A novel composite film and a preparation method and application thereof

Abstract

The present disclosure discloses a composite film, which comprises: a polymer skeleton layer; and alginate-polyacrylamide gel layers arranged on both sides of the polymer skeleton layer. The composite film of the present disclosure has a three-layer structure of an elastic film, is based on a novel ultra-thin film material of the composite film, is superior to the hydrophilicity of the prior polydimethylsiloxane polymer material, and can realize three-dimensional deformation, truly simulate the inflation and contraction of alveoli in the breathing process, and improve the information exchange and gas exchange efficiency at the barrier structure of the existing organ chip.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomaterials and tissue engineering, and in particular to a composite film comprising a polyvinyl alcohol gel layer and a alginate-polyacrylamide gel layer, and a preparation method and application thereof. BACKGROUND

[0002] The gas exchange model chip is a kind of bionic respiratory cell micro-culture system, which can simulate the physiological process of lung organ in vivo to a certain extent, predict drug response, avoid the problems of ethics, high cost and species difference caused by animal experiments, and provide a powerful auxiliary tool for mechanism exploration, preclinical research and drug development of lung diseases.

[0003] The gas exchange model chip adopts micro-nano scale processing technology, integrates cells, culture medium perfusion system, mechanical strain system and the like on the chip, and constructs a stretchable lung alveolus-lung capillary gas-blood exchange barrier structure, which simulates the dynamic respiratory microenvironment in vivo to a certain extent. However, the existing gas exchange model chip still has some defects. For example, the classic gas exchange model chip only produces stretching and shrinking in a single direction in two-dimensional scale, which is difficult to truly simulate the three-dimensional expansion and contraction of lung alveoli driven by the movement of diaphragm and pleural cavity in vivo.

[0004] The barrier structure of the classic gas exchange model chip depends on polymethylsiloxane, polycarbonate, polyethylene terephthalate and polypropylene materials. Such materials have poor hydrophilic property and poor deformation ability in response to pressure, and the thickness is 100 times or more than that of the physiological gas-blood barrier (about 0.5 μm), which has not been able to simulate the in-vivo highly hydrophilic, highly soft, highly permeable, highly porous and ultra-thin extracellular matrix environment, which is not conducive to uniform information exchange between upper and lower cells, rapid exchange of oxygen-carbon dioxide and the like.

[0005] Therefore, there is an urgent need for a three-dimensional gas exchange model chip model that can highly realistically simulate the in-vivo gas-blood barrier and lung respiratory environment. SUMMARY

[0006] In order to solve one of the above technical problems in the prior art, the present application provides a sandwich structure composite film material capable of simulating three-dimensional deformation of lung respiration in vivo, a preparation method thereof, and a three-dimensional gas exchange model chip model using the composite film material and its application.

[0007] According to one aspect of the present disclosure, a composite film is provided, comprising: a polymer skeleton layer; and alginate-polyacrylamide gel layers arranged on both sides of the polymer skeleton layer.

[0008] In some embodiments, the alginate-polyacrylamide gel layer can be prepared from an alginate, an acrylamide monomer, and a crosslinking agent. In some embodiments, the crosslinking agent can include a first crosslinking agent for polymerization of the acrylamide monomer, and a second crosslinking agent for crosslinking of the alginate.

[0009] In some embodiments, the alginate-polyacrylamide gel layer can be prepared by the following steps:

[0010] 1) providing a pre-polymerization solution comprising an acrylamide monomer and 0.1 wt% to 5.0 wt% of an alginate, the mass ratio of alginate to acrylamide monomer being (1:0) to (1:12);

[0011] 2) adding a first crosslinking agent to facilitate formation of a polyacrylamide network structure from the acrylamide monomer, to obtain a first crosslinking product; and

[0012] 3) immersing the first crosslinking product in a solution containing a second crosslinking agent to facilitate crosslinking of the alginate, to obtain an alginate-polyacrylamide gel layer.

[0013] In some embodiments, the alginate can be selected from one or more of sodium alginate, calcium alginate, strontium alginate, and barium alginate. In specific embodiments, the alginate can be sodium alginate.

[0014] In some embodiments, the pre-polymerization solution can comprise 0.5 wt% to 4.0 wt% of the alginate. In some embodiments, the pre-polymerization solution can comprise about 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, or 4.0 wt% of the alginate.

[0015] In some embodiments, the mass ratio of alginate to acrylamide monomer can be (1:3) to (1:12). In some embodiments, the mass ratio of alginate to acrylamide monomer can be about 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, or 1:12. In some embodiments, the mass ratio of alginate to acrylamide monomer can be (1:6) to (1:12).

[0016] In some embodiments, the acrylamide monomer can be selected from one or more of acrylamide, N-substituted methacrylamide, and N,N'-substituted methacrylamide. In some embodiments, the acrylamide monomer can be acrylamide.

[0017] In some embodiments, the first crosslinking agent can be selected from one or more of N,N'-methylenebisacrylamide, N,N'-diallyltartardiamide, divinylbenzene, polyethylene glycol diacrylate, and polyethylene glycol dimethacrylate. In some embodiments, the first crosslinking agent can be N,N'-methylenebisacrylamide.

[0018] In some embodiments, the mass ratio of the acrylamide monomer to the first crosslinking agent can be (1 :0)~(1 :0.1). In some embodiments, the mass ratio of the acrylamide monomer to the first crosslinking agent can be (1 :0.0001)~(1 :0.01). In some embodiments, the mass ratio of the acrylamide monomer to the first crosslinking agent can be about 1 :0.0001, 1 :0.0002, 1 :0.0003, 1 :0.0004, 1 :0.0005, 1 :0.0006, 1 :0.0007, 1 :0.0008, 1 :0.0009, 1 :0.001, 1 :0.002, 1 :0.003, 1 :0.004, 1 :0.005, 1 :0.006, 1 :0.007, 1 :0.008, 1 :0.009, or 1 :0.01.

[0019] In some embodiments, the prepolymer solution can further comprise an initiator. In some embodiments, the initiator can be selected from one or more of peroxo glutaric acid, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0020] In some embodiments, the prepolymer solution can further comprise a catalyst. In some embodiments, the catalyst can be tetramethylethylenediamine. In some embodiments, the mass ratio of the catalyst to the acrylamide monomer is (0.002~0.025):1. In a particular embodiment, the mass ratio of the catalyst to the acrylamide monomer is 0.0025:1.

[0021] In some embodiments, in step 2), after adding the first crosslinking agent, ultraviolet light irradiation is performed to obtain a first crosslinking product. In some embodiments, the ultraviolet light irradiation is maintained for 20 min to 5 h, preferably for 0.5 h to 4 h, for example, for 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, or 4.0 h.

[0022] In some embodiments, the second crosslinking agent can be a solution containing a polyvalent cation. In some embodiments, the polyvalent cation can be a divalent or trivalent cation. In some embodiments, the polyvalent cation can be selected from Fe 3+ , Mg 2+Al 3+ Ca 2+ 、Sr 2+ Ba 2+ One or more of the following. In some embodiments, the multivalent cation may be Ca. 2+ In some embodiments, the second crosslinking agent may be a solution containing CaCl2. In some embodiments, the second crosslinking agent may be a solution containing 0.1 wt% to 5.0 wt% CaCl2. In some embodiments, the second crosslinking agent may be a solution containing 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, or 5.0 wt% CaCl2.

[0023] In some embodiments, the composite film may be treated with a buffer solution to reduce its thickness. In some embodiments, the composite film may be degraded using 1× phosphate (PBS) buffer, 2× PBS, 3× PBS, 4× PBS, 5× PBS, or higher concentrations of PBS buffer to remove some of the cross-linked network structures.

[0024] In some embodiments, the polymer backbone layer may be made of one or more of aliphatic polyesters, polystyrene, polyolefins, polysaccharides, collagen, corn gluten, gelatin, sericin, polyvinylpyrrolidone, hydroxypropyl methylcellulose, polyethylene oxide, polyethyleneimine, polyvinyl alcohol, polyamide, or polyurethane. In some embodiments, the aliphatic polyester is polycaprolactone, polylactic acid, polyethylene glycol, polydioxane, polyhydroxyalkanoates, or copolymers of the aforementioned polyesters.

[0025] In some embodiments, the polymer backbone layer may be made of polyvinyl alcohol. In some embodiments, the polymer backbone layer may be a polyvinyl alcohol gel layer.

[0026] In some embodiments, the polyvinyl alcohol gel layer can be prepared by the following steps:

[0027] i) Freezing and solidifying a polyvinyl alcohol-gelatin solution, wherein the polyvinyl alcohol-gelatin solution contains 5 to 20 wt% polyvinyl alcohol;

[0028] ii) Soak the frozen polyvinyl alcohol in a 1.0-2.0 mol / L citrate solution for 1-5 days, then remove it and wash it to remove excess citrate.

[0029] iii) optionally, repeating steps i) and ii).

[0030] In some embodiments, the 5-20 wt% polyvinyl alcohol solution is freeze- coagulated under conditions below -20 °C. In specific embodiments, a metal sheet (e.g., a copper sheet) is inserted into the polyvinyl alcohol solution, and one end of the metal sheet is inserted into a -25 °C to -20 °C refrigerator or liquid nitrogen to form a temperature gradient to induce gradient freeze-coagulation of the polyvinyl alcohol solution.

[0031] In some embodiments, the mass ratio of polyvinyl alcohol to gelatin in the polyvinyl alcohol-gelatin solution is (5-20):2.

[0032] In some embodiments, the freeze-coagulated polyvinyl alcohol is soaked in a citrate solution at 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L, or 2.0 mol / L.

[0033] In some embodiments, the citrate can be selected from one or more of calcium citrate, potassium citrate, ammonium citrate, sodium dihydrogen citrate, and sodium citrate. In some embodiments, the citrate can be sodium citrate.

[0034] In some embodiments, steps i) and ii) can be repeated 1-7 times.

[0035] In some embodiments, the composite film can be impregnated with at least one member of the group of fibronectin, collagen (e.g., type I collagen, type IV collagen, or type V collagen), laminin, fibronectin, gelatin, perlecan, entactin, proteoglycan, osteopontin, tenascin, nidogen, basement membrane matrix, and polylysine. In some embodiments, the hydrogel can be impregnated with one or more of collagen, fibronectin, and laminin.

[0036] In some embodiments, the composite film can have a thickness of 5-50 μιη. In some embodiments, the composite film can have a thickness of about 5 μιη, 10 μιη, 15 μιη, 20 μιη, 25 μιη, 30 μιη, 35 μιη, 40 μιη, 45 μιη, or 50 μιη.

[0037] In some embodiments, the composite film can be in the form of a sheet, a disc, a wafer, a block, a disc, a sheet, a strip, etc.

[0038] The composite film of the present disclosure is composed of a three-layer structure, including a polymer skeleton layer and elastic material layers arranged on both sides thereof. The composite film of the present disclosure improves the degradation speed of the material as a whole, wherein the degradation speed of the skeleton layer is slower, and the degradation speed of the elastic material layer is faster, and the combination of the two can control / regulate the degradation speed of the composite film as a whole.

[0039] Existing materials, such as polydimethylsiloxane materials, cannot be degraded, and cannot achieve thickness reduction, and too fast degradation is also not conducive to early cell growth. The present disclosure can achieve control of the degradation speed through the polymer skeleton layer and the elastic material layers on both sides thereof.

[0040] The composite film of the present disclosure gradually degrades during cell culture, and the thickness gradually decreases, and an ultra-thin lung gas-blood barrier can be obtained. Since the composite film of the present disclosure itself has the characteristics of high softness, high permeability, high porosity, and high hydrophilicity, etc., it does not need to be micropillar arrayed to realize the diffusion of substances therein, unlike the processing of polydimethylsiloxane porous membranes which need to construct a through-hole array therein. In the gas exchange model chip, the double-layer cells introduced can form a natural gas-blood barrier after the natural degradation of the composite film of the present disclosure.

[0041] According to still another aspect of the present disclosure, a method for preparing a composite film is provided, the method comprising:

[0042] 1) providing a polymer skeleton layer;

[0043] 2) soaking the polymer skeleton layer in a pre-polymerization solution, wherein the pre-polymerization solution comprises acrylamide monomers and 0.1wt% to 5.0wt% of alginate, and the mass ratio of alginate to acrylamide monomers is (1:0) to (1:12); and

[0044] 3) adding a cross-linking agent to obtain a composite film with alginate-polyacrylamide gel layers arranged on both sides of the polymer skeleton layer.

[0045] In some embodiments, step 3) can include: adding a first cross-linking agent to promote the formation of a polyacrylamide network structure by the acrylamide monomers to obtain a first composite cross-linking product; and soaking the first composite cross-linking product in a solution containing a second cross-linking agent to promote the cross-linking of the alginate to obtain the composite film.

[0046] In some embodiments, the alginate can be selected from one or more of sodium alginate, calcium alginate, strontium alginate, and barium alginate.

[0047] In some embodiments, the pre-polymerization solution can contain 0.5wt% to 4.0wt% of alginate.

[0048] In some embodiments, the mass ratio of the alginate salt to the acrylamide monomer can be (1:3)~(1:12).

[0049] In some embodiments, the acrylamide monomer can be selected from one or more of acrylamide, N-substituted methacrylamide, and N,N'-substituted methacrylamide.

[0050] In some embodiments, the first crosslinking agent can be selected from one or more of N,N'-methylenebisacrylamide, N,N'-diallyltartardiamide, divinylbenzene, polyethylene glycol diacrylate, and polyethylene glycol dimethacrylate.

[0051] In some embodiments, the mass ratio of the acrylamide monomer to the first crosslinking agent can be (1:0)~(1:0.1).

[0052] In some embodiments, the prepolymer solution can further comprise an initiator. In preferred embodiments, the initiator can be selected from one or more of peroxo glutaric acid, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0053] In some embodiments, the prepolymer solution can further comprise a catalyst. In preferred embodiments, the catalyst can be tetramethylethylenediamine.

[0054] In some embodiments, the mass ratio of the catalyst to the acrylamide monomer can be (0.002~0.025):1.

[0055] In some embodiments, the second crosslinking agent can be a solution containing a polyvalent cation. In preferred embodiments, the polyvalent cation can be a divalent or trivalent cation. In more preferred embodiments, the polyvalent cation is selected from one or more of Fe 3+ , Mg 2+ , Al 3+ , Ca 2+ , Sr 2+ , and Ba 2+ .

[0056] In some embodiments, the method further comprises rinsing the composite film with a buffer solution to reduce the thickness of the composite film. In some embodiments, the composite film can be rinsed with 1x phosphate buffered saline (PBS) buffer solution, 2x PBS, 3x PBS, 4x PBS, 5x PBS, or higher concentration of PBS buffer solution to degrade part of the crosslinked network structure, thereby reducing the thickness of the composite film.

[0057] In some embodiments, the polymeric backbone layer can be made of one or more of aliphatic polyester, polystyrene, polyolefin, polysaccharide, collagen, zein, gelatin, sericin, polyvinylpyrrolidone, hydroxypropyl methylcellulose, polyethylene oxide, polyethylene imine, polyvinyl alcohol, polyamide or polyurethane.

[0058] In preferred embodiments, the polymeric backbone layer is made of polyvinyl alcohol. In more preferred embodiments, the polymeric backbone layer can be a polyvinyl alcohol gel layer.

[0059] In some embodiments, the polymeric backbone layer can be prepared by the following steps:

[0060] i) freeze coagulating a polyvinyl alcohol-gelatin solution, wherein the polyvinyl alcohol-gelatin solution comprises 5-20 wt% of polyvinyl alcohol;

[0061] ii) immersing the freeze coagulated polyvinyl alcohol in a citrate solution of 1.0-2.0 mol / L for 5-7 days, removing, and washing to remove excess citrate;

[0062] iii) optionally, repeating steps i) and ii).

[0063] In some embodiments, the method can further comprise a step of impregnating the surface of the composite film. In some embodiments, the surface of the composite film can be impregnated with at least one of fibronectin, collagen (e.g., type I collagen, type IV collagen or type V collagen), laminin, fibronectin, gelatin, perlecan, entactin, proteoglycan, osteopontin, tenascin, nidogen, basement membrane matrix and polylysine. In some embodiments, the surface of the composite film can be impregnated with one or more of collagen, fibronectin and laminin.

[0064] In some embodiments, after adding the first crosslinking agent, the first composite crosslinking product is obtained by UV irradiation. In some embodiments, the UV irradiation is maintained for 20 min to 5 h, preferably for 0.5 h to 4 h, for example, for 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h or 4.0 h.

[0065] In some embodiments, the wavelength of the UV light is 10 nm-400 nm, preferably 100-400 nm. In some embodiments, the wavelength of the UV light is, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm or 400 nm.

[0066] The composite film of the present disclosure has very good transparency, good elasticity and appropriate toughness, can absorb water and reach swelling equilibrium in a short time, and can realize deformation recovery under multiple stretching conditions, which is conducive to the shape and volume of the hydrogel to remain stable in a short period of time. In addition, the composite film of the present disclosure has good biocompatibility and cell adhesion.

[0067] According to another aspect of the present disclosure, an organ chip comprising the composite film of the present disclosure is provided.

[0068] In some embodiments, the organ chip comprises: an in / out liquid layer (1) provided with a first channel in / out liquid port (7) and a second channel in / out liquid port (8); a first cell culture layer (2) provided with an anti-overflowing rubber ring (9) and a first cell culture cavity and connecting flow channel (10); a composite film layer (3); a second cell culture layer (4) provided with a second cell culture cavity and connecting flow channel (11); an actuating membrane (5); and, a chamber layer (6) provided with an air outlet (12) and an air chamber (13).

[0069] In some embodiments, the actuating membrane (5) can be selected from, but not limited to, polymethyl methacrylate, polydimethylsiloxane, polycarbonate, polyethylene terephthalate, glass, quartz, polystyrene, polyethylene, silicon wafer, epoxy resin, urethane resin, styrene thermoplastic elastomer, olefin thermoplastic elastomer, acrylic thermoplastic elastomer, polyvinyl alcohol, etc.

[0070] In some embodiments, the composite film can be partially degraded or dissolved in the elastic layer of the chip. In some embodiments, the composite film can be partially degraded in the chip by a PBS solution (e.g., 5×PBS).

[0071] In some embodiments, the composite film can be coated with a biological macromolecule. In some embodiments, the composite film can be coated with at least one selected from the group consisting of fibronectin, collagen (e.g., type I collagen, type IV collagen or type V collagen), laminin, fibronectin, gelatin, basement membrane proteoglycan, nidogen, proteoglycan, osteonectin, tenascin, nephrin, basement membrane matrix and polylysine. In some embodiments, the composite film can be impregnated with one or more of collagen, fibronectin and laminin.

[0072] In some embodiments, the first cell culture layer and the second cell culture layer are each provided with one or more cell culture chambers.

[0073] In some embodiments, the organ-on-a-chip of the present disclosure can be used to mimic the lung gas-blood barrier structure. In such embodiments, the first cell culture layer can be seeded with lung vascular endothelial cells, such as microvascular endothelial cells. In such embodiments, the second cell culture layer can be seeded with alveolar epithelial cells.

[0074] Alternatively, the second cell culture layer can be seeded with lung vascular endothelial cells, such as microvascular endothelial cells. In such embodiments, the first cell culture layer can be seeded with alveolar epithelial cells.

[0075] In some embodiments, the air suction port is connected to an air suction pump to drive the deformation of the cell membrane in the first cell culture layer and the second cell culture layer. In some embodiments, the air suction pump is connected to a controller to simulate normal respiratory movement by adjusting the pressure. In some embodiments, the air suction pump can adjust the pressure change in any form known to those skilled in the art. In some embodiments, the air suction pump can provide a sine wave, a straight wave or a square wave. In some embodiments, the air suction pump can provide a pressure of -800-0 mbar.

[0076] In some embodiments, the first cell culture layer can also be provided with an anti-overflow structure, such as an anti-overflow rubber ring.

[0077] The present disclosure develops a composite membrane that can simulate the three-dimensional deformation of the lung in the body. The new ultra-thin membrane material is prepared using a skeleton layer and two elastic layers as the matrix, which is superior to the hydrophilicity of existing high molecular materials (such as polydimethylsiloxane), can achieve three-dimensional deformation, is not easy to break, can truly simulate the inflation and contraction of alveoli during the breathing process, and can improve the information exchange and gas exchange efficiency at the barrier structure of the existing gas exchange model.

[0078] Using the composite membrane of the present disclosure, the gas-blood barrier can be constructed by means of the ultra-thin membrane in both two-dimensional scale of stretching and shrinking, and the inflation and contraction process of alveoli in the breathing process can be simulated by the three-dimensional deformation of the membrane.

[0079] The present disclosure improves the biological information exchange and effective gas exchange efficiency of the gas exchange model chip, constructs a more bionic disease model, such as infectious diseases (such as COVID-19, influenza), cancer, obstructive pulmonary disease, asthma and acute lung injury / acute respiratory distress syndrome disease model, and provides a reliable technical platform for preclinical research and drug development of respiratory diseases. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 The composite membrane of the present disclosure and the process of its bio-macromolecule dyeing are exemplarily shown.

[0081] Figure 2 An exemplary organ chip structure diagram according to an embodiment of the present disclosure is shown. The organ chip comprises: an inlet and outlet layer 1; a first cell culture layer 2; a composite membrane layer 3; a second cell culture layer 4; a polydimethylsiloxane actuating membrane 5; a chamber layer 6; an anti-overflow rubber ring 9; a first cell culture chamber and connecting flow channel 10; a second cell culture chamber and connecting flow channel 11; a gas suction port 12; a gas chamber 13.

[0082] Figure 3 An exemplary organ chip according to an embodiment of the present disclosure is shown. In the figure, Figure 3 A shows a perspective view of a finished product of a lung gas exchange model chip, Figure 3 B shows an enlarged view of an intermediate culture chamber and flow channel.

[0083] Figure 4 A microstructure electron microscope image of a polyvinyl alcohol skeleton membrane according to an embodiment of the present disclosure is shown.

[0084] Figure 5 Photos of elastic membrane layers prepared from different proportions of raw materials are shown.

[0085] Figure 6 A swelling curve of an elastic layer material prepared according to an embodiment of the present disclosure is shown, in which the abscissa represents time (minutes) and the ordinate represents the swelling rate (%).

[0086] Figure 7 A composite membrane (left) and a microstructure electron microscope image (right) according to an embodiment of the present disclosure are shown.

[0087] Figure 8 A three-dimensional deformation of a composite membrane according to an embodiment of the present disclosure is shown. In the figure, Figure 8 A shows the three-dimensional deformation state of the composite membrane, Figure 8 B shows the relationship between the deformation amount of the composite membrane and the pressure (the control group is polydimethylsiloxane).

[0088] Figure 9 Live cell staining results and quantification results of surface cultured cells of a composite membrane according to an embodiment of the present disclosure are shown. In the figure, Figure 9 A shows live cell fluorescence staining results of cells cultured on a surface of a composite membrane without immersion (blank); Figure 9 B shows live cell fluorescence staining results of cells cultured on a surface of a composite membrane immersed with type I collagen; Figure 9 C shows cell survival rate statistical results of a composite membrane without immersion (blank) and a composite membrane immersed with type I collagen after 1, 3, and 5 days of culture, in which the abscissa represents time (days) and the ordinate represents cell survival rate.

[0089] Figure 10 A lung alveolar epithelial cell and an endothelial cell are shown to form a tight junction on a composite membrane surface of a chip according to one embodiment of the present disclosure. DETAILED DESCRIPTION

[0090] Existing methods establish a low degree of alveolar organ simulation, the porous membrane is thick, and only one direction of stretching and shrinking can be achieved in two-dimensional scale. The composite membrane of the present disclosure is based on a degradable, elastic and highly permeable material, which realizes the construction of an ultra-thin barrier and three-dimensional deformation. The composite membrane of the present disclosure is conducive to uniform information exchange and gas exchange between lung alveolar epithelial cells and vascular endothelial cells, and truly simulates the physiological inflation and contraction of alveoli in the breathing process and biological information exchange and effective gas exchange. It can provide a reliable technical platform for preclinical research and drug development of respiratory diseases.

[0091] Figure 1 An example shows a process of a composite membrane and its bio-macromolecule immersion according to one embodiment of the present disclosure. First, a skeleton membrane, i.e., a polymer skeleton layer, is prepared by directional freezing. The obtained polymer skeleton layer is immersed and ultraviolet cured to introduce elastic layers, i.e., alginate-polyacrylamide gel layers, on both sides, to obtain a composite membrane with a three-layer structure. The obtained composite membrane is immersed in PBS (for example, 5×PBS) to partially degrade the elastic layer (for example, the part shown in blue), and then immersed with a protein solution to obtain a composite membrane with a protein-immersed surface. Figure 1 An example shows a process of a composite membrane and its bio-macromolecule immersion according to one embodiment of the present disclosure. First, a skeleton membrane, i.e., a polymer skeleton layer, is prepared by directional freezing. The obtained polymer skeleton layer is immersed and ultraviolet cured to introduce elastic layers, i.e., alginate-polyacrylamide gel layers, on both sides, to obtain a composite membrane with a three-layer structure. The obtained composite membrane is immersed in PBS (for example, 5×PBS) to partially degrade the elastic layer (for example, the part shown in blue), and then immersed with a protein solution to obtain a composite membrane with a protein-immersed surface.

[0092] Figure 2 An example shows a structure schematic diagram of an organ chip according to one embodiment of the present disclosure. The organ chip comprises: an in / out liquid layer 1, provided with a first channel in / out liquid port 7 and a second channel in / out liquid port 8; a first cell culture layer 2, provided with an anti-overflowing rubber ring 9, and a first cell culture cavity and a connecting flow channel 10; a composite membrane layer 3; a second cell culture layer 4, provided with a second cell culture cavity and a connecting flow channel 11; an actuating membrane 5; and a chamber layer 6, provided with a gas outlet 12 and a gas chamber 13.

[0093] In the case of simulating the lung gas-blood barrier, the first cell culture layer 2 can be used to culture lung epithelial cells. The first channel liquid inlet 7 can be used to connect the pipeline for perfusing the epithelial cell culture medium to provide continuous perfusion of the culture medium, and the first channel liquid outlet 7 is used to connect the liquid outlet pipeline to discharge the liquid in the cell culture cavity, which can be collected for subsequent detection.

[0094] The bottom of the first cell culture layer 2 is provided with one or more recessed lung epithelial cell culture cavities 10.

[0095] The second cell culture layer 4 is provided with one or more vascular endothelial cell culture cavities and connecting flow channels 11.

[0096] The chamber layer 6 is provided with one or more air chambers 13 and an air outlet 12. The chamber layer 6 can also include a connecting channel in fluid communication with the second cell culture outlet.

[0097] In some embodiments, the first cell culture layer 2 can be used to culture vascular endothelial cells, and the second cell culture layer can be used to culture lung epithelial cells.

[0098] Figure 3 Exemplary chip and culture chamber are shown in the figures.

[0099] The depth of the upper cell culture chamber and channel is less than or equal to the thickness of the upper chip, and the depth of the inlet and outlet is equal to the thickness of the chip; the depth of the middle cell culture chamber, channel and inlet and outlet is equal to the thickness of the chip; the depth of the lower air chamber and channel is less than the thickness of the lower chip. The three-layer chip structure has a certain matching relationship, the lower surface of the epithelial cell culture chamber overlaps the upper surface of the endothelial cell culture chamber, and the two surfaces are separated by a hydrogel base film; the air chambers are located on both sides of the endothelial cell culture chamber, staggered and not overlapping, and the lower surface of the endothelial cell culture chamber is separated from the upper surface of the air chamber by a polydimethylsiloxane film. The chip layers can be fixed and sealed by clamps, hot pressing, adhesive tape and glue.

[0100] The preparation method of the polydimethylsiloxane film can be molding, spin coating, etc.; the material of the new composite film can be natural and synthetic polymer materials, the natural polymer can be protein, polysaccharide (such as gelatin, alginate, collagen, silk fibroin, agarose, dextran), and the synthetic polymer can be polyester, polyamide, polyvinyl alcohol (such as polylactic acid, polycaprolactone, polyacrylamide), etc. By selecting different types and proportions of polymer chains, a high-elasticity film preparation material can be obtained, and the film forming method can be directional freezing, molding, spin coating, electrospinning, etc. Combined with the selected material, a high-elasticity, high-biocompatibility and high-transparency film can be processed. The composite film material is degradable and can degrade under certain conditions, gradually reducing the thickness during cell culture, obtaining an air-blood barrier with a thickness less than the initial thickness, thereby constructing an ultra-thin lung air-blood barrier. Due to the high softness, high permeability, high porosity and high hydrophilicity of the high-permeability material, the ultra-thin composite film does not need to be microporous to achieve the diffusion of substances therein, unlike the porous polydimethylsiloxane film which needs to construct a through-hole array therein.

[0101] For constructing the lung gas-blood barrier, cells are used as (primary) lung cells, and a single-cell suspension from alveolar epithelial cells is introduced into the upper cell culture chamber to form a gas-blood barrier epithelial cell layer, and an endothelial cell suspension from lung microvessels is introduced into the lower cell culture chamber to form a gas-blood barrier endothelial cell layer. The height of the vascular endothelial cell culture chamber and the alveolar epithelial cell culture chamber can be 100-1000 pm. After the cells are planted in the chip, different pressures can be applied through the air outlet at the bottom and the connecting channel to drive the polydimethylsiloxane film to deform in three dimensions. Since the volume of the fluid in the lower cell culture chamber is constant, the newly developed hydrogel matrix film assembled in the upper layer can be driven to produce three-dimensional deformation at the same frequency.

[0102] The conditions in the culture process chip, such as cell type, culture medium composition, culture medium flow rate, three-dimensional deformation amount, temperature, pH, and oxygen concentration, can be changed, and can be used to study the effects of different conditions on cell behavior.

[0103] In addition to the necessary epithelial cells and endothelial cells contained in the gas-blood barrier, other lung cells can also be introduced to establish a respiratory microenvironment close to the physiological structure of the lung; the middle layer chip and the lower layer chip are separated by a layer of non-porous film, which can be a polydimethylsiloxane non-porous film; the lower layer chip includes an air chamber, an air outlet, and a connecting channel that provides deformation pressure, and the pressure applied by this layer structure can drive the polydimethylsiloxane non-porous film to deform in three dimensions; since the volume of the liquid in the second layer is not compressible under cell culture conditions, the three-dimensional deformation of the polydimethylsiloxane non-porous film between the middle layer chip and the lower layer chip will drive the three-dimensional deformation of the porous film between the upper layer and the middle layer chip; the materials of the upper, middle, and lower layers of the chip can be polymethyl methacrylate, polydimethylsiloxane, polycarbonate, and glass; due to the high elasticity, high softness, high permeability, high porosity, and high hydrophilicity of the ultra-thin hydrogel film supporting the construction of the gas-blood barrier, three-dimensional deformation can be achieved through the design of the chip structure, which can more simulate the inflation and deflation of alveoli during the breathing process, and improve the biological information exchange and effective gas exchange efficiency of the lung organ chip.

[0104] The composite film of the present disclosure can achieve three-dimensional deformation, truly simulate the inflation and deflation of alveoli during the breathing process, and improve the information exchange and gas exchange efficiency at the barrier structure of the existing organ chip. The composite film of the present disclosure can be used to construct more biomimetic disease models, such as infectious diseases (e.g. COVID-19, influenza), cancer, obstructive lung disease, asthma, and acute lung injury / acute respiratory distress syndrome disease models, to provide a reliable technical platform for preclinical research and drug development of respiratory diseases.

[0105] In order to make the objects, technical solutions, and advantages of the present application clearer, the following further describes the present application in conjunction with embodiments. The specific embodiments described herein are intended for explanation only and are not intended to constitute any limitation of the present application. Furthermore, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concept of the present disclosure. Such structures and techniques are described in many publications.

[0106] Definitions

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein, and carry the anticipated meaning per the context of this application. For purposes of interpreting this specification, the words "comprises," "comprising," "includes," "including," "has," "having" or the like are inclusive and are determined without the use of the word "only." As used herein, the terms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0108] The expressions "a" and "an" as used herein include plural references unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and the like.

[0109] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0110] As used herein, the terms "gel" or "hydrogel" are used interchangeably and include a network of hydrophilic polymer chains. Hydrogels (also called "aqueous gels") sometimes exist as colloidal gels, in which water is the dispersion medium. Hydrogels have a degree of flexibility very similar to natural tissues due to their high water content. Hydrogels are composed of cross-linked polymers and are a biocompatible polymeric matrix. Such biocompatible polymeric matrix is fully biodegradable or partially biodegradable. Examples of materials that can form hydrogels include, for example, alginate and alginate derivatives, polylactic acid, polyglycolic acid, polylactic-glycolic acid copolymer (PLGA), gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids (e.g., polypeptides, especially poly(lysine)), polyesters (such as polyhydroxybutyrate and poly-epsilon-caprolactone), polyanhydrides; polyphosphazines, poly(vinyl alcohol), poly(alkylene oxides), especially poly(ethylene oxide), poly(allylamine) (PAM), poly(acrylate), modified polystyrene (such as poly(4-aminomethylstyrene), complex polyols, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of the foregoing.

[0111] As used herein, the term "alginate" refers to a class of polysaccharides extracted from brown algae, which are random block copolymers of β-1,4-D-mannuronic acid (M units) and a-1,4-L-guluronic acid (G units). Generally, M and G units in alginate are linked by 1,4-glycosidic bonds in M-M, G-G or M-G combinations to form block copolymers. The chemical formula of alginate is (C6H8O6)n, and its molecular weight is generally 4-1500 kDa. As used herein, "alginate salt" refers to a salt formed from alginate, including but not limited to sodium alginate, calcium alginate, strontium alginate, barium alginate, etc.

[0112] The term "biocompatibility" herein refers to having no toxic or harmful effects on biological functions.

[0113] Examples and drawings are provided below to help understand the present application. However, it should be understood that these examples and drawings are only for illustration of the present application, but do not constitute any limitation. The actual protection scope of the present application is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present application.

[0114] Example 1. Preparation of skeleton layer film material

[0115] Polyvinyl alcohol is selected to prepare a tough skeleton film material. The polyvinyl alcohol solution is subjected to repeated freeze-thaw, directional freezing and salting-out steps (Mutian Hua et al., Nature, Vol. 590, 594-599 (2021)) to obtain a polyvinyl alcohol hydrogel having a fibrous network with closely arranged and highly anisotropic molecular arrangement. Figure 4 The obtained polyvinyl alcohol hydrogel is used as a skeleton layer film material in subsequent examples. Such fibrous network structure has strong tensile and fatigue resistance, and is suitable for the construction of alveolar air-blood barrier structure, wherein the concentration of polyvinyl alcohol and gelatin can be selected in the range of 5% to 10%, and after uniform mixing to form a uniform solution, the solution is injected into a mold of corresponding thickness to press into a film. Among them, the polyvinyl alcohol hydrogel can be soaked in 1.5 mol / L sodium citrate solution for 1 to 5 days, repeated freeze-thaw for 5 to 7 cycles at -20°C to 25°C, directional freezing with liquid nitrogen as cold source and copper sheet as heat conduction base material. The tough skeleton film obtained is washed with pure water, Hanks balanced salt (HBSS), PBS solution (GIBCO) to remove unreacted monomers, and then used for subsequent chip assembly.

[0116] Example 2. Preparation of elastic film layer material

[0117] In this example, a double network hydrogel composed of alginate and polyacrylamide is prepared as an elastic layer, and the properties of the gel prepared under different alginate concentrations and mass ratios of alginate to acrylamide monomer are studied.

[0118] The preparation method comprises the following steps: dissolving sodium alginate (Alg), acrylamide, 1wt% ketopentanedioic acid in water at room temperature, adding methylene bisacrylamide with a mass of 0.006 of the mass of acrylamide, and adding tetramethylethylenediamine with a mass of 0.0025 of the mass of acrylamide to obtain a hydrogel pre-polymer solution. After the hydrogel pre-polymer solution is uniformly mixed, a film is pressed in a glass mold. The pre-polymer is irradiated with ultraviolet light (253.7 nm) for 3 hours to form a first layer network of polyacrylamide. Then, it is soaked in a 1wt% CaCl2 solution to form a second layer network of ion-crosslinked alginate.

[0119] The hydrogel pre-polymer solution with a final concentration of 0.5wt%, 1wt%, 2wt%, and 4wt% of sodium alginate, and the mass ratio of sodium alginate to acrylamide being 1:0, 1:3, 1:6, and 1:12, respectively, is tested for hydrogel forming performance and transparency. The results are shown in Figure 5

[0120] From the results of Figure 5 , it can be seen that when the viscosity of the monomer solution is large, for example, under the conditions of 4wt% sodium alginate and the mass ratio of sodium alginate to acrylamide being 1:3, 1:6, and 1:12 (indicated by the blue box), the reaction is too fast, making it difficult for bubbles to overflow, and the transparency is poor; when the density of the acrylamide cross-linking network in the hydrogel is too low, such as when the acrylamide concentration is 0, 0.5% sodium alginate, and the mass ratio of sodium alginate to acrylamide is 1:12, 0.5%, 1%, and 2% sodium alginate, and the mass ratio of sodium alginate to acrylamide is 1:0, 1:3, and 0.5%, 1% sodium alginate, and the mass ratio of sodium alginate to acrylamide is 1:6 (indicated by the green box), the gel forming effect is poor.

[0121] At the same time, the results also show that when the mass ratio of 1wt% sodium alginate to acrylamide is 1:12, and the mass ratio of 2wt% sodium alginate to acrylamide is 1:6, the forming effect is good, as shown in the red box in Figure 5

[0122] Example 3. Optimization of the elastic film layer material

[0123] In this example, the elastic film layer material is further optimized to understand the related use performance of the elastic layer material and to ensure that it can be stably used when assembled in the chip.

[0124] 1) Preparation of the elastic layer pre-polymer solution

[0125] ​​Sodium alginate (Alg), acrylamide, and 1 wt% ketoglutarate were dissolved in water, and tetramethylethylenediamine was added at a mass ratio of 0.0025 of the acrylamide mass. The swelling properties of the hydrogel were investigated by varying the amount of crosslinking agent N,N'-methylenebisacrylamide added, with mass ratios of crosslinking agent methylenebisacrylamide to monomer acrylamide being 0.0001, 0.0006, 0.001, or 0.006.

[0126] 2) Swelling performance test of elastic materials: 300 μL of hydrogel prepolymer (sodium alginate:acrylamide = 1:6) was used to prepare hydrogel pieces of similar shape and size (n = 4). These pieces were air-dried at room temperature until their mass remained constant, and the initial mass was recorded. They were then immersed in 5 mL of Hanks' balanced salt buffer solution for 0, 1, 5, 15, 30, 60, 120, 240, 540, 1440, 4320, and 7200 min, respectively. After immersion, the surface moisture was wiped dry, the mass was weighed and recorded, and a hydrogel swelling curve was plotted based on the measured data. Figure 6 This allows us to determine whether the hydrogel can absorb water quickly and maintain a constant mass without causing the chip to crack due to swelling, thus facilitating chip assembly. The shorter the time required for the hydrogel to reach swelling equilibrium and the less water it absorbs, the more beneficial it is for the hydrogel to maintain stable shape and volume in the short term.

[0127] according to Figure 6 The results show that good swelling equilibrium was achieved at all four tested mass ratios, making it suitable for constructing organ-on-a-chip internal barrier structures. It can also be seen that when the crosslinking agent concentration ratio is 0.006 (main monomer mass ratio), it reaches swelling equilibrium earlier than the other groups and remains stable thereafter. Based on its swelling performance, it is more suitable for constructing organ-on-a-chip internal barrier structures.

[0128] 3) Porosity detection of the elastic layer: A block hydrogel was prepared according to a mass ratio of acrylamide monomer to methylenebisacrylamide crosslinking agent of 0.006. The hydrogel was then frozen at -40℃ and freeze-dried. The fracture surface of the freeze-dried elastic material was sputter-coated with gold using a gold sputtering machine, and the surface structure was observed using a scanning electron microscope. The results are as follows: Figure 7 As shown in the right figure, the elastic layer material is highly transparent and has a porous network structure, which provides a basis for high-efficiency imaging observation and mass exchange.

[0129] Example 4. Preparation of composite films and detection of biocompatibility

[0130] The polyvinyl alcohol skeleton film obtained in Example 1 was immersed in an elastic film layer material prepolymer solution, wherein the elastic film layer material prepolymer solution contained 2 wt% of sodium alginate, the mass ratio of sodium alginate to acrylamide was 1:6, the mass ratio of crosslinking agent methylene bisacrylamide to monomer acrylamide was 0.006, and the addition of tetramethyl ethylenediamine was 0.0025 of the mass of acrylamide. The composite film was obtained by irradiation curing for 3 h using ultraviolet light (253.7 nm).

[0131] The deformation test (-30 mbar) results are shown in Figure 8 The composite film and the existing polydimethylsiloxane film were assembled in the chip structure shown in Figure 2 , negative pressures of -10 mbar, -20 mbar, -30 mbar and -40 mbar were applied, and the film sag height was measured and the area expansion ratio was calculated. Figure 8 A shows that the composite film is in a three-dimensional expansion state. Figure 8 B shows that the composite film can achieve three-dimensional expansion and contraction, and the deformation is larger than that of the existing polydimethylsiloxane film, which is beneficial to subsequent assembly into a three-dimensional deformable lung organ chip.

[0132] In addition, the biocompatibility of the obtained composite film was detected using type I collagen. The obtained composite film was macerated with a 0.3 mg / mL type I collagen solution. Briefly, the composite film was immersed in a 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide (EDC / NHS) solution, then a 0.3 mg / mL type I collagen solution was added, and after standing for 4 h, the composite film was taken out and washed with Hanks' balanced salt solution to remove unattached molecules and the EDC / NHS solution, and washed 3-5 times, then the film was immersed in DMEM (Dulbecco's modified eagle medium, Gibco). A549 cells were seeded on the composite film (blank) or the composite film with type I collagen macerated on the surface at a density of 54000 cells / cm 2 TM The results are shown in Figure 9 A, Figure 9 B and Figure 9 ​In Figure C, it can be seen that the survival rate of A549 cells on both the composite membrane (blank) and the composite membrane surface impregnated with type I collagen reached 90% or higher, indicating that the composite membrane impregnated with biomolecules is suitable for cell culture and has good biocompatibility. Furthermore, microscopic observation of the collagen-impregnated composite membrane surface shows that cell morphology is more conducive to expansion.

[0134] Example 5. Preparation of organ-on-a-chip using composite thin films

[0135] An organ-on-a-chip was constructed using the composite film obtained in Example 3. This organ-on-a-chip consists of a four-layer chip with two thin films and a fixing film, wherein the composite film 3 and the polydimethylsiloxane film 5 are respectively disposed. To ensure the light transmittance of the chip, chip components 1, 2, 4, and 6 made of polymethyl methacrylate were used. The assembled organ-on-a-chip is shown below. Figure 3 As shown.

[0136] The preparation process of polydimethylsiloxane film 5 mainly includes: mixing adhesive A and adhesive B (SYLGARD) TM The 184siliconeElastomer kit was used to prepare polydimethylsiloxane prepolymer at a ratio of 10:1. The hydrophobically treated silicon wafer was placed in a spin coater and a suitable thickness of polydimethylsiloxane prepolymer was spin-coated. After curing at 80°C, a non-porous polydimethylsiloxane film with a thickness of less than 100 μm was obtained.

[0137] The polydimethylsiloxane film 5 is bonded to the endothelial cell culture layer 4 and the chamber layer 6, and then cleaned using plasma. The specific assembly process is as follows: a polydimethylsiloxane film is spin-coated onto the surface of the endothelial cell culture layer 4 and the chamber layer 6 components. After curing at 60°C, the side of the endothelial cell culture layer 4 with the polydimethylsiloxane film coated on it is bonded to the polydimethylsiloxane film 5 after plasma surface treatment. It is then placed in a 60°C oven for curing and removed. This process is then repeated to bond and seal the side of the chamber layer 6 with the polydimethylsiloxane film coated on it. The assembly order of the endothelial cell culture layer 4 and the chamber layer 6 can be interchanged.

[0138] Epithelial cell culture layer 2 and endothelial cell culture layer 4 were bonded together using adhesive, thermoforming, or clamp-assisted bonding, and a composite film was placed between them. The resulting organ-on-a-chip appeared as follows. Figure 3 As shown.

[0139] Example 6. Construction of a three-dimensional deformable intra-chip lung air-blood barrier structure

[0140] The organ chip assembled in Example 4 was subjected to ultraviolet sterilization, and then the composite film thickness was thinned and the biological macromolecule was immersed and dyed in a sterile environment. Briefly, 5xPBS (7.2-7.4) was introduced into the flow channel, and after 30 minutes of treatment, the flow channel was flushed with sterile water to complete the thickness reduction process. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide (EDC / NHS) solution was introduced into the flow channel, followed by a composite solution composed of 100 μg / mL collagen I / collagen IV (Sigma), 100 μg / mL fibronectin (Sigma), and 50 μg / mL laminin (Sigma). After standing for 2 hours or more, the unattached molecules and residual EDC / NHS solution were removed by flushing with 5xPBS buffer, and the washing was repeated 3-5 times. Then, the film was immersed in DMEM (Gibco) to complete the biological macromolecule immersion process.

[0141] Human lung microvascular endothelial cells (Sciencell) were injected into the chip through the lower channel inlet / outlet 8( Figure 2 ) and the chip was placed upside down in the cell incubator. After the endothelial cells settled and adhered to the lower surface of the composite film, a constant flow of fresh endothelial cell culture medium (Sciencell) was provided by connecting the syringe pump. After observing the good adhesion of the endothelial cells under a microscope, the chip was turned over and human alveolar epithelial cells (Sciencell) were injected from the upper channel inlet / outlet 7( Figure 2 ). Similarly, the chip was placed in the cell incubator, and after the epithelial cells settled and adhered to the upper surface of the composite film, a constant flow of fresh iCell epithelial cell culture medium (Sciencell) was provided by connecting the syringe pump.

[0142] Realization of three-dimensional deformation of the chip: After the human lung microvascular endothelial cells and alveolar epithelial cells on both sides of the film grew to form good tight junctions, the gas chamber exhaust port 12( Figure 2 ) was connected to an exhaust pump and a controller providing negative pressure. Under the driving of a sinusoidal wave, the film with attached cells was deformed. In order to realize the consistency with the breathing frequency in vivo, a sinusoidal negative pressure was used to drive the film deformation during normal breathing motion, and the pressure range was -800-0 mbar. By applying negative pressure, the in vivo lung pressure simulation under the breathing environment was realized. After the composite film with surface-implanted cells was deformed, it was placed under a microscope for deformation monitoring.

[0143] Example 7. Lung air-blood barrier verification experiment

[0144] This example verifies the necessity of biological macromolecule immersion of the composite film for the formation of the lung air-blood barrier structure in the chip by immunofluorescence staining of tight junction proteins.

[0145] After the medium in the chip was aspirated, the cells were fixed by injecting 4% paraformaldehyde for 15 min, and then washed with PBS buffer for 3 times, 5 min each time. The cells were treated with blocking permeabilization solution (0.5% Triton X-100 and 5% BSA / ovine blocking serum) for 30 min at room temperature, and then labeled with vascular endothelial cadherin (VE-Cadherin) antibody (Abeam) for endothelial cells and epithelial cadherin (E-Cadherin) antibody (Proteintech) for epithelial cells. After the antibodies were diluted, they were injected into the chip channel for staining treatment, and incubated at room temperature for 1 h or at 4°C overnight. The results are shown in Figure 10 Fig. 2, and the cells can form good tight junction on the surface of the composite film immersed in collagen.

[0146] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A composite film, characterized by, The composite membrane comprises: a polymer backbone layer; and alginate-polyacrylamide gel layers disposed on both sides of the polymer backbone layer, wherein the alginate-polyacrylamide gel layers are prepared by the following steps: 1) providing a pre-polymerization solution comprising acrylamide monomers and alginate, wherein: the pre-polymerization solution comprises 0.5 wt% ~ 1 wt% alginate, and the mass ratio of the alginate to the acrylamide monomers is (1:3) ~ (1:12); or, the pre-polymerization solution comprises 0.5 wt% ~ 2.0 wt% alginate, and the mass ratio of the alginate to the acrylamide monomers is (1:3) ~ (1:6); or, the pre-polymerization solution comprises 0.5 wt% ~ 4.0 wt% alginate, and the mass ratio of the alginate to the acrylamide monomers is 1:3; 2) adding a first cross-linking agent to facilitate the formation of a polyacrylamide network structure by the acrylamide monomers to obtain a first cross-linking product; and 3) immersing the first cross-linking product in a solution containing a second cross-linking agent to facilitate the cross-linking of the alginate to obtain alginate-polyacrylamide gel layers, wherein the polymer backbone layer is prepared by the following steps: i) freeze coagulating a polyvinyl alcohol-gelatin solution, wherein the polyvinyl alcohol-gelatin solution comprises 5 ~ 20 wt% polyvinyl alcohol, and the freeze coagulation is a step of repeated freeze-thawing and directional freezing; ii) after immersing the freeze coagulated polyvinyl alcohol in a 1.0 ~ 2.0 mol / L citrate solution for 5 ~ 7 days, taking it out, and washing to remove excess citrate; and iii) optionally, repeating steps i) and ii).

2. The composite film according to claim 1, characterized in that, the alginate is sodium alginate; and / or the acrylamide monomers are selected from acrylamide; and / or the first cross-linking agent is selected from one or more of N,N'-methylene bisacrylamide, N,N'-diallyl tartaric acid diamide, divinylbenzene, polyethylene glycol diacrylate, and polyethylene glycol dimethacrylate.

3. The composite film according to claim 1, wherein The mass ratio of the acrylamide monomers to the first cross-linking agent is (1:0.0001) ~ (1:0.1).

4. The composite film of claim 1, wherein The pre-polymerization solution further comprises an initiator, and / or The pre-polymerization solution further comprises a catalyst.

5. The composite film according to claim 4, wherein The initiator is selected from one or more of peroxoglutaric acid, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ammonium persulfate, potassium persulfate, and sodium persulfate.

6. The composite film of claim 4, wherein The catalyst is tetramethylethylenediamine.

7. The composite film according to claim 4, wherein The mass ratio of the catalyst to the acrylamide monomers is (0.002 ~ 0.025):

1.

8. The composite film of claim 1, wherein The second cross-linking agent is a solution containing a polyvalent cation.

9. The composite film of claim 8, wherein, The polyvalent cation is a divalent or trivalent cation.

10. The composite film of claim 8, wherein The polyvalent cations are selected from one or more of Fe 3+ , Mg 2+ , Al 3 + , Ca 2+ , Sr 2+ , Ba 2+ .

11. The composite film of claim 1, wherein The composite membrane is treated with a buffer solution to reduce the thickness of the composite membrane.

12. The composite film of claim 1, wherein The composite membrane is degraded using 1 × PBS, 2 × PBS, 3 × PBS, 4 × PBS, or 5 × PBS buffer solution to remove part of the cross-linked network structure.

13. The composite film of claim 1, wherein The surface of the composite film is impregnated with at least one of fibronectin, collagen, laminin, gelatin, entactin, proteoglycan, osteopontin, tenascin, nidogen, and polylysine.

14. The composite film of claim 1, wherein The surface of the composite film is impregnated with one or more of collagen, fibronectin, and laminin.

15. The composite film of claim 1, wherein The surface of the composite film is impregnated with a basement membrane matrix.

16. The composite film of claim 1, wherein The surface of the composite film is impregnated with a basement membrane proteoglycan.

17. The composite film of claim 1, wherein The thickness of the composite film is 5-100 μm.

18. A method of making a composite film, characterized by, The method comprises the following steps: 1) providing a polymer backbone layer; 2) soaking the polymer backbone layer in a pre-polymerization solution, wherein the pre-polymerization solution comprises acrylamide monomers and alginate, wherein: the pre-polymerization solution comprises 0.5 wt%-1% wt% alginate, and the mass ratio of the alginate to the acrylamide monomers is (1:3)-(1:12); or, the pre-polymerization solution comprises 0.5 wt%-2.0 wt% alginate, and the mass ratio of the alginate to the acrylamide monomers is (1:3)-(1:6); or, the pre-polymerization solution comprises 0.5 wt%-4.0 wt% alginate, and the mass ratio of the alginate to the acrylamide monomers is 1:3; and 3) adding a cross-linking agent to obtain a composite film with alginate-polyacrylamide gel layers arranged on both sides of the polymer backbone layer, wherein the polymer backbone layer is prepared by the following steps: i) freeze coagulating a polyvinyl alcohol-gelatin solution, wherein the polyvinyl alcohol-gelatin solution comprises 5-20 wt% polyvinyl alcohol, wherein the freeze coagulation is a step of repeated freeze-thawing and directional freezing; ii) after soaking the freeze coagulated polyvinyl alcohol in a 1.0-2.0 mol / L citrate solution for 5-7 days, removing it, and washing to remove excess citrate; and iii) optionally, repeating steps i) and ii).

19. The method of claim 18, wherein, Step 3) comprises: adding a first cross-linking agent to promote the acrylamide monomers to form a polyacrylamide network structure to obtain a first composite cross-linking product; and soaking the first composite cross-linking product in a solution containing a second cross-linking agent to promote cross-linking of the alginate to obtain a composite film.

20. The method of claim 19, wherein: the alginate is sodium alginate; and / or the acrylamide monomers are selected from acrylamide; and / or the first cross-linking agent is selected from one or more of N,N'-methylenebisacrylamide, N,N'-diallyltartardiamide, divinylbenzene, polyethylene glycol diacrylate, and polyethylene glycol dimethacrylate.

21. The method of claim 19, wherein, The mass ratio of the acrylamide monomers to the first cross-linking agent is (1:0.0001)-(1:0.1).

22. The method of claim 18, wherein, The pre-polymerization solution further comprises an initiator.

23. The method of claim 22, wherein, The initiator is selected from one or more of peroxoglutaric acid, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ammonium persulfate, potassium persulfate, and sodium persulfate.

24. The method of claim 18, wherein, The pre-polymerization solution further comprises a catalyst.

25. The method of claim 24, wherein, The catalyst is tetramethylethylenediamine.

26. The method of claim 24, wherein, The mass ratio of the catalyst to the acrylamide monomer is (0.002-0.025):

1.

27. The method of claim 19, wherein, The second crosslinking agent is a solution containing a multi-valence cation.

28. The method of claim 27, wherein, The multi-valence cation is a divalent or trivalent cation.

29. The method of claim 27, wherein, The polyvalent cations are selected from one or more of Fe 3+ , Mg 2+ , Al 3+ , Ca 2+ , Sr 2+ , Ba 2+ .

30. The method of claim 18, wherein, The method further comprises a step of rinsing the composite film with a buffer solution to reduce the thickness of the composite film.

31. The method of claim 30, wherein, The composite film is rinsed with 1×PBS, 2×PBS, 3×PBS, 4×PBS, 5×PBS buffer solution to remove part of the crosslinked network structure.

32. The method of claim 18, wherein, The method further comprises a step of impregnating the surface of the composite film.

33. The method of claim 18, wherein, The method further comprises a step of impregnating the surface of the composite film with at least one of fibronectin, collagen, laminin, gelatin, entactin, proteoglycan, osteopontin, tenascin, nephrin and polylysine.

34. The method of claim 18, wherein, The method further comprises a step of impregnating the surface of the composite film with one or more of collagen, fibronectin and laminin.

35. The method of claim 18, wherein, The method further comprises a step of impregnating the surface of the composite film with a basement membrane matrix.

36. The method of claim 18, wherein, The method further comprises a step of impregnating the surface of the composite film with a basement membrane proteoglycan.

37. An organ-on-a-chip, comprising: The organ chip comprises the composite film of any one of claims 1-17.

38. Organ-chip according to claim 37, characterized in that The organ chip comprises: an inlet / outlet layer (1) provided with a first channel inlet / outlet port (7) and a second channel inlet / outlet port (8); a first cell culture layer (2) provided with an anti-overflowing rubber ring (9), a first cell culture cavity and a connecting flow channel (10); a composite film layer (3); a second cell culture layer (4) provided with a second cell culture cavity and a connecting flow channel (11); an actuating membrane (5); and a chamber layer (6) provided with an air suction port (12) and an air chamber (13).

39. Organ-chip according to claim 38, characterized in that The actuating membrane (5) is selected from one or more of polydimethylsiloxane, styrene thermoplastic elastomer, olefin thermoplastic elastomer and acrylic thermoplastic elastomer.

40. The organ-chip of claim 38, wherein, The first cell culture layer (2) is inoculated with lung vascular endothelial cells, and the second cell culture layer (4) is inoculated with alveolar epithelial cells; or the second cell culture layer (4) is inoculated with lung vascular endothelial cells, and the first cell culture layer (2) is inoculated with alveolar epithelial cells.

41. The organ-chip of claim 38, wherein, The air suction port (12) is connected to an air suction pump to drive the deformation of the cell membranes in the first cell culture layer (2) and the second cell culture layer (4).

42. Use of the composite film of any one of claims 1-17 or the organ chip of any one of claims 37-41 in simulating a lung gas-blood barrier structure.

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