Membrane, preparation method and organ chip thereof

Abstract

The invention discloses a membrane, a preparation method and an organ chip thereof, and the membrane comprises a substrate layer (10) which is provided with a pore structure penetrating through the surfaces of the two sides; the culture layers (11 and 11 ') respectively cover the two side surfaces of the substrate layer (10) and are used for cell culture, and the hardness of the substrate layer (10) is greater than that of the culture layers (11 and 11'). According to the membrane provided by the invention, due to the arrangement of the culture layer, the hydrophilicity of the membrane is improved, cell attachment is facilitated, the relatively hard substrate layer can provide stronger structural support and is crucial for maintaining the integrity and stability of the membrane, and the repeatability of an experiment is improved; meanwhile, nutrient substances, oxygen, carbon dioxide, signal molecules or other substances are allowed to penetrate through the pore structure of the substrate layer, cells of different types or states can be cultured on the two sides of the membrane at the same time, a complex tissue model is established, and a similar in-vivo growth environment is provided for the cells.

Description

Technical Field

[0001] The present application relates to the field of organ chips, and specifically to a membrane and a preparation method and an organ chip thereof. Background Art

[0002] Organ-on-a-chip technology represents cutting-edge innovation in the field of biomedical engineering. Through microfluidic platforms and precision engineering design, it can simulate the physiological functions and intercellular microenvironment of human organs, aiming to provide a more accurate, physiologically relevant human organ model.

[0003] In organ chip technology, basement membrane plays a vital role. Basement membrane is a special form of extracellular matrix, which is located between epithelial cells and connective tissue. It not only provides physical support, but also regulates cell behavior, such as cell adhesion, migration and differentiation. At present, in organ chips, the commonly used basement membranes include organic materials and biological membranes. Organic material membranes have high mechanical strength and are prone to plastic deformation during mechanical loading. At the same time, their surface properties are quite different from human tissues, which is not conducive to cell attachment and growth. Natural hydrogel materials are similar to human tissues in terms of mechanical strength and biocompatibility, but their softness and natural swelling and shrinkage properties make them difficult to be accurately processed and utilized.

[0004] Therefore, how to provide a membrane with high mechanical strength and good biocompatibility is a technical problem that needs to be solved in the field of organ chips. Summary of the invention

[0005] In view of this, the present application proposes a membrane, a preparation method and an organ chip thereof.

[0006] According to the first aspect of the present application, a membrane is proposed, which includes: a base layer having a pore structure penetrating both side surfaces; and a culture layer, which is respectively covered on both side surfaces of the base layer and is used for cell culture, wherein: the hardness of the base layer is greater than the hardness of the culture layer.

[0007] Specifically, the material of the base layer is at least one of polycarbonate, polyethersulfone, polydimethylsiloxane, polyethylene terephthalate, polylactic acid or polycaprolactone.

[0008] Specifically, the culture layer is hydrophilic, and is a polymer and / or a biological layer, preferably a biological layer.

[0009] Specifically, the biological layer is at least one of extracellular matrix protein, polypeptide, gelatin or hydrophilic protein. Preferably, the biological layer is extracellular matrix protein.

[0010] Specifically, the extracellular matrix protein includes collagen and / or laminin.

[0011] Specifically, the membrane further comprises a support layer, and the support layer is arranged between the culture layer and the base layer.

[0012] Specifically, both the interior and the surface of the support layer have a porous structure.

[0013] Specifically, the material of the support layer includes a biological material support layer and / or a non-biological material support layer; preferably, the material of the support layer is a biological material support layer.

[0014] Specifically, the material of the biomaterial support layer is at least one of hyaluronic acid, gelatin, GelMa, CoMa, collagen or laminin fibronectin; preferably, the material of the support layer is at least one of GelMa, CoMa, collagen and / or laminin fibronectin.

[0015] Specifically, the hardness of the base layer ranges from 10 MPa to 200 MPa; and / or the hardness of the culture layer ranges from 10 Kpa to 100 Kpa; and / or the hardness of the support layer ranges from 0.1 MPa to 5 MPa.

[0016] Specifically, the thickness of the substrate layer is in the range of 5 μm-10 μm; and / or the thickness of the culture layer is in the range of 100 nm-500 nm; and / or the thickness of the support layer is in the range of 20 μm-50 μm.

[0017] According to a second aspect of the present application, a method for preparing a membrane is proposed, the method comprising: covering the culture layer on both side surfaces of the substrate layer.

[0018] Specifically, the preparation method includes: covering the two side surfaces of the base layer with a support layer, with the first surface of the support layer in contact with the base layer, and then covering the culture layer on the second surface of the support layer, or covering the culture layer on the second surface of the support layer, and covering the first surface of the support layer on the two side surfaces of the base layer.

[0019] Specifically, the support layer is formed by preparing a mixed solution of GelMa, CoMa, collagen and / or laminin, and performing electrostatic spinning and solidification.

[0020] Specifically, the volume concentration of GelMa and CoMa in the mixed solution is 5%-12%, and the final concentration of collagen and laminin in the mixed solution is 1 mg / ml-5 mg / ml.

[0021] Specifically, the electrospinning conditions are positive and negative pressure of 10KV-20KV, spinning distance of 10cm-15cm, left and right and up and down moving range of 30cm, extrusion speed of 0.01ml / min-ml / min, needle rotation range of 1cm-3cm, and air humidity of 70%-90%.

[0022] Specifically, the curing includes one of photocuring, chemical curing, thermal curing, enzymatic curing and pH curing, preferably photocuring.

[0023] Specifically, the light curing is ultraviolet light curing, the exposure intensity of the ultraviolet light curing is 30μW / cm2-50μW / cm2, the exposure time is 10s-30s, and the exposure distance is 0.8cm-1.2cm.

[0024] Specifically, the mixed solution is cured by ultraviolet light under the action of a photoinitiator, and the ratio of the photoinitiator to the mixed solution is 7.5-8.5:0.5-1.5.

[0025] Specifically, the culture layer is 1 mg / ml-5 mg / ml collagen and / or laminin.

[0026] According to the third aspect of the present application, an organ chip is proposed, which includes a bottom layer, a middle layer and a top layer, wherein the bottom layer and the top layer have at least one fluid channel corresponding to each other up and down; the top layer has at least two inlets and two outlets; the middle layer includes a cell culture area and the above-mentioned membrane, and the membrane is horizontally arranged in the middle of the cell culture area to separate the cell culture area into an upper culture area and a lower culture area.

[0027] Specifically, the cells cultured in the upper culture area of ​​the organ chip are intestinal epithelial cells, and the cells cultured in the lower culture area are stromal cells and / or vascular endothelial cells, or

[0028] The cells cultured in the upper culture area of ​​the organ chip are alveolar epithelial cells, and the cells cultured in the lower culture area are endothelial cells, or

[0029] The cells cultured in the upper culture area of ​​the organ chip are glomerular endothelial cells and / or podocytes, and the cells cultured in the lower culture area are renal tubular epithelial cells, or

[0030] The cells cultured in the upper culture area of ​​the organ chip are neurons and / or glial cells, and the cells cultured in the lower culture area are endothelial cells, pericytes and / or astrocytes, or

[0031] The cells cultured in the upper culture area of ​​the organ chip are gastric epithelial cells, and the cells cultured in the lower culture area are mesenchymal cells and / or vascular endothelial cells, or

[0032] The cells cultured in the upper culture area of ​​the organ chip are cardiomyocytes, and the cells cultured in the lower culture area are endocardial cells, endothelial cells and / or interstitial cells, or

[0033] The cells cultured in the upper culture area of ​​the organ chip are hepatocytes, and the cells cultured in the lower culture area are endothelial cells, hepatic stellate cells and / or fibroblasts, or

[0034] The cells cultured in the upper culture area of ​​the organ chip are bile duct epithelial cells, and the cells cultured in the lower culture area are vascular endothelial cells and / or interstitial cells, or

[0035] The cells cultured in the upper culture area of ​​the organ chip are epidermal cells, and the cells cultured in the lower culture area are fibroblasts, endothelial cells and / or immune cells, or

[0036] The cells cultured in the upper culture area of ​​the organ chip are testicular supporting cells and / or spermatogonia, and the cells cultured in the lower culture area are endothelial cells.

[0037] The membrane proposed in the present application has a culture layer that increases the hydrophilicity of the membrane and facilitates cell attachment. The harder base layer can provide stronger structural support, which is essential for maintaining the integrity and stability of the membrane and increases the repeatability of the experiment. At the same time, the pore structure of the base layer allows nutrients, oxygen, carbon dioxide, signaling molecules or other substances to pass through, and cells of different types or states can be cultured on both sides of the membrane at the same time, establishing a complex tissue model and providing a growth environment similar to that in vivo for the cells.

[0038] Other features and advantages of the present application will be described in detail in the subsequent detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present application, and the schematic implementation modes and descriptions of the present application are used to explain the present application. In the accompanying drawings:

[0040] Figure 1 is a schematic diagram of a membrane according to the present application.

[0041] Figure 2 Schematic diagram of an organ chip according to the present application comprising the membrane of the present application.

[0042] Figure 3 The diagram shows the cell status of the kidney organ chip cultured using the membranes prepared in Comparative Example 1 and Examples 1-3 of the present application.

[0043] Figure 4 The diagram shows the status of HK-2 cells cultured on the membranes prepared according to Comparative Examples 2-3 and Example 4 of the present application.

[0044] Figure 5 For the evaluation of the barrier function of the membrane according to this application.

[0045] Figure 6 For the evaluation of the reabsorption function of the membrane according to this application. DETAILED DESCRIPTION

[0046] The technical solution of the present application will be described in detail below with reference to the accompanying drawings and in combination with the implementation modes.

[0047] In vivo, the basement membrane can provide structural support for cells, maintain the structural integrity of tissues, and constitute a selective barrier and signal transmission function. In organ chips, the basement membrane plays a vital role in the successful establishment of organ chips and in restoring the growth conditions of organs or tissues in vivo. Therefore, the key to successfully constructing organs or tissues in vitro that are highly similar to those in vivo is to establish a membrane that is highly similar to the basement membrane in vivo.

[0048] According to the first aspect of the present application, a membrane is proposed, which includes: a base layer 10, the base layer 10 has a pore structure penetrating the two side surfaces; and culture layers 11, 11', the culture layers 11, 11' are respectively covered on the two side surfaces of the base layer 10, and are used for cell culture, wherein: the hardness of the base layer 10 is greater than the hardness of the culture layers 11, 11'.

[0049] The pore diameter of the base layer 10 is 200nm-4μm. The pore diameter of the base layer 10 can be adjusted according to the specific application. For example, a larger pore size is conducive to faster material exchange, and a smaller pore is suitable for experiments that require fine control. The pores of the base layer 10 can be evenly distributed. Evenly distributed pores are conducive to uniform material flow and cell distribution. The pores of the base layer 10 can also be a specific arrangement pattern to simulate the microenvironment of a specific tissue. The pores of the base layer 10 can be of appropriate shapes, such as round, square, mesh, etc.

[0050] The membrane structure in this application cleverly achieves the optimization of mechanical stability and cell culture environment by combining a base layer with higher hardness and a softer culture layer. The higher hardness of the base layer provides reliable support to prevent the membrane from deforming or breaking during use. At the same time, its through-pore structure allows the substances on both sides of the culture layer to exchange freely, thereby simulating the substance transfer in the body. The softer culture layer covering both sides of the base layer provides a soft environment close to the extracellular matrix in the body, promotes the healthy growth and functional expression of cells, effectively separates the structural support and cell culture functions, and improves the stability of cell culture and experimental repeatability.

[0051] In order to provide sufficient mechanical support for the membrane, specifically, the material of the base layer 10 can be at least one of polycarbonate, polyethersulfone, polydimethylsiloxane, polyethylene terephthalate, polylactic acid or polycaprolactone, preferably polylactic acid. The base layer made of polylactic acid has high biocompatibility and can also adjust the shape and function of the base layer to provide stable support. Secondly, polylactic acid has high processability and high transparency.

[0052] In order to promote the attachment of cells to the membrane surface and improve the biocompatibility of the membrane, specifically, the culture layer 11, 11' is hydrophilic, and the culture layer 11, 11' is a polymer and / or a biological layer, preferably a biological layer. The hydrophilicity of the culture layer can enhance the adhesion ability of cells on its surface, and the hydrophilic surface can maintain a good moist environment and optimize cell culture conditions. The hydrophilicity is conducive to material exchange and improves the efficiency of cell growth and metabolism; the use of a biological layer can better simulate the extracellular matrix environment in the body, improve biocompatibility, enhance cell-matrix interaction, and optimize cell behavior, such as proliferation, differentiation and migration.

[0053] In order to reproduce the structure and function of in vivo tissue as much as possible, specifically, the biological layer can be at least one of extracellular matrix proteins, polypeptides, gelatin or hydrophilic proteins. Preferably, the biological layer is an extracellular matrix protein. Extracellular matrix proteins have significant advantages as culture layers, including enhancing cell adhesion, expansion and proliferation, simulating the microenvironment of in vivo tissues, and thus providing more realistic growth conditions. Specifically, the extracellular matrix protein can include collagen and / or laminin, preferably collagen, and the above collagen can be at least one of type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, and type VII collagen, preferably type I collagen, and type I collagen can provide an extracellular matrix environment similar to that in vivo, support cell adhesion, differentiation and functionalization, and help simulate cell behavior closer to the physiological state. In addition, it can also enhance the mechanical stability of the basal layer of the organ chip, especially maintain the integrity of the cell layer in a dynamic fluid environment, and can be compatible with other matrix proteins, further optimize the cell microenvironment, thereby improving the physiological relevance and predictability of the experiment.

[0054] In order to optimize the cell growth environment and ensure the stability of the experiment, specifically, the membrane may also include a support layer 12, 12', which may be disposed between the culture layer 11, 11' and the base layer 10. The support layer 12, 12' has a porous structure both inside and on the surface. The hardness of the support layer 12, 12' is lower than that of the base layer 10. In the organ chip, the support layer 12, 12' having a lower hardness than the culture layer is disposed between the culture layer and the base layer, and has a porous structure both inside and on the surface, which can effectively buffer the mechanical stress from the harder base layer and prevent the stress from being directly transmitted to the cells in the culture layer, thereby avoiding changes in cell morphology and function. The elastic environment provided by the soft support layer helps to simulate the natural flexibility of tissues in the body, promote cell growth, expansion and differentiation, while reducing the negative effects of mechanical stimulation and maintaining the stability of cells during the experiment. In addition, the low hardness and porous structure of the support layer can enhance the effect of three-dimensional cell culture, support the formation of more complex three-dimensional tissue structures, avoid direct contact between the culture layer and the basal layer, ensure the integrity and functional performance of the cell layer, and thus improve the physiological relevance and accuracy of the experiment.

[0055] In order to simulate the in vivo environment more realistically, specifically, the material of the support layer 12, 12' may include a biological material support layer and / or a non-biological material support layer; preferably, the material of the support layer 12, 12' is a biological material support layer. The biological material support layer has high biocompatibility, can provide an environment closer to the natural extracellular matrix, support cell adhesion, expansion and growth, help maintain the normal morphology and functional performance of cells, and can better simulate the microstructure and mechanical properties of in vivo tissues.

[0056] In order to better simulate the cell matrix in the body, specifically, the material of the biomaterial support layer 12, 12' can be at least one of hyaluronic acid, gelatin, GelMa, CoMa, collagen or laminin; preferably, the material of the support layer 12, 12' is at least one of GelMa, CoMa, collagen and / or laminin. In the present application, the support layer uses GelMa, CoMa, collagen and laminin as the support layer; GelMa is a modified gelatin, a synthetic hydrogel generated by reacting gelatin with methacrylic anhydride. It combines the natural biocompatibility of gelatin and the photo-crosslinking ability of methacryloyl, so that it can form a stable three-dimensional network structure under light. CoMa is a methacrylamide type III collagen, which gives collagen the ability to photo-crosslink, so that it can form a stable three-dimensional network structure under ultraviolet or visible light, thereby enhancing its mechanical properties and stability. Methacrylamide type III collagen not only retains the biocompatibility and function of natural collagen, but also can optimize its physical properties by adjusting the degree of crosslinking. The above-mentioned collagen is type I collagen. In this application, GelMa, CoMa, collagen and laminin fibronectin are used as support layers to provide multi-level support for the cell microenvironment. The photocrosslinking ability of GelMa allows precise control of mechanical properties and stability; CoMa retains the biological properties of natural collagen while enhancing its stability; type I collagen provides excellent biocompatibility and support; laminin fibronectin can promote cell adhesion and tissue formation. The combined use of these materials can achieve precise control of the support layer, improve the physiological relevance of the experiment, support cell growth and functional performance, and meet the needs of various biomedical applications.

[0057] In order to simulate the hardness of the extracellular matrix in vivo, specifically, the hardness of the base layer 10 can range from 10 MPa to 200 MPa; and / or the hardness of the culture layers 11, 11' can range from 10 Kpa to 100 Kpa; and / or the hardness of the support layers 12, 12' can range from 0.1 MPa to 5 MPa. This can provide support and barrier functions for cells or tissues cultured on the membrane, simulate the rigidity of the in vivo environment, and facilitate the attachment of cells to the membrane.

[0058] In order to simulate the thickness of the extracellular matrix in vivo, specifically, the thickness of the base layer 10 is in the range of 5 μm-10 μm; and / or the thickness of the culture layer 11, 11' is in the range of 100 nm-500 nm; and / or the thickness of the support layer 12, 12' is in the range of 20 μm-50 μm. This fully simulates the extracellular matrix environment in vivo, is easy to prepare, and restores the growth environment of cells in vivo to the greatest extent.

[0059] According to a second aspect of the present application, a method for preparing a membrane is provided. The method comprises: covering culture layers 11 , 11 ′ on both side surfaces of a substrate layer 10 .

[0060] Specifically, the preparation method includes: covering the two side surfaces of the base layer 10 with support layers 12, 12', the first surface of the support layers 12, 12' is in contact with the base layer 10, and then covering the culture layers 11, 11' on the second surface of the support layers 12, 12', or covering the culture layers 11, 11' on the second surface of the support layers 12, 12', and covering the first surface of the support layers 12, 12' on the two side surfaces of the base layer 10.

[0061] In order to form a three-dimensional network structure with certain mechanical properties, specifically, the support layer 12, 12' can be prepared by preparing GelMa, CoMa, collagen and / or laminin into a mixed solution, and then formed by electrospinning and curing. By first electrospinning and then light curing, a uniform fiber network can be formed in the support layer, improving its mechanical properties and ensuring the uniformity and control of the curing process.

[0062] In order to obtain a matrix structure similar to that in vivo, specifically, the volume concentration of GelMa and CoMa in the mixed solution is 5%-12%, and the final concentration of collagen and laminin in the mixed solution is 1 mg / ml-5 mg / ml.

[0063] In order to prepare an extracellular matrix similar to that in vivo, specifically, the electrospinning conditions can be 10KV-20KV positive and negative pressure, 10cm-15cm spinning distance, 30cm left and right and up and down movement range, 0.01ml / min-0.4ml / min extrusion speed, 1cm-3cm needle rotation range, 70%-90% air humidity. Under these conditions, a uniform fiber network can be effectively formed, the fiber size and shape can be controlled, the fiber arrangement and distribution can be optimized, and the structural stability and biocompatibility of the support layer can be improved.

[0064] In order to obtain a more stable support layer, specifically, the curing may include one of photocuring, chemical curing, thermal curing, enzymatic curing and pH curing, preferably photocuring. Specifically, the photocuring is ultraviolet light curing, and the exposure intensity of the ultraviolet light curing is 30μW / cm2-50μW / cm2, the exposure time is 10s-30s, and the exposure distance is 0.8cm-1.2cm. Photocuring allows precise spatial and temporal control of the curing process to achieve high-resolution production of complex three-dimensional structures. Photocuring usually reacts quickly and can complete curing within a few seconds to a few minutes without the need for additional chemical curing agents or solvents, reducing chemical contamination and cytotoxicity. By adjusting the lighting conditions, the physical properties of the hydrogel can be flexibly adjusted to improve biocompatibility, while simplifying the operation, which is suitable for efficient and precise structure production in biomedical applications.

[0065] Specifically, the mixed solution is cured by ultraviolet light under the action of a photoinitiator, and the ratio of the photoinitiator to the mixed solution is 7.5-8.5:0.5-1.5.

[0066] Specifically, the culture layers 11, 11' are 1 mg / ml-5 mg / ml collagen and / or laminin.

[0067] According to the third aspect of the present application, an organ chip is proposed, which includes a bottom layer, a middle layer and a top layer, wherein the bottom layer and the top layer have at least one fluid channel corresponding to each other up and down; the top layer has at least two inlets and two outlets; the middle layer includes a cell culture area and the above-mentioned membrane, and the membrane is horizontally arranged in the middle of the cell culture area to separate the cell culture area into an upper culture area and a lower culture area.

[0068] The above-mentioned organ chip can be used to construct an air-blood barrier, a blood barrier, a blood-brain barrier, a blood-testis barrier, a skin mucosal barrier, etc. Specifically, the cells cultured in the upper culture area of ​​the organ chip may be intestinal epithelial cells, and the cells cultured in the lower culture area may be stromal cells and / or vascular endothelial cells, which are used to simulate the intestinal blood barrier, or the cells cultured in the upper culture area of ​​the organ chip may be alveolar epithelial cells, and the cells cultured in the lower culture area may be endothelial cells, which are used to simulate the air-blood barrier, or the cells cultured in the upper culture area of ​​the organ chip are glomerular endothelial cells and / or podocytes, and the cells cultured in the lower culture area are renal tubular epithelial cells, which are used to simulate the renal filtration barrier of the glomerulus, or the cells cultured in the upper culture area of ​​the organ chip are neuronal cells and / or glial cells, and the cells cultured in the lower culture area are vascular endothelial cells, pericytes and / or astrocytes, which are used to simulate the blood-brain barrier, or the cells cultured in the upper culture area of ​​the organ chip are gastric epithelial cells, and the cells cultured in the lower culture area are interstitial cells. Cells and / or vascular endothelial cells are used to simulate the blood barrier of the stomach, or the cells cultured in the upper culture area of ​​the organ chip are cardiomyocytes, and the cells cultured in the lower culture area are endocardial cells, endothelial cells and / or interstitial cells, which are used to simulate the blood barrier of the heart, or the cells cultured in the upper culture area of ​​the organ chip are hepatocytes, and the cells cultured in the lower culture area are endothelial cells, hepatic stellate cells and / or fibroblasts, which are used to simulate the blood barrier of the liver, or the cells cultured in the upper culture area of ​​the organ chip are bile duct epithelial cells, and the cells cultured in the lower culture area are vascular endothelial cells and / or interstitial cells, which are used to simulate the blood barrier of the bile duct, or the cells cultured in the upper culture area of ​​the organ chip are epidermal cells, and the cells cultured in the lower culture area are fibroblasts, endothelial cells and / or immune cells, which are used to simulate the skin mucosal barrier, or the cells cultured in the upper culture area of ​​the organ chip are testicular supporting cells and / or spermatogonia, and the cells cultured in the lower culture area are endothelial cells, which are used to simulate the blood-testis barrier.

[0069] The membrane, preparation method and organ chip of the present application are described below with reference to examples.

[0070] 1. Example of the method for preparing the membrane

[0071] Example 1

[0072] A1. Prepare GelMa and CoMa hydrogel solutions with a volume concentration of 5%, and add collagen and laminin solutions to the hydrogel solutions to make the final concentration of 1 mg / ml.

[0073] A2. Use a rheometer (Kinexus Lab+, NETZSCH Instrument Manufacturing Co., Ltd., Germany) to measure the viscosity. When the viscosity range is 800mPa.s-2000mPa.s, put it into a 5 ml syringe for use.

[0074] A3. The hydrogel solution was electrospun. The positive electrode of the electrospinning equipment (ET-2535M, Beijing Yongkang Leye Technology Development Co., Ltd.) was connected to the needle tip of the syringe, and the negative electrode was connected to the mesh receiver. The electrospinning parameters were positive and negative pressure 10KV, spinning distance 10cm, left and right and up and down moving range 30cm, extrusion speed 0.01mL / min, needle moving range 1cm, and air humidity 70%.

[0075] A4. The spinning time is controlled according to the required stacking thickness, which is generally more than 12 hours (thickness > 10μm).

[0076] A5. After spinning is completed, the mesh receiver with the hydrogel film is exposed to ultraviolet light at an exposure intensity of 30 μW / cm 2 , exposure time is 10s, and exposure distance is 0.8cm.

[0077] A6. After UV curing is completed, remove the hydrogel film from the receiver and cut it into a specific shape.

[0078] A7. Adhere the hydrogel film to the mesh structure, then transfer both to a culture dish and incubate a layer of type I collagen at a concentration of 1 mg / ml.

[0079] A8. After incubation, encapsulate it into the organ chip for later use.

[0080] Example 2

[0081] The film preparation method of Example 1 is adopted, wherein:

[0082] A1. Prepare GelMa and CoMa hydrogel solutions with a volume concentration of 12%, and add collagen and laminin solutions to the hydrogel solutions to make the final concentration of 5 mg / ml.

[0083] A3. The electrospinning parameters are positive and negative pressure 20KV, spinning distance 15cm, left and right and up and down moving range 30cm, extrusion speed 0.4mL / min, needle moving range 3cm, and air humidity 90%.

[0084] A5. After spinning is completed, the mesh receiver with the hydrogel film is exposed to ultraviolet light at an exposure intensity of 50 μW / cm 2 , exposure time is 30s, and exposure distance is 1.2cm.

[0085] A7. Adhere the hydrogel film to the mesh structure, then transfer both to a culture dish and incubate a layer of type I collagen at a concentration of 5 mg / ml.

[0086] Example 3

[0087] The film preparation method of Example 1 is adopted, wherein:

[0088] A1. Prepare GelMa and CoMa hydrogel solutions with a volume concentration of 8%, and add collagen and laminin solution to the hydrogel solution to make the final concentration of 3 mg / ml.

[0089] A3. The electrospinning parameters are positive and negative pressure 15KV, spinning distance 12cm, left and right and up and down moving range 30cm, extrusion speed 0.2mL / min, needle moving range 2cm, and air humidity 80%.

[0090] A5. After spinning is completed, the mesh receiver with the hydrogel film is exposed to UV light at an exposure intensity of 40 μW / cm 2 , exposure time is 20s, and exposure distance is 1cm.

[0091] A7. Adhere the hydrogel film to the mesh structure, then transfer both to a culture dish and incubate a layer of type I collagen at a concentration of 3 mg / ml.

[0092] Example 4

[0093] The film preparation method of Example 1 is adopted, wherein:

[0094] A1. Prepare GelMa and CoMa hydrogel solutions with a volume concentration of 10%, and add collagen and laminin solution to the hydrogel solution to make the final concentration of 2 mg / ml.

[0095] A3. The electrospinning parameters are positive and negative pressure 10KV, spinning distance 10cm, left and right and up and down moving range 30cm, extrusion speed 0.4mL / min, needle moving range 1cm, and air humidity 90%.

[0096] A5. After spinning is completed, the mesh receiver with the hydrogel film is exposed to ultraviolet light at an exposure intensity of 30 μW / cm 2 , exposure time is 15s, and exposure distance is 1cm.

[0097] A7. Adhere the hydrogel film to the mesh structure, then transfer both to a culture dish and incubate a layer of type I collagen at a concentration of 2 mg / ml.

[0098] Comparative Example 1

[0099] The preparation method of Example 1 is adopted, wherein GelMa, CoMa, collagen and laminin are not included.

[0100] Comparative Example 2

[0101] The preparation method of Example 1 is adopted, wherein the support layer does not contain CoMa.

[0102] Comparative Example 3

[0103] The preparation method of Example 1 was adopted, wherein the incubation with type I collagen was not performed.

[0104] 2. Experimental Design

[0105] In this application, the construction of a kidney chip is taken as an example.

[0106] The kidney chip was constructed by using the preparation methods of the membranes in the embodiment and the comparative example, and HK-2 cells and HUVEC cells were inoculated in the chip at a density of 1×10 5 cells / cm 2 , the upper cell culture area was cultured with Advanced RPMI 1640 medium (12633012, Thermo Fisher Scientific), and the lower cell culture area was cultured with endothelial cell culture medium (1001, ScienCell Research Laboratories).

[0107] 3. Test results

[0108] 3.1 Physical properties of membranes

[0109] The physical properties of each embodiment and comparative example in the present application are shown in Table 1. The thickness of the membrane prepared in Example 1 is 25.1 μm and the hardness is 10.11 MPa. The thickness of the membrane prepared in Example 2 is 45.3 μm and the hardness is 205.1 MPa. The thickness of the membrane prepared in Example 3 is 78 μm and the hardness is 107.555 MPa. The thickness of the membrane prepared in Example 4 is 40.2 μm and the hardness is 115.1 MPa; the thickness of the membrane prepared in Comparative Example 1 is 5.3 μm and the hardness is 10.01 MPa, the thickness of the membrane prepared in Comparative Example 2 is 15.2 μm and the hardness is 10.05 MPa, and the thickness of the membrane prepared in Example 3 is 23 μm and the hardness is 10.08 MPa. Therefore, the hardness and thickness of the membrane prepared in the embodiment are greater than those in the comparative example. The physical properties of the membrane in the embodiment are better, which can provide support for cells, and are similar to the stiffness and thickness of the extracellular matrix in vivo.

[0110] Table 1 Physical properties of the films

[0111]

[0112] 3.2 Evaluation of cell status

[0113] like Figure 3As shown, the cell states after culturing HK-2 cells and HUVEC cells for 5 days on both sides of the membranes prepared by the preparation methods of Examples 1-3 and Comparative Example 1 of the present application are shown in FIG. Figure 3 It can be seen that compared with Comparative Example 1, the density of the two types of cells in Examples 1-3 is higher, the cells are tightly connected without gaps, the cells are evenly distributed on the membrane, the edges are clear, and the cells are in good condition.

[0114] The state of the membranes prepared by the methods of Comparative Example 2, Comparative Example 3 and Example 4 after culturing HK-2 cells for 5 days is as follows: Figure 4 As shown by Figure 4 It can be seen that the HK-2 cells cultured in the membrane without CoMa added to the support layer in Comparative Example 2 and the membrane without type I collagen incubation in Comparative Example 3 are relatively sparse, and no tight junctions are formed between cells. The HK-2 cells in Example 4 have good vitality, regular cell morphology, tight junctions, and regular cell morphology.

[0115] It can be seen from this that the biocompatibility of the membrane after incubation using the support layer and adding CoMa and type I collagen to the support layer in the present application is high, which is conducive to cell attachment and proliferation.

[0116] 3.3 Barrier function evaluation

[0117] Barrier function was evaluated by measuring the apparent permeability of FITC-labeled dextran (4 kDa and 40 kDa).

[0118] Figure 5 The barrier function of the membranes prepared in Comparative Examples 1-3 and Examples 1-4 in this application is compared. Figure 5 It can be seen that the permeability of the membrane prepared in the example of the present application to small molecules is much greater than the permeability of the membrane prepared in the comparative example, indicating that the permeability of the membrane in the present application is relatively high, and can allow nutrients, oxygen, carbon dioxide, signal molecules and other substances to pass through; the membrane prepared in the example has a low permeability to macromolecules and can effectively prevent the penetration of macromolecules.

[0119] 3.4 Evaluation of reabsorption function

[0120] The reabsorption function was evaluated by measuring the apparent permeability of albumin.

[0121] Figure 6 The reabsorption function of the membranes prepared in Comparative Examples 1-3 and Examples 1-4 in this application is compared. In this application, the reabsorption of albumin is taken as an example. Figure 6 It can be seen that the permeability of the membrane prepared in the example to albumin reabsorption is higher, which is significantly higher than the permeability of the membrane prepared in the comparative example to albumin.

[0122] It can be seen that the membrane proposed in this application is used in organ chips and can fully simulate the functions of organs or tissues in the body.

[0123] The membrane proposed in the present application has a culture layer that increases the hydrophilicity of the membrane and facilitates cell attachment. The harder base layer can provide stronger structural support, which is essential for maintaining the integrity and stability of the membrane and increases the repeatability of the experiment. At the same time, the pore structure of the base layer allows nutrients, oxygen, carbon dioxide, signaling molecules or other substances to pass through, and cells of different types or states can be cultured on both sides of the membrane at the same time, establishing a complex tissue model and providing a growth environment similar to that in vivo for the cells.

[0124] The preferred embodiments of the present application are described in detail above; however, the present application is not limited to the specific details in the above embodiments. Within the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the protection scope of the present application.

[0125] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0126] In addition, the various implementation modes of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A membrane, characterized in that The membrane includes: A base layer (10), the base layer (10) having a pore structure penetrating through both side surfaces; and Culture layers (11, 11'), which are respectively covered on both side surfaces of the base layer (10) and are used for cell culture, wherein: The hardness of the base layer (10) is greater than the hardness of the culture layer (11, 11').

2. The film according to claim 1, characterized in that The material of the base layer (10) is at least one of polycarbonate, polyethersulfone, polydimethylsiloxane, polyethylene terephthalate, polylactic acid or polycaprolactone; The culture layer (11, 11') is hydrophilic and is a polymer and / or a biological layer, preferably a biological layer.

3. The film according to claim 2, characterized in that The biological layer is at least one of extracellular matrix protein, polypeptide, gelatin or hydrophilic protein, Preferably, the biological layer is an extracellular matrix protein; The extracellular matrix proteins include collagen and / or laminin.

4. The membrane according to any one of claims 1 to 3, characterized in that The membrane further comprises a support layer (12, 12'), wherein the support layer (12, 12') is arranged between the culture layer (11, 11') and the base layer (10); The support layer (12, 12') has a porous structure both inside and on the surface.

5. The film according to claim 4, characterized in that The material of the support layer (12, 12') includes a biological material support layer and / or a non-biological material support layer; Preferably, the support layer (12, 12') is made of a biological material support layer; The material of the biomaterial support layer is at least one of hyaluronic acid, gelatin, GelMa, CoMa, collagen or laminin; Preferably, the material of the support layer (12, 12') is at least one of GelMa, CoMa, collagen and / or laminin.

6. The film according to claim 4, characterized in that The hardness of the base layer (10) is in the range of 10 MPa-200 MPa; and / or the hardness of the culture layer (11, 11') is in the range of 10 Kpa-100 Kpa; and / or the hardness of the support layer (12, 12') is in the range of 0.1 MPa-5 MPa; The thickness of the substrate layer (10) is in the range of 5 μm-10 μm; and / or the thickness of the culture layer (11, 11') is in the range of 100 nm-500 nm; and / or the thickness of the support layer (12, 12') is in the range of 20 μm-50 μm.

7. A method for preparing a membrane, characterized in that: The method comprises: covering the culture layers (11, 11') on both side surfaces of the base layer (10).

8. The preparation method according to claim 7, characterized in that: The preparation method comprises: The support layer (12, 12') is covered on both sides of the base layer (10), the first surface of the support layer (12, 12') is in contact with the base layer (10), and the culture layer (11, 11') is covered on the second surface of the support layer (12, 12'), or The culture layer (11, 11') is covered on the second surface of the support layer (12, 12'), and the first surface of the support layer (12, 12') is covered on both side surfaces of the base layer (10).

9. The preparation method according to claim 8, characterized in that: The support layer (12, 12') is formed by preparing a mixed solution of GelMa, CoMa, collagen and / or laminin, and performing electrostatic spinning and solidification; Preferably, the volume concentration of GelMa and CoMa in the mixed solution is 5%-12%. The final concentration of collagen and laminin in the mixed solution is 1 mg / ml-5 mg / ml.

10. The preparation method according to claim 9, characterized in that: The electrospinning conditions are as follows: positive and negative pressure 10KV-20KV, spinning distance 10cm-15cm, left and right and up and down moving range 30cm, extrusion speed 0.01ml / min-0.4ml / min, needle rotation range 1cm-3cm, air humidity 70%-90%; The curing comprises one of light curing, chemical curing, thermal curing, enzymatic curing and pH curing, preferably light curing; Preferably, the light curing is ultraviolet light curing, and the exposure intensity of the ultraviolet light curing is 30 μW / cm 2 -50μW / cm 2 , exposure time is 10s-30s, exposure distance is 0.8cm-1.2cm; The mixed solution is cured by ultraviolet light under the action of a photoinitiator, and the volume ratio of the photoinitiator to the mixed solution is 7.5-8.5:0.5-1.

5.

11. The preparation method according to claim 7 or 8, characterized in that: The culture layer (11, 11') is 1 mg / ml-5 mg / ml of collagen and / or laminin.

12. An organ chip, characterized in that The organ chip comprises a bottom layer, a middle layer and a top layer, wherein the bottom layer and the top layer have at least one fluid channel corresponding to each other; the top layer has at least two inlets and two outlets; the middle layer comprises a cell culture area and a membrane according to any one of claims 1 to 6, wherein the membrane is horizontally arranged in the middle of the cell culture area to separate the cell culture area into an upper culture area and a lower culture area; The cells cultured in the upper culture area of ​​the organ chip are intestinal epithelial cells, and the cells cultured in the lower culture area are stromal cells and / or vascular endothelial cells, or The cells cultured in the upper culture area of ​​the organ chip are alveolar epithelial cells, and the cells cultured in the lower culture area are endothelial cells, or The cells cultured in the upper culture area of ​​the organ chip are glomerular endothelial cells and / or podocytes, and the cells cultured in the lower culture area are renal tubular epithelial cells, or The cells cultured in the upper culture area of ​​the organ chip are neurons and / or glial cells, and the cells cultured in the lower culture area are endothelial cells, pericytes and / or astrocytes, or The cells cultured in the upper culture area of ​​the organ chip are gastric epithelial cells, and the cells cultured in the lower culture area are mesenchymal cells and / or vascular endothelial cells, or The cells cultured in the upper culture area of ​​the organ chip are cardiomyocytes, and the cells cultured in the lower culture area are endocardial cells, endothelial cells and / or interstitial cells, or The cells cultured in the upper culture area of ​​the organ chip are hepatocytes, and the cells cultured in the lower culture area are endothelial cells, hepatic stellate cells and / or fibroblasts, or The cells cultured in the upper culture area of ​​the organ chip are bile duct epithelial cells, and the cells cultured in the lower culture area are vascular endothelial cells and / or interstitial cells, or The cells cultured in the upper culture area of ​​the organ chip are epidermal cells, and the cells cultured in the lower culture area are fibroblasts, endothelial cells and / or immune cells; or The cells cultured in the upper culture area of ​​the organ chip are testicular supporting cells and / or spermatogonia, and the cells cultured in the lower culture area are endothelial cells.

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