KR101352411B1 - Cell carrier for immunosuppression, and method for preparing the same - Google Patents

Abstract

The present invention relates to a cell carrier for immunosuppression, comprising a cell, a hydrophilic polymer in which the cell is dispersed, and a biodegradable porous membrane coated on the hydrophilic polymer, and a method of manufacturing the same.
According to the present invention, the hydrophilic polymer solution or hydrogel particles in which the cells are dispersed and fixed are coated with a biodegradable polymer to facilitate the permeation of the bioactive substances secreted by the cells, while having selective permeability to block immune response factors. In addition, it can be effectively used as a cell carrier with enhanced physical properties.

Description

Cell carrier for immunosuppression and preparation method thereof {Cell carrier for immunosuppression, and method for preparing the same}

The present invention relates to a cell carrier for immunosuppression and a method for producing the same.

Diabetes is a disease caused by a deficiency in glucose metabolism due to the lack of insulin, a hormone that regulates glucose in the blood, or the failure of insulin to function in each organ of the body.

This type of diabetes is caused by pancreatic islet cells that secrete insulin from the pancreas and destroyed by autoimmune mechanisms. Type 1 diabetes is caused by insulin deficiency. It is divided into type 2 diabetes, which is insulin-independent due to inability to function properly.

The treatment of type 1 diabetes can reduce the complications of diabetes due to hyperglycemia even though insulin administration is currently the main treatment or insulin fortification.

Type 2 diabetes also seeks to reduce insulin resistance through diet and exercise therapy and relieve relative insulin deficiency, but diet, exercise and drug therapy alone cannot cure diabetes.

Methods for treating diabetes include oral hypoglycemic agents, insulin secretagogues, and the like, and recently, pancreatic islet cell transplantation has been suggested as an ideal method for treating diabetes. Diabetes Control and Complications Trial (DCCT), published in 1993, showed that in patients with type 1 diabetes, tight blood sugar control can significantly reduce the risk of microvascular complications. However, thorough blood sugar control more than three times the frequency of hypoglycemia compared to conventional treatment.

Therefore, insulin alone is difficult to respond to subtle changes in blood sugar caused by diet or exercise, and the most physiological glycemic control method to date is the only pancreatic or pancreatic transplantation.

In pancreatic islet cell transplantation, allogeneic transplantation has the advantage that the procedure can be simplified and the insulin can be completely stopped by the second or third repetitive procedure even if the insulin administration is not completely stopped by one islet cell transplant. However, immunosuppressive agents must be used continuously to suppress the immune response, and the number of islet cells extracted from a donor is only about 30% of the total number of separable islet cells. Therefore, if the immune response of the xenotransplantation is overcome, it will be possible to continuously supply islet cells using pigs.

A method for suppressing the immune response following islet cell transplantation, and isolating islet cells from the host's immune system by microencapsulation with biocompatible materials to overcome rejection without the use of toxic immunosuppressants. And research has been actively conducted to protect and mimic the environment in the pancreas.

For example, there is a method of introducing and transplanting pancreatic islet cells into a crosslinked alginate hydrogel, which is a natural polymer, and in this case, the crosslinked unlinked alginate hydrogel is unbroken after transplantation in the body, thereby preventing the islet cells from being protected from the external immune system. there is a problem.

In order to overcome this problem, encapsulation of pancreatic islet cells in an alginate-polylysine-alginate multilayer structure in which the anions on the surface of the alginate hydrogel carrying the islet cells and the cations of polylysine are physically bonded in multiple layers. After transplantation, it is reported that blood glucose control is possible by blocking the immune response from the host's immune cells or cytokines. However, it is known that the encapsulation of pancreatic islet cells using alginate / polylysine, which is currently used in the clinic, may cause damage due to the weak physical properties of the capsule membrane, and damage to the membrane when the external shock is applied, resulting in leakage of the pancreatic islet cells and an immune response. have.

Therefore, in the use of pancreatic or pancreatic islet transplantation as a method for regulating physiological blood sugar, it is urgent to develop a pancreatic islet cell carrier which can effectively protect pancreatic islet cells from the immune system.

I. K. Jeong, et al., Islet transplantation and the use of the stem cell as a new source of β-cell in the treatment of diabetes mellitus, Med. Postgrad., 29, 255-268 (2011) 2. R. G. Bretzel, et al., Improved survival of intraportal pancreatic islet cell allografts in patients with type-1 diabetes mellitus by refined peritransplant management, J. Mol. Med., 77, 140-143 (1999) 3.J. J. Altman, et al., Long-term plasma glucose normalization in experimental diabetic rats with macroencapsulated implants of benign human insulinomas, Diabetes, 35, 625-633 (1986)

Disclosure of Invention An object of the present invention is to solve various problems in a conventional cell carrier, and has physical properties that can safely protect various cells including islet cells, while attacking from external immune cells or immune responses from cytokines. The present invention provides an immunosuppressive cell carrier having selective permeability to block silver and supply nutrients necessary for cell survival, and to release bioactive substances secreted by cells (eg, insulin, dopamine, etc.).

Another object of the present invention is to provide a method for producing an immunosuppressive cell carrier having the selective permeability as described above.

An immunosuppressive cell carrier according to an embodiment for achieving the object of the present invention is characterized in that it comprises a cell, a hydrophilic polymer in which the cells are dispersed, and a biodegradable porous membrane coated on the hydrophilic polymer in which the cells are dispersed. .

According to one embodiment of the invention, the hydrophilic polymer is alginate, hyaluronate, chondroitin, carrageenan, pectin, gellan gum, xanthan gum, collagen, elastin, polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide-polylactic acid Copolymer, polyethylene oxide-polylactic glycolic acid copolymer, polyethylene oxide-polycaprolactone copolymer, polyethylene oxide, polyvinyl alcohol, polyoxyethylene alkyl ethers, polyoxyethylene caster oil derivatives, polyoxyethylene sorbitan fatty acid At least one selected from the group consisting of esters and polyoxyethylene stearate can be preferably used.

The hydrophilic polymer according to the present invention may be a solution or hydrogel particles.

According to one embodiment of the invention, the biodegradable porous membrane is polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polydioxanone, polycaprolactone, polylactic acid-caprolactone copolymer, polydioxane One or more water-insoluble polymers selected from the group consisting of on-caprolactone copolymers, polyhydroxybutyric acid-hydroxyvaleric acid copolymers and polyphosphoesters can be preferably used.

According to an embodiment of the present invention, the porous membrane preferably has an asymmetric structure including two or more layers having different pore sizes from each other.

Accordingly, the porous membrane having an asymmetric structure according to the present invention preferably has an average pore size of 5 to 500 μm, and an inside thereof has an average pore size of 0.1 to 3,000 nm.

In addition, the method for producing an immunosuppressive cell carrier according to an embodiment for achieving another object of the present invention comprises the steps of dispersing the cells in a hydrophilic polymer, and coating a biodegradable porous membrane on the cell dispersed polymer It may include.

According to the present invention, it is easy to release various bioactive substances (eg, insulin, dopamine, etc.) that cells secrete by coating a hydrophilic polymer solution or hydrogel particles in which various cells including islets cells are dispersed with a biodegradable polymer. In the meantime, immune response factors can be effectively used as cell carriers with enhanced physical properties.

1 is a view illustrating a manufacturing process of a cell carrier coated with a polycaprolactone (PCL) porous membrane on the surface of an alginate hydrogel spherical particle of Example 1 of the present invention,
Figure 2 shows the manufacturing process of the cell carrier coated with a PCL porous membrane surface of the alginate solution droplet of Example 2 of the present invention,
3 is an SEM photograph of the cell carrier according to Example 1,
4 is a graph showing the release behavior of FITC-BSA in the cell carrier according to Example 1 and Comparative Example 1,
5 is a cytotoxicity measurement graph in the cell carrier according to Example 1 and Comparative Example 1,
6 is a result of evaluating the stability (form change, 14 days) in the buffer solution (PBS) of the cell carrier according to Example 1 and Comparative Example 1.

Hereinafter, the present invention will be described in detail.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a,""an," and "the" include singular forms unless the context clearly dictates otherwise. Also, " comprise "and / or" comprising "when used herein should be interpreted as specifying the presence of stated shapes, numbers, steps, operations, elements, elements, and / And does not preclude the presence or addition of one or more other features, integers, operations, elements, elements, and / or groups.

The present invention relates to an immunosuppressive cell carrier which stably delivers cells to a desired region of the body and does not cause an immune response from external cells or factors.

The cell carrier according to the present invention has a structure including a cell, a hydrophilic polymer in which the cell is dispersed, and a biodegradable porous membrane coated on the hydrophilic polymer.

The cells included in the cell carrier of the present invention are not particularly limited as long as they are fixed in a stable carrier and can be transplanted to a desired area, and include, for example, pancreatic islet cells used for transplanting pancreatic islets for diabetes treatment. , Embryonic stem cells, adult stem cells and induced pluripotent stem cells, etc. may be, but is not limited thereto.

In the present invention, the cells are dispersed in a polymer, wherein the hydrophilic polymer used is alginate, hyaluronate, chondroitin, carrageenan, pectin, gellan gum, xanthan gum, collagen, elastin, polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide -Polylactic acid copolymer, polyethylene oxide-polylactic glycolic acid copolymer, polyethylene oxide-polycaprolactone copolymer, polyethylene oxide, polyvinyl alcohol, polyoxyethylene alkyl ethers, polyoxyethylene caster oil derivatives, polyoxy One or more polymers selected from the group consisting of ethylene sorbitan fatty acid esters and polyoxyethylene stearate may be preferably used, of which alginate is most preferred.

The hydrophilic polymers must be water soluble because they must be mixed with cells dispersed in an aqueous solution (buffer solution or cell culture solution).

The hydrophilic polymer according to the present invention may be used in a solution state or in the form of hydrogel particles.

That is, according to one embodiment of the present invention, the hydrophilic polymers are dissolved in water to prepare an aqueous solution, cells are dispersed therein, and the cell dispersed aqueous solution is directly sprayed onto the biodegradable polymer solution for forming a porous membrane. The biodegradable porous membrane may be coated on the surface of the droplet including cells.

In addition, according to another embodiment of the present invention, the water-soluble hydrophilic polymers are prepared in an aqueous solution state by dissolving in water, cells are dispersed therein, and then the cell-dispersed aqueous solution is dissolved in an appropriate crosslinking agent using a specific nozzle. It may be prepared in the form of hydrogel particles and coated with a porous membrane on the surface of the hydrogel particles.

The term "hydrogel particles" used in the present invention may be defined as a matrix in the form of particles in which the water-soluble polymer is crosslinked by a specific reaction and contains a large amount of water.

The method for producing the hydrogel particles, as shown in Figure 1, may be made by first uniformly dispersing the cells in a hydrophilic polymer aqueous solution, and then dropping the hydrophilic aqueous polymer solution in a crosslinking solution.

The concentration of the hydrophilic polymer aqueous solution and the crosslinking solution is not particularly limited, and may be used within a known concentration range.

A solution containing a divalent cation such as calcium (Ca) may be used as the crosslinking solution of the hydrophilic polymer aqueous solution, and the cells are immobilized in the hydrogel particles by a crosslinking reaction between the anion of the hydrophilic polymer and the cation in the crosslinking solution. do.

The cell carrier according to the present invention is coated with a biodegradable porous membrane again on the surface of the aqueous solution in which the cells are dispersed or the hydrogel particles to which the cells are immobilized to block the attack from external immune cells or the immune response from cytokines. Let's do it.

According to one embodiment of the invention, the biodegradable porous membrane is polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polydioxanone, polycaprolactone, polylactic acid-caprolactone copolymer, polydioxane One or more polymers selected from the group consisting of on-caprolactone copolymers, polyhydroxybutyric acid-hydroxyvaleric acid copolymers and polyphosphoesters can be preferably used, among which polylactic acid and polylactic acid -Glycolic acid copolymer, polycaprolactone can be preferably used. In addition, the porous membrane contains a hydrophilic polymer within 30% by weight of the total porous membrane material in addition to the biodegradable polymers listed above to impart hydrophilicity to the membrane coated on the surface of the bead, the physiological activity generated by nutrients and cells essential for cell survival It is also possible to improve the permeation properties of the substance (eg insulin, dopamine, etc.).

The biodegradable polymers used as the porous membrane of the present invention should be precipitated / coated on the surface of the aqueous solution itself or the hydrogel particles containing a large amount of aqueous solution, and thus should have water insoluble (precipitation formation upon contact with aqueous solution). .

According to one embodiment of the present invention, it is preferable that the porous membrane has an asymmetric structure including two or more layers having different pore sizes from each other. That is, the surface (outside exposed surface) of the porous membrane having the asymmetric structure has a relatively large pore structure with a large average pore size, and the inside (surface in contact with the solution containing the cell or the hydrogel beads) is relatively A polymer membrane having a compact structure having a small average pore size is used.

Specifically, the surface of the biodegradable porous membrane having the asymmetric structure of the present invention has an average pore size of 5 to 500 µm, more preferably 50 to 200 µm. Since the average pore size of the surface has a structure that is relatively larger than the inside thereof, the prepared asymmetric porous membrane not only has a brittleness and flexibility, but also allows blood vessels to penetrate into the pores, thereby being present in the blood. Nutrient / oxygen can be delivered to the cells quickly and efficiently can have the effect of increasing the viability of the cell.

In addition, the interior of the biodegradable porous membrane having the asymmetric structure has an average pore size of 0.1 to 3,000 nm, more preferably 5 to 100 nm. Therefore, it may have an effect of inhibiting the penetration of cells and cytokines causing an immune response, but facilitating the penetration of bioactive substances (eg, insulin, dopamine, etc.) produced by the cells.

The porous membrane of the asymmetric structure according to the present invention may be prepared through a process of precipitation (phase transition) by contact with an aqueous solution of a biodegradable polymer solution, and first, the asymmetric porous membrane is prepared by dispersing the hydrophilic polymer solution or hydrogel particles in which the cells are dispersed. Immerse in biodegradable polymer solution for preparation. Thereafter, the biodegradable polymer-coated particles may be separated and washed in distilled water to prepare a cell carrier coated with a biodegradable polymer porous membrane having an asymmetric structure on the surface of the hydrophilic polymer solution or hydrogel particles.

Therefore, the method of preparing an immunosuppressive cell carrier according to the present invention may include dispersing cells in a hydrophilic polymer, and coating a biodegradable porous membrane on the cell dispersed hydrophilic polymer.

According to one embodiment of the invention, the hydrophilic polymer may be a solution or hydrogel particles.

According to an embodiment of the present invention, when the biodegradable porous membrane is coated using the hydrogel particles, the porous membrane may be prepared by immersing the hydrogel particles in a biodegradable polymer solution for forming the porous membrane. Can be. This process is as shown in FIG.

According to another embodiment of the present invention, when the surface of the hydrophilic polymer solution is coated with a biodegradable porous membrane, the porous membrane is prepared by spraying a hydrophilic polymer solution through a nozzle to prepare a polymer droplet, and the hydrophilic polymer droplet It may be prepared by immersing in a biodegradable polymer solution for forming a porous membrane on the surface. This process is as shown in FIG. 2.

That is, the cell carrier according to the present invention may form a hydrophilic polymer in a solution state in which cells are uniformly dispersed into a large number of droplets to form a porous membrane on the surface of the droplet, and hydrogel particles in a solution state in which cells are uniformly dispersed. And a porous membrane on the surface of the hydrogel particles.

Through this process, in the cell carrier according to the present invention, cells (eg, islet cells, stem cells, etc.) are dispersed in a hydrophilic polymer solution or hydrogel particles, and the surface of the hydrophilic polymer solution or hydrogel particles has an asymmetric structure. It has a structure coated with a biodegradable polymer porous membrane of.

The cell carrier according to the present invention has a 'selective permeability' that is easy to permeate the bioactive substances secreted by the cells, while blocking the immune response factors.

Accordingly, the cell carriers according to the present invention can be used in various applications such as diabetes treatment (using islets and various stem cells), Parkinson's disease and Alzheimer's treatment (using nerve-related cells and various stem cells) to stably protect cells. Not only can it be delivered, but it can also stably protect the cells from the external immune system.

The immunosuppressive cell carrier according to the present invention described above will be described in more detail by the following examples. The embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art, and the following examples can be modified in various other forms, and the scope of the present invention is It is not limited to an Example. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art.

Example  One : Hydrogel  Preparation of Cell Carrier Using Spherical Particle Polymers

In the exemplary embodiment of the present invention, fibroblasts (NIH 3T3 fibroblasts) were used as model cells, and albumin [FITC-bovine serum albumin (BSA)] was used as a model of a bioactive substance produced by the cells, as shown in FIG. The cell carrier was prepared by the procedure.

1) albumin ( FITC - BSA Polymers, including Alginate ) Preparation of Solution and Spherical Particles

5 mg of albumin (FITC-BSA) was added to 5 ml of 1.5 wt% sodium alginate aqueous solution, followed by uniform mixing. For hydrogelization of the mixture, the mixture was sprayed onto a 2wt% CaCl ₂ solution using a nozzle and maintained at room temperature for 10 minutes for their hydrogelation. The prepared hydrogel particles were separated and washed with distilled water to obtain alginate hydrogel spherical particles.

2) porous polymer ( Polycaprolactone , PCL A) membrane coating

The alginate hydrogel particles in a polycaprolactone mixed solution containing a polyethylene oxide-polypropylene oxide copolymer [in N-methyl-2-pyrrolidone (NMP); Total polymer concentration 15 wt%; Polyethylene oxide-polypropylene oxide copolymer / polycaprolactone, 5/95 (w / w)] and was immersed at room temperature for 3 minutes for precipitation / coating of the polymer membrane on the surface of the hydrogel particles. The particles surrounded by the polymer membrane and the non-precipitated polymer solution were separated and washed with NMP and washed with distilled water to obtain beads coated with the porous polymer membrane.

Example  2: Preparation of Cell Carrier Using Polymer in Solution State

5 mg of albumin (FITC-BSA) was added to 5 ml of 1.5 wt% sodium alginate aqueous solution, followed by uniform mixing. The mixed solution was prepared by spraying directly onto a polycaprolactone mixed solution containing a polyethylene oxide-polypropylene oxide copolymer using a nozzle devised by the present inventors. Hold at room temperature for 3 minutes for coating.

The particles surrounded by the porous membrane and the non-precipitated polymer solution were separated and washed with NMP and washed with distilled water to obtain particles coated with the porous polymer membrane. Next, a cell carrier was prepared by the process as shown in FIG. 2.

Comparative Example  One

In Example 1, the alginate hydrogel particles subjected to the process of 1) were immersed in 0.1% poly-L-lysine (PLL) solution for 10 minutes, phosphate buffer saline (PBS) buffer solution Washing to induce the coating of PLL on the surface of the alginate particles.

The PLL-coated beads were also immersed in 0.3% sodium alginate solution for 10 minutes and washed with PBS buffer to form multiple layers (alginate layer / PLL layer / alginate layer) on the surface of the alginate particles. (The method used in the study in which pancreatic islets were introduced into existing alginate beads), and this was used as a control.

Experimental Example  One : PCL Coated Alginate  Observe the shape of the particles

The shape of the alginate particles coated with PCL prepared in Example 1 was observed using a scanning electron microscope (SEM), and the results are shown in FIG. 3.

Next, as shown in Figure 3, it can be seen that a thin PCL film is formed on the surface of the alginate solution or hydrogel particles, the surface of the PCL membrane has a micro-sized pores, the inside of the asymmetric having a nanometer size pores It was confirmed that the structure is a porous membrane.

In addition, it was confirmed that a porous membrane having an asymmetric structure similar to that of Example 1 was also formed in the cell carrier prepared according to Example 2.

The formation of a porous membrane of such an asymmetric structure allows the penetration of blood vessels into the pores, thereby enabling fast and efficient delivery of nutrients / oxygen present in the blood to the cells, thereby increasing the survival rate of the cells as well as immunity. It can be expected to have an effect of inhibiting the penetration of the cells and cytokines causing the reaction, but facilitates the penetration of the bioactive material produced by the cells.

Experimental Example  2: albumin ( FITC - BSA )of Penetration  evaluation

Cell carrier particles comprising FITC-BSA prepared in accordance with Example 1 and Comparative Example 1, respectively, were impregnated in 1 ml of buffer solution (PBS, pH 7.4), and then rotated in a thermostat at 37 ° C. and 50 rpm. It was stored for a period of time (2 months). Samples were collected by carefully sampling the entire buffer solution at a set time and then replacing the new buffer solution. The amount of FITC-BSA eluted from the obtained buffer solution was analyzed by 490 nm excitation and 525 nm absorption using a fluorescence analyzer (RF-5301 PC, Shimadzu, Japan). Indicated.

Next, as shown in FIG. 4, it was confirmed that both of the alginate particles coated with PLL (Comparative Example 1) and the alginate particles coated with PCL (Example 1) were continuously released from the particles regardless of time. That is, through the surface (membrane) of the polymer-coated particles prepared in the present invention, it was confirmed that the permeation of the bioactive material produced from the cell is easy.

In addition, it was confirmed that the cell carrier prepared according to Example 2 exhibited similar release characteristics as those of Example 1 above.

Experimental Example  3: PCL Coated Alginate  Cytotoxicity Assessment of Particles

Cytotoxicity was analyzed to evaluate the effect of NMP used as a solvent on the cells during the precipitation / coating of the surface of the alginate solution or particles containing cells with PCL.

First, 1 × 10 ⁵ cells / ml of fibroblast was uniformly mixed in the alginate solution, and the alginate particles and PLL-coated particles having PCL precipitated / coated were prepared through the same procedure as in Example 1-2) and Comparative Example 1. . Each prepared particles were cultured in a cell culture solution containing serum (RPMI 1640, 10% FBS, 1% antibiotics, 4ml; 6-well plate) for a certain period of time (2, 7 days) and then cytotoxic through MTS assay (Fig. 5) was compared and analyzed.

As can be seen in Figure 5, the cell viability in Example 1 and Comparative Example 1 is similar, that is, cytotoxicity is not induced during the precipitation / coating process of PCL on the surface of the alginate of Example 1 using the organic solvent NMP I could confirm it.

In addition, in the cell carrier prepared according to Example 2, it was confirmed that cytotoxicity was not induced during the coating of the porous PCL membrane on the alginate surface as in Example 1.

Experimental Example  4 : PLL  And PCL Coated Alginate  Evaluation of Particle Stability

In order to evaluate / compare the stability of the cell carrier particles prepared according to Example 1 and Comparative Example 1, the prepared cell carrier particles were each impregnated in 1 ml of buffer solution (PBS, pH 7.4), and then, 37 ° C., It was stored for a period of time in a thermostat rotating at a speed of 50rpm. After 14 days, the shape of each particle was observed using an inverted microscope, and the results are shown in FIG. 6.

Next, as shown in FIG. 6, the PLL-coated alginate particles (Comparative Example 1) were able to observe the phenomenon that the particles burst due to the weak physical properties of the PLL coating after a certain time, but the alginate particles coated with the PCL porous membrane (Example 1) can be observed to exist stably without a change in shape. That is, the coating of the PCL porous membrane was able to solve weak physical properties, which is one of the biggest problems of the conventional immunosuppressive cell carriers.

From these results, the cell carrier according to the present invention is easy to permeate the physiologically active substances secreted by the cells, while having a selective permeability to block the immune response factors, and excellent physical properties as a carrier for carrying various cells effectively Can be used.

Claims (7)

One or more cells, including islet cells, embryonic stem cells, adult stem cells, and induced pluripotent stem cells,
Alginate hydrophilic polymers in which the cells are dispersed, and
Cell carrier comprising a biodegradable porous membrane selected from polycaprolactone, polylactic acid, polyglycolic acid, polydioxaneone coated on the hydrophilic polymer.
delete The method of claim 1,
The hydrophilic polymer is a cell carrier in the form of a solution or hydrogel particles.
delete The method of claim 1,
The biodegradable porous membrane is a cell carrier, characterized in that it has an asymmetric structure comprising two or more layers having different pore sizes from each other.
The method of claim 5,
Biodegradable porous membrane having the asymmetric structure,
The surface has an average pore size of 5 to 500 μm,
The interior of the cell carrier, characterized in that it has an average pore size of 0.1 ~ 3,000nm.
Dispersing one or more cells, including islets, embryonic stem cells, adult stem cells, and induced pluripotent stem cells, to the alginate hydrophilic polymer, and
And coating a biodegradable porous membrane selected from polycaprolactone, polylactic acid, polyglycolic acid, and polydioxane on the hydrophilic polymer in which the cells are dispersed.

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