A polyurethane ultra-thin hierarchical porous membrane for cell encapsulation and its manufacturing method and application

Abstract

The present invention discloses a polyurethane ultra-thin hierarchical porous membrane that can be used for cell encapsulation and its manufacturing method and application. The method comprises the following steps: (1) polyurethane is dissolved in dimethyl sulfoxide, and then deaerated; (2) an aluminum plate is pre-cooled using a refrigeration cycle machine; (3) the obtained polyurethane solution is added dropwise on a glass plate, and then excess solution is scraped off with a film-laying device; (4) the glass plate is placed on a pre-cooled aluminum plate; (5) after observing that the solution crystallizes, the glass plate is placed in warm water; (6) the film is taken out of the water and dried. The method of the present invention is simple, overcomes the problem that the unidirectional freezing aperture is larger, and the obtained microporous membrane pore diameter is within the range of 500nm-5μm, and the film-making conditions are adjusted to obtain the desired pore diameter. The porous membrane of the present invention has a large number of longitudinal through-holes, and in an ultra-thin thickness (100μm), a hierarchical structure is realized, with a supporting layer with a large aperture, and a barrier layer with a small aperture, which is conducive to controlling the permeation isolation molecular weight.

Description

Technical Field

[0001] The present invention belongs to the technical field of PU film production, and in particular relates to a polyurethane ultra-thin graded porous film that can be used for cell encapsulation, and a manufacturing method and application thereof. Background Art

[0002] With the continuous development of regenerative medicine and cell therapy technologies, mesenchymal stem cells (MSCs) have attracted widespread attention due to their unique biological properties and broad application prospects. MSC cell encapsulation technology, as an important part of the field of cell therapy, is of great significance for improving cell survival and promoting cell proliferation and differentiation. Currently, MSC cell encapsulation mainly adopts technologies such as microencapsulation and microspherization, but in practical applications, there are still problems such as low encapsulation efficiency, loss of cell viability, immunogenicity, and non-retrievability. Therefore, exploring new cell encapsulation technologies and materials is of great significance for improving the efficacy of cell therapy. Macroscopic encapsulation methods, encapsulating cells in thicker polymer layers and larger diameter polymers, can improve the stability of the implant and make it easier to retrieve the capsule when needed, but may hinder the diffusion of oxygen and nutrients into the capsule and the release of waste, thereby impairing cell viability. Therefore, as an important component of cell encapsulation and bioreactors, the performance of membrane materials directly affects the cell living environment and therapeutic efficacy.

[0003] For example, Tae HK et al. encapsulated MSCs in a polylactic-co-glycolic acid copolymer semipermeable membrane and implanted them into the subcutaneous area of ​​the dorsal liver or interlobular space to study the functional enhancement of stem cells or the priming effect of hypoxia induced by macro-encapsulation. Macro-encapsulated MSCs reduced liver inflammation and fibrosis by upregulating hypoxia-induced growth hormone secretion (Tae HK, Kiseok J, Won DJ, et al. Macro-encapsulation of mesenchymal stem cells in acute and chronic liver injury animal models. [J]. Journal of gastroenterology and hepatology, 2021, 36(7): 1997-2007). Wang Kai et al. encapsulated adipose-derived mesenchymal stem cells in a macroencapsulation device and verified the production of MSC exosomes in the pouch, which effectively promoted angiogenesis and reduced device fibrosis (Wang K, Yu L, Jiang L, et al. The paracrine effects of adipose-derived stem cells on neovascularization and biocompatibility of a macroencapsulation device [J]. Acta Biomaterialia, 2015, 1565-76). Suman B et al. used photolithography to prepare different master molds and cast PDMS to obtain cell encapsulation semipermeable membranes with different pore sizes. They observed that membranes with a pore size of 1 μm allowed host macrophages to migrate into the device without losing the transplanted cells, while membranes with a pore size of <0.8 μm prevented them from being infiltrated by immune cells. (Suman, R LV, Devina T, et al. A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells. [J]. Nature biomedical engineering, 2020, 4 (8): 814-826) However, the semipermeable membrane used in the above encapsulation device does not have fine pore size and permeability control on the one hand, and on the other hand, precise control of the pore size has high costs and complicated steps.

[0004] To address the complex production process and tortuous pores and poor permeability of porous membranes for cell encapsulation devices, a simple, additive-free, ultra-thin, hierarchical polyurethane porous membrane with a two-level structure, ordered pore size, and good permeability was developed. The effects of different production conditions on pore size and pore structure were investigated. This provides a simple membrane manufacturing method reference for the production of porous membranes for cell encapsulation. Summary of the Invention

[0005] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a preparation method for a polyurethane ultra-thin hierarchical porous film. The present invention uses thermoplastic polyurethane as raw material, dissolves polyurethane in DMSO, and adopts the operating method of combining unidirectional freezing and non-solvent induced phase separation to obtain a porous membrane with longitudinal through-holes. Compared with electrospinning membrane and PTFE membrane, this polyurethane porous membrane has good biocompatibility, simple production, high permeability, and can achieve thickness within 100 μm and still have the advantages of good mechanical strength, and the porous membrane pore size is controllable, and can achieve holes in the nanometer to micrometer range, thereby improving the feasibility of cell encapsulation technology.

[0006] Another object of the present invention is to provide an ultra-thin hierarchical porous polyurethane film obtained by the above preparation method and its application.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0009] (1) dissolving polyurethane (PU) in dimethyl sulfoxide (DMSO) to obtain a polyurethane solution, and then degassing;

[0010] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate;

[0011] (3) adding the polyurethane solution dropwise onto a glass plate, and scraping off excess solution with a film spreader;

[0012] (4) placing the glass plate on a pre-cooled aluminum plate;

[0013] (5) After the solution crystallizes, place the glass plate in water;

[0014] (6) Take the membrane out of the water and dry it;

[0015] The concentration of the polyurethane solution is 10-30 wt %; the pre-cooling temperature of the refrigeration cycle machine is -50-0° C.; and the film laying thickness of the film laying device is 50-500 μm.

[0016] Preferably, the concentration of the polyurethane in the polyurethane solution in step (1) is 12-18 wt%.

[0017] Preferably, the pre-cooling temperature of the refrigeration cycle machine in step (2) is -5-20°C.

[0018] Preferably, in step (3), the film laying device lays a film with a thickness of 75-250 μm.

[0019] Preferably, the temperature of water in step (5) is 15-25°C, more preferably 20°C.

[0020] Further preferably, the concentration of the polyurethane solution is 14 wt %; the pre-cooling temperature of the refrigeration cycle machine is -20°C; and the film laying thickness of the film laying device is 100 μm.

[0021] Preferably, the stirring speed of the PU solution in step (1) is 300-500 rpm.

[0022] Preferably, the thickness of the glass plate in step (3) is 1 mm.

[0023] Preferably, in step (4), the aluminum plate is placed in a semi-enclosed space.

[0024] Preferably, the degassing is carried out in an oven at 40°C for 24 hours.

[0025] An ultra-thin hierarchical polyurethane porous membrane prepared by any of the above manufacturing methods.

[0026] Application of the above-mentioned polyurethane ultra-thin graded porous membrane in cell encapsulation.

[0027] Preferably, the polyurethane ultra-thin graded porous membrane is used in a cell encapsulation device.

[0028] The polyurethane porous film of the present invention has a pore size that gradually decreases with increasing polyurethane concentration within the range of 12-18wt%, and increases with decreasing concentration; and has a pore size that gradually decreases with increasing thickness within the range of 50-250μm, and increases with decreasing thickness.

[0029] Compared with the prior art, the present invention has the following technical advantages and beneficial effects:

[0030] (1) The present invention uses PU and DMSO as raw materials and adopts a unidirectional freezing method to prepare a polyurethane ultra-thin graded porous membrane without using other additives.

[0031] (2) The present invention overcomes the problem of large aperture of one-way freezing.

[0032] (3) The pores obtained by the present invention have a large number of longitudinal through holes, which improves the penetration efficiency.

[0033] (4) The pore size of the microporous membrane produced by the present invention can be in the range of 500 nm-5 μm, and the desired pore size can be obtained by adjusting the membrane production conditions.

[0034] (5) The porous membrane prepared in the present invention has a hierarchical structure, comprising a support layer with a large pore size and an isolation layer with a small pore size, which is beneficial for controlling the permeation molecular weight cut-off. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope image of the microporous membrane prepared in Comparative Example 4, where surface I is close to the glass surface, surface II is away from the glass surface, and CS is a cross-sectional view of the membrane.

[0036] Figure 2 These are scanning electron microscope images of the microporous membranes prepared in Example 7 and Comparative Example 5, where surface I is close to the glass surface and surface II is away from the glass surface.

[0037] Figure 3 These are scanning electron microscope images of the microporous membranes prepared in Example 7 and Comparative Example 5, where CS is a cross-sectional view, I-CS is the side close to the glass plate, and II-CS is the side away from the glass plate.

[0038] Figure 4 These are scanning electron microscope images of the microporous membranes prepared in Examples 1, 2, 3, and 4, where surface I is the surface close to the glass, surface II is the surface away from the glass, and CS is a cross-sectional view of the membrane.

[0039] Figure 5 The pore size images of the microporous membranes prepared in Examples 1, 2, 3, and 4 were measured using Image J based on scanning electron microscopy images.

[0040] Figure 6 The stress-strain curves and tensile strength diagrams of the microporous membranes prepared in Examples 1, 2, 3, and 4 are shown.

[0041] Figure 7 These are scanning electron microscope images of the microporous membranes prepared in Examples 2, 5, 6, and 7, where surface I is the surface close to the glass, surface II is the surface away from the glass, and CS is a cross-sectional view of the membrane.

[0042] Figure 8 The pore size images of the microporous membranes prepared in Examples 2, 5, 6, and 7 were measured using Image J based on scanning electron microscopy images.

[0043] Figure 9 These are the stress-strain curves and tensile strength diagrams of the microporous membranes prepared in Examples 2, 5, 6, and 7.

[0044] Figure 10 These are scanning electron microscope images of the microporous membranes prepared in Examples 5, 8, 9, and 10, where surface I is the surface close to the glass, surface II is the surface away from the glass, and CS is a cross-sectional view of the membrane.

[0045] Figure 11 The pore size images of the microporous membranes prepared in Examples 5, 8, 9, and 10 were measured using Image J based on scanning electron microscopy images.

[0046] Figure 12 These are the stress-strain curves and tensile strength diagrams of the microporous membranes prepared in Examples 5, 8, 9, and 10.

[0047] Figure 13 Experimental setup used for permeate retention testing.

[0048] Figure 14 This is the 1 / 3 / 5h lysozyme permeability graph of Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 5.

[0049] Figure 15 This is a graph showing the 1 / 3 / 12 hBSA retention rate for Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 5. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0051] Sources of the following raw materials are explained: polyurethane is purchased from BASF, and dimethyl sulfoxide is purchased from Maclean.

[0052] Example 1

[0053] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0054] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 12 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0055] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0056] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 75 μm film spreader to scrape off the excess solution.

[0057] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0058] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0059] (6) Take the membrane out of the water and dry it;

[0060] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0061] Example 2

[0062] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0063] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0064] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0065] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 75 μm film spreader to scrape off the excess solution.

[0066] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0067] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0068] (6) Take the membrane out of the water and dry it;

[0069] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0070] Example 3

[0071] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0072] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 16 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0073] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0074] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 75 μm film spreader to scrape off the excess solution.

[0075] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0076] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0077] (6) Take the membrane out of the water and dry it;

[0078] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0079] Example 4

[0080] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0081] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a PU / DMSO solution with a concentration of 18 wt%, and place it in an oven for 24 hours to degas before use;

[0082] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0083] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 75 μm film spreader to scrape off the excess solution.

[0084] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0085] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0086] (6) Take the membrane out of the water and dry it;

[0087] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0088] Example 5

[0089] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0090] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0091] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0092] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 100 μm film spreader to scrape off the excess solution.

[0093] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0094] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0095] (6) Take the membrane out of the water and dry it;

[0096] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0097] (8) Cut the membrane obtained in step (6) and place it in a permeation retention device for lysozyme permeation test.

[0098] (9) Take 800 μL of the prepared 1 mg / mL lysozyme solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0099] (10) At 1 h, 3 h, and 5 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The permeability (P) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0100]

[0101] (11) The membrane obtained in step (6) was cut and placed in a permeation retention device for a bovine serum albumin (BSA) retention test.

[0102] (12) Take 800 μL of the prepared 1 mg / mL BSA solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0103] (13) At 1 h, 3 h, and 12 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The retention rate (R) (%) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0104]

[0105] Example 6

[0106] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0107] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0108] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0109] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 150 μm film spreader to scrape off the excess solution.

[0110] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0111] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0112] (6) Take the membrane out of the water and dry it;

[0113] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0114] Example 7

[0115] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0116] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0117] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0118] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 250 μm film spreader to scrape off the excess solution.

[0119] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0120] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0121] (6) Take the membrane out of the water and dry it;

[0122] (7) Cut the membrane into dumbbell shape and use a universal tensile testing machine to test its mechanical properties.

[0123] Example 8

[0124] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0125] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0126] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -15°C;

[0127] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 100 μm film spreader to scrape off the excess solution.

[0128] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0129] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0130] (6) Take the membrane out of the water and dry it;

[0131] (7) The membrane obtained in step (6) is subjected to a permeation isolation test.

[0132] Example 9

[0133] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0134] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0135] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -10°C;

[0136] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 100 μm film spreader to scrape off the excess solution.

[0137] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0138] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0139] (6) Take the membrane out of the water and dry it;

[0140] (7) The membrane obtained in step (6) is subjected to a permeation isolation test.

[0141] Example 10

[0142] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0143] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0144] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -5°C;

[0145] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 100 μm film spreader to scrape off the excess solution.

[0146] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0147] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0148] (6) Take the membrane out of the water and dry it;

[0149] (7) The membrane obtained in step (6) is subjected to a permeation isolation test.

[0150] Comparative Example 1

[0151] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0152] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0153] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -15°C;

[0154] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 150 μm film spreader to scrape off the excess solution.

[0155] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0156] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0157] (6) Take the membrane out of the water and dry it;

[0158] (7) Cut the membrane obtained in step (6) and place it in a permeation retention device for lysozyme permeation test.

[0159] (8) Take 800 μL of the prepared 1 mg / mL lysozyme solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0160] (9) At 1 h, 3 h, and 5 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The permeability (P) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0161]

[0162] (10) The membrane obtained in step (6) was cut and placed in a permeation retention device for a bovine serum albumin (BSA) retention test.

[0163] (11) Take 800 μL of the prepared 1 mg / mL BSA solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0164] (12) At 1 h, 3 h, and 12 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The retention rate (R) (%) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0165]

[0166] Comparative Example 2

[0167] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0168] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 16 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0169] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0170] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 150 μm film spreader to scrape off the excess solution.

[0171] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0172] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0173] (6) Take the membrane out of the water and dry it;

[0174] (7) Cut the membrane obtained in step (6) and place it in a permeation retention device for lysozyme permeation test.

[0175] (8) Take 800 μL of the prepared 1 mg / mL lysozyme solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0176] (9) At 1 h, 3 h, and 5 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The permeability (P) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0177]

[0178] (10) The membrane obtained in step (6) was cut and placed in a permeation retention device for a bovine serum albumin (BSA) retention test.

[0179] (11) Take 800 μL of the prepared 1 mg / mL BSA solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0180] (12) At 1 h, 3 h, and 12 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The retention rate (R) (%) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0181]

[0182] Comparative Example 3

[0183] A method for manufacturing an ultra-thin graded polyurethane porous membrane comprises the following steps:

[0184] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 16 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0185] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -15°C;

[0186] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 75 μm film spreader to scrape off the excess solution.

[0187] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0188] (5) After observing that the solution has crystallized, quickly place the glass plate in water;

[0189] (6) Take the membrane out of the water and dry it;

[0190] (7) Cut the membrane obtained in step (6) and place it in a permeation retention device for lysozyme permeation test.

[0191] (8) Take 800 μL of the prepared 1 mg / mL lysozyme solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0192] (9) At 1 h, 3 h, and 5 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The permeability (P) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0193]

[0194] (10) The membrane obtained in step (6) was cut and placed in a permeation retention device for a bovine serum albumin (BSA) retention test.

[0195] (11) Take 800 μL of the prepared 1 mg / mL BSA solution and add it to the solution reservoir of the device, and add an equal amount of PBS solution to the other side.

[0196] (12) At 1 h, 3 h, and 12 h, 50 μL of the permeate solution was collected from the PBS solution and placed in an EP tube. The protein content in the permeate was determined using a BCA protein assay kit. The retention rate (R) (%) of the different porous membranes was calculated using the following formula, where C0 and C1 represent the protein concentrations of the original permeate solution and the permeate, respectively.

[0197]

[0198] Comparative Example 4

[0199] The method for manufacturing a polyurethane porous membrane by thermally induced phase separation comprises the following steps:

[0200] (1) Polyurethane (PU) was dissolved in dimethyl sulfoxide (DMF, good solvent) and water (H2O, non-good solvent) at 70°C (m DMSO :m H2O =10:1) mixed solvent to obtain PU / DMSO solutions with concentrations of 12, 14, and 18 wt%, which were placed in a 70°C oven for 12 h to degas before use;

[0201] (2) Preheat the heating plate, film spreader, and glass plate to 70°C;

[0202] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to 0°C;

[0203] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 100 μm film spreader to scrape off the excess solution.

[0204] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0205] (5) After observing the phase separation of the solution, quickly place the glass plate in ice water;

[0206] (6) Take the membrane out of the water and dry it.

[0207] Comparative Example 5

[0208] The conventional method for manufacturing a polyurethane porous membrane by one-way freezing comprises the following steps:

[0209] (1) Dissolve polyurethane (PU) in dimethyl sulfoxide (DMSO) at room temperature to obtain a 14 wt% PU / DMSO solution, which is then placed in an oven for 24 h for degassing before use;

[0210] (2) Use a refrigeration cycle machine to pre-cool the aluminum plate to -20°C;

[0211] (3) Take a small amount of polyurethane solution with a dropper and drop it onto a glass plate. Use a 250 μm film spreader to scrape off the excess solution.

[0212] (4) Place the glass plate quickly on a pre-cooled aluminum plate;

[0213] (5) After observing that the solution has crystallized, quickly place the glass plate in ice water (0°C);

[0214] (6) Take the membrane out of the water and dry it.

[0215] Figure 1 This is a scanning electron microscope image of the microporous membrane prepared in Comparative Example 4. Surface I is the surface close to the glass, surface II is the surface away from the glass, and CS is a cross-sectional view of the membrane. Experiments have shown that water can easily pass through the membrane with a concentration of 12 wt%. Water can pass through the membrane prepared with a concentration of 14 wt%, but with some difficulty. Water cannot pass through the membrane prepared with a concentration of 18 wt%. Figure 1 The SEM images of membranes with PU concentrations of 12, 14, and 18 wt% show a typical nucleation-growth (NG) mechanism. The 12 wt% membrane exhibits insufficient connectivity between the polymer-rich phase and the membrane matrix during phase separation, resulting in numerous bead-like particles adhering to the membrane matrix. Due to surface tension, the surface PU concentration is relatively high during the membrane formation process, resulting in a low surface porosity. Furthermore, the partially disconnected polymer-rich phase results in large pores with diameters up to 8 μm. The membrane formed with the 18 wt% PU solution, however, has time to grow and connect at this cooling rate, forming a closed foam structure with pores approximately 4 μm in diameter. The CS plot shows that the pores are mostly closed, which explains the impermeability of the 18 wt% PU membrane. The 14 wt% PU membrane exhibits a more pronounced fibrous structure, indicating that the critical PU solution concentration used in this experiment is around 14 wt%. However, the coarse fibers collapse, and after solvent removal, there are almost no interconnected pores.

[0216] Figure 2 The scanning electron microscope images of the microporous membranes prepared in Example 7 and Comparative Example 5 are shown. The I-side is close to the glass surface, and the II-side is away from the glass surface. The traditional one-way freezing method is to use DMSO as the casting solvent. The DMSO is crystallized by cooling, and the PU is pushed open to form the pore wall. The crystallized membrane is immersed in ice water and the DMSO is extracted to obtain vertically arranged microchannels. Figure 2 , macropores are formed on the surface of the membrane at 0℃, such as Figure 2 , among which the I-surface close to the glass surface is less affected by water and presents a shape squeezed by DMSO crystals. After the II-surface (II-surface) far from the glass surface, i.e. the air surface, is immersed in water, since the temperature of the entire coagulation bath is lower than the melting point of the solvent, most of the DMSO remains crystallized inside the membrane, while the part of the DMSO in contact with water on the surface dissolves, destroying the original surface structure and forming a relatively closed membrane structure with large cell pores. When used to encapsulate cells, it may cause cell leakage or infiltration of cells by immune cells, which is not conducive to cell growth. By changing the temperature of the extractant, the steps before crystallization of the membrane preparation process are the same. After the membrane is crystallized, the crystallized membrane is quickly immersed in a water bath with a temperature slightly higher than the melting point of DMSO (20°C). It is surprising to find that the retention capacity of the membrane is increased, and it also has permeability. The surface structure of the membrane is as follows Figure 2 As shown, the I-side has nanoscale pores with a diameter of about 570μm, and the II-side has a micron-scale pore diameter in the range of 1-10μm. The large pores formed on the II-side are due to the rapid dissolution of DMSO in the one-way frozen surface in water. After being immersed in water, the heat transfer of the part of the membrane close to the glass is faster than the mass transfer, resulting in partial dissolution of the I-side of the membrane, and then through slow mass transfer, that is, non-solvent-induced phase separation, smaller pores are formed.

[0217] Figure 3 The scanning electron microscope images of the microporous membranes prepared in Example 7 and Comparative Example 5 are shown. CS is the cross-sectional view, I-CS is the side close to the glass plate, and II-CS is the side away from the glass plate. The cross-sectional view I-CS / II-CS of the CS image close to the I-surface and II-surface is shown in FIG. Figure 3 As shown in Figure 2, the pore diameter is basically the same, about 10 μm; while increasing the coagulation bath temperature to 20 ° C, the resulting membrane structure is as follows Figure 3 As shown, the porous membrane presents a hierarchical structure, with one part consisting of a relatively loose structure and the other part consisting of a relatively tight structure. The membrane structures obtained by the general single-phase freezing method are all ordered and porous. However, based on previous research experience, in order to obtain small pores in the range of 500nm-2μm, one can only use a lower freezing temperature or increase the thickness of the membrane. However, this not only wastes energy, but also the diffusion limit distance is 200μm. When the thickness of the membrane is too thick, it will cause cell death due to lack of oxygen. Therefore, through unidirectional freezing and non-solvent-induced phase separation to form pores, and exploring the balance of heat transfer and mass transfer of the frozen membrane in the extractant, an ultra-thin porous membrane with a hierarchical structure, longitudinally arranged macropores, and surface-controllable micropores is obtained.

[0218] Figure 4 The scanning electron microscope images of the microporous membranes prepared in Examples 1, 2, 3, and 4 are shown. The I-surface is close to the glass surface, the II-surface is away from the glass surface, and CS is the cross-sectional view of the membrane. Figure 1 The microporous membrane shows uniform pore size, which decreases with increasing concentration. At low concentrations, the pore structure is longitudinally through-hole, but crystallization is hindered as the concentration continues to increase. When the polymer concentration increases to 18 wt%, DMSO crystallization is restricted, resulting in relatively small pores. During the subsequent DMSO extraction process, the small pores slow solute exchange and form nanoscale pores on the bottom layer of the membrane, the I-side.

[0219] Figure 5Figure 1 shows the pore size of the microporous membranes prepared in Examples 1, 2, 3, and 4, as measured using Image J based on scanning electron microscopy images. The membranes have a narrow pore size distribution, with similar pore sizes on the upper and lower surfaces. However, when the concentration increases to a point where the resulting pores are relatively small, such as when the membrane is laid with an 18wt% PU solution, the portion of the membrane closest to the glass melts due to slower mass transfer than heat transfer, partially dissolving the membrane structure and forming pores with a diameter of approximately 1.2μm through non-solvent-induced phase separation.

[0220] Figure 6 The stress-strain curves and tensile strength diagrams of the microporous membranes prepared in Examples 1, 2, 3, and 4 are shown. Figure 6 As shown in the figure, as the concentration increases, the pore wall of the membrane becomes thicker, the pore size becomes smaller, and a denser structure appears on the I-side, which increases the strength of the membrane with increasing concentration. These differences in the membrane also make it more difficult for the membrane to deform. It can be seen from the stress-strain curve of the membrane that the increase in concentration reduces the elongation at break of the membrane.

[0221] Figure 7 The following are scanning electron micrographs of the microporous membranes prepared in Examples 2, 5, 6, and 7. Side I is the surface close to the glass, side II is the surface away from the glass, and CS is a cross-sectional view of the membrane. It can be observed that as the membrane thickness increases, the dense portion of the membrane becomes increasingly thicker. This is because the thicker the membrane, the longer it takes for the internal DMSO to exchange substances with the external surface. Heat exchange is much faster than substance exchange, so the thicker portions cannot exchange substances in time and can only undergo non-solvent-induced phase separation. The 75 μm membrane, being relatively thin, can complete mass transfer within a moment of immersion, thus retaining the macropores formed by DMSO crystallization and extrusion on both sides. When the membrane thickness increases to 100 μm, signs of a dense layer appearing on the membrane. When the membrane thickness increases to 150 μm, a dense layer appears on the porous membrane. At this point, the dense layer is 3 μm thick, and the membrane surface pore size decreases to 1 μm. As the membrane thickness continues to increase, the dense layer becomes thicker. There are also slight differences on the other side, namely the II-side. This is because during the freezing process, as the thickness of the membrane increases, it becomes increasingly difficult for DMSO crystals to push away the top PU, and the holes formed on the II-side will become smaller. After the crystallized membrane is immersed in water, the original change trend of the hole diameter is retained.

[0222] Figure 8 Figure 1 shows the pore size of the microporous membranes prepared in Examples 2, 5, 6, and 7, as measured using Image J based on scanning electron micrographs. It can be seen that the membrane structure has few influencing factors and interactions, resulting in a narrow pore size distribution. The range of pore size differences for the I-face of each membrane group is less than 0.5 μm, and the pore size of each II-face is approximately 3 μm.

[0223] Figure 9The stress-strain curves and tensile strength graphs of the microporous membranes prepared in Examples 2, 5, 6, and 7 show that the membrane strength increases with increasing membrane thickness, which is mainly due to the increase in thickness and the dense part. When the membrane thickness increases from 150 μm to 250 μm, the thickness of the dense part increases by 10 μm, and the membrane strength increases from 7.2 MPa to 38.1 MPa. The difference in dense thickness is not significant, and the strength of the membranes of 100 μm and 150 μm only differs by 2.5 MPa. The strength of the membranes of 75 μm and 100 μm thickness increases from 1.2 MPa to 4.7 MPa only due to the appearance of the thin dense layer.

[0224] Figure 10 The scanning electron microscope images of the microporous membranes prepared in Examples 5, 8, 9, and 10 are shown. The I-side is close to the glass surface, the II-side is away from the glass surface, and the CS is the cross-sectional view of the membrane. The membrane structure formed at -20 / -15 / -10 / -5°C is as follows: Figure 10 , observing the cross-sectional structure of the membrane, it can be seen that within the temperature range of -20℃ to -10℃, the higher the temperature, the less DMSO nucleates, the larger the volume of crystal growth, and the larger the pores. However, at -5℃, due to the high temperature, DMSO is difficult to crystallize, and the formed crystals are uneven and relatively small, and the final pores are also smaller. Under such crystallization conditions, a more open membrane is formed at -10℃, and the surface diameters of both the I-face and II-face increase with temperature. At -5℃, due to the difficulty of crystallization, the membrane structure is not open enough, resulting in difficulty in bottom mass transfer and the formation of smaller pores on the I-face.

[0225] Figure 11 Figure 1 shows the pore size of the microporous membranes prepared in Examples 5, 8, 9, and 10, as measured using Image J based on scanning electron micrographs. With the exception of the membrane produced at -5°C, which exhibits a wider pore size distribution due to uneven crystallization, the pore size distributions of the remaining membranes are narrow. The difference in pore size between the upper and lower surfaces is minimal. The pore size of the loose layer can be adjusted by changing the temperature; a small temperature change can lead to a significant change in the pore size of the loose layer. Furthermore, the membrane production temperature should not be too high, otherwise the membrane structure will be uneven, making it difficult to predict and control.

[0226] Figure 12 Figures 2 and 3 show the stress-strain curves and tensile strength of the microporous membranes prepared in Examples 5, 8, 9, and 10. Within the temperature range of -20°C to -10°C, the membrane strength increases with increasing temperature, but the difference is not significant. This increase is likely due to thicker pore walls. The larger pore diameter gives the membrane greater deformation capacity, and the fracture strain also increases slightly. The structure of the Moral porous membrane at -5°C is relatively dense, and its strength is significantly increased compared to the membrane prepared at -20°C.

[0227] Figure 13The experimental setup for permeation retention testing. The prepared porous membrane was cut into 2cm x 2cm dimensions and sandwiched between two silicone rubber sheets with openings of 1.5cm x 1.5cm. Two 0.5cm thick silicone rubber sheets with openings of the same size were then sandwiched on either side. The outermost layer was a complete silicone rubber sheet, creating two 1.125mL solution reservoirs. The permeation solution was added to one side, while an equal amount of PBS solution was placed in the other. Four clamps were used to secure the membrane. During use, the openings were simply sealed with tin foil.

[0228] Figure 14 The following are the 1 / 3 / 5 hour lysozyme permeability graphs for Comparative Examples 1, 2, 3, and Example 5. A 150 μm thick membrane, combined with the previous membrane structure, has a thicker, dense layer, poor permeability, and slow material exchange. Lysozyme permeability within 5 hours is only 17%, and temperature and concentration have little effect on permeability. A 100 μm thick porous membrane has slightly lower permeability than a 75 μm thick membrane. The 16 / 75 / -15 membrane reaches over 90% permeability in just 3 hours. The 14 / 100 / -20 membrane achieves 56% lysozyme permeability within 3 hours and 90% within 5 hours.

[0229] Figure 15 This is a graph showing the 1 / 3 / 12 hBSA retention rate for Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 5.

[0230] The retention rate of BSA by the 150μm thick membrane was above 98% within 12h, and the retention rate of BSA by the 14 / 100 / -20 membrane was above 98%.

[0231] The 12-hour retention rate was 70%, demonstrating that it effectively prevents immune cells and large-molecular-weight immune molecules from entering the membrane and causing cell damage. The 16 / 75 / -15 membrane exhibited poor retention performance, with a 12-hour retention rate of only 32%. This indicates that the 14 / 100 / -20 polyurethane porous membrane can meet the requirements for nutrient permeation and immune isolation in cell encapsulation.

Claims

1. A method for producing an ultra-thin graded polyurethane porous membrane, characterized in that: The steps include: (1) dissolving polyurethane in dimethyl sulfoxide to obtain a polyurethane solution, and then degassing; (2) Use a refrigeration cycle machine to pre-cool the aluminum plate; (3) dropping the polyurethane solution onto a glass plate, and then scraping off the excess solution with a film spreader; (4) placing the glass plate on a pre-cooled aluminum plate; (5) After observing that the solution has crystallized, place the glass plate in warm water; (6) Take the membrane out of the water and dry it; The concentration of the polyurethane solution is 12-18wt%; the pre-cooling temperature of the refrigeration cycle machine is -5-20°C; the film laying thickness of the film laying device is 50-500μm; and the warm water is 15-25°C.

2. The method for producing an ultra-thin hierarchical polyurethane porous membrane according to claim 1, wherein: In step (3), the film thickness of the film laying device is 75-250 μm.

3. The method for producing an ultra-thin hierarchical polyurethane porous membrane according to claim 1, wherein: The concentration of the polyurethane solution is 14 wt %; the pre-cooling temperature of the refrigeration cycle machine is -20°C; the film laying thickness of the film laying device is 100 μm; and the temperature of the warm water is 20°C.

4. The method for producing an ultra-thin hierarchical polyurethane porous membrane according to claim 1, wherein: The thickness of the glass plate in step (3) is 1 mm.

5. The method for manufacturing an ultra-thin hierarchical polyurethane porous membrane according to claim 1, wherein: The degassing step is to place the mixture in an oven at 40° C. for 24 hours.

6. The polyurethane ultra-thin hierarchical porous membrane prepared by the manufacturing method according to any one of claims 1 to 5.

7. Use of the polyurethane ultra-thin graded porous membrane prepared by the manufacturing method according to claim 3 in cell encapsulation.

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