Artificial pancreas and preparation method and application thereof

Abstract

The invention discloses an artificial pancreas and a preparation method and application thereof, and belongs to the technical field of biomedical engineering and biological materials. The preparation method of the device comprises the following steps: preparing a cavity by taking a cellulose acetate microporous film as a shell, reserving a filling opening, mixing a sodium alginate or polyacrylamide hydrogel precursor solution with exogenous living cells with a glucose responsive insulin secretion function, filling the mixed solution into the cavity, sealing the filling opening, and sealing the filling opening. The whole device is soaked in a calcium chloride solution for crosslinking, or after exogenous living cells with a glucose-responsive insulin secretion function are encapsulated by calcium alginate hydrogel or polyacrylamide hydrogel, a cellulose acetate microporous film is used as a shell for packaging, so that the artificial pancreas is formed. The preparation method has a wide application prospect in the field of preparation of implantable artificial pancreas devices for treating diabetes cells.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and biomaterials technology, specifically to an immune isolation device for cell encapsulation, transplantation, and function, its preparation method, and its application, particularly for the preparation of an implantable artificial pancreas device for the treatment of diabetes after encapsulating pancreatic islets and related cells. Background Technology

[0002] Islet transplantation, as a potential functional cure for type 1 diabetes mellitus (T1DM), has the core advantage of replacing damaged islets with transplanted islet cells. Acting as an artificial pancreas, it autonomously responds to changes in the patient's blood glucose levels and secretes insulin on demand. The challenges in its development lie in two aspects: firstly, the cell culture environment (cell culture medium) needs to provide the free transfer of nutrients required for islet cell survival while ensuring the free diffusion of blood glucose and insulin; secondly, it must ensure the isolation of islet cells from the patient's immune system. To simultaneously achieve these two requirements, artificial pancreas based on encapsulated functional islet cells has become a research hotspot. Its basic principle is to physically isolate islet cells from the host's immune system through encapsulation devices, while allowing the free exchange of nutrients, glucose, and insulin. Representative technologies are as follows: (1) Using hydrogel materials as islet encapsulation devices: Chinese patent application CN120699283A discloses the preparation of hydrogel microspheres using a mixed solution of gelatin and sodium alginate crosslinked with calcium chloride to encapsulate islets as artificial pancreatic grafts, which improves the immunoisolation, mechanical strength and toughness compared with single polymer hydrogels. However, the calcium chloride crosslinked gelatin / sodium alginate hydrogel is not stable, and its pore size cannot completely block immune cells. As the literature "3D Bioplotting of Gelatin / Alginate Scaffolds for Tissue Engineering: Influence of Crosslinking Degree and Pore Architecture on Physicochemical Properties" points out: the calcium chloride crosslinked gelatin / sodium alginate hydrogel has a degradation rate of up to 40% after 15 days, and the material pore size is between 100 and 250 μm. To address the stability issue, this paper supplemented the calcium chloride-crosslinked gelatin / sodium alginate crosslinking with glutaraldehyde, reducing the 15-day degradation rate to 20%. However, this increased the material pore size to over 500 μm, resulting in poorer immunoassay. Furthermore, glutaraldehyde itself is a toxic substance. Therefore, fabricating islet encapsulation devices using only hydrogel materials presents challenges in simultaneously addressing stability, immunoassay, and biosafety.

[0003] (2) Fabrication of composite islet encapsulation devices using hydrogel-microporous films: To overcome the above problems, the literature "Qingsheng Liu, et al. "A Zwitterionic Polyurethane Nanoporous Device with Low Foreign-Body Response for Islet" The "encapsulation" approach shifted towards a composite structure design. A hydrogel formed by crosslinking sodium alginate with calcium and barium ions was used as a cell culture medium to encapsulate the islets of Langerhans. Simultaneously, a zwitterionic polyurethane microporous film, prepared by electrospinning, was coated onto the outer layer of the hydrogel. This hydrogel-microporous film composite islet encapsulation device initially created an "artificial pancreas" capable of autonomously responding to changes in the patient's blood glucose levels and secreting insulin as needed. However, while this approach represents a step forward in structural design, it introduced new problems. First, polyurethane materials have poor biocompatibility, and the residual catalyst stannous octoate from the preparation process is a toxic substance. Once dissolved after implantation, it can cause neurotoxicity and other problems in the human body. Furthermore, the barium ions used to crosslink sodium alginate are highly toxic, and their slow dissolution can also produce chronic toxicity. Second, electrospinning technology is complex, difficult to scale up, and costly. Therefore, this composite device still has shortcomings in terms of biosafety, process feasibility, and material safety. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to address the difficulty in balancing biosafety, process complexity, effectiveness of immune isolation, and long-term stability in existing artificial pancreas devices.

[0005] The present invention solves the above-mentioned technical problems through the following technical means: A first aspect of the present invention provides a method for preparing an artificial pancreas, comprising the following steps: (1) Mix the solution with the cells to obtain a hydrogel precursor solution for encapsulating cells; (2) The hydrogel precursor solution for encapsulating cells was encapsulated in a cellulose acetate membrane to obtain the device; (3) Immerse the device in the crosslinking agent to obtain the desired product; or (a) The cells were digested with enzymes, resuspended, and mixed with the solution to obtain a mixture; (b) The mixture was added dropwise to the cross-linking agent for cross-linking, and washed to obtain a hydrogel encapsulating cells; (c) The hydrogel containing the cells is sealed into a cellulose acetate membrane to obtain the final product.

[0006] Preferably, in steps (1) and (a), the solution is either sodium alginate solution or polyacrylamide solution.

[0007] Preferably, the concentration of the sodium alginate solution is 1-5% (w / v); more preferably 1.5-4% (w / v).

[0008] The optimal concentration is 2-3% (w / v).

[0009] Preferably, the concentration of the polyacrylamide solution is 3-10% (w / v); more preferably 4-7% (w / v); and even more preferably 4-5% (w / v).

[0010] Preferably, in steps (1) and (a), the cells are exogenous live cells with glucose-responsive insulin secretion function, more preferably one of allogeneic islets, xenogeneic islets, or islet-like cell clusters derived from stem cell differentiation; more preferably mouse islet tumor MIN6 cells.

[0011] Preferably, in steps (1) and (a), the cell density is 1×10⁻⁶. 6 ~1×10 7 cells / mL.

[0012] A further preferred method for preparing islets includes the following steps: S1: Collagenase IV was dissolved in Hank's balanced salt solution (HBSS) to prepare collagenase IV / HBSS; S2: The pancreas was minced and digested with collagenase IV / HBSS, and the digestion was terminated with RPMI-1640 medium containing fetal bovine serum (FBS). The pancreas was then sieved and the islets were collected. S3: Primary islets were obtained by culturing islets in RPMI-1640 medium containing fetal bovine serum (FBS) and penicillin-streptomycin (PS).

[0013] Preferably, in step S1, the final concentration of collagenase IV / HBSS is 1.0 mg / mL.

[0014] Preferably, in step S2, the digestion specifically involves digesting with 15 mL of 1.0 mg / mL collagenase IV / HBSS in a water bath at 37°C for 15 min.

[0015] Preferably, in step S2, the RPMI-1640 medium containing fetal bovine serum (FBS) is specifically an RPMI-1640 medium containing 10% fetal bovine serum (FBS).

[0016] Preferably, in step S2, the sieving specifically involves first coarsely filtering through a 70-mesh cell sieve, centrifuging and resuspending the filtrate, then filtering through a 200-mesh sieve, and collecting the islets by inverting the filter screen.

[0017] Preferably, in step S3, the RPMI-1640 medium containing fetal bovine serum (FBS) and penicillin-streptomycin (PS) is specifically an RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS).

[0018] Preferably, in step (2), the sealing is to prepare a cavity with a cellulose acetate membrane as the outer shell and reserve an infusion port, inject the hydrogel precursor solution for encapsulating cells into the cavity, and seal the infusion port.

[0019] Preferably, the specific steps of sealing are as follows: align two cellulose acetate membranes of the same size or roll a cellulose acetate membrane into a tube, prepare a cavity by instantaneous hot pressing, inject the hydrogel precursor solution for encapsulating cells into the cavity, and seal the injection port by instantaneous hot pressing.

[0020] Preferably, the hot pressing conditions are 100~180℃, pressure is 8~12N, and time is 8~12s; more preferably, it is 10N and 10s.

[0021] Preferably, in steps (2) and (c), the micropore size of the cellulose acetate membrane is 0.1~5μm; more preferably 0.15~3μm.

[0022] Preferably, the device is in the shape of a disc, a square disc, or a tube.

[0023] Preferably, in steps (3) and (b), the crosslinking agent is a CaCl2 solution.

[0024] Preferably, the concentration of the CaCl2 solution is 90~210 mmol / L.

[0025] Preferably, in steps (3) and (b), the crosslinking time is 5 to 12 minutes.

[0026] Preferably, in step (a), the enzyme is pancreatic enzyme.

[0027] Preferably, in step (b), the washing specifically involves washing three times with RPMI-1640.

[0028] Preferably, in step (c), the specific steps of sealing are as follows: spread the hydrogel in the center of the cellulose acetate membrane, cover it with another cellulose acetate membrane of the same size, align the edges, and perform heat sealing.

[0029] Preferably, the hot pressing conditions are 100~180℃, pressure is 8~12N, and time is 8~12s; more preferably, it is 10N and 10s.

[0030] A second aspect of the present invention provides an artificial pancreas prepared based on the above-described preparation method.

[0031] A third aspect of the present invention provides an application of the above-described preparation method in the preparation of an implantable artificial pancreas device for treating diabetes.

[0032] The beneficial effects of this invention are as follows: 1. This invention provides an artificial pancreas device based on a hydrogel-cellulose acetate membrane composite structure: (1) The cavity of the artificial pancreas is prepared with a cellulose acetate microporous membrane as the shell and an infusion port is reserved. Exogenous living cells with glucose-responsive insulin secretion function (including but not limited to allogeneic islets, xenogeneic islets, islet-like cell clusters derived from stem cell differentiation, etc.) are mixed with sodium alginate or polyacrylamide hydrogel precursor solution and then infused into it. After sealing the infusion port, the entire device is immersed in calcium chloride solution for cross-linking to form an artificial pancreas.

[0033] (2) After encapsulating exogenous living cells with glucose-responsive insulin secretion function (including but not limited to allogeneic islets, xenogeneic islets, islet-like cell clusters derived from stem cell differentiation, etc.) with calcium alginate hydrogel or polyacrylamide hydrogel, an artificial pancreas is formed by encapsulating it with a cellulose acetate microporous membrane as the outer shell.

[0034] 2. Long-term functional stability of the device: In a type 1 diabetic mouse model, after implantation of a device encapsulating primary pancreatic islets, the glucose-responsive insulin secretion function of the recovered device showed no significant difference compared to before implantation. This demonstrates that the artificial pancreas device of this invention possesses long-term (120 days) stable in vivo function.

[0035] 3. Biosafety: The core materials used (sodium alginate, calcium chloride, polyacrylamide, and cellulose acetate) are all recognized as having excellent biocompatibility, ensuring the biosafety of the device. In vitro cytotoxicity (MTT assay) and cell co-culture experiments confirmed that the calcium alginate hydrogel and polyacrylamide hydrogel used in this invention have no significant cytotoxicity and can effectively support the survival and proliferation of encapsulated cells. After 120 days of in vivo implantation, the encapsulation membrane surrounding the device is thin and transparent, accompanied by neovascularization. This indicates that the device has good tissue compatibility, and the neovascularization facilitates the efficient exchange of nutrients and metabolites, providing an ideal microenvironment for the long-term survival and functional performance of the encapsulated cells.

[0036] 4. Physical immune isolation provided by the cellulose acetate membrane shell: Using a cellulose acetate membrane with a suitable pore size as the outer shell of the device effectively blocks the penetration of host immune cells (such as lymphocytes and macrophages), thus achieving immune isolation physically.

[0037] 5. The preparation process is simple, the cost is low, and it is easy to standardize and scale up.

[0038] 6. The device of this invention is based on a composite of calcium alginate hydrogel or polyacrylamide hydrogel and cellulose acetate membrane. This invention combines the good biocompatibility of polymer hydrogels with the selective permeability of synthetic membrane materials to prepare an implantable cell encapsulation platform with glucose-responsive insulin secretion function and low immune rejection. It is suitable for the encapsulation, transplantation, and functionalization of exogenous living cells with glucose-responsive insulin secretion function (including but not limited to allogeneic islets, xenogeneic islets, and islet-like cell clusters derived from stem cell differentiation). It has broad application prospects in the field of preparing implantable artificial pancreas devices for diabetic cell therapy.

[0039] 7. This invention provides various device forms such as circular, square, and tubular shapes, as well as two optional assembly processes (crosslinking before encapsulation and encapsulation before crosslinking). It can be flexibly selected and optimized according to different application scenarios (such as peritoneal implantation and subcutaneous implantation) and cell types. The process has strong repeatability and is easy to standardize and scale up.

[0040] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the structure of the packaged device of the present invention; Figure 2 This is to verify the cell compatibility of the calcium alginate and polyacrylamide hydrogel material in Example 8 of the present invention; Figure 3 This is the average fluorescence intensity diagram of the cell adhesion experiment in Example 9 of the present invention, n=5; Figure 4 This is a diagram of the static response experiment of glucose in Example 10 of the present invention; Figure 5 This is a static glucose response test diagram before and after the artificial pancreas was implanted in Example 11 of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0043] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0044] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0045] like Figure 1 As shown, the artificial pancreas device of the present invention includes: (1) Hydrogel layer: the contents, which consists of calcium alginate or polyacrylamide hydrogel that encapsulates multiple pancreatic islet cell clusters.

[0046] (2) Microporous film layer: This is the outer shell, which is sealed by a cellulose acetate membrane through an instantaneous heat-sealing method at the edges.

[0047] (3) Artificial pancreas device: The device is round, square, or tubular in shape, with a large specific surface area. It has high permeability to nutrients, glucose, and insulin, while blocking immune cells and acting as an immune barrier. It can be implanted into the recipient's peritoneal cavity or subcutaneous tissue (e.g., Figure 2 As shown in the figure, it autonomously responds to changes in glucose levels and secretes insulin.

[0048] Example 1: Preparation of calcium alginate hydrogel Weigh 1.5 g of sodium alginate powder under sterile conditions, dissolve it in 100 mL of sterile physiological saline to prepare a 1.5% (w / v) sodium alginate solution, stir magnetically until completely dissolved, and irradiate with ultraviolet light overnight to obtain the sodium alginate solution.

[0049] Healthy mouse pancreatic islet tumor MIN6 cells were digested with trypsin, resuspended, and mixed with sodium alginate solution to achieve a cell density of 5 × 10⁻⁶. 6 Cells / mL. Using a microinjection pump and a 20G needle, the cell-sodium alginate mixture was added dropwise to a 200 mmol / L CaCl2 crosslinking solution. The dropwise distance was 10 cm, and the crosslinking time was 10 minutes. Cell-containing calcium alginate hydrogel microspheres with a diameter of approximately 1 mm were formed. After washing three times with RPMI-1640, the cell-encapsulated calcium alginate hydrogel was obtained.

[0050] Example 2: Preparation of sodium alginate hydrogel precursor solution Under sterile conditions, weigh 3 g of sodium alginate powder, dissolve it in 100 mL of RPMI-1640 to prepare a 3% (w / v) sodium alginate solution, stir magnetically until completely dissolved, and irradiate with ultraviolet light overnight to obtain the sodium alginate solution.

[0051] Collagenase IV was dissolved in Hank's balanced salt solution (HBSS) to a final concentration of 1.0 mg / mL, yielding 1.0 mg / mL collagenase IV / HBSS. Kunming mice were sacrificed, their pancreas isolated and minced, and then digested with 15 mL of 1.0 mg / mL collagenase IV / HBSS in a 37°C water bath for 15 min. Digestion was terminated with an equal volume of RPMI-1640 medium containing 10% fetal bovine serum (FBS) (10% FBS / RPMI-1640). The pancreas was first coarsely filtered through a 70-mesh sieve, the filtrate was centrifuged and resuspended, then filtered through a 200-mesh sieve, and the islets were collected by inverting the sieve. The islets were then routinely cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin (PS). Primary islets from Kunming mice were obtained after passing morphological observation and glucose-stimulated insulin secretion experiments.

[0052] The islets of Langerhans were mixed with sodium alginate solution to achieve a cell density of 3 × 10⁻⁶. 6 The sodium alginate hydrogel precursor solution for encapsulating cells was obtained by measuring cells / mL.

[0053] Example 3: Preparation of Polyacrylamide Hydrogel Acrylic acid and acrylamide were free-radical copolymerized at a molar ratio of 1:4, with a total monomer concentration of 5 wt%. The pH was adjusted to neutral with 1M sodium bicarbonate solution. A viscous polymer solution was synthesized via free-radical copolymerization using 0.5 wt% ammonium persulfate as an initiator. The polymerization reaction was carried out at 70°C under nitrogen protection for 2 h. After the reaction, the product was precipitated with anhydrous ethanol and washed repeatedly to thoroughly remove unreacted monomers and byproduct impurities. The product was redissolved in RPMI-1640 to obtain a 7% polyacrylamide solution.

[0054] Healthy mouse pancreatic islet tumor MIN6 cells were digested with trypsin, resuspended, and mixed with polyacrylamide solution to achieve a cell density of 1×10⁻⁶. 6 The cell-polyacrylamide mixture was poured into a 100 mmol / L CaCl2 crosslinking solution and the crosslinking time was 10 minutes to form a cell-containing polyacrylamide hydrogel sheet with a thickness of approximately 5 mm. After washing three times with RPMI-1640 and trimming the edges, the cell-encapsulated polyacrylamide hydrogel was obtained.

[0055] Example 4: Preparation of polyacrylamide hydrogel precursor solution Acrylic acid and acrylamide were free-radical copolymerized at a molar ratio of 1:5, with a total monomer concentration of 7 wt%. The pH was adjusted to neutral by adding 1 mol / L sodium bicarbonate solution. A viscous polymer solution was synthesized via free-radical copolymerization using 0.2 wt% ammonium persulfate as an initiator. The polymerization reaction was carried out at 70°C under nitrogen protection for 2 h. After the reaction, the product was precipitated with anhydrous ethanol and washed repeatedly to thoroughly remove unreacted monomers and byproduct impurities. The product was redissolved in RPMI-1640 to obtain a 5% polyacrylamide solution. The preparation method of primary islets of Kunming mice was the same as in Example 2. The islets were mixed with the polyacrylamide solution to achieve a cell density of 1 × 10⁻⁶ cells / mL. 7 The cells / mL yielded a polyacrylamide hydrogel precursor solution for encapsulating cells.

[0056] Example 5: Assembly and heat sealing of a circular artificial pancreas device A 150 μm thick cellulose acetate membrane with a 3 μm pore size was cut into discs approximately 15 mm in diameter. A calcium alginate hydrogel microsphere encapsulated with cells from Example 1 was placed in the center of the disc, excess culture medium was removed, and another disc of the same size was placed on top, ensuring the edges were aligned and the microspheres were completely encapsulated. The discs were then heat-sealed at 140°C, 10 N, and 10 s, with approximately 2 mm of the edges remaining. After trimming, a disc-shaped artificial pancreas device was obtained.

[0057] Example 6: Assembly and heat sealing of a square-shaped artificial pancreas device A 120 μm thick cellulose acetate membrane with a 0.45 μm pore size was cut into squares of approximately 10 mm × 20 mm. Another cellulose acetate membrane square of the same size was then placed on top, ensuring the edges were aligned. The two long sides and one short side were then instantaneously heat-pressed at 150°C, 10 N, and for 10 s. The sodium alginate hydrogel precursor solution used in Example 2 to encapsulate cells was poured into the tube, and the infused end was then instantaneously heat-pressed to seal it. The assembled device was then immersed in 150 mM CaCl2 for 7 minutes for crosslinking. After trimming, a square-shaped artificial pancreas device was obtained.

[0058] Example 7: Assembly and heat sealing of a tubular artificial pancreas device A 100 μm thick cellulose acetate membrane with a 0.15 μm pore size was cut into square sheets of approximately 15 mm × 33 mm and rolled into a tube with an overlap of approximately 3 mm on the sides. The tubes were then sealed by instantaneous heat pressing, followed by instantaneous heat pressing at one end at 160°C, 10 N, and 10 s. The polyacrylamide hydrogel precursor solution for cell encapsulation from Example 4 was infused into the tube, and the infused end was then sealed by instantaneous heat pressing. The assembled device was then immersed in 120 mmol / L CaCl2 for 7 minutes for crosslinking. After trimming, a tubular artificial pancreas device was obtained.

[0059] Example 8: Cell compatibility of hydrogels Cytotoxicity assay: The hydrogels obtained in Examples 1 and 3 were co-cultured with HeLa cells for 3 days, and the cytotoxicity of the materials was detected by the MTT assay. The results showed that no cytotoxicity was detected in either sample. Figure 2 ).

[0060] Cell co-culture test: Following the methods in Examples 1 and 3, GFP-HeLa cells were encapsulated in hydrogels and cultured for 7 days as usual. Both hydrogels could well support the growth and proliferation of GFP-HeLa cells.

[0061] Example 9: Validation of the in vitro immunoisolation performance of cellulose acetate membrane Evaluation of the anti-protein adsorption performance of the material: Cellulose acetate membranes were incubated with phosphate-buffered saline (2.5% FBS / PBS) containing 2.5% fetal bovine serum at 37°C for 8 h. The change in protein content in the solution before and after incubation was measured using the BCA method to quantitatively analyze the anti-protein adsorption performance of the cellulose acetate membranes. The results showed that the protein adsorption capacity of cellulose acetate membranes of different thicknesses and pore sizes reached saturation at around 5 h, and the protein adsorption capacity was below 180 g / cm³ at 8 h. 2 .

[0062] Evaluation of the anti-cell adhesion properties of the material: GFP-HeLa was inoculated onto the cellulose acetate membrane and glass slide control of Example 6 and cultured for 1, 2, and 4 days. Fluorescence microscopy observation showed that no live cells were observed on the surface of the cellulose acetate membrane, and no cell adhesion or growth was observed on the surface. Figure 3 ).

[0063] Evaluation of the cell isolation performance of the material as a physical barrier: GFP-HeLa was seeded onto the top side of the cellulose acetate membrane of Example 6 and cultured for 7 days. The results showed that the thickness of the cellulose acetate membrane had a negligible effect on the cell isolation performance, while the pore size of the cellulose acetate membrane had a significant effect on the cell isolation performance.

[0064] Example 10: In vitro functional verification of artificial pancreas In Example 6, the device was used to encapsulate mouse pancreatic islet tumor MIN6 cells or primary islets from Kunming mice. These were then incubated for 1 hour in low-glucose (2 mmol / L) and high-glucose (20 mmol / L) media, respectively, and insulin secretion was detected by ELISA. Figure 4 As shown, the devices encapsulating MIN6 cells or primary islets exhibited similar glucose concentration responsiveness to the same amount of MIN6 cells or primary islets, meaning that insulin secretion under high glucose stimulation was significantly higher than that under low glucose stimulation, confirming that the device did not affect the function of encapsulated cells and that the device has good in vitro glucose responsiveness.

[0065] Example 11: In vivo functional verification of artificial pancreas in mice Kunming mice were used to select type 1 diabetes models by intraperitoneal injection of streptozotocin (STZ, 50 mg / kg) and screening mice with a fasting blood glucose level exceeding 16.7 mmol / L for 5 consecutive days. After anesthesia with isoflurane, the mice underwent laparotomy to implant a device encapsulating primary pancreatic islet cells.

[0066] On day 120, the artificial pancreas device was removed, and a thin, transparent tissue membrane with angiogenesis was observed to have formed around the device. This neovascularization effectively provides the microenvironment necessary to maintain device function, enabling the artificial pancreas to sense blood glucose changes in real time and effectively transport insulin secreted by the artificial pancreas.

[0067] In vitro glucose static response tests were performed on the recovered artificial pancreas. Compared with the artificial pancreas before implantation, the device's function of secreting insulin in response to changes in glucose concentration did not change significantly. Figure 5 ).

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an artificial pancreas, characterized in that, This includes the following steps: (1) Mix the solution with the cells to obtain a hydrogel precursor solution for encapsulating cells; (2) The hydrogel precursor solution for encapsulating cells was encapsulated in a cellulose acetate membrane to obtain the device; (3) Immerse the device in the crosslinking agent to obtain the desired product; or (a) The cells were digested with enzymes, resuspended, and mixed with the solution to obtain a mixture; (b) The mixture was added dropwise to the cross-linking agent for cross-linking, and washed to obtain a hydrogel encapsulating cells; (c) The hydrogel containing the cells is sealed into a cellulose acetate membrane to obtain the final product.

2. The preparation method according to claim 1, characterized in that, In steps (1) and (a), the solution is either sodium alginate solution or polyacrylamide solution; the concentration of the sodium alginate solution is 1-5%; and the concentration of the polyacrylamide solution is 3-10%.

3. The preparation method according to claim 1, characterized in that, In steps (1) and (a), the cells are exogenous, living cells with glucose-responsive insulin secretion function.

4. The preparation method according to claim 1, characterized in that, In step (2), the sealing process involves preparing a cavity with a cellulose acetate membrane as the outer shell and reserving an infusion port, injecting the hydrogel precursor solution for encapsulating cells into the cavity, and then sealing the infusion port.

5. The preparation method according to claim 1, characterized in that, In steps (2) and (c), the micropore size of the cellulose acetate membrane is 0.1~5μm.

6. The preparation method according to claim 1, characterized in that, In steps (3) and (b), the crosslinking agent is a CaCl2 solution; the crosslinking time is 5-12 min.

7. The preparation method according to claim 1, characterized in that, In step (c), the specific steps of sealing are as follows: spread the hydrogel in the center of the cellulose acetate membrane, cover it with another cellulose acetate membrane of the same size, align the edges, and perform heat sealing.

8. The preparation method according to claim 7, characterized in that, The hot-pressing conditions are 100~180℃, pressure 8~12N, and time 8~12s.

9. The artificial pancreas prepared by the preparation method according to any one of claims 1-8.

10. The application of the preparation method according to any one of claims 1-8 in the preparation of an implantable artificial pancreas device for treating diabetes.

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