Method for preparing islet microspheres by using gas microfluidic, islet microspheres and application

Abstract

The application discloses a method for preparing islet microspheres by using gas microflow, the islet microspheres and application, and the preparation process of the islet microspheres is as follows: after centrifugation of islet cells, the islet cells are resuspended by using a core layer ion crosslinking type hydrogel material A to obtain a cell-containing hydrogel material A; the cell-containing hydrogel material A and gas are respectively pushed out through a core needle and a shell needle of a coaxial needle head, under the shearing of the gas, the cell-containing hydrogel material A is cut into microsphere droplets, and the microsphere droplets fall into a receiving area C to be solidified to obtain core microspheres; the core microspheres are resuspended by using a shell layer amphiphilic ion hydrogel material B containing a photoinitiator to obtain a core microsphere-containing hydrogel material B, the core microsphere-containing hydrogel material B and gas are respectively pushed out through the core needle and the shell needle of the coaxial needle head, under the shearing of the gas, the core microsphere-containing hydrogel material B droplets fall into a receiving area D to be photo-cured to obtain islet microspheres. The islet microspheres disclosed by the application are controllable in diameter and have good biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials and biomedical engineering, and in particular to a method for preparing pancreatic islet microspheres by utilizing gas microfluidics, the pancreatic islet microspheres and applications. Background Art

[0002] Diabetes is a major global chronic disease, and years of diabetes can lead to numerous complications. Conventional insulin replacement therapy currently fails to achieve physiological blood sugar control and can even cause fatal hypoglycemia. Islet transplantation is a highly effective treatment, but factors such as severe host immune rejection can significantly reduce the survival rate of transplanted islets. Treatment with immunosuppressive drugs can significantly reduce the host's immune system and cause other unpredictable illnesses. Therefore, avoiding immunosuppressive drugs is a key prerequisite for optimization.

[0003] Encapsulating pancreatic islet cells using hydrogels is an effective strategy, mitigating immune rejection through physical isolation. Based on the diameter of the encapsulated device, it can be categorized as nanoencapsulation, microencapsulation, and macroencapsulation. The degree of immune protection is directly proportional to the encapsulation thickness and inversely proportional to the material exchange capacity. Relatively speaking, microencapsulation offers the greatest balance between immune protection and material exchange. However, existing technologies still face two major challenges that urgently need to be addressed. First, some microencapsulation designs only consider physical isolation to protect cells, failing to prevent nonspecific immune rejection, particularly foreign body reactions. Shortly after transplantation, the graft becomes encapsulated by a dense fibrotic capsule, leading to rapid cell death due to ischemia and hypoxia. Second, current microencapsulation technologies present multiple challenges. Traditional oil-cut microfluidics suffers from low throughput and poor biocompatibility. While the advent of gas microfluidics has significantly improved these issues, the limited range of materials currently supported by gas microfluidics makes them inadequate for diverse applications. The development of new microfluidic methods and the synthesis of microencapsulated microspheres that effectively prevent foreign body reactions remain challenging. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing pancreatic islet microspheres using gas microfluidics to address the technical defects in the prior art.

[0005] Another aspect of the present invention provides pancreatic islet microspheres.

[0006] The present invention also provides an application of pancreatic islet microspheres in preparing transplants for treating diabetes.

[0007] The technical solution adopted to achieve the purpose of the present invention is:

[0008] A method for preparing pancreatic islet microspheres using gas microfluidics, comprising the following steps:

[0009] Step 1: After centrifugation, the pancreatic islet cells are resuspended in a nuclear layer ion-crosslinked hydrogel material A to obtain a hydrogel material A containing pancreatic islet cells; the hydrogel material A containing pancreatic islet cells and gas are respectively pushed out through the core needle and shell needle of a coaxial needle. Under the shear of the gas, the hydrogel material A containing pancreatic islet cells is cut into microsphere droplets, which fall into a receiving area C and solidify to obtain core microspheres;

[0010] In step 2, the core microspheres prepared in step 1 are resuspended with the shell zwitterionic hydrogel material B to obtain the hydrogel material B containing the core microspheres. The hydrogel material B containing the core microspheres and the gas are pushed out through the core needle and the shell needle of the coaxial needle respectively. Under the shear of the gas, the droplets of the hydrogel material B containing the core microspheres fall into the receiving area D and are photocured to obtain pancreatic islet microspheres.

[0011] In the above technical solution, the core layer ion-crosslinked hydrogel material A in step 1 is prepared by dissolving sodium alginate, chitosan, sodium carboxymethyl cellulose or carrageenan in water, and the mass fraction of the sodium alginate, chitosan, sodium carboxymethyl cellulose or carrageenan is 1.0~3.0%.

[0012] In the above technical solution, the gas in step 1 and step 2 is nitrogen, the gas flow rate in step 1 is 1.5~3.0 L / min, and the gas flow rate in step 2 is 1.0~2.5 L / min.

[0013] In the above technical solution, the core needle in step 1 is 20~25G, and the shell needle is 15~19G; the core needle in step 2 is 18~22G, and the shell needle is 13~17G.

[0014] In the above technical solution, the receiving domain C in step 1 is an aqueous solution of calcium chloride, barium chloride, potassium chloride, sodium tripolyphosphate, ferric chloride or aluminum chloride with a mass fraction of 1.5-5.0%.

[0015] In the above technical solution, the shell zwitterionic hydrogel material B in step 2 is prepared by dissolving sulfobetaine methacrylate or polycarboxybetaine methacrylate in water and adding a photoinitiator, wherein the mass fraction of the sulfobetaine methacrylate or polycarboxybetaine methacrylate is 20-40%, and the photoinitiator is LAP, and its mass fraction is 0.1-1.0%.

[0016] In the above technical solution, the receiving domain D in step 2 is a polydimethylsiloxane or trimethylsiloxysilicate solution with a viscosity of 5 to 50 cs.

[0017] In the above technical solution, in step 2, after the core microspheres are resuspended in the shell zwitterionic hydrogel material B, they are pre-cured by ultraviolet light, and the pre-curing time is 5 to 40 seconds.

[0018] In another aspect of the present invention, the islet microspheres obtained by the method described above have a diameter of 20-250 μm for the core microspheres and a diameter of 200-400 μm for the islet microspheres.

[0019] Another aspect of the present invention is the use of the pancreatic islet microspheres in preparing transplants for treating diabetes.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses gas as shear force, avoiding the use of mineralized oil in traditional microfluidics and improving the biocompatibility of the preparation process;

[0022] 2. The present invention broadens the types of materials used in gas microfluidics, making shell zwitterionic materials also suitable for gas microfluidics, rather than being limited to ion-crosslinked hydrogel materials;

[0023] 3. The pancreatic islet microspheres of the present invention do not involve special reactions and have strong universal applicability. The core layer material has excellent biocompatibility and can provide a good microenvironment for transplanted cells. Compared with ion-crosslinked hydrogels, the zwitterionic material of the shell layer has anti-fouling properties, which can reduce foreign body reactions in the transplant without immunosuppressants, thereby improving transplant survival rate.

[0024] 4. The present invention makes the diameter of the prepared pancreatic islet microspheres highly controllable by selecting a suitable receiving area and controlling the gas flow rate, thereby preventing the pancreatic islet microspheres from affecting the nutrient exchange of cells due to an excessively large diameter. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is a schematic diagram of a method for preparing pancreatic islet microspheres using gas microfluidics according to the present invention.

[0026] Figure 2 Shown are the pancreatic islet microspheres prepared in Example 1 of the present invention.

[0027] Figure 3 The diagram shows the blood compatibility results of the pancreatic islet microspheres prepared in Example 1 of the present invention, wherein (a) is a physical image and (b) is a bar graph.

[0028] Figure 4 The figures show the anti-cell adhesion results of the pancreatic islet microspheres prepared in Example 1 of the present invention, wherein (a) is a physical picture and (b) is a bar graph.

[0029] Figure 5 Shown is a diagram showing the cell function results of the pancreatic islet microspheres prepared in Example 1 of the present invention.

[0030] Figure 6 Shown are the pancreatic islet microspheres prepared in Comparative Example 1 of the present invention.

[0031] Figure 7 Shown are the pancreatic islet microspheres prepared in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Example 1

[0034] Reference Figure 1 A method for preparing pancreatic islet microspheres using gas microfluidics comprises the following steps:

[0035] Step 1, preparation of core microspheres:

[0036] Pancreatic islet cells Min6 were resuspended in 1.5% sodium alginate solution to obtain a 1.5% sodium alginate solution containing Min6, and the 1.5% sodium alginate solution containing Min6 was aspirated into a syringe. The 1.5% sodium alginate solution containing Min6 was slowly pushed out using a syringe pump. The outlet of the syringe was connected to a coaxial needle through an extension tube, wherein the core needle was 22 G and the shell needle was 18 G. Nitrogen gas was connected to the shell needle after passing through a gas flow meter, and the gas flow rate was controlled at 2.0 L / min. The 1.5% sodium alginate solution containing Min6 formed tiny droplets under the shearing of nitrogen gas, and the droplets fell into a 2% calcium chloride solution to form core microspheres, the diameter of which was 240 μm.

[0037] Step 2, preparation of islet microspheres:

[0038] After dissolving sulfobetaine methacrylate in water, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was added to obtain a 20% sulfobetaine methacrylate solution. The core microspheres prepared in step 1 were resuspended in this 20% sulfobetaine methacrylate solution to obtain a 20% sulfobetaine methacrylate solution containing the core microspheres. This 20% sulfobetaine methacrylate solution containing the core microspheres was placed in a 1.5 mL EP tube and pre-cured by irradiating the tube wall with UV light for 15 seconds. A peristaltic pump was used to evenly distribute the 20% sulfobetaine methacrylate solution containing the core microspheres. The outlet of the peristaltic pump silicone tubing was connected to a coaxial needle with a 19 G core needle and a 15 G shell needle. Nitrogen gas was passed through a gas flow meter and then connected to the shell needle, with a controlled gas flow rate of 1.6 L / min. The 20% sulfobetaine methacrylate solution containing core microspheres forms tiny droplets under nitrogen shearing. The droplets fall into a polydimethylsiloxane solution with a viscosity of 5 cs and form microspheres under ultraviolet light. Figure 2The islet microspheres shown have a diameter of 360 μm.

[0039] Example 2

[0040] A method for preparing pancreatic islet microspheres using gas microfluidics, comprising the following steps:

[0041] Step 1, preparation of core microspheres:

[0042] Min6 was resuspended in a 2.0% sodium alginate solution to obtain a 2% sodium alginate solution containing Min6. The 2% sodium alginate solution containing Min6 was sucked into a syringe, and the 2% sodium alginate solution containing Min6 was slowly pushed out using a syringe pump. The outlet of the syringe was connected to a coaxial needle through an extension tube, wherein the core needle was 23 G and the shell needle was 17 G. Nitrogen gas was connected to the shell needle after passing through a gas flow meter, and the gas flow rate was controlled at 2.5 L / min. The 2% sodium alginate solution containing Min6 formed tiny droplets under the shear of nitrogen gas, and the droplets fell into a 3% calcium chloride solution to form core microspheres, wherein the diameter of the core microspheres was 200 μm;

[0043] Step 2, preparation of islet microspheres:

[0044] After dissolving sulfobetaine methacrylate in water, the photoinitiator LAP was added to obtain a 30% sulfobetaine methacrylate solution. The core microspheres were resuspended in this 30% sulfobetaine methacrylate solution to obtain a 30% sulfobetaine methacrylate solution containing core microspheres. This 30% sulfobetaine methacrylate solution containing core microspheres was placed in a 1.5 mL EP tube and pre-cured by irradiating the tube wall with UV light for 20 seconds. A peristaltic pump was used to evenly distribute the 30% sulfobetaine methacrylate solution containing core microspheres. The outlet of the peristaltic pump silicone tubing was connected to a coaxial needle with an 18-gauge core needle and a 13-gauge shell needle. Nitrogen gas was passed through a gas flow meter and then connected to the shell needle, with a controlled gas flow rate of 1.8 L / min. The 30% sulfobetaine methacrylate solution containing core microspheres forms tiny droplets under nitrogen shearing. The droplets fall into a polydimethylsiloxane solution with a viscosity of 10 cs and form microspheres under ultraviolet light. Figure 2 The islet microspheres shown have a diameter of 330 μm.

[0045] Example 3

[0046] A method for preparing pancreatic islet microspheres using gas microfluidics, comprising the following steps:

[0047] Step 1, preparation of core microspheres:

[0048] Min6 was resuspended with a 2.5% sodium alginate solution to obtain a 2.5% sodium alginate solution containing Min6, and the 2.5% sodium alginate solution containing Min6 was aspirated into a syringe. The 2.5% sodium alginate solution containing Min6 was slowly pushed out using a syringe pump. The outlet of the syringe was connected to a coaxial needle through an extension tube, wherein the core needle was 24 G and the shell needle was 19 G. Nitrogen gas was connected to the shell needle after passing through a gas flow meter, and the gas flow rate was controlled at 2.8 L / min. The 2.5% sodium alginate solution containing Min6 formed tiny droplets under the shearing of nitrogen, and the droplets fell into a 5% calcium chloride solution to form core microspheres, the diameter of which was 180 μm.

[0049] Step 2, preparation of islet microspheres:

[0050] After dissolving sulfobetaine methacrylate in water, the photoinitiator LAP was added to obtain a 40% sulfobetaine methacrylate solution. The core microspheres prepared in step 1 were resuspended in this 40% sulfobetaine methacrylate solution to obtain a 40% sulfobetaine methacrylate solution containing the core microspheres. This 40% sulfobetaine methacrylate solution containing the core microspheres was placed in a 1.5 mL EP tube and pre-cured by irradiating the tube wall with UV light for 30 seconds. A peristaltic pump was used to evenly distribute the 40% sulfobetaine methacrylate solution containing the core microspheres. The outlet of the peristaltic pump silicone tubing was connected to a coaxial needle with a 21-gauge core needle and a 14-gauge shell needle. Nitrogen gas was passed through a gas flowmeter and then connected to the shell needle, with a controlled gas flow rate of 2.0 L / min. A 40% sulfobetaine methacrylate solution containing core microspheres formed tiny droplets under nitrogen shear. The droplets fell into a polydimethylsiloxane solution with a viscosity of 20 cs and formed pancreatic islet microspheres under ultraviolet light. The diameter of the pancreatic islet microspheres was 310 μm.

[0051] Comparative Example 1

[0052] A method for preparing pancreatic islet microspheres using gas microfluidics, comprising the following steps:

[0053] Step 1, preparing core microspheres: the preparation of core microspheres is the same as in Example 1;

[0054] Step 2, preparation of islet microspheres:

[0055] After dissolving sulfobetaine methacrylate in water, the photoinitiator LAP was added to obtain a 20% sulfobetaine methacrylate solution. The core microspheres prepared in step 1 were resuspended in this 20% sulfobetaine methacrylate solution. This 20% sulfobetaine methacrylate solution containing the core microspheres was placed in a 1.5 mL EP tube and pre-cured by irradiating the tube wall with UV light for 15 seconds. A peristaltic pump was used to evenly distribute the 20% sulfobetaine methacrylate solution containing the core microspheres. The outlet of the peristaltic pump silicone tubing was connected to a coaxial needle with a 19-gauge core needle and a 15-gauge shell needle. Nitrogen gas was passed through a gas flow meter and then connected to the shell needle, with a controlled gas flow rate of 1.6 L / min. A 20% sulfobetaine methacrylate solution containing core microspheres was sheared by nitrogen to form tiny droplets, which then fell into a 10% polyethylene oxide solution and formed pancreatic islet microspheres under ultraviolet light. Figure 6 As shown, because the sulfobetaine methacrylate solution and the polyethylene oxide solution cannot be stably separated, the sulfobetaine methacrylate solution will diffuse freely when entering the polyethylene oxide solution, and the shape of the islet microspheres cannot be controlled and is solidified into a random shape, which cannot meet the transplantation requirements.

[0056] Comparative Example 2

[0057] A method for preparing pancreatic islet microspheres using gas microfluidics, comprising the following steps:

[0058] Step 1, preparing core microspheres: the preparation of core microspheres is the same as in Example 1;

[0059] Step 2, preparation of islet microspheres:

[0060] After dissolving sulfobetaine methacrylate in water, the photoinitiator LAP was added to obtain a 20% sulfobetaine methacrylate solution. The core microspheres prepared in step 1 were resuspended in this 20% sulfobetaine methacrylate solution to obtain a 20% sulfobetaine methacrylate solution containing the core microspheres. This 20% sulfobetaine methacrylate solution containing the core microspheres was placed in a 1.5 mL EP tube and pre-cured by irradiating the tube wall with UV light for 15 seconds. A peristaltic pump was used to evenly distribute the 20% sulfobetaine methacrylate solution containing the core microspheres. The outlet of the peristaltic pump silicone tubing was connected to a coaxial needle with a 19 G core needle and a 15 G shell needle. Nitrogen gas was passed through a gas flow meter and then connected to the shell needle, with a controlled gas flow rate of 1.6 L / min. A 20% sulfobetaine methacrylate solution containing core microspheres was sheared by nitrogen to form tiny droplets, which fell into a polydimethylsiloxane solution with a viscosity of 1 cs and formed pancreatic islet microspheres under ultraviolet light. Figure 7 As shown, due to the low viscosity of the receiving area, the islet microsphere droplets fell too quickly during the receiving process, sticking together due to the "like dissolves like" principle, making their shape uncontrollable. Because shape directly affects the distance between cells and the external environment for nutrient exchange, sticking together prevented optimal transplantation.

[0061] Test Example 1

[0062] The performance of the pancreatic islet microspheres prepared in Examples 1-3 is similar, and the performance of the pancreatic islet microspheres prepared in Example 1 is described below as an example.

[0063] 1. Blood compatibility verification:

[0064] Step 1: Place the sterile pancreatic islet microspheres prepared in Example 1 in physiological saline and extract at 37°C for 24 hours to obtain a microsphere extract;

[0065] Step 2: Blood was collected from the abdominal aorta of the rat using a blood collection tube with an anticoagulant coating. The blood was mixed and centrifuged at 3500 rpm for 15 min. The red blood cells in the lower layer were removed and thoroughly washed with normal saline.

[0066] Step 3. After adding 200 μL of red blood cells obtained in step 2 to a 1.5 mL EP tube, add 800 μL of normal saline, 0.1% Triton X-ray dilution, and islet microsphere extract, respectively. Mix gently and incubate at 37°C for 1 h. After incubation, centrifuge at 3500 rpm for 15 min to obtain the supernatant. Use a microplate reader to measure the absorbance of each group at 540 nm and calculate the hemolysis rate of the islet microsphere group.

[0067] Reference Figure 3 0.1% Triton X-ray was used as the positive control group (hemolysis rate was 100%), and normal saline was used as the negative control group (hemolysis rate was 0%). The hemolysis rate of the islet microsphere group was calculated, and the quantitative results showed that the hemolysis rate was 2%, which was less than the hemolysis standard value of 5%. The supernatant of the islet microsphere group was colorless and transparent, and had good blood compatibility.

[0068] 2. Anti-adhesion characterization:

[0069] Step 1: Add 1 mL of sodium alginate, 800 μL of sodium alginate + 200 μL of sulfobetaine methacrylate (referred to as core-shell gel), and 1 mL of methacrylated gelatin to a confocal microplate. After solidification, add 1 mL of L929 cell suspension to each dish and incubate in a cell culture incubator for 24 h.

[0070] Step 2: After incubation, gently rinse the gel surface with preheated PBS. Fix with 4% paraformaldehyde for 20 minutes and then wash three times with PBS. Permeabilize the membrane with 0.1% Triton X-ray for 10 minutes and then wash three times with PBS. Finally, add 1 mL of DAPI-containing anti-fluorescence quencher and observe under a confocal microscope.

[0071] Reference Figure 4 Compared with sodium alginate and methacrylated gelatin, the fluorescence intensity of the core-shell gel was the weakest. Quantitative results showed that the average fluorescence intensities of methacrylated gelatin, sodium alginate, and core-shell gel were 5.189, 0.872, and 0.540, respectively. The core-shell gel had the lowest average fluorescence intensity, indicating good anti-cell adhesion properties. This is beneficial for reducing foreign body reactions after transplantation.

[0072] 3. Insulin secretion experiment:

[0073] In order to characterize that the cell encapsulation process does not affect its cell secretion function, glucose-stimulated insulin secretion experiments were performed to verify.

[0074] Step 1: naked islet cells or islet microspheres were added to KRB buffer containing 16.7 mM glucose and incubated in an incubator for 1 h to obtain naked islet groups and islet microsphere groups, respectively;

[0075] Step 2: After the incubation, the supernatant was collected and the insulin content was detected using an ELISA kit.

[0076] Reference Figure 5 The insulin secretion levels of the naked cell group and the islet microsphere group were 1.715 pg / mL and 1.631 pg / mL, respectively, with no significant difference, indicating that the encapsulation of the shell and nuclear layer did not affect the secretory function of islet cells.

[0077] When verifying in mice or rats, the pancreatic islet microspheres (gel in macroscopic view) are transplanted into the epididymal fat pad of the rat or mouse, and the fat pad is spread out. In actual clinical application, the pancreatic islet microspheres can be transplanted into the greater omentum of humans to physiologically regulate the host's blood sugar.

[0078] When islet microspheres are used as transplants for treating diabetes, the islet microspheres (in a macroscopic state of gel) are transplanted into the epididymal fat pad of rats or mice or the greater omentum of humans. The fat pad is spread out, and the islet microspheres prepared by the present invention are placed in the center, and then the islet microspheres are covered with the fat pad.

[0079] In summary, the present invention successfully prepared pancreatic islet microspheres. These are characterized by excellent blood compatibility, which can prevent hemolysis after transplantation. Furthermore, they exhibit good anti-cell adhesion, which helps reduce foreign body reactions after transplantation. Finally, the encapsulation of pancreatic islet cells using core and shell hydrogels does not affect the secretory function of the islet cells, facilitating physiological regulation of host blood glucose after transplantation.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing pancreatic islet microspheres using gas microfluidics, characterized in that: The following steps are involved: Step 1: After centrifugation, the pancreatic islet cells are resuspended in a nuclear layer ion-crosslinked hydrogel material A to obtain a hydrogel material A containing pancreatic islet cells; the hydrogel material A containing pancreatic islet cells and gas are respectively pushed out through the core needle and shell needle of a coaxial needle. Under the shear of the gas, the hydrogel material A containing pancreatic islet cells is cut into microsphere droplets, which fall into a receiving area C and solidify to obtain core microspheres; Step 2: resuspending the core microspheres prepared in step 1 with a shell zwitterionic hydrogel material B containing a photoinitiator to obtain a hydrogel material B containing core microspheres. The hydrogel material B containing core microspheres and gas are respectively pushed out through the core needle and shell needle of the coaxial needle. Under the shear of the gas, the droplet of the hydrogel material B containing core microspheres falls into the receiving area D and is photocured to obtain pancreatic islet microspheres. The shell zwitterionic hydrogel material B is prepared by dissolving sulfobetaine methacrylate or polycarboxybetaine methacrylate in water and adding a photoinitiator, wherein the mass fraction of the sulfobetaine methacrylate or polycarboxybetaine methacrylate is 20-40%; The receiving domain D is a polydimethylsiloxane or trimethylsiloxysilicate solution with a viscosity of 5 to 50 cs.

2. The method for preparing pancreatic islet microspheres using gas microfluidics according to claim 1, wherein: The core layer ion-crosslinked hydrogel material A in step 1 is prepared by dissolving sodium alginate, chitosan, sodium carboxymethyl cellulose or carrageenan in water, wherein the mass fraction of the sodium alginate, chitosan, sodium carboxymethyl cellulose or carrageenan is 1.0-3.0%.

3. The method for preparing pancreatic islet microspheres using gas microfluidics according to claim 1, wherein: The gas in step 1 and step 2 is nitrogen, the gas flow rate in step 1 is 1.5~3.0 L / min, and the gas flow rate in step 2 is 1.0~2.5 L / min.

4. The method for preparing pancreatic islet microspheres using gas microfluidics according to claim 1, wherein: The core needle in step 1 is 20-25 G and the shell needle is 15-19 G. The core needle in step 2 is 18-22 G and the shell needle is 13-17 G.

5. The method for preparing pancreatic islet microspheres using gas microfluidics according to claim 1, wherein: The receiving domain C in step 1 is an aqueous solution of calcium chloride, barium chloride, potassium chloride, sodium tripolyphosphate, ferric chloride or aluminum chloride with a mass fraction of 1.5-5.0%.

6. The method for preparing pancreatic islet microspheres using gas microfluidics according to claim 1, wherein: Step 2: After the core microspheres are resuspended in the shell zwitterionic hydrogel material B, they are pre-cured by ultraviolet light, and the pre-curing time is 5 to 40 seconds.

7. The pancreatic islet microspheres obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The diameter of the core microspheres is 20-250 μm, and the diameter of the islet microspheres is 200-400 μm.

8. Use of the pancreatic islet microspheres according to claim 7 in preparing a transplant for treating diabetes.

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