to deliver the fluid to an injection module for injecting one or more substances into each of the cells; and a fourth channel fluidly communicable with the third channel to convey the cells to an outlet for collection.

[0020] In one or more embodiments, the array includes a plurality of loops formed by successive lengths of the second channel.

[0021] In one or more embodiments, the apparatus or the microfluidic device as described above is for use in the manufacture of an extracellular vesicle-based therapeutic for the treatment of a neurological disease or disorder.

Brief Description of Drawings

[0022] Exemplary embodiments of the present disclosure will now be described, by way of examples only, with reference to the accompanying description and drawings in which:

[0023] FIG. 1 is a schematic diagram of an apparatus for production of engineered cells according to an embodiment, showing simplified representations of components of the apparatus;

[0024] FIG. 2 is a partial, longitudinally sectioned view of an injection module of the apparatus of FIG. 1;

[0025] FIG 2A is a detail view of a portion of the injection module of FIG. 2;

[0026] FIG. 3 is a simplified schematic diagram of a workflow of the injection module of FIG. 2;

[0027] FIG. 4 is a schematic diagram of a cell sorting chamber according to an embodiment of the apparatus of FIG. 1;

[0028] FIG. 4A is a schematic diagram of a serialised cell sorting chamber according to another embodiment of the apparatus of FIG. 1;

[0029] FIG. 5 is a schematic diagram of a microinjection chamber according to an embodiment of the apparatus of FIG. 1; [0030] FIG. 6 is a schematic diagram of a workflow of an extrusion chamber according to an embodiment of the apparatus of FIG. 1;

[0031] FIG. 7 shows the process of extrusion from a loaded red blood cell to a red blood cell- derived extracellular vesicle (RBC-EV) in (A) according to the present disclosure. Histograms show the results of nanotracking analysis (NTA) on size distribution of RBC-EVs after extrusion; focused size spectra from zero to 200 nanometres in (B); and full-size spectra up to 1000 nanometres in (C). Zetasizer results of size distribution of RBC-EVs after extrusions in (D);

[0032] FIG. 8 shows the RBC-EVs are stable over freeze thaw cycles. The stability over four freezing cycles is depicted in (A), through upper graph which shows a normalised count of vesicles as a percent of the control while the lower graph depicts the count of particles per millilitre. This experiment was conducted n=2 times. (B) describes the size distribution of the RBC-EVs for each specific freeze/thaw cycle (technical replicates n=5);

[0033] FIG. 9 shows in vitro evaluation of the ability of the RBC-EVs to deliver the payload, in comparison with commercial lipid nanoparticles. Transfection efficacy evaluated measuring the amount of fluorescent tagged siRNA in HSJD-DIPG007 (A) and in U87 (B) cells. Cell death evaluated as percentage of PI positive cells in HSJD-DIPG007 and U87 after transfection with lipofectamine RNAiMax and RBC-EVs created with TrUTh protocol (C). P-values are denoted as: * = p < 0,1; ** = p < 0,05; *** = p < 0,01; ****p < 0.005; ns= non-significant. Data is mean ± sd;

[0034] FIG. 10 shows in vitro evaluation of the ability of the RBC-EVs to deliver RNA interfering with brain tumour cells and affecting cell growth and drug sensitivity. Real-time PCR performed to evaluate the level of c-MYC downregulation for HJSD-DIPG007 and U87 cell lines (A). To evaluate the sensitivity to TEPA after c-MYC knockdown viability is normalised to their untreated controls thus viability is expressed as a percentage reduction from the control (WT, lipofectamine RNAiMAX KD, and RBC-EV c-MYC KD (B). The effect of a prolonged transfection (96 hours instead of 48 hours) on HSJD-DIPG007 cell line (C).

Technical replicates n=3. P-values are denoted as: * = p < 0,1; ** = p < 0,05; *** = p < 0,01; ****p < 0.005; ns= non-significant. Graph displaying the effects of increasing TEPA concentrations on both WT and RBC- EV c-MYC knockdown HSJD-DIPG007 cells (D); [0035] FIG. 11 shows in vivo data at 2 and 4 hours post injection (upper and bottom views, respectively). Mouse on the left has been treated with TYE705 loaded RBC-EVs, whilst the mouse on the right has been treated with empty RBC-EVs. Mice have been monitored during the week post injection; no signal of distress has been noticed in either of the treated and the untreated mouse; and

[0036] FIG. 12 shows the ability of the RBC-EVs to cross the blood-brain barrier (BBB) and accumulate in the brain of DIPG bearing mice. Quantification of signal over background in live imaging, at 2 and 4 hours post tail injection of empty vesicles and vesicles loaded with a red fluorescent (TYE705) scramble oligo of 30bp size. Quantification of the signal in Brain, Liver and Kidneys. This preliminary data shows a specific accumulation of the signal inside mice brains confirming the ability of the RBC-EVs to cross the blood brain barrier.

Description of Embodiments

[0037] Referring firstly to FIG. 1 of the drawings, an apparatus 100 for production of engineered immune cells 102 (shown in FIG. 3) according to an embodiment is depicted. The apparatus 100 is primarily configured to transfect or transduce mammalian cells, particularly immune effector cells, with recombinant receptors, such as chimeric antigen receptors (CARs), for clinical applications, such as immunotherapy, and will be described with reference to this application. However, the apparatus 100 may also be utilised to perform plasmid transduction or gene engineering on eukaryotic cells, such as red blood cells (RBCs) to produce red blood cell- derived extracellular vesicles (RBC-EVs), for therapeutic research and development or the like.

[0038] As shown in FIG. 1, the apparatus 100 may be configured as a compartmentalised machine or instrument having one or more components which operate to facilitate the production of engineered cells, for example engineered immune cells 102. In the embodiment depicted, the apparatus 100 includes four main components each of which are configured to operate successively as will be discussed below: The four main components include: a sorting block 104 to separate the cells, for example immune cells 105 (shown in FIG. 3), to be engineered from a blood sample or other medium; a microinjection block 106 to receive the sorted immune cells 105 from the sorting block 104 and to inject material into the immune cell 105 to modulate cell receptor expression; a filtering block 108 to purify the engineered immune cells 102; and an incubator 110 for storing, cultivating and maintaining the purified engineered immune cells for collection.

[0039] The components 104, 106, 108, 110 may be serialised and automated (via robotics, for example) to streamline and standardise the production of engineered immune cells 102 and enhance yield thereof. In other embodiments, the sorting block 104, the filtering block 108 and/or the incubator 110 may be optional components of the apparatus 100 such that the microinjection block 106 operates to produce engineered immune cells 102 as a primary function of the apparatus 100.

[0040] With reference to FIG. 4, in one embodiment, the sorting block 104 may include a cell sorting chamber 107 employing inertial focusing using Archimedean spiral microfluidic channels or other curved channel geometries. The sorting chamber 107 is optimised to sort cells by size from a blood sample, cell culture or other biofluids. In one example, biofluids are pumped into the cell sorting chamber 107 via an initial spiral microchannel A. The cells flow toward the central region of the spiral microchannel whereby the cells are sorted by size according to their differential focusing by virtue of hydrodynamic forces acting on the fluid flow. Upon reaching the center, cells of a specific size are separated upon branching into either a waste channel W or a collection channel B of the cell sorting chamber 107. In some embodiments, the cell sorting chamber 107 may be serialised to sort different populations. For example, as shown in FIG. 4A, whole blood can enter a primary spiral SI of the cell sorting chamber 107a via an initial spiral microchannel A to cause depletion of platelets through the waste channel W, whilst RBCs and immune cells migrate to a secondary spiral S2, via a linkage channel linking the primary and secondary spirals SI, S2, whereby RBCs and immune cells are subsequently divided into either an additional waste channel W2 or a collection channel B depending on the intended application.

[0041] The microinjection block 106 includes an array of microinjection chambers 112 and a microhydraulic system 114 operatively coupled to the microinjection chambers 112. The microhydraulic system 114 is configured to receive and convey the sorted immune cells 105 from the sorting block 104 to a first selection of the microinjection chambers 112. The first selection of the microinjection chambers 112 includes one or more of the microinjection chambers 112 which are each configured to hold a variable quantity of the immune cells 105 to be engineered in a buffer solution. The first selection of the microinjection chambers 112 may include means for maintaining the variable quantity of the immune cells 105 in steady-state movement within the buffer solution. A second selection of the microinjection chambers 112 includes one or more of the microinjection chambers 112 which are each configured to hold a variable quantity of the engineered immune cells 102.

[0042] As shown in FIG. 3, the microinjection block 106 further includes an injection module 116 operatively associated with the microinjection chambers 112 to 1) collect one of the immune cells 105 from one of the microinjection chambers 112 of the first selection; 2) engineer the collected immune cell 105; and 3) release the engineered immune cell 102 into one of the microinjection chambers 112 of the second selection. In some embodiments, the microinjection block 106 may include a plurality of the injection modules 116 to increase throughput and/or efficiency of the apparatus 100.

[0043] With particular reference to FIGs 2 and 2 A, the injection module 116 includes an aspiration unit 118 having an elongate body 120 providing an interior, and an inlet 122 formed at an operative end of the body 120. The inlet 122 is sized to exclusively permit one of the immune cells 105 to be engineered to pass therethrough, at least partially into the interior upon application of suction through the aspiration unit 118. In one embodiment, the inlet 122 has a diameter in the range of about 4-7 pm. In other embodiments, the diameter of the inlet 122 may be adjustable or variable depending on the size of the immune cell 105 to be collected. The body 120 includes a sidewall 124 which extends from the inlet 122 and is generally frustoconical in shape so as to form a tapered tube circumferentially surrounding the interior of the body 120.

[0044] The injection module 116 also includes a porous filter 126 arranged within the interior of the body 120 at a location which is spaced from the inlet 122. The filter 126 is coupled or otherwise fixed to an internal surface of the sidewall 124 to sealingly engage the sidewall 124. In other embodiments, the injection module 116 may include a separate seal between the filter 126 and the sidewall 124. Together, the filter 126 and the sidewall 124 partly surround a working chamber 128 adjacent the inlet 122 for receiving the immune cell 105 after passing through the inlet 122. The filter 126 includes a plurality of pores each having a pore size to permit fluid and extraneous material to pass therethrough whilst preventing passage of the immune cell 105, thereby selectively retaining at least a portion of the immune cell 105 within the chamber 128 upon the application of suction through the aspiration unit 118. In some embodiments, the filter 126 is axially spaced from the inlet 122 by a distance in the range of about 4-7 pm. In other embodiments, the filter 126 may be axially spaced from the inlet 122 by a distance which is adjustable or variable depending on the size of the immune cell 105 to be received. In the embodiment depicted, the filter 126 is in the form of a disc or ring comprised of a nanoporous membrane having a thickness of about 0.5 pm. In other embodiments, the pore size and/or shape of the membrane may vary depending on the application.

[0045] The injection module 116 further includes an injector 130 which projects through the filter 126, particularly through the centre of the filter 126, and terminates at a tip located within the working chamber 128. In the embodiment depicted, the injector 130 is in the form of a needle (such as a microinjector or a nanoinjector) for penetrating the immune cell 105 within the working chamber 128 for delivering or injecting one or more substances into the immune cell 105. In some embodiments, the injector 130 is configured to administer a microinjection, a nanoinjection, a high pressure flux injection, or an elastic recoil injection. The one or more substances may be macromolecules including RNA, shRNA, siRNA, ASO, mRNA, plasmids, DNA oligos, peptides, proteins, small molecule drugs or a combination thereof which are designed to cause the immune cell 105 to express chimeric antigen receptors on its surface for binding to specific proteins, or antigens, on the surface of target cells. In some embodiments, the injector 130 and the filter 126 form an integrated structure housed within the aspiration unit 118.

[0046] By virtue of the structural arrangement of the injection module 116, the apparatus 100 permits engineering of immune cells 105 via a mechanical process (that is, using physical stress) as opposed to a process involving viral vectors or chemical transfection.

[0047] Optionally, the microinjection block 106 may further include a vacuum unit (not shown) operatively associated with the injection module 116 to provide suction or negative pressure through the aspiration unit 118.

[0048] Operation of the apparatus 100 for production of engineered immune cells 102 will now be described.

[0049] In a first phase, a blood sample or other biological medium containing immune cells 105 to be engineered is conveyed through the sorting block 104. The sorting block 104 may include a plurality of meshes of variable dimensions to progressively filter and sort the sample or medium passing therethrough into various cell populations based on size. In other embodiments, the sorting block 104 may include the cell sorting chamber 107 employing inertial focusing as described above. At the end of the first phase, only the portion of blood or medium containing the immune cells 105 to be engineered is conveyed from the sorting block 104 to the microinjection block 106 (via, for example, pumps or other hydraulic systems). The sorted immune cells 105 are transported into one or more of the microinjection chambers 112 of the first selection which may contain reagents or other buffer solutions.

[0050] In a second phase, the one or more injection modules 116 are operated by firstly immersing the inlet 122 of the aspiration unit 118 in fluid communication with the buffer solution containing the immune cells 105, and secondly applying vacuum (negative) pressure or suction through the aspiration unit 118. By virtue of the combinational arrangement of the inlet 122, the working chamber 128 and the porous filter 126, the application of suction causes only one of the immune cells 105 to be isolated from the buffer solution at a given time. Optionally, changes in negative pressure may be sensed by a sensor (not shown) of the microinjection block 106 to indicate that one of the immune cells 105 has been captured. As the suction is applied, the negative pressure within the working chamber 128 causes the immune cell 105 to bear against the porous filter 126, causing the tip of the needle 130 to pierce the immune cell 105. Simultaneously upon piercing or otherwise, the one or more substances or macromolecules are then delivered into the immune cell 105 via the tip of the needle 130 to engineer the immune cell 105. The injection module 116 is then operated to move and release the engineered immune cell 102 into one of the microinjection chambers 112 of the second selection which may contain sterile media, regents and/or is otherwise maintained in a temperature stable environment. The processes of the second phase are repeated until a desired number of engineered immune cells 102 is reached. In another embodiment, the microinjection chamber 112a may include the arrangement as depicted in FIG. 5, whereby the injection module 116 is integrated with a microfluidic chip 117 comprising an array or loops of lengths L of microchannels to impart fluidic resistance to stabilize the flux and modulate the flow rate, enabling cell manipulation downstream. In use, the sorted cells enter channel A via an inlet, whilst PBS enters channel P, for migration downstream through the lengths L toward the injection module 116 for subsequent injection of the substance into the cell. The loaded/engineered cell then flows through the channel B for exiting the microinjection chamber 112a via an outlet for subsequent collection. In this way, aspiration is not required by virtue of the arrangement of the lengths L being integrated with the injection module 116 on the microfluidic chip 117. Optionally, a fluorescent marker may be added to the material/sub stance to inject, to image and sort the final population according to fluorescence. In some embodiments, the microinjection chamber may have a resistor length of 250 mm.

[0051] In a third phase, the engineered immune cells 102 are transported from the microinjection block 106 to the filtering block 108 where the engineered immune cells 102 may be passed through various filters under pressure to purify the engineered immune cells 102 from other biological or extraneous media (see FIG. 6). In this optional step, the loaded cells coming from the previous phase can be collected for further processing or cultured. In some applications, the loaded cells can either be kept as whole cells (T cells engineered to be CAR T- cell, RBCs loaded with lipophilic drugs for controlled release of the drug in the bloodstream) or transformed into vesicles (RBCs derived vesicles used as a targeted drug delivery system, see FIG. 7). According to the present disclosure, the extruded RBCs do not suffer batch to batch preparation, resulting in a similar size distribution over the various biological replicates, regardless of the payload.

[0052] In a fourth phase, the purified engineered immune cells 102 are passed from the filtering block 108 to the incubator 110 where temperature and nourishment is regulated for storing, cultivating and maintaining the purified engineered immune cells 102 for collection.

[0053] The apparatus 100 may provide a more homogeneous final product, remove the requirement for sorting populations (since all the immune cells that are collected in the second selection of the microinjection chambers are engineered), fine tune the quantity of molecules injected (that is, the concentration of the material in the buffer to inject, quantity of material injected), enable injection of different types of molecules simultaneously (for example, proteins and RNA, CRISPR/cas machinery etc.), and/or minimise complicated and costly purification steps compared to existing procedures (since the apparatus 100 does not rely on viral infection or chemical transduction). As shown in FIGs. 7 to 12, the present inventors have demonstrated safety and efficacy in the utility of the present disclosure for the production of RBC-EVs.

[0054] Although specific embodiments of the invention are illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternative and/or equivalent implementations exist. It should be appreciated that the exemplary embodiment or exemplary embodiments are examples only and are not intended to limit the scope, applicability, or configuration in any way. Rather, the foregoing summary and detailed description will provide those skilled in the art with a convenient road map for implementing at least one exemplary embodiment, it being understood that various changes may be made in the function and arrangement of elements described in an exemplary embodiment without departing from the scope as set forth in the appended claims and their legal equivalents. Generally, this application is intended to cover any adaptations or variations of the specific embodiments discussed herein.

[0055] It will also be appreciated that in this document the terms "comprise", "comprising", "include", "including", "contain", "containing", "have", "having", and any variations thereof, are intended to be understood in an inclusive (i.e. non-exclusive) sense, such that the process, method, device, apparatus, assembly or system described herein is not limited to those features or parts or elements or steps recited but may include other elements, features, parts or steps not expressly listed or inherent to such process, method, device, apparatus, assembly or system. Furthermore, the terms "a" and "an" used herein are intended to be understood as meaning one or more unless explicitly stated otherwise. Moreover, the terms "first", “primary”, "second", “secondary” etc. are used merely as labels, and are not intended to impose numerical requirements on or to establish a certain ranking of importance of their objects.

Claims

1. An apparatus for production of engineered cells, the apparatus including: at least one injection module to be at least partly immersed in a fluid mixture containing cells to be engineered, the module including: an aspiration unit for isolating one of the cells from the fluid mixture, the aspiration unit including: a chamber to receive said one of the cells; and an inlet configured to convey at least a portion of said one of the cells from the fluid mixture to the chamber upon application of suction through the aspiration unit; a porous filter arranged with respect to the aspiration unit to selectively retain said one of the cells at least partially within the chamber upon the application of suction; and an injector projecting through the porous filter and terminating at a tip located within the chamber for penetrating said one of the cells upon the application of suction to deliver one or more substances into said one of the cells to cause said one of the cells to express a recombinant receptor.

2. The apparatus of claim 1, wherein the injector is configured to administer a microinjection, a nanoinjection, a high pressure flux injection, or an elastic recoil injection.

3. The apparatus of claim 1 or 2, wherein the porous filter is formed of a nanoporous membrane.

4. The apparatus of any one of the preceding claims further including a vacuum unit communicable with the module to provide suction through the aspiration unit.

5. The apparatus of any one of the preceding claims further including a sensor operatively associated with the module to sense changes in negative pressure within the aspiration unit.

6. The apparatus of any one of the preceding claims further including: a sorting block to separate the cells to be engineered from a blood sample or other medium; a microinjection block to receive the cells from the sorting block, the microinjection block including the at least one injection module; a filtering block to receive the engineered cells from the microinjection block for purifying the engineered cells; and an incubator to receive the engineered cells from the filtering block for storing, cultivating and maintaining the engineered cells for collection.

7. The apparatus of claim 6, wherein the at least one injection module includes a plurality of injection modules.

8. The apparatus of any one of the preceding claims, wherein the porous filter and the injector form an integrated structure within the aspiration unit.

9. The apparatus of any one of the preceding claims, wherein the inlet has a variable diameter.

10. The apparatus of any one of the preceding claims, wherein the inlet has a diameter in the range of about 4-7 pm.

11. The apparatus of any one of the preceding claims, wherein the porous filter is axially spaced from the inlet by a variable distance.

12. The apparatus of any one of the preceding claims, wherein the porous filter is axially spaced from the inlet by a distance in the range of about 4-7 pm.

13. A microfluidic device for production of engineered cells, the device including: a first channel providing an inlet for introduction of a fluid containing cells to be engineered; a second channel fluidly communicable with the first channel, the second channel being arranged to resist flow of the fluid to modulate a flow rate thereof, enabling manipulation of each of the cells; a third channel fluidly communicable with the second channel to deliver the fluid to an injection module for injecting one or more substances into each of the cells; and a fourth channel fluidly communicable with the third channel to convey the cells to an outlet for collection.

14. The microfluidic device of claim 13, wherein the array includes a plurality of loops formed by successive lengths of the second channel.

15. The apparatus of any one of claims 1 to 12 or the microfluidic device of claim 13 or 14, for use in the manufacture of an extracellular vesicle-based therapeutic for the treatment of a neurological disease or disorder, a solid or liquid tumour, or a genetic disease or disorder.