WO2021005531A1

WO2021005531A1 - Decellularized human chorion membrane as a new substrate to mimic biological barriers, method, kit and uses thereof

Info

Publication number: WO2021005531A1
Application number: PCT/IB2020/056426
Authority: WO
Inventors: Laura PASSOS MORGADO FRANCO FRAZÃO; Joana VIEIRA DE CASTRO; Rui L. REIS; Nuno MELEIRO ALVES DAS NEVES
Original assignee: Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) - Associação
Priority date: 2019-07-08
Filing date: 2020-07-08
Publication date: 2021-01-14

Abstract

The present disclosure relates to the field of medicine, namely to the use of placenta for the production of decellularizing an isolated human chorion membrane (HCM) and its use to mimic BBs.

Description

DECELLULARIZED HUMAN CHORION MEMBRANE AS A NEW SUBSTRATE TO MIMIC

BIOLOGICAL BARRIERS, METHOD, KIT AND USES THEREOF

Technical field

[0001] The present disclosure relates to the field of medicine, namely to the use of placenta for the production of decellularizing an isolated human chorion membrane (HCM).

Background

[0002] The human chorion membrane (HCM) composes the fetal part of the human placenta and it is located between the amnion (fetal side) and the maternal decidua.

[0003] The decellularization protocol was optimized by quantifying and analyzing the presence and distribution of cell nuclei, DNA, and components of the extracellular matrix (ECM). Moreover, dHCM mechanical properties were assessed and metabolic and proliferation tests with an endothelial cell line were performed.

[0004] The HCM was successfully decellularized and the main ECM proteins were preserved. dHCM is a membrane, mostly composed by collagen type I, with two different sides: dHCM A and dHCM B. In dHCM A, the basement membrane is conserved being composed by collagen type IV, fibronectin and laminin. Regarding the dHCM B, it is constituted by nanofibers and proteins such as collagen type I and laminin. The mechanical properties of dHCM demonstrate that this membrane can be used as a substrate to mimic BBs. Additionally, in both sides of dHCM, an endothelium-like structure was formed by EA.hy926 cells, reinforcing the suitability of dHCM to mimic BBs.

[0005] Biological barriers (BBs) separate different microenvironments in the human body, having as main functions the protection of the organism from the external environment and the maintenance of homeostasis for physiological functions. [0006] There are a wide variety of BBs, including skin (a well-defined physical barrier), connective tissue around nerves and Schwann cells around some types of axons (both considered poorly defined physical barriers). BBs may be free of living cells, such as the outer layer of skin, or can be composed by living cells (such as connective tissue and endothelium). Furthermore, as it happens in the blood-brain barrier and in the epithelium, cells that compose BBs might have specific connections between them, like tight junctions, strongly limiting its permeability, making it harder to cross them. The vascularization degree is also very variable in different BBs [1], [2].

[0007] The study of BBs is of great interest not only for the development of strategies to replace a damaged BB but also to study disease's mechanisms associated to the disruption of these barriers. Moreover, by mimicking BBs, it is possible to better simulate the function of organs in vitro and consequently to create an alternatives for avoiding animal testing, according to the 3R (replace, refine, reduce) principle [l]-[4]

[0008] Currently, the gold standard for modeling BBs in vitro are microporous membranes mainly composed by polyester or polycarbonate, mounted in adequate supports (e.g. Traswell^® inserts) [2] However, these models lack some important characteristics present in BBs such as the proteins of the extracellular matrix (ECM) [4] In fact, it is of great interest to mimic the ECM in vitro since it creates a specific cell microenvironment and can guide cell behavior [5] Moreover, the ECM is important for organ development and stability and for barrier function since it provides structural support to the tissue and functional input to modulate cell performance and function [6]

[0009] The human placenta is considered a biological waste. Nevertheless, it is also a source of ECM proteins and is consistently available from full-term births [7] The human placenta has two membranes, the amnion (HAM) and the chorion (HCM) membranes [8] Dehydrated human amnion/chorion membranes have been shown to recruit circulating progenitor cells when implanted subcutaneously [9], to regulate stem cell activity in vitro [10], and to be useful in the treatment of chronic-wounds [11]

[0010] The HAM alone is widely characterized and studied and has been shown to induce osteogenic differentiation of human dental apical papilla cells [12], to be a good chondrocyte substrate/carrier [13], and to promote epithelization [14]— [16]. Moreover, decellularized HAM have been used in tissue engineering in cell-matrix adhesion studies, to produce skin equivalents, and as a pericardial substitute [8], [15], [17]— [19].

[0011] The HCM composes the fetal part of the human placenta and it is located between the amnion (fetal side) and the maternal decidua. [14], [20] The potential of HCM alone is still largely unexplored, it has only been used as a reservoir of stem [21] and pro-angiogenic [22] cells, and as a source of small-diameter vascular grafts [7] The HCM is formed by three different layers (from the fetal to the maternal side): 1) Reticular Layer that contacts with the amnion, representing the majority of the chorion thickness and composed of collagens I, III, IV, V and VI; 2) Basement Membrane that anchors the reticular layer and the trophoblasts and is composed by collagen IV, fibronectin and laminin; 3) Trophoblasts layers (3-5 cell layers) that provide nutrients to the embryo by digesting proteins before passing them into the fetal blood [23], [24]

[0012] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

General Description

[0013] It is disclosed the use of an acellular HCM to mimic BBs. A decellularization of HCM was performed which allowed the maintenance of the HCM original architectural rearrangement and composition.

[0014] In the present disclosure, the HCM was successfully decellularized, according with the stablished criteria for decellularized tissues to avoid cell and host adverse effects [28], preserving the main proteins of ECM. dHCM is a membrane, mostly composed by collagen type I, with two different sides: dHCM A and dHCM B. I n dHCM A, the basement membrane was conserved, and it is composed by collagen type IV, fibronectin and laminin, while dHCM B is constituted by nanofibers and proteins such as collagen type I and laminin. The mechanical properties of dHCM make it a suitable substrate to mimic BBs, since its young's modulus is similar to the one described for basement membranes [36] and it is stable in a wide range of frequencies. In both sides of the dHCM, an endothelium-like structure was formed by EA.hy926 cells, reinforcing the suitability of dHCM to develop models of BBs.

[0015] The present invention discloses a method for decellularizing an isolated human chorion membrane (HCM) comprising the following steps:

submitting the isolated HCM to freezing/thawing cycle(s) in order to decellularize the HCM;

washing the HCM with an anionic surfactant;

bathing the HCM overnight with an anionic surfactant;

washing the HCM with a buffer solution;

scrapping both sides of the HCM;

exposing the HCM to DNase I;

washing the HCM with an anionic surfactant and washing the decellularized membrane with a buffer solution until the resulting membrane is substantially or completely free of throphoblast layer and nuclei for the reticular layer.

[0016] In an embodiment of the present invention it is disclosed a method comprising the following steps:

submitting an isolated HCM to at least two freezing/thawing cycles of around

- 80 °C/37 °C, in order to decellularize the HCM;

treating the HCM with an anionic surfactant;

washing the HCM at least three times with an anionic surfactant; bathing the HCM overnight with an anionic surfactant;

washing the HCM at least three times with Triton-X100 in PBS;

scrapping both sides of the HCM;

exposing the HCM to DNase I at 0.001 mg/mL at 37 °C;

washing the HCM with an anionic surfactant at 4 °C;

washing the decellularized membrane with PBS at least three times at 4°C; wherein the resulting membrane is substantially or completely free of throphoblast layer and nuclei for the reticular layer.

[0017] In a further embodiment of the present invention it is disclosed a method comprising the following steps: submitting an isolated HCM to at least two freezing/thawing cycles of around - 80 °C/37 °C, in order to decellularize the HCM;

treating the HCM with an anionic surfactant solution at 0.5% and with an SDS solution at 0.1%(wt/v);

washing the HCM at least three times with an anionic surfactant at 0.5 %(wt/v);

bathing the HCM overnight with SDS at 0.1%(wt/v);

washing the HCM three times with Triton-X100 in PBS at 1 % (wt/v); scrapping both sides of the HCM;

exposing the HCM to DNase I at 0.001 mg/mL during 30 min at 37 °C; washing the HCM with an anionic surfactant at 0.1 % (wt/v) at 4 °C; washing the decellularized membrane with PBS at least three times at 4°C; wherein the resulting membrane is substantially or completely free of throphoblast layer and nuclei for the reticular layer.

[0018] In a further embodiment of the present invention it is disclosed a method wherein the anionic surfactant is sodium dodecyl sulfate.

[0019] In a further embodiment of the present invention it is disclosed a method wherein all the steps are performed under sterile conditions and the water of said aqueous solutions is ultra-pure water.

[0020] In a further embodiment of the present invention it is disclosed a method wherein said three washes with SDS at 0.5% are washes of around 2 hours each.

[0021] In a further embodiment of the present invention it is disclosed a method wherein said three washes with Triton-X100 in PBS are washes of around 15 minutes each.

[0022] In a further embodiment of the present invention it is disclosed a method wherein said exposition to DNase I lasts around 30 minutes.

[0023] In a further embodiment of the present invention it is disclosed a method wherein said at least three washes with PBS are washes of around 2 hours each.

[0024] In a further embodiment of the present invention it is disclosed a method wherein the following steps were performed at 4 °C in an orbital shaker at 110 rpm: treating the HCM with an anionic surfactant solution at 0.5% and with an SDS solution at 0.1%(wt/v);

washing the HCM at least three times with an anionic surfactant at 0.5 %(wt/v); bathing the HCM overnight with SDS at 0.1%(wt/v);

washing the HCM three times with Triton-X100 in PBS at 1 % (wt/v);

scrapping both sides of the HCM;

washing the HCM with an anionic surfactant at 0.1 % (wt/v);

washing the decellularized membrane with PBS at least three times;

[0025] In an embodiment of the present invention it is disclosed a method wherein the membranes are scrapped in both sides and marked with a knot with a suture line to identify both membrane sides.

[0026] In a further embodiment of the present invention it is disclosed a method wherein after decellularization, membranes are stored in PBS with an antibiotic/antimycotic solution at 1-2 %(wt/v), at 4 °C.

[0027] In an embodiment of the present invention it is disclosed a method wherein after decellularization, membranes are object to a recellularization step.

[0028] In a particular embodiment of the present invention it is disclosed a decellularized human chorion membrane obtainable by the process of the present invention wherein the decellularized human chorion membrane is substantially or completely free of throphoblast layer and nuclei for the reticular layer.

[0029] In an embodiment of the present invention it is disclosed a method forevaluating blood-brain barrier permeability of a test substance, test cell or test protein comprising exposing said test substance, test cell or test protein to the decellularized membrane of the present invention.

[0030] In an embodiment of the present invention it is disclosed a drug screening, distribution and permeability kit for drugs comprising the decellularized human chorion membrane of the present invention.

[0031] In a further embodiment of the present invention it is disclosed the use of the decellularized human chorion membrane of the present invention as a cell scaffold. [0032] In a further embodiment of the present invention it is disclosed the use of the decellularized human chorion of the present invention as an in vitro model normal or disease-related of human blood-brain barrier.

[0033] In a particular embodiment of the present invention it is disclosed the use of a decellularized human chorion of the present invention as a substrate to mimic biological barriers for same or different types of cells.

[0034] In a particular embodiment of the present invention it is disclosed the use of decellularized human chorion as a substrate to mimic basement membranes.

Brief Description of Drawings

[0035] Figure 1 - Native and Decellularized Human Chorion Membrane. Representative transversal sections of Hematoxylin/Eosin staining of native human chorion membrane (HCM) (A) and decellularized HCM (dHCM) (B). Representative SEM micrographs of the dHCM from the amnion side - dHCM A (C) and trophoblast side - dHCM B (D). Representative transversal sections of DAPI staining from native (E) and dHCM (F). Double-stranded DNA (dsDNA) quantification in native and dHCM (G). Agarose gel electrophoresis of DNA extracted from native and dHCM (H). Thickness of air-dried native and dHCM measured with a picometer in, at least, three different sites (I). Top view of native (J) and dHCM (K) cut into pieces of 2x2 cm. Swelling behavior of dHCM in culture medium and DPBS (L). Where, ** p < 0.01 and *** p < 0.001.

[0036] Figure 2 - Composition of both Native and decellularized HCM. Representative transversal sections of Masson's Trichrome, Alcian Blue and Safranin-0 staining of native and dHCM (A). Quantification of collagen (B) and sulfated glycosaminoglycans (GAGs) (C) content in native and dHCM. Representative dot blot results for collagen type I, collagen type IV, fibronectin and lamini n, in both native and dHCM (D). Representative transversal sections of native and dHCM immunolocalization of collagen type I, collagen type IV, fibronectin and laminin (E). SDS-PAGE gel of the digested native and dHCM (F). Where, * p < 0.05 and **** p < 0.0001. [0037] Figure 3 - Mechanical Properties of native and decellularized HCM. Stress-strain curves of native HCM (A) and dHCM (B). Static mechanical properties: ultimate tensile strength (C) and Young's modulus (D) of both native and dHCM. Dynamic mechanical properties: storage modulus (E) and tan (F) of both native and dHCM. Where, *** p < 0.001.

[0038] Figure 4 - Seeding of EA.hy926 cells on dHCM. EA.hy926 cells' metabolic activity (A) and proliferation (B) in control (CTR), dHCM A and dHCM B membranes (results are normalized to control condition (A) or represented in number of cells (B)). Representative images of phalloidin (red) staining in EA.hy926 cells seeded on CTR membranes or in both sides of dHCM. DAPI (blue) was used to stain nuclei. Where, * p < 0.05; ** p < 0.01; *** p < 0.001 and; **** p < 0.0001.

[0039] Figure 5 - The suitability of dHCM for a BBB in vitro model: Metabolic activity and phalloidin/DAPI staining of bEnd.3 (A) and C8-D1A (B) cells in commercial inserts, dHCM trophoblast side and dHCM amnion side in both mono and co-culture conditions at day 3 and 7 of culture.

[0040] Figure 6 - BBB model validation: TEER values were obtained for bEnd.3 monolayer (A) and for the system composed by bEnd.3 monolayer and dHCM (B) in both mono and co-culture conditions. Diffusion assays with 4 kDa (C) and 70 kDa (D) FITC- dextran were also performed in three different conditions: dHCM alone (without cells); monoculture, and co-culture, at three different timepoints (day 1, 3 and 7).

[0041] Figure 7 - Functional BBB permeability: Different molecules were used to exemplify the different transport systems at the BBB. Caffeine was used for membrane diffusion (A), glucose for carrier-mediated transport (B), cholesterol as an example of a BBB non-crossing molecule (C), and transferrin for receptor-mediated transport (C).

[0042] Figure 8 - GLUT1 and transferrin receptor: GLUT1 (A) and transferrin receptor (B) expression were assessed over time (day 1, 3 and 7) in both conditions (mono and co culture), in order to demonstrate the functional transportation of glucose and transferrin across the BBB. Detailed Description

[0043] In an embodiment, human placentas used in the present disclosure were collected from cesarean sections performed on Hospital de Braga (SECVS 136/2015, CESH 030/2016). An informed consent was signed by all the donors. Sterile conditions were maintained during all processing steps. Placentas were stored in Dulbecco's phosphate-buffered saline (D-PBS) with 10 % antibiotic/antimycotic (#15240062, Thermo Fisher Scientific) at 4 °C for a maximum of 1 day. The chorion membrane was removed (and separated from the amnion), washed with Phosphate Buffered Saline (PBS) to remove the blood and stored at - 80 °C until further use.

[0044] In another embodiment, in order to perform the decellularization of the chorion membrane, chorion membranes were submitted to two freezing/thawing cycles (- 80 °C and 37 °C, respectively) and subsequently were treated with different concentrations (0.5% and 0.1%) of Sodium Dodecyl Sulfate (SDS) (#MB18101, NZYTech) solution in ultra- pure water. Membranes were submitted to three washes with 0.5 % SDS of 2h. Then, membranes were washed overnight with 0.1% SDS. Three washes of 15 min were performed with 1 % Triton-X100 (#A16046, Thermo Fisher Scientific) in PBS. All treatments were done at 4 °C in an orbital shaker at 110 rpm. Membranes were then scrapped in both sides and marked with a knot with suture line in order to identify both membrane sides. Then, the membrane was treated with 0.001 mg/mL DNase I (#A3778 PanReac AppliChem ITW Reagents), for 30 min at 37 °C, followed by a 30min wash with 0.1 % SDS at 4 °C. Finally, membranes were washed with PBS at least three times, during 2 h each, at 4 °C. All the process was done under sterile conditions. After decellularization, membranes were stored in PBS with 1-2 % antibiotic/antimycotic at 4 °C.

[0045] In an embodiment, for DNA extraction and quantification, membranes were air dried and weighed and total DNA from both native and decellularized HCM (dHCM) was extracted using the DNeasy Blood and Tissue kit (#69504, Qiagen), according to the manufacturer's instructions. The quantification of double-strand DNA (dsDNA) was performed using Quant-IT PicoGreen dsDNA Assay kit according to the manufacturer's instructions (#P7589, Invitrogen). Electrophoresis was performed to assess the size of the DNA fragments, using a 1 % Agarose gel and GeneRuler DNA Ladder Mix (#SM0334, Thermo Fisher Scientific). Four independent samples were used in each condition.

[0046] In an embodiment, histological analysis was performed according to the following procedure: native and decellularized tissues were fixed in 10 % neutral- buffered formalin at 4 °C (for at least 24h), embedded in paraffin and transversely sectioned at 5 pm. Histochemical stains such as Hematoxylin and Eosin (H&E), Masson's Trichrome, Alcian Blue, and Safranin-0 were all performed. For H&E staining, samples were stained with hematoxylin (#7212, Thermo Fisher Scientific) for 1 min, washed for 30 s and stained with eosion for 10 min (#71204, Thermo Fisher Scientific). For Masson's Trichrome staining, slides were submitted to Azure B solution for 5 min, stained with hematoxylin for 5 min and washed in picric ethanol for 5 min. After that, samples were stained in Biebrich Scarlet-Acid Fuchsin for 15 min, submitted to 1 % phosphomolybdic acid for 5 min, and to Aniline Blue for 4 min. The Alcian Blue staining was performed by rinsing the samples in 3 % acetic acid and keeping them in 1 % alcian blue solution (#A3157, Sigma-Aldrich) for 1 h. A counterstain with hematoxylin was done for 3 min. Regarding safranin-0 staining, slides were incubated in hematoxylin for 8 min, dipped in 0.5 % ethanolic acetic acid and immersed in 0.02 % Fast Green staining solution for 5 min. After that, slides were immersed i n 0.1 % safranin-0 staining solution for 6 min. After each staining, all slides were washed with water, let to dry, and rinsed with alcohol, cleared in xylene, and mounted in Entellan rapid (#107960, Merck). Slides were observed under an optical microscope with a coupled camera (DM750, Leica).

[0047] In a further embodiment, immunolocalization of different proteins, such as collagen type I, collagen type IV, fibronectin and laminin, was performed in paraffin- embedded samples sectioned at 5 pm, as previously described for collagen type II [25] Briefly, samples were incubated with mouse anti-collagen type I 1:100 (#ab90395, abeam); rabbit anti-collagen type IV, 1:50 (#ab6311, abeam); mouse anti-laminin, 1:300 (#L8271, Sigma-Aldrich), rabbit anti-fibronectin 1:300 (#ab45688, abeam), overnight at 4 °C in a humidified atmosphere. As a secondary antibody, R.T.U. VECTASTAIN^® Universal ABC Elite^® Kit (#PK-7200, Vector Laboratories) was used, in accordance with manufacturer instructions. Incubation was revealed by using Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories). Samples were counterstained with hematoxylin and mounted in an aqueous mounting medium. Slides were observed in an optical microscope with a coupled camera (DM750, Leica).

[0048] In an embodiment, for fluorescence imaging, phalloidin staining was performed in fresh tissue, fixed with 10 % formalin for, at least, 24 h, at 4 °C. Briefly, fixed samples were incubated with 1:200 Phalloidin-TRITC, (#P1951, Sigma-Aldrich), at room temperature for 1 h. Nuclei were stained with DAPI (#40009, VWR), for 1-5 min at room temperature. For paraffin embedded tissues and formalin-fixed dHCM A and B, DAPI was used at 1:10000. For PET membranes from millicell hanging cell culture inserts (#MCHT24H48, Millicell), DAPI was used at 1:1000. Stainings were observed under fluorescence microscope with a coupled camera (Axio Imager Zlm, Zeiss). Images were analyzed with Zeiss Zen microscope software.

[0049] Sulfated glycosaminoglycans (GAGs) quantification of native and decellularized tissue was quantified as previously described [26] Samples were digested with 0.5 mg/mL papain (#P4762, Sigma-Aldrich) and the supernatant was stained with 1,9- dimethylmethylene blue (DMB). A dilution series of chondroitin sulfate in distilled water (50 pg/mL) was used as the standard solution. The samples were diluted 1:10 before the measurement. Twenty pL of the standards and diluted samples were mixed with 250 pL of DMB in a 96-well plate. Absorbance was measured immediately at 525nm. Three independent samples per condition were analyzed.

[0050] The collagen quantification of native and decellularized tissues was extracted and quantified using SIRCOL Collagen Assay Kit (#S5000, biocolor) as described before [27] and according to manufacturer's instructions. Three independent samples were used in each condition.

[0051] Soluble protein was extracted from native and decellularized tissue using Tissue Extraction Reagent I (#FNN0071, Thermo Fisher Scientific) with a Protease Inhibitor Cocktail (#P8340, Sigma-Aldrich). The quantification of the soluble protein content was determined using Protein Assay Dye Reagent Concentrate (#5000006, Biorad) according to manufacturer's instructions.

[0052] The SDS-PAGE Gel Preparation Kit (#08091, Sigma-Aldrich) was used to prepare the 4 % stacking gel and 9 % running gel. For each sample, 0.5 mg/mL of protein were loaded in the respective well. Following SDS-PAGE, the gel was stained with Coomassie blue R-250 (HS-604, National Diagnostics) and the image was obtained using a Transilluminator (Biospectrum ac chemi hr 410, UVP).

[0053] In an embodiment, for dot-blot analysis, one drop of each sample of soluble protein was placed in a nitrocellulose membrane. After drying, membranes were washed with 5 % BSA, 1 hour, with agitation, at room temperature. Subsequently, membranes were incubated, overnight, with mouse anti-collagen type I 1:1000 (#ab90395, abeam); rabbit anti-collagen type IV, 1:500 (#ab6311, abeam); mouse anti laminin, 1:500 (#L8271, Sigma-Aldrich), rabbit anti-fibronectin 1:500 (#ab45688, abeam). After 3 washes of 5 minutes with TBS-tween 20, R.T.U. VECTASTAIN^® Universal ABC Elite^® Kit (#PK-7200, Vector Laboratories) was used as a secondary antibody, in accordance with manufacturer instructions. Finally, incubation was revealed by using Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories).

[0054] For Scanning Electron Microcopy (SEM), samples were fixed with 2.5 % glutaraldehyde in PBS. Following washing with PBS, samples were dehydrated with increasing concentrations of ethanol. Samples were air dried and mounted in SEM pins using carbon tape. The samples were coated with gold using a Sputter Coater (#EM ACE600, Leica). Images were collected with a Scanning Electron Microscope with EDS (#JSM-6010 LV, JEOL).

[0055] The swelling assay was performed according to the following procedure: samples (n = 6) were initially weighted and after that, were immersed in D-PBS or culture medium. At different time-points (30 min; lh30; 2h30; 3h30; 4h30; 5h30), samples were weighted immediately after the excess of liquid was removed by putting them between two pieces of filter paper. Samples were re-immersed in the liquid until the next time- point. The process was repeated until the equilibrium was reached (percentage of water uptake constant).

[0056] Static mechanical properties were assessed using Universal mechanical testing equipment (#5543, INSTRON) equipped with a 1 kN load cell. Nine samples of native and decellularized HCM were cut in pieces of 20 x 5 mm and mounted in specific cassettes (to prevent the clamping system from damaging the samples). After that, the specimens were hydrated. The strain rate was defined at 5 mm/min and a 10 mm gauge length was used in the tensile tests. Tests were finished when the specimens were ruptured. Dynamic Mechanical Analysis (DMA) was performed using Tritec 2000B equipment in a tensile mode. Samples were air dried and cut with a width of 5 mm and a length of 20 mm. For each sample, the width and the thickness were characterized in at least three different points. For the tests, samples were clamped with a grip distance of 5 mm and with total immersion of the sample in reservoir containing D-MEM culture medium at 37 °C. After equilibration at 37 °C, the DMA spectra were obtained using stress mode and following a cycle of increasing frequency from 0.1 to 20 Hz (3 points per decade). At least, five samples were tested per condition (dHCM and native HCM).

[0057] Citotoxicity analysis was performed according the following procedure: human umbilical vein endothelial cell line EA.hy926 was cultured in complete medium (DMEM with 10 % fetal bovine serum (FBS; #A3160801, Thermo Fisher Scientific) and 1% Penicillin/Streptomycin (#15240062, Thermo Fisher Scientific) in T150 flasks at 37 °C in a humidifier incubator with 5 % CO2 to reach 80 % of confluence before being transferred into the inserts. dHCM were mounted in cell crown inserts for 24-wells plate (#Z742380- 12EA, Sigma). Millicell hanging cell culture inserts, PET 0.4 pm for 24-well plates (#MCHT24H48, Millicell) were used as control. Both inserts were immersed in culture medium overnight before cell seeding. EA.hy926 cells were seeded on the inserts with a density of 20xl0³ cells/cm². Cell proliferation was evaluated by DNA quantification (Quant-IT PicoGreen dsDNA assay, Invitrogen, Alfagene) normalized to number of cells and metabolic activity by MTS assay (CellTiter 96 AQueous One Solution, Promega) according to the manufacturer's instructions. Three independent assays were performed.

IB [0058] Statistical analysis was performed using Graph Pad Prism 7. Shapiro-Wilk test was used to assess the data normality. When data followed a normal distribution, parametric tests were used, namely unpaired t- test and two-way ANOVA test followed by Turkey's multiple comparisons test. When data did not follow a normal distribution, the Mann-Whitney test was used. A p < 0.05 was considered statistically significant in the analysis of the results.

[0059] In the present disclosure, HCM was successfully decellularized and dHCM retained the basement membrane and the main proteins of the ECM.

[0060] In an embodiment, the efficiency of the decellularization method for decellularizing of the present disclosure was assessed (Fig. 1). As demonstrated by H&E there is no nuclei in decellularized human chorion membrane (dHCM) when compared with native tissue (Fig. 1A, IB). Moreover, through a chemical and physical decellularization process, it was possible to completely remove the HCM's trophoblast layer and nuclei from the reticular layer (Fig. 1A, IB). SEM images revealed that the dHCM is composed by nanofibers (dHCM B) (Fig. 1C). However, a thin compact layer (basement membrane) covers the dHCM only in the throphoblast layer's side (dHCM A) (Fig. ID). Cellular removal in dHCM is also corroborated by DAPI staining (Fig. IE, IF). Moreover, DNA quantification and length of DNA fragments showed a significant removal of DNA ( p = 0.0002) in the dHCM when compared with the native HCM (Fig. 1G). The dHCM presented a DNA content around 10 ng of dsDNA/mg of dry tissue (Fig. 1G) and its remaining DNA fragments presented less than 200 bp (Fig. 1H). Additionally, the thickness measurement of air-dried native HCM and dHCM was performed. After the decellularization method of the present disclosure the thickness membrane becomes 5 times thinner (119.50 ± 34.32 miti of native HCM compared to 24.50 ± 3.11 miti of dHCM) (p = 0.0150; Fig. II). Although, having large differences in tissue thickness between native and decellularized tissue, a compact membrane was obtained (Fig. 1J, IK).

[0061] In an embodiment, the swelling behavior of dHCM in culture medium and PBS is shown in Fig. 1L. In the first 30 min, a n increase of 230-240 % of dHCM's weight was observed. After this time, the swelling behavior stabilized around 300-350 %. Moreover, when it is wet, dHCM's thickness increased to 36.00 ± 8.03 mih ( p= 0.0159; data not shown).

[0062] In an embodiment, dHCM's composition was assessed using immunolocalization of proteins, SDS-PAGE and dot blot. Moreover, quantitative analyzes were performed to verify collagen and GAGs content. Histological sections of dHCM with Masson's Trichrome, Alcian Blue and Safranin-0 showed the presence of collagen and sulfated GAGs, respectively (Fig. 2A). The quantification of these components revealed a significant difference in collagen and GAGs content between native tissue and dHCM (p = 0.0139 and p < 0.0001, respectively) (Fig. 2B and 2C). Collagen content was 6.33 ± 0.92 pg/mg of dry tissue in native HCM and 3.70 ± 0.46 pg/mg of dry tissue in dHCM (Fig. 2B). Regarding sulfated GAGs quantification, 17.07 ± 1.08 pg/mg of dry tissue were present in the native tissue and 2.64 ± 0.19 pg/mg of dry tissue composed the dHCM (Fig. 2C).

[0063] In an embodiment, native and dHCM were stained to show the presence and distribution of collagen type I, collagen type IV, fibronectin and laminin in both, the sections and the digested tissue (dot blot). By dot blot the presence of those proteins in both native and decellularized tissue was confirmed (Fig. 2D). In tissue sections, it was possible to verify that while collagen type I was spread along the dHCM, collagen type IV and fibronectin were focused on the basement membrane (dHCM A). Laminin was present in both peripheries of dHCM (Fig. 2E). SDS-PAGE gel corroborates these results, showing a similar pattern between all dHCM samples and preserving higher molecular weight species when compared to the native HCM (Fig. 2F).

[0064] In an embodiment, the mechanical properties of the HCM obtain by the method of the present disclosure make it a suitable substrate to mimic BBs: a. The mechanical properties of dHCM's were assessed in hydrated samples.

Nine stress-strain curves are represented in Fig. 3A and 3B for both native and decellularized tissue, respectively. At low strain levels, the stress varied linearly with the strain in accordance with Hooks law. At higher strain levels, membranes show a strain hardening behavior. The average ultimate tensile strength (Fig. 3C) was 1.793 ± 0.284 MPa for native tissue and 5.327 ± 0.414 MPa for dHCM. The average Young's Modulus (Fig. 3D) was 1.686 ± 0.187 MPa for the native and 5.936 ± 0.466 MPa for the decellularized tissue. Both, ultimate tensile strength and Young's Modulus, were significantly higher in dHCM (p < 0.0001). In Fig. 3E are depicted the storage module curves of native and dHCM, both curves presented a stable behavior along the increasing frequencies (1-15.85Hz). Storage modulus value of the native tissue was 5.008 ± 0.812 MPa while in the dHCM was 28.841 ± 5.972 MPa (p<0,0001), showing a similar trend of several times (approximately 6x) higher stiffness of the decellularized membrane compared to the native one. Tan-d curves are represented in Fig. 3F and the stable behavior is also observed in both native and decellularized tissue. The value of tan-d for native tissue was 0.567 ± 0.179 and for dHCM was 0.376 ± 0.106, showing a slightly lower viscoelastic behavior of the dHCM. Between 1.00-6.30 Hz, there were no statistically significant differences concerning native and decellularized tissue. b. To evaluate dHCM cytocompatibility, cytotoxicity of dHCM, metabolic activity and cell proliferation assays were performed. Commercial inserts were used as controls (CTR). As demonstrated in Fig. 4A, the metabolic activity of endothelial cells was maintained overtime, in all conditions tested, with exception of day 6. At that time-point, the metabolic activity decreases when endothelial cells are seeded in the side A of dHCM (p < 0.001). Moreover, at day 1, metabolic activity is higher in dHCM A compared with dHCM B (p = 0.0353). The same was observed at day 3 where the metabolic activity of EA.hy926 was higher in dHCM A compared with both CTR (p = 0.0112) and dHCM B (p = 0.0004). At day 6, cells' metabolic activity is maintained in CTR, however in both dHCM A (p= 0.0198) and dHCM B (p = 0.0341) a decrease in cells' metabolic activity occurred, being more pronounced in dHCM B.

[0065] In an embodiment, regarding cell proliferation (Fig. 4B and supplementary graphic 1), the number of cells increased significantly in dHCM A, over time (p = 0.0101 (day 1 vs. day 3); p < 0.0001 (day 3 vs. day 6)). In CTR, only between day 3 and day 6 an increase on cell proliferation was observed (p = 0.4630 (day 1 vs. day 3); p < 0.0001(day 3 vs. day 6)). Regarding dHCM B, no differences were observed (p = 0.9362 (day 1 vs. day 3); p = 0.6128 (day 3 vs. day 6)). On day 3, dHCM A and CTR presented a higher number of cells than dHCM B (p = 0.0036 and p = 0.0482, respectively). On day 6, the CTR presented a higher number of cells than dHCM A (p = 0.0220) and dHCM B (p < 0.0001). Also, at day 6, cells seeded on dHCM A presented a significantly higher proliferation compared to dHCM B (p < 0.0001).

[0066] Fluorescence images of endothelial cells seeded on CTR and in both sides of dHCM were performed. As demonstrated in Fig. 4C, an increase on the number of cells from day 1 to day 3 was observed, in all the conditions. However, on day 1, it seems that endothelial cells have a more rounded morphology in dHCM B than in CTR and dHCM A. On day 3, EA.hy.926 seem to form a monolayer in dHCM B, what was not observed in dHCM A and CTR. Nevertheless, in dHCM A cells tend to acquire a rounded morphology while in CTR, cells maintain a more stretched morphology. On day 6, it appears that there was an overlap of cell monolayers in CTR and dHCM A. Conversely, in dHCM B it seems that cells' detachment has occurred.

[0067] In an embodiment, the method of the present disclosure successfully decellularized HCM since it is in accordance with the established criteria to decellularized tissues. In order to avoid cell and host adverse response, it is important that the decellularized tissue: 1) lacks visible nuclear material (stained with DAPI); 2) has DNA fragments less than < 200 bp; and 3) presents amount of dsDNA/mg of dry tissue less than 50 ng [28] In fact, all these criteria were achieved by the dHCM of the present disclosure (Fig. 1). Moreover, as shown in SEM micrographs (Fig. 1C and ID), after the decellularization method of the present disclosure the basement membrane in the dHCM was preserved (dHCM A). The maintenance of the basement membrane is of great interest to mimic BBs, since it is a specialized ECM that is found basolateral to all cell monolayers in the body that separates them from the underlying connective tissue. Moreover, its main functions are to provide structural support to the tissue and to offer functional signals to modulate cell behavior and function [29], [30] The maintenance of the basement membrane in dHCM is also supported by the results obtained in the immunohistochemistry's tissue sections (Fig. 2E), where it is possible to observe that collagen type IV, fibronectin and laminin are present in dHCM A. These proteins along with nidogen and perlecan compose the major components of the basement membrane [29], [30]

[0068] In an embodiment, the trophoblast layer was lost during the decellularization process. Trophoblasts have an important role during pregnancy, due to their ability to produce growth factors and hormones that support and regulate placental and fetal development and growth. In the first trimester, trophoblasts are crucial for angiogenesis, since they can invade maternal myometrial spiral arteries. On the second and third trimester, the placenta is vascularized by growth factors produced by trophoblasts [31]. Given its particular function and taking into account one of the goals of the present disclosure, there was no benefit in maintaining the trophoblast layer in the decellularized tissue. However, the significant decrease in the quantity of sulfated GAGs and collagen in dHCM (when compared to native tissue) is probably associated with the loss of most of the content of this layer.

[0069] In an embodiment, the main proteins of ECM were maintained in the dHCM, as demonstrated by dot blot analysis (Fig. 2D). Moreover, by SDS-PAGE (Fig. 2F) we confirmed, not only the reproducibility of the decellularization method of the present disclosure (the bands are similar between different dHCM), but also that the higher molecular weight species were preserved in the dHCM. This is very important for us since the proteins that characterize the ECM have around 130-400 kDa [32]

[0070] In an embodiment, collagen type I is present along all dHCM. This type of collagen is the major collagen of tendons, skin, ligaments, cornea and several interstitial connective tissues (with the exception of tissues such as hyaline cartilage, brain and vitreous body). In most organs, collagen type I is associated with tensile stiffness, load bearing capacity, tensile strength, and torsional stiffness [33] Young's modulus of collagen fibrils is around 1-1.5 GPa [34] In dHCM the young's modulus is considerably lower (5.936 ± 0.466 MPa) (Fig. 3D) probably due to the presence of other proteins in the dHCM such as fibronectin and laminin (Fig. 2D and 2E). In fact, although there is a significant loss of collagen in dHCM, the young's modulus is higher in dHCM than in native tissue (p < 0.0001, Fig. 3D). This stiffness and strength may be associated with the presence of cells in the native tissue, that mainly bound to the ECM through focal adhesions [35] Moreover, the young's modulus of dHCM is similar to the one described for basement membranes (l-4MPa) [36] So, dHCM is stiffer than native tissue and also has a higher ultimate tensile strength (p<0.0001, Fig. 3C), making it more resistant to fracture.

[0071] In an embodiment, taking into account that water retention is one of the major functional characteristics of GAGs within a tissue, some studies have shown that the removal of GAGs from a scaffold may have a negative effect on its viscoelastic behavior [37] Although there is a significant loss of sulfated GAGs i n dHCM (p < 0.0001, Fig. 2C), no statistically significant differences occurred in tan-d concerning native and decellularized tissue between 1.00 and 6.30 Hz. Differences were only observed in lower (0.20-0.63 Hz) and higher (10-15.85 Hz) frequencies (Fig. 3D). Moreover, the storage modulus of dHCM is stable after the range of applied frequencies (Fig. 3E), suggesting that dHCM can be implanted in the human body, even in areas subjected to significant stresses over extended frequency ranges (e.g. muscle).

[0072] In an embodiment, the swelling behavior is an important parameter in the characterization of the dHCM since it is related with the stability of the membrane in aqueous medium. To perform this analysis, culture medium and PBS were used to characterize the stability of dHCM during cell culture and storage, respectively (Fig. 1L). Since we observed that after 30 min the swelling behavior of dHCM normalized, we decided that before each seeding, both membranes (dHCM and CTR) were immersed in culture medium overnight.

[0073] In an embodiment, the cytocompatibility of dHCM was investigated using EA.hy926 cell line. Human endothelial cells were used since the endothelium is a major component of BBs [lj. CTR inserts were used as a reference. Differences between dHCM A and dHCM B were expected due to its different composition (Fig, 2E) and structure (Fig. 1C and ID). In Fig. 4, we observed that both sides of dHCM were cytocompatible. However, dHCM B seems to be associated with a less metabolic activity and with a lower proliferation of cells. The morphology of the cells was also different between the different sides of dHCM. In the early stages of cell culture (from day 1), cells presented a more rounded morphology in dHCM B and only in later stages (from day 3), cells in dHCM A begin to acquire this morphology. The detachment of EA.hy926 observed in dHCM B's phalloidin/DAPI staining on day 6 (Fig. 4C) was not corroborated by both metabolic activity and proliferation assays (Fig. 4A and 4B).

[0074] In an embodiment, the different composition of dHCM A and dHCM B validate these results. dHCM B is mainly composed by collagen type I and laminin. High laminin content promotes proliferation and migration of endothelial cells into a vessel-like morphology [38], justifying the rapid organization of EA.hy926 cells in dHCM B. However, fibronectin has an important role in cell adhesion, since it interacts and activates cell surface integrins that are associated with the formation of focal adhesions [39] The absence of fibronectin in dHCM B may be associated with cell detachment.

[0075] In an embodiment, the use of a membrane from the human placenta has intrinsic advantages such as its privileged immune tolerance. Given its origin, it is expected that dHCM will be useful to model and mimic BBs and it will be well tolerated by the human body, without severe immune response, when considered for implantation.

[0076] In an embodiment, it was show that dHCM trophoblast side is more suitable for a BBB in vitro model. To evaluate the suitability of dHCM for a BBB in vitro model, metabolic activity of bEnd.3 and C8-D1A cell lines was analysed and compared with commercial inserts in both mono and co-culture conditions. Moreover, as previously described, dHCM has two different sides (amnion side and trophoblast side) that were also compared (Fig. 5). When dHCM side is mentioned it always corresponds to the side of dHCM in the upper side of the insert. For bEnd.3 cell line in monoculture (in the upper side of the insert) (Fig. 5A), a higher metabolic activity is observed in dHCM trophoblast side in both timepoints (day 3 and day 7). Nevertheless, statistically significant differences are only observed on day 7 (p = 0.0002 (Commercial insert vs. dHCM trophoblast side) and p = 0.0021 (dHCM trophoblast side vs. dHCM amnion side)). Regarding the co-culture of bEnd.3 cells with C8-D1A cells, dHCM outperformed commercial inserts for bEnd.3 cell line metabolic activity on day 3 (p = 0.0003 (Commercial insert vs. dHCM trophoblast side) and p = 0.0019 (Commercial insert vs. dHCM amnion side)) and day 7 (p < 0.0001 (Commercial insert vs. dHCM trophoblast side) and p < 0.0001 (dHCM trophoblast side vs. dHCM amnion side)) (Fig. 5A). Although a slight increase in metabolic activity was observed in dHCM trophoblast side when compared to dHCM amnion side, no statistically significant differences were observed (p = 0.8538 (day 3) and p = 0.9824 (day 7)). The same tendency was observed for C8- D1A cell line in monoculture (seeded in the bottom of the well where an insert without cells was then placed) (Fig. 5B). So, a higher metabolic activity for C8-D1A cells was observed in both sides of dHCM when compared with commercial insert in monoculture on day 3 (p < 0.0001 (Commercial insert vs. dHCM trophoblast side) and p < 0.0001 (Commercial insert vs. dHCM amnion side)) and day 7 (p = 0.0033 (Commercial insert vs. dHCM trophoblast side) and p = 0.0012 (dHCM trophoblast side vs. dHCM amnion side)). No differences were observed between dHCM sides (p = 0.9506 (day 3) and p = 0.0861 (day 7)). For C8-D1A in co-culture with bEnd.3 cells, no differences were observed on day 3 (p = 0.9989 (Commercial insert vs. dHCM trophoblast side), p = 0.9932 (Commercial insert vs. dHCM amnion side) and p = 0.9833 (dHCM trophoblast side vs. dHCM amnion side)) (Fig. 5B). Nevertheless, on day 7 the metabolic activity of dHCM was higher than in commercial inserts (p < 0.0001 (Commercial insert vs. dHCM trophoblast side), p < 0.0001 (Commercial insert vs. dHCM amnion side)). Although a slight increase in C8-D1A cells metabolic activity was observed in dHCM trophoblast side in comparison with dHCM amnion side, no statistically significant differences were observed (p = 0.0861). Phalloidin/DAPI staining show that a monolayer of bEnd.3 cells is achieved on day 3 in all conditions (CTR, dHCM trophoblast side and dHCM amnion side) (Fig. 5A). Moreover, a F-actin increase is observed in dHCM trophoblast side and amnion side. In contrast, commercial inserts show a decrease in F-actin. An increase in C8-D1A cells confluency is observed from day 3 to day 7 in all conditions (CTR, dHCM trophoblast side and dHCM amnion side) (Fig. 5B). Taking into account this results, further assays were performed with dHCM trophoblast side in contact with bEnd.3 cell line in monoculture and in non-contact co-culture with C8-D1A cells. [0077] In an embodiment, to validate the BBB model of the of the present disclosure TEER measurements (Fig. 6A-B), and diffusion assays (Fig. 6C-D) were performed. As expected, TEER values for bEnd.3 monolayer and for the system (bEnd.3 monolayer with dHCM) behaved in the same way (Fig. 6A-B). In general, monoculture condition (mean 19.21.16 ± 2.98 W/cm² (bEnd.3 monolayer); mean 105.08 ± 5.00 W/cm² (system)) was associated with slightly lower TEER values when compared to co-culture condition (mean 22.76 ± 7.27 W/cm² (bEnd.3 monolayer); mean 116.90 ± 12.77 W/cm² (system)). Nevertheless, statistically significant differences between mono and co-culture conditions were only observed in system measures on day 3 (p = 0.0196). This may be associated with the TEER increase observed on day 3 in co-culture condition in both bEnd.3 monolayer and system. Moreover, on day 7, a TEER decrease is observed in all conditions, being more prominent in co-culture condition, achieving mono-culture values in both bEnd.3 monolayer (p = 0.0416 (day 3 vs. day 7)) and system (p = 0.0126 (day 1 vs. day 7); p = 0.0027 (day 3 vs. day 7)).

[0078] To further assess the integrity of the BBB model of the of the present disclosure, diffusion assays with FITC-dextran with 4 kDa and 70 kDa were performed (Fig. 6C-D). As expected, both in mono and co-culture conditions, the model of the present disclosure was more permeable to 4 kDa FITC dextran (8.39 ± 0.35 x 10-6 cm/s (monoculture), 9.24 ± 1.10 x 10 ⁶ cm/s (co-culture)) than to 70 kDa FITC-dextran (6.57 ± 0.92 x 10 ⁶ cm/s (monoculture), 6.83 ± 0.53 x 10 ⁶ cm/s (co-culture)) (Fig. 6C-D). Moreover, the permeability coefficient was lower in mono and co-culture conditions when compared with blank (without cells) (11.83 ± 0.71 x 10 ⁶ cm/s (4 kDa), 9.82 ± 0.94 x 10 ⁶ cm/s (70 kDa)). In general, FITC-dextran diffusion was stable over time and no statistically significant differences were observed. Nevertheless, a slight increase in permeability coefficient was observed on day 3 for 4 kDa FITC-dextran in both without cells and co-culture conditions. Interestingly, the opposite trend is observed for monoculture. The same happens with 70 kDa FITC-dextran, but on day 7. While without cells and co-culture conditions present the same curve behavior, monoculture has a slight increase of the permeability coefficient on day 7. Between conditions, statistically significant differences were observed with 4 kDa on day 1 (p = 0.0123 (without cells vs. co-culture)), day 3 (p < 0.0001 (without cells vs. monoculture), p = 0.0216 (without cells vs. co-culture), p = 0.0492 (monoculture vs. co-culture)), and day 7 (p = 0.0221 (without cells vs. monoculture). For 70 kDa, these differences were observed on day 1 (p = 0.0181 (without cells vs. monoculture)) and day 3 (p = 0.0003 (without cells vs. monoculture), p = 0.0016 (without cells vs. co-culture)).

[0079] In an embodiment, for functional permeability assays, molecules known to cross or not the BBB were used. For caffeine (Fig. 7A), the condition without cells has a similar behaviour than the one observed with 4 kDa and 70 kDa FITC-dextran, with an increase on day 3 (p = 0.0069 (day 3 vs. day 7)). All conditions (without cells, mono and co-culture) have a similar permeability coefficient to caffeine on day 1 (7.13 ± 0.14 x lO ⁶ cm/s (without cells), 7.06 ± 0.26 x 10 ⁶ cm/s (monoculture), 7.06 ± 0.27 x 10 ⁶ cm/s (co culture)). Nevertheless, on day 3, while the permeability coefficient increased in monoculture (p = 0.0074 (day 1 vs. day 3)), similarly to what happened with without cells, it slightly decreased on co-culture condition (p = 0.0010 (monoculture vs. co culture), p = 0.0056 (without cells vs. co-culture)). On day 7, in contrast with without cells condition, the permeability coefficient to caffeine was maintained in mono and co culture conditions (p = 0.0051 (without cells vs. monoculture), p = 0.0116 (monoculture vs. co-culture)). The overall values suggest that the monoculture condition had the highest permeability coefficient to caffeine (7.40 ± 0.25 x lO ⁶ cm/s) when compared to without cells (7.18 ± 0.24 x 10 ⁶ cm/s) and co-culture (7.01 ± 0.04 x 10 ⁶ cm/s). Glucose permeability coefficient was relatively stable in the condition without cells and increased over time in mono (p = 0.0326 (day 1 vs. day 7)) and co-culture conditions (p = 0.0277 (day 1 vs. day 7)) (Fig. 7B). At day 1 the permeability coefficient of without cells (2.42 ± 0.19 x 10 ⁵ cm/s) was higher than mono (1.95 ± 0.24 x 10 ⁵ cm/s) and co-culture (1.65 ± 0.67 x 10 ⁵ cm/s; p = 0.0007 (vs. without cells). Eventually, this difference faded along time. Although all conditions had similar permeability coefficients to glucose, co culture presented the lowest (1.99 ± 0.36 x lO ⁵ cm/s) when compared with monoculture (2.25 ± 0.31 x 10 ⁵ cm/s) and without cells (2.49 ± 0.08 x 10 ⁵ cm/s). Transferrin uptake is represented on Fig. 7D. Differences between mono and co-culture were not observed, with the exception of day 1, where no transferrin vesicles were observed in monoculture. The highest transferrin uptake was observed on day 3 while the lowest transferrin uptake occurred on day 1. Cholesterol concentration in the upper chamber of the insert was relatively stable over time in all conditions (Fig. 7C). As expected, dHCM alone (without cells) was associated with lower cholesterol concentration in the upper chamber of the insert, suggesting a higher cholesterol permeability (93.86 ± 3.50 %), when compared to co-culture (95.56 ± 1.60 %) and monoculture (97.84 ± 0.30 %) that presented the lowest cholesterol permeability.

[0080] In an embodiment, glucose and transferrin cross the BBB through a carrier and a transporter, respectively. So, to demonstrate the functional transportation of these molecules across the BBB, both GLUT1 and transferrin receptor expression were assessed. It was observed that GLUT1 expression was very similar between mono and co-culture and that it increased over time, corroborating the results of glucose permeability coefficient (Fig. 8A). On the other hand, on day 1, the expression of transferrin receptor was higher in co-culture when compared to monoculture. The opposite is observed on day 3. Regarding day 7 no differences were observed between mono and co-culture (Fig. 8A). Altogether, these results support the ones observed for transferrin uptake (Fig. 7D).

[0081] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0082] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. [0083] The above described embodiments are combinable.

[0084] The following claims further set out particular embodiments of the disclosure.

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Claims

C L A I M S

1. Method for decellularizing an isolated human chorion membrane (HCM) comprising the following steps:

submitting the isolated HCM to at least a freezing/thawing cycle in order to decellularize the HCM;

washing the HCM with an anionic surfactant;

bathing the HCM overnight with an anionic surfactant;

washing the HCM with a buffer solution;

scrapping both sides of the HCM;

exposing the HCM to DNase I;

2. Method according to the previous claim comprising the following steps:

submitting an isolated HCM to at least two freezing/thawing cycles of around

- 80 °C/37 °C, in order to decellularize the HCM;

treating the HCM with an anionic surfactant;

washing the HCM at least three times with an anionic surfactant;

bathing the HCM overnight with an anionic surfactant;

washing the HCM at least three times with Triton-X100 in PBS;

scrapping both sides of the HCM;

exposing the HCM to DNase I at 0.001 mg/mL at 37 °C;

washing the HCM with an anionic surfactant at 4 °C;

3. Method according to any of the previous claims comprising the following steps: submitting an isolated HCM to at least two freezing/thawing cycles of around

- 80 °C/37 °C, in order to decellularize the HCM; treating the HCM with an anionic surfactant solution at 0.5% and with an SDS solution at 0.1%(wt/v);

washing the HCM at least three times with an anionic surfactant at 0.5 %(wt/v);

bathing the HCM overnight with an anionic surfactant at 0.1%(wt/v);

washing the HCM three times with Triton-X100 in PBS at 1 % (wt/v);

scrapping both sides of the HCM;

exposing the HCM to DNase I at 0.001 mg/mL during 30 min at 37 °C;

washing the HCM with an anionic surfactant at 0.1 % (wt/v) at 4 °C;

4. Method according to any of the previous claims wherein the anionic surfactant is sodium dodecyl sulfate.

5. Method according to any of the previous claims wherein all the steps are performed under sterile conditions and the water of said aqueous solutions is ultra-pure water.

6. Method according to any of the previous claims wherein said three washes with SDS at 0.5% are washes of around 2 hours each.

7. Method according to any of the previous claims wherein said three washes with Triton-X100 in PBS are washes of around 15 minutes each.

8. Method according to any of the previous claims wherein said exposition to DNase I lasts around 30 minutes.

9. Method according to any of the previous claims wherein said at least three washes with PBS are washes of around 2 hours each.

10. Method according to any of the previous claims wherein the following steps are performed at 4 °C in an orbital shaker at 110 rpm:

washing the HCM at least three times with an anionic surfactant at 0.5 %(wt/v);

BO bathing the HCM overnight with SDS at 0.1%(wt/v);

washing the HCM three times with Triton-X100 in PBS at 1 % (wt/v);

scrapping both sides of the HCM;

washing the HCM with an anionic surfactant at 0.1 % (wt/v);

washing the decellularized membrane with PBS at least three times.

11. Method according to any of the previous claims wherein the membranes are scrapped in both sides and marked with a knot with a suture line to identify both membrane sides.

12. Method according to the any of the previous claims wherein after decellularization, membranes are stored in PBS with an antibiotic/antimycotic solution at 1-2 %(wt/v), at 4 °C.

IB. Method according to any of the previous claims wherein after decellularization, membranes are object to a recellularization step.

14. Decellularized human chorion membrane obtainable by the process according to any of the previous claims wherein the decellularized human chorion membrane is substantially or completely free of throphoblast layer and nuclei for the reticular layer.

15. A method for evaluating blood-brain barrier permeability of a test substance, test cell or test protein comprising:

exposing said test substance, test cell or test protein to the decellula rized membrane described in claim 14.

16. Drug screening, distribution, and permeability kit for drugs comprising the decellularized human chorion membrane according to any of the previous claims.

17. Use of decellularized human chorion described in any one of the previous claims as a cell scaffold.

18. Use of decellularized human chorion described in any one of the previous claims as an in vitro model of normal or disease-related human blood-brain barrier.

19. Use of decellularized human chorion described in any one of the previous claims as a substrate to mimic biological barriers for same or different cell types.

20. Use of decellularized human chorion described in any one of the previous claims as a substrate to mimic basement mem branes.