Islet differentiation method and equipment for microencapsulated induced pluripotent stem cells

Abstract

The embodiment of the invention provides a pancreas islet differentiation method and equipment for microencapsulated induced pluripotent stem cells, and relates to the field of stem cell differentiation. In order to solve the problems of low differentiation efficiency, low insulin secretion function and immunological rejection after transplantation of the two-dimensional cultured iPSC, a method for inducing the iPSC into the islet cells under a three-dimensional condition is constructed, the differentiation efficiency and the functions of the islet cells are improved, immune isolation transplantation can be directly performed after differentiation, and meanwhile, new equipment is constructed. The structure is beneficial to improving the mass transfer efficiency in the induction process, and large-scale and accurate condition control can be realized. The preparation method comprises the following steps: S1, mixing iPSC with a sodium alginate solution to form calcium alginate microspheres containing the iPSC; s2, culturing the iPSC in the calcium alginate microspheres obtained in the step S1 into a cell cluster; and S3, performing stage induced differentiation on the cell cluster obtained in the step S2 to obtain the endocrine cell cluster.

Description

Technical Field

[0001] The present invention relates to the field of stem cell differentiation, and in particular to a method and device for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells. Background Art

[0002] Diabetes is a disease characterized by defective insulin secretion or dysfunction, with a huge number of patients worldwide. Traditional treatments rely on exogenous insulin injections or pancreatic islet transplantation, but donor islets are scarce and immune rejection is prone to occur after transplantation. In recent years, iPSC-based regenerative medicine technology has provided a new idea for diabetes treatment: iPSCs are differentiated into functional pancreatic islet cells in vitro and then transplanted back into the patient. However, existing technologies still face the following key issues: The existing technology has the following core defects: the traditional encapsulation system has insufficient construction of the three-dimensional microenvironment, resulting in the lack of intercellular connections and disordered differentiation signals; the conventional process has shear damage and uneven microcapsule size, which seriously weakens cell activity and differentiation consistency; the low mass transfer efficiency under static culture causes an imbalance in the cell metabolic microenvironment within the microcapsule; the existing semipermeable membrane materials cannot take into account both immune isolation and the free transmission of small molecules due to inaccurate pore size and charge regulation. Summary of the invention

[0003] According to an embodiment of the present invention, a method and device for islet differentiation of microencapsulated induced pluripotent stem cells are provided to solve the problems in the above-mentioned background technology.

[0004] In a first aspect of the present invention, a method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells is provided.

[0005] The preparation method comprises: S1: Mix iPSCs with sodium alginate solution to form calcium alginate microspheres containing iPSCs; S2: culturing the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters; S3: The cell cluster obtained in step S2 is subjected to stage-by-stage induction differentiation to obtain an endocrine cell cluster.

[0006] Preferably, step S1 also includes: The calcium alginate microspheres are mixed with a film-forming solution, the calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with a sodium alginate solution diluted ten times, and washed with a sodium chloride solution.

[0007] Preferably, the weight average molecular weight of the sodium alginate solution is in the range of 100,000-500,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is (1:2)-(2:1), and the mass concentration range is 1%-3%.

[0008] Preferably, the seeding density of the iPSC in the sodium alginate solution is in the range of (0.5-3)×10 6 cells / mL alginate solution.

[0009] Preferably, after the iPSC and the sodium alginate solution are evenly mixed, the sodium alginate mixture is passed through a high voltage electric field and added dropwise to the calcium chloride solidification solution for solidification, thereby obtaining calcium alginate microspheres containing iPSC, wherein the concentration of the calcium chloride solidification solution is 50 mM-200 mM; and the solidification time of the sodium alginate mixture is 10-30 minutes.

[0010] Preferably, the film-forming liquid is PLL, and the mass concentration percentage of the PLL solution is 0.05-0.2%; the time for mixing the calcium alginate microspheres and the PLL film-forming liquid to react is 5-15 minutes.

[0011] Preferably, iPSCs in microspheres are cultured with iPSC culture medium to form cell clusters. The in vitro culture time range is 1-10 days. DE differentiation induction culture medium, PP differentiation induction medium and EN differentiation induction medium are used in sequence to induce differentiation of iPSCs in calcium alginate microspheres or microcapsules.

[0012] In a second aspect of the present invention, a device for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells is provided.

[0013] The pancreatic islet differentiation device of microencapsulated induced pluripotent stem cells comprises a shell, a feeding mechanism, a motor and a stirring rod; the output end of the motor is connected to the stirring rod, and the stirring rod is rotatably connected to the shell; The feeding mechanism comprises a liquid outlet pipe, a pump body, a main pipe, four first solenoid valves, a second solenoid valve, a first tank body and four second tank bodies; The liquid outlet pipe is connected to the output end of the pump body, the liquid outlet pipe passes through the shell, the input end of the pump body is connected to one end of the main pipe, the other end of the main pipe is connected to the first tank body through the second solenoid valve, the main pipe is connected to four first solenoid valves, and the four first solenoid valves are respectively connected to four second tank bodies; The feeding mechanism further comprises an annular tube and a plurality of nozzles; the annular tube is connected to the liquid outlet pipe, and the plurality of nozzles are connected to the annular tube; An oxygen sensor is connected to the interior of the housing, a monitoring end of the oxygen sensor extends into the culture medium, and an oxygen injection mechanism is provided on the housing; The oxygen injection mechanism comprises an oxygen tank, a third solenoid valve and a pipe body; The oxygen tank is connected to the shell, the oxygen tank is connected to the third solenoid valve, the third solenoid valve is connected to the tube body, and the tube body passes through the shell.

[0014] Preferably, it further comprises a control mechanism, wherein the control mechanism comprises a first gear, a second gear, a shaft, an inner ring, a plurality of protrusions, a plate, a rotating plate, a rod, a torsion spring, an outer ring, two first blocks, a second block, a vertical cylinder, a first cylinder, a second cylinder, a first air outlet and a second air outlet; The first gear is connected to the first gear, the first gear is meshed with the second gear, the second gear is connected to the shaft body, the shaft body is rotatably connected to the outer shell, the plate body is connected to the shaft body, the plate body is connected to the rod body, the rotating plate is rotatably connected to the rod body, the torsion spring is sleeved on the rod body, the two ends of the torsion spring are respectively connected to the rod body and the rotating plate, the inner side of the inner ring is connected to the plurality of protrusions, the inner ring is connected to the vertical cylinder, the vertical cylinder is connected to the first cylinder, the first cylinder is rotatably connected to the second cylinder through a bearing, the second air outlet is processed on the first cylinder, the first air outlet is processed on the second cylinder, the tube body passes through the second cylinder, the second stopper is connected to the vertical cylinder, the two first stoppers are connected to the inner side of the outer ring, the outer ring is connected to the inner wall of the outer shell, and the second cylinder is connected to the inner wall of the outer shell.

[0015] Preferably, a discharge mechanism is provided inside the shell; The discharge mechanism includes an annular plate, a microporous filter, a first connecting rod, a convex block, a frame, a second connecting rod, a block, a sliding rod, a first spring, a support plate and a contact rod; The annular plate is connected to the inner wall of the outer shell, the annular plate is rotatably connected to the microporous filter, the microporous filter is rotatably connected to the first connecting rod, the first connecting rod is connected to the protrusion, the protrusion is slidably connected to the frame, the frame is connected to the block through the second connecting rod, the block is connected to the sliding rod, the sliding rod is slidably connected to the support plate, the support plate is connected to the inner wall of the outer shell, the stirring rod passes through the center of the support plate, the first spring is sleeved on the sliding rod, the two ends of the first spring are respectively connected to the bottom of the support plate and the top of the block, and the contact rod is arranged below the block.

[0016] One or more technical solutions provided in this application have at least the following technical effects or advantages: 1. The present invention provides a method and device for islet differentiation of microencapsulated induced pluripotent stem cells. The present invention prepares calcium alginate microspheres based on the high-voltage electrostatic droplet method, and forms a composite semipermeable membrane system through layer-by-layer self-assembly of film-forming liquids such as polylysine (PLL) and chitosan, systematically solving the key problems in the clinical transformation of iPSC-derived islet cells. Its core advantages are: using the biocompatibility and three-dimensional pore structure of sodium alginate to construct a bionic microenvironment to support intercellular connection and directional differentiation, significantly improving the functional maturity of islet-like cell clusters; achieving uniformity of microcapsule size and controllability of membrane thickness by optimizing the electrostatic molding process, and combining dynamic mass transfer design to ensure oxygen partial pressure and metabolic balance in the microcapsule, effectively maintaining cell survival rate; the composite semipermeable membrane achieves precise immune isolation through molecular weight screening and charge regulation, while blocking immune cell infiltration, ensuring efficient bidirectional transmission of small molecules such as glucose and insulin.

[0017] It should be understood that the contents described in the summary of the invention are not intended to limit the key or important features of the embodiments of the present invention, nor are they intended to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other features, advantages and aspects of the embodiments of the present invention will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. In the accompanying drawings, the same or similar reference numerals represent the same or similar elements, wherein: Figure 1 A schematic diagram of the three-dimensional connection structure of a pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 2 An exploded view of a pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 3 A partial cross-sectional view of a pancreatic islet differentiation device of microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 4 A schematic diagram showing the connection structure of the trigger mechanism and the discharge mechanism of the pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 5 A schematic diagram showing the connection structure of the stirring rod and the control mechanism of the pancreatic islet differentiation device of microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 6 A schematic diagram of the connection structure of the first gear, the second gear and the outer ring of the pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 7A schematic diagram of the connection structure of the first barrel, the first air outlet and the second barrel of the pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Figure 8 A schematic diagram of the connection structure of the second air outlet, the second stopper and the first stopper of the pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Fig. 9 A schematic diagram of the connection structure of a rotating plate, a rod body and a torsion spring of a pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Fig.10 A plan view showing a control mechanism of a pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention; Fig.11 A schematic diagram showing the connection structure of a trigger mechanism of a pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Fig.12 A schematic diagram showing the connection structure of a discharge mechanism of a pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Fig.13 A schematic diagram of the connection structure of the annular plate, the microporous filter and the first connecting rod of the pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown; Fig.14 A schematic diagram of the connection structure of the sealing plate, the platform and the inclined rods of the pancreatic islet differentiation device of microencapsulated induced pluripotent stem cells according to an embodiment of the present invention is shown.

[0019] Description of reference numerals: 1-housing, 2-motor, 3-feeding mechanism, 301-annular tube, 302-nozzle, 303-pump body, 304-main tube, 305-first solenoid valve, 306-second tank body, 307-first tank body, 308-liquid outlet pipe, 309-second solenoid valve, 4-oxygen injection mechanism, 401-oxygen tank, 402-tube body, 403-third solenoid valve, 5-stirring rod, 6-oxygen sensor, 7-trigger mechanism, 701-thread, 702-sleeve, 703-extension rod, 704-contact ring, 705-limiting sleeve, 706-third spring, 707-block, 708-electric push rod, 709-side plate, 710-plug plate, 711-first limiting rod, 712-second limiting rod, 713-second spring, 714-transmission rod, 715-slot, 716-blocking, 8-discharging mechanism, 801-annular plate, 802-microporous filter, 803-first connecting rod, 804-bump, 805-frame, 806-second connecting rod, 807-block, 808-sliding rod, 809-first spring, 810-support plate, 811-contact rod, 9-control mechanism, 901-first gear, 902-second gear, 903-shaft, 904-inner ring, 905-protrusion, 906-outer ring, 907-vertical cylinder, 908-first cylinder, 909-first air outlet, 910-second cylinder, 911-second air outlet, 912-second stopper, 913-first stopper, 914-plate body, 915-rotating plate, 916-rod body, 917-torsion spring, 10-discharge pipe, 11-sealing plate, 12-platform, 13-inclined rod, 14-processor. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In addition, the term "and / or" in this article is only a description of the association relationship between the associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0022] The pancreatic islet differentiation method of the microencapsulated induced pluripotent stem cells: Mix iPSCs with sodium alginate solution. iPSCs include but are not limited to human and various mammalian sources. A high-voltage electrostatic droplet generator can drop the mixed solution into a calcium chloride solution for solidification. Calcium alginate microspheres containing iPSCs are formed. Sodium alginate is composed of M unit α-L-mannuronic acid and G unit β-D-guluronic acid. These two units have certain differences in structure and properties, which together determine the various physical and chemical properties and biological functions of sodium alginate. The G / M value is the ratio of guluronic acid (G) to mannuronic acid (M) in the sodium alginate molecule. The main structure of the high-voltage electrostatic droplet generator includes but is not limited to a needle and a high-voltage electric field. Sodium alginate solution is contained inside the needle. The sodium alginate solution dripping from the needle will pass through the high-voltage electric field and fall into the calcium chloride solution. Under the action of the high-voltage electric field, the sodium alginate solution forms a Taylor Cone at the tip of the needle. The Taylor Cone is a unique phenomenon caused by the electric field force acting on the liquid surface. Its formation indicates that the liquid is about to be electrosprayed. And thus, charged sodium alginate droplets are generated. At the same time as the Taylor cone is formed, the high-voltage electric field will further affect the sodium alginate solution, causing the sodium alginate solution to spray out fine droplets from the tip of the Taylor cone. These droplets are charged due to the action of the electric field. Due to the Coulomb repulsion of the same poles of the surface charge of the droplet, a mutually repulsive force will be generated between the droplets. At this time, the electric field force and the electrostatic repulsion work together to cause the originally larger droplets to split into multiple smaller and more uniform droplets. The charged droplets move downward in the direction of the electric field under the action of the electric field force. Since these droplets carry the same charge, there is Coulomb repulsion and electrostatic repulsion between them. This repulsion keeps the droplets at a certain distance during movement and prevents the droplets from agglomerating. This process ensures further dispersion of the droplets, making them evenly distributed in space. The electric field force not only pushes the droplets downward, but also guides the trajectory of the droplets. The small droplets fall into the beaker of calcium chloride solution through the high-voltage electric field. When these charged droplets fall into the beaker containing calcium chloride solution, the calcium ions Ca²⁺ in the calcium chloride solution undergo an ion exchange reaction with the sodium ions Na⁺ in the sodium alginate. This reaction results in the formation of calcium alginate Ca-alginate, a water-insoluble gel. This reaction causes the surface of the droplets to solidify rapidly to form stable microcapsules. The size of the needle tip on the needle affects the particle size of the calcium alginate microspheres.

[0023] The obtained calcium alginate microspheres are directly cultured, or the calcium alginate microspheres are mixed with PLL or chitosan film-forming solution to form a membrane. The calcium alginate microspheres after film formation are washed with 0.9% sodium chloride solution, and then the calcium alginate microspheres are mixed with a ten-fold diluted low-concentration sodium alginate solution to neutralize excess charges, and then washed again with 0.9% sodium chloride solution to form microcapsules that block immune cells and allow nutrients to pass through, and then cell culture is performed; iPSC culture medium is used to culture iPSC in calcium alginate microspheres or microcapsules to form cell clusters. iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The size of the cell clusters formed in the three-dimensional microcapsules or microspheres ranges from 30-100 μm.

[0024] The iPSCs in the calcium alginate microspheres or microcapsules are sequentially induced to differentiate using DE differentiation induction medium, PP differentiation induction medium and EN differentiation induction medium. The composition of the DE differentiation induction medium includes: iPSC differentiation into DE stage: Culture medium composition: Basic culture medium: DMEM / F12 + 0.1%-0.5% (volume percentage, v / v) bovine serum albumin (BSA) + 25-100 ng / mL Activin A + 1-5 μM CHIR99021 + 1-5 mM lithium chloride (LiCl).

[0025] Duration: 1 day Description: BSA is added to stabilize cell adhesion, ActivinA induces endoderm differentiation signals, CHIR99021 inhibits GSK3β to promote pluripotency maintenance, and LiCl activates the Wnt / β-catenin pathway to synergistically regulate DE formation.

[0026] PP induced differentiation stage: Culture medium composition: Basal culture medium DMEM + 0.5%-2% (volume percentage, v / v) B27 (nerve growth factor supplement) + 1-5 μM retinoic acid (Retinoic Acid) + 5-15 μM SB431542 (BMP signal inhibitor) + 5-15 ng / mL bFGF (basic fibroblast growth factor) + 200-300 nM KAAD-cyclopalmine (Wnt pathway activator).

[0027] Duration of action: 6 days Description: B27 provides nutrition for neural differentiation, Retinoic Acid promotes forebrain development, SB431542 inhibits BMP signaling, bFGF maintains cell proliferation, and KAAD-cyclopalmine enhances Wnt pathway activity to induce foregut-type progenitor cells.

[0028] EN induced differentiation stage: Phase 1 (first 4 days) Culture medium composition: Basal culture medium DMEM + 0.5%-2% (volume percentage, v / v) B27 + 25-100 μg / mL ascorbic acid + 1-5 μM Dorsomorphin + 5-15 μM SB431542 (BMP / Smad and TGF-β signaling inhibitor) + 5-15 μM DAPT (γ-secretase inhibitor).

[0029] Duration of action: 4 days Description: Ascorbic Acid acts as an antioxidant to support cellular metabolism, Dorsomorphin selectively inhibits BMP receptors, DAPT blocks Notch signaling, and SB431542 dually inhibits BMP / Smad and TGF-β / ALK4 / 5 pathways, synergistically activating EN lineage gene expression.

[0030] Phase 2 (next 4 days): Culture medium composition: Basal culture medium CMRL1066 + 0.5%-2% (volume percentage, v / v) Penicillin-Streptomycin + 0.5%-2% (volume percentage, v / v) B27 + 20-50 mM Glucose + 200-500 μM Dibutyryl-cAMP + 5-15 μM Exendin-4 + 1-5 μMDorsomorphin + 5-15 μM SB431542 + 5-15 mM Nicotinamide + 20-100 μg / mL Ascorbic Acid.

[0031] Duration of action: 4 days Description: CMRL1066 optimizes energy metabolism, Glucose concentration gradient simulates the embryonic development microenvironment, Dibutyryl-cAMP activates the cAMP / PKA pathway to promote neural crest differentiation, Exendin-4 enhances GLP-1 signaling, Nicotinamide mediates epigenetic modification through SIRT1, and multiple components synergistically induce EN neuron maturation.

[0032] The volume ratio of calcium alginate microspheres or microcapsules to culture medium is in the range of 1:10-1:4. Through the culture in multiple culture media, iPSCs are differentiated into DE cells, PP cells and EN cells in sequence, and finally EN cell clusters embedded in calcium alginate microspheres or microcapsules with glucose-responsive insulin secretion function are obtained.

[0033] iPSCs have the potential for pluripotent differentiation, and need to be directed to differentiate through culture media containing different cytokines. Differentiation of iPSCs into pancreatic islet cells with insulin secretion function requires multiple stages, namely DE, PP, and EN. Therefore, different culture media are used in a three-dimensional microcapsule environment to differentiate iPSCs into cells of various stages and finally into pancreatic islet cells.

[0034] Embodiment 1: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 200,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 2:1, and the mass concentration range is 1.5%. The seeding density range of the iPSCs in the sodium alginate solution is (1.5)×10 6 cells / ml sodium alginate solution. After the iPSCs and the sodium alginate solution were evenly mixed, the mixture of cells and sodium alginate was added to the calcium chloride solidified solution through the needle tip using a high-voltage electrostatic droplet generator. The voltage of the high-voltage electric field was 6 kV, the inner diameter of the needle tip was 0.18 mm, and the distance from the needle tip to the liquid surface was 3 cm. Calcium alginate microspheres containing iPSCs were obtained, and the concentration of the calcium chloride solidified solution was 100 mM; the solidification time was 20 minutes. Calcium alginate microspheres containing iPSCs were formed.

[0035] The calcium alginate microspheres are mixed with a film-forming solution, the film-forming solution is PLL, and the mass concentration of the PLL solution is 0.1%; the reaction time is 5 minutes. The calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with 0.15% sodium alginate, reacted for 5 minutes, washed with a sodium chloride solution, and then performed step S2; S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using an iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 5 days.

[0036] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microcapsules ranges from 30 to 70 μm.

[0037] Through experimental verification and theoretical analysis, the key parameters in this technical solution have the following synergistic mechanism on microsphere preparation and biocompatibility: Control of polymer material properties: Sodium alginate molecular weight (2.0×10 5) shows a significant correlation with the rheological properties of the solution: when the molecular weight is lower than this threshold, the solution viscoelasticity is insufficient to maintain the stability of the microsphere morphology (sphericity parameter <90%); while too high a molecular weight causes the system viscosity to exceed 3000 mPa·s, significantly affecting the uniformity of cell dispersion (coefficient of variation >15%) and microsphere forming efficiency (needle clogging rate >30%). By constructing a mathematical model of the G / M ratio and thermal stability, it was found that when the G unit accounted for ≥65%, the molecular weight retention rate of the solution under 121°C wet heat sterilization conditions could reach 95%±2%, effectively maintaining the stability of the microsphere preparation process.

[0038] Optimization of preparation process parameters: The optimal process parameter combination was determined by a sixth-order orthogonal experiment: under the action of a pulsed electric field with an electric field strength of 6 kV / mm, with the parameter settings of a needle inner diameter of 0.18±0.01 mm and a liquid level spacing of 30±1 mm, the microsphere particle size distribution CV value ≤5% (D50=350±10 μm) can be achieved, while ensuring a cell survival rate of ≥98%. A gradient solidification strategy was adopted. In the first stage, 100mM CaCl2 solution was cross-linked for 20 minutes to achieve complete solidification (swelling rate ≤5%). In the second stage, a ten-fold diluted sodium alginate solution was used for secondary coating for 5 minutes, which reduced the surface Zeta potential from -35 mV to -8 mV, significantly reducing the macrophage activation rate (the proportion of CD68+ cells <5%).

[0039] Mechanism of improving biocompatibility: The thickness of the PLL membrane layer was regulated by surface modification technology: when the PLL concentration was 0.1% and the action time was 5 minutes, the membrane thickness was stable at 15±2 nm (measured by AFM), which could effectively block macromolecular immune factors such as IgG (retention rate>99%), while maintaining permeability to nutrients such as glucose (permeability≥95%). In vitro simulation experiments showed that the secretion of foreign body reaction-related factors (TNF-α, IL-6) of microspheres under this parameter combination was 80%±5% lower than that of the control group 72 hours after transplantation.

[0040] This technical solution achieves a synergistic improvement in microsphere structural parameters (sphericity > 95%, pore size 30±5 nm), cell activity (proliferation rate ≥ 300% after 7 days of culture) and immune isolation effect (number of infiltrating lymphocytes < 50 / mm²) through systematic optimization of material properties, preparation process and biological effects, providing a reliable technical guarantee for cell transplantation therapy.

[0041] Embodiment 2: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 100,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 1:1, and the mass concentration range is 1%. The seeding density range of the iPSCs in the sodium alginate solution is (0.5)×10 6 cells / ml sodium alginate solution. After iPSCs and sodium alginate solution are evenly mixed, a high-voltage electrostatic droplet generator is used to drop the mixture of cells and sodium alginate into the calcium chloride solidification liquid through a needle tip. The voltage of the high-voltage electric field is 4 kV, the inner diameter of the needle tip is 0.06 mm, and the distance from the needle tip to the liquid surface is 2 cm. Calcium alginate microspheres containing iPSCs are obtained. The concentration of the calcium chloride solidification liquid is 150 mM; the solidification time is 10 minutes. Calcium alginate microspheres containing iPSCs are formed.

[0042] The calcium alginate microspheres are mixed with a film-forming solution, wherein the film-forming solution is PLL and the mass concentration percentage of the polylysine solution is 0.05%; the reaction time is 10 minutes. The calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with 0.1% sodium alginate, reacted for 5 minutes, washed with a sodium chloride solution, and then proceeded to step S2; S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 10 days.

[0043] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microcapsules ranges from 70 to 100 μm.

[0044] Embodiment three: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 500,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 1:2, and the mass concentration range is 3%. The seeding density range of the iPSCs in the sodium alginate solution is (3)×10 6 After iPSCs and sodium alginate solution were mixed evenly, the mixture of cells and sodium alginate was added to the calcium chloride solidification solution through the needle tip using a high-voltage electrostatic droplet generator. The voltage of the high-voltage electric field was 9 kV, the inner diameter of the needle tip was 0.25 mm, and the distance from the needle tip to the liquid surface was 5 cm. Calcium alginate microspheres containing iPSCs were obtained. The concentration of the calcium chloride solidification solution was 200 mM; the solidification time was 30 minutes. Calcium alginate microspheres containing iPSCs were formed.

[0045] The calcium alginate microspheres are mixed with a film-forming solution, wherein the film-forming solution is PLL and the mass concentration percentage of the polylysine solution is 0.2%; the reaction time is 15 minutes. The calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with 0.3% sodium alginate, reacted for 5 minutes, washed with a sodium chloride solution, and then proceeded to step S2; S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using an iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 1 day.

[0046] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microcapsules ranges from 70 to 90 μm.

[0047] Embodiment 4: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 200,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 2:1, and the mass concentration range is 1.5%. The seeding density range of the iPSCs in the sodium alginate solution is (1.5)×10 6 cells / ml sodium alginate solution. After iPSCs and sodium alginate solution were evenly mixed, the mixture of cells and sodium alginate was added to the calcium chloride solidification solution through the needle tip using a high-voltage electrostatic droplet generator. The voltage of the high-voltage electric field was 4 kV, the inner diameter of the needle tip was 0.06 mm, and the distance from the needle tip to the liquid surface was 2 cm. Calcium alginate microspheres containing iPSCs were obtained. The concentration of the calcium chloride solidification solution was 50 mM; the solidification time was 10 minutes. Calcium alginate microspheres containing iPSCs were formed.

[0048] The calcium alginate microspheres are mixed with a film-forming solution, the film-forming solution is a chitosan solution, and the mass concentration of the chitosan solution is 1%; the reaction time is 30 minutes. The calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with 0.15% sodium alginate, reacted for 5 minutes, washed with a sodium chloride solution, and then carried out step S2; S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 10 days.

[0049] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microcapsules ranges from 70 to 80 μm.

[0050] Embodiment five: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 500,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 1:2, and the mass concentration range is 3%. The seeding density range of the iPSCs in the sodium alginate solution is (3)×10 6 cells / ml sodium alginate solution. After iPSCs and sodium alginate solution were evenly mixed, the mixture of cells and sodium alginate was added to the calcium chloride solidification solution through the needle tip using a high-voltage electrostatic droplet generator. The voltage of the high-voltage electric field was 9 kV, the inner diameter of the needle tip was 0.25 mm, and the distance from the needle tip to the liquid surface was 5 cm. Calcium alginate microspheres containing iPSCs were obtained. The concentration of the calcium chloride solidification solution was 200 mM; the solidification time was 30 minutes. Calcium alginate microspheres containing iPSCs were formed.

[0051] The calcium alginate microspheres are mixed with a film-forming solution, the film-forming solution is a chitosan solution, and the mass concentration of the chitosan solution is 3%; the reaction time is 5 minutes. The calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with 0.3% sodium alginate, reacted for 5 minutes, washed with a sodium chloride solution, and then carried out step S2; S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using an iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 1 day.

[0052] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microcapsule ranges from 50 to 80 μm.

[0053] Embodiment six: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 500,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 1:2, and the mass concentration range is 3%. The seeding density range of the iPSCs in the sodium alginate solution is (3)×10 6 cells / ml sodium alginate solution. After iPSCs and sodium alginate solution are evenly mixed, a high-voltage electrostatic droplet generator is used to drop the mixture of cells and sodium alginate into the calcium chloride solidification liquid through a needle tip. The voltage of the high-voltage electric field is 9 kV, the inner diameter of the needle tip is 0.25 mm, and the distance from the needle tip to the liquid surface is 5 cm. Calcium alginate microspheres containing iPSCs are obtained. The concentration of the calcium chloride solidification liquid is 200 mM; the solidification time is 30 minutes. Calcium alginate microspheres containing iPSCs are formed.

[0054] The calcium alginate microspheres are mixed with a film-forming solution, the film-forming solution is a chitosan solution, and the mass concentration of the chitosan solution is 2%; the reaction time is 23 minutes. The calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with 0.3% sodium alginate, reacted for 5 minutes, washed with a sodium chloride solution, and then carried out step S2; S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using an iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 5 days.

[0055] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microcapsules ranges from 40 to 80 μm.

[0056] Embodiment seven: S1: iPSCs are mixed with a sodium alginate solution, wherein the weight average molecular weight (Mw) of the sodium alginate solution is 500,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is 1:2, and the mass concentration range is 3%. The seeding density range of the iPSCs in the sodium alginate solution is (3)×10 6 After iPSCs and sodium alginate solution were mixed evenly, the mixture of cells and sodium alginate was added to the calcium chloride solidification solution through the needle tip using a high-voltage electrostatic droplet generator. The voltage of the high-voltage electric field was 9 kV, the inner diameter of the needle tip was 0.25 mm, and the distance from the needle tip to the liquid surface was 5 cm. Calcium alginate microspheres containing iPSCs were obtained. The concentration of the calcium chloride solidification solution was 200 mM; the solidification time was 30 minutes. Calcium alginate microspheres containing iPSCs were formed.

[0057] S2: Cultivate the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters. Cultivate the iPSCs in the microspheres using an iPSC culture medium to form cell clusters. The iPSC culture medium is a culture medium that can maintain the stemness of iPSCs. The in vitro culture time is 1 day.

[0058] S3: The cell cluster obtained in step S2 is subjected to stage-induced differentiation to obtain an endocrine cell cluster, wherein the size of the microspheres ranges from 30 to 40 μm.

[0059] like Figures 1 to 14As shown, the present invention also provides a microencapsulated induced pluripotent stem cell islet differentiation device for implementing the above method, comprising a housing 1, a feeding mechanism 3, a motor 2 and a stirring rod 5. The output end of the motor 2 is connected to the stirring rod 5 through a coupling, and the stirring rod 5 is rotatably connected to the housing 1 through a deep groove ball bearing. The feeding mechanism 3 includes a liquid outlet pipe 308, a pump body 303, a main pipe 304, four first solenoid valves 305, a first tank body 307 and four second tank bodies 306. The liquid outlet pipe 308 is sealed and connected to the output end of the pump body 303 through a 316L stainless steel flange. The liquid outlet pipe 308 passes through the PTFE anti-wear bushing on the side wall of the shell 1. The input end of the pump body 303 is connected to one end of the main pipe 304 through a quick-plug connector. The other end of the main pipe 304 is connected to the bottom outlet of the first tank body 307 through the second solenoid valve 309. The main pipe 304 is connected to the inlet ends of the four first solenoid valves 305. The outlet ends of the four first solenoid valves 305 are respectively connected to the top outlets of the four second tank bodies 306 through hoses.

[0060] In actual use, the processor 14 controls the opening and closing sequence of the four first solenoid valves 305 in sequence according to the preset differentiation stage program: close three first solenoid valves 305 and open one of them, the pump body 303 extracts the culture medium in the corresponding second tank body 306 at a flow rate of 200 ml / min through the main pipe 304, and injects it into the shell 1 through the liquid outlet pipe 308; after the current stage of cultivation is completed, all first solenoid valves 305 are closed and the second solenoid valve 309 is opened, and the pump body 303 extracts 0.9% saline in the first tank body 307 at a flow rate of 300 ml / min to flush the inside of the shell 1 to avoid the residual culture medium from interfering with the subsequent stage. By storing the culture medium of different induction stages in separate tanks and cooperating with programmed control, fully automatic multi-stage induction of stem cell differentiation is achieved; the saline cleaning mechanism effectively reduces cross contamination and improves the success rate of cell differentiation.

[0061] In this embodiment, the feeding mechanism 3 further includes an annular tube 301 and a plurality of nozzles 302. The annular tube 301 is sealedly connected to the liquid outlet pipe 308 via an M12×1.5 thread, and the plurality of nozzles 302 are connected to the annular tube 301 via a quick-release stainless steel joint. The nozzles 302 adopt a fan-shaped atomization structure, with a spray angle of 60°, an aperture of 0.5 mm, and are fixed to the circumference of the annular tube 301 at an inclination of 35°.

[0062] In actual use, after the pump body 303 delivers the culture medium or cleaning liquid to the annular tube 301, the liquid forms an atomized liquid film through 12 circumferentially evenly distributed nozzles 302, covering the microcapsules on the microporous filter 802 along the tangent direction of the inner wall of the shell 1; the inclined design of the nozzle 302 reduces the liquid flow rate to 0.2 m / s, avoiding direct impact on the microcapsule and causing structural damage. The annular spraying design achieves uniform distribution of the culture medium, reduces local concentration gradient differences, and ensures the uniformity of stem cell differentiation in the microcapsule; the inclined nozzle reduces the influence of fluid shear force on the integrity of the microcapsule.

[0063] In this embodiment, an oxygen sensor 15 is connected to the interior of the housing 1, and the monitoring end of the oxygen sensor 15 extends to 3 cm below the liquid surface of the culture medium, which can ensure the normal monitoring of the oxygen concentration in the culture medium. An oxygen injection mechanism 4 is provided on the housing 1. The oxygen injection mechanism 4 includes an oxygen tank 401, a third solenoid valve 403 and a tube body 402. The oxygen tank 401 is fixed to the side wall of the housing 1 by M8 bolts, the outlet of the oxygen tank 401 is connected to the inlet of the third solenoid valve 403 through a high-pressure hose, the outlet of the third solenoid valve 403 is connected to the tube body 402 by a ferrule joint, and the tube body 402 extends through the rubber sealing ring at the top of the housing 1 to 2 cm above the liquid surface.

[0064] In actual use, the oxygen sensor 15 monitors the dissolved oxygen concentration of the culture medium in real time. When the detected value is lower than 20% saturation, the processor 14 synchronously opens the third solenoid valve 403 and starts the motor 2; the motor 2 drives the stirring rod 5 to rotate at a speed of 60 rpm, and at the same time, through the 3:1 reduction meshing transmission of the first gear 901 and the second gear 902, drives the rotating plate 915 to periodically move the protrusion 905 at a speed of 20 rpm, so that the second air outlet 911 on the vertical cylinder 907 is aligned with the first air outlet 909 of the second cylinder 910 every 5 seconds, and the oxygen in the oxygen tank 401 is released into the housing 1 at a flow rate of 0.1L / min. The stirring rod 5 is arranged above the microporous filter 802. When the stirring rod 5 rotates, the culture medium will flow and will not contact the microcapsule, which can prevent the microcapsule from being damaged. The coordinated control of stirring and oxygen supplementation is realized by setting a gear linkage mechanism. The pulsed oxygen supply is combined with stirring to improve the dissolved oxygen efficiency and avoid cell peroxidation damage caused by continuous oxygen supply; the closed-loop control of oxygen concentration ensures the stability of the differentiation microenvironment.

[0065] In this embodiment, a control mechanism 9 is also included, and the control mechanism 9 includes a first gear 901, a second gear 902, a shaft 903, an inner ring 904, a plurality of protrusions 905, a plate 914, a rotating plate 915, a rod 916, a torsion spring 917, an outer ring 906, two first blocks 913, a second block 912, a vertical cylinder 907, a first cylinder 908, a second cylinder 910, a first air outlet 909 and a second air outlet 911. The first gear 901 is keyed to the output shaft of the motor 2, the first gear 901 is meshed with the second gear 902, the second gear 902 is connected to the shaft 903 through a flat key, the shaft 903 is rotatably connected to the housing 1 through a double-row angular contact ball bearing, the plate 914 is welded to the end of the shaft 903, the plate 914 is fastened to the rod 916 through a thread, the rotating plate 915 is rotatably connected to the rod 916 through a pin, the torsion spring 917 is sleeved on the rod 916 and limited by the slot, the two ends of the torsion spring 917 are respectively engaged with the grooves of the rod 916 and the rotating plate 915, and the inner side of the inner ring 904 is fixed by bolts with four height 2 mM protrusion 905, the inner ring 904 is connected to the vertical cylinder 907 by welding, the vertical cylinder 907 is connected to the first cylinder 908 by a flange, the first cylinder 908 is rotatably connected to the second cylinder 910 by a deep groove ball bearing, the second air outlet 911 is processed on the circumference of the first cylinder 908, the aperture is 1 mm, the spacing is 90°, the first air outlet 909 is processed on the circumference of the second cylinder 910, the aperture is 1 mm, the spacing is 90°, the tube 402 passes through the sealing stuffing box at the center of the second cylinder 910, the second stopper 912 is welded to the outer wall of the vertical cylinder 907, the two first stoppers 913 are fixed to the inner side of the outer ring 906 by screws, the outer ring 906 is connected to the inner wall of the outer shell 1 by welding, and the second cylinder 910 is fixed to the inner wall of the outer shell 1 by bracket bolts.

[0066] In actual use, when the motor 2 drives the stirring rod 5 to rotate forward, the first gear 901 drives the second gear 902 to rotate at a reduced speed of 60 rpm at a speed of 180 rpm, and the shaft 903 drives the rod body 916 and the rotating plate 915 to rotate synchronously through the plate body 914; the rotating plate 915 swings outward under the action of centrifugal force and moves the protrusion 905, and the torsion spring 917 accumulates elastic potential energy; when the rotating plate 915 is separated from the protrusion 905, the torsion spring 917 releases the potential energy to reset the rotating plate 915, and the inner ring 904 drives the vertical cylinder 907 to rotate 45° until the second stop block 912 contacts the first stop block 913, at which time the first air outlet 909 is aligned with the second air outlet 911 to realize oxygen release; when rotating in the reverse direction, the second stop block 912 contacts another first stop block 913, and the air holes are dislocated by 90° to close the oxygen passage. The mechanical intermittent oxygen supply mechanism is used to avoid reliability problems caused by long-term high-frequency operation of electronic components; and the limiting design of the first stopper 913 and the second stopper 912 can control the oxygen release time to ensure the dynamic balance of dissolved oxygen.

[0067] In this embodiment, a discharge mechanism 8 is provided inside the housing 1. The discharge mechanism 8 includes an annular plate 801, a microporous filter 802, a first connecting rod 803, a protrusion 804, a frame 805, a second connecting rod 806, a block 807, a sliding rod 808, a first spring 809, a support plate 810 and a contact rod 811. The annular plate 801 is connected to the inner wall of the housing 1 by welding, and the annular plate 801 is connected to the microporous filter 802 by hinge rotation. The microporous filter 802 is made of polyethersulfone, which is a high-performance thermoplastic engineering plastic. The pore size is 20μm and the diameter is 30cm. The microporous filter 802 is rotatably connected to the first connecting rod 803 through a pin shaft. The first connecting rod 803 is fixed to the protrusion 804 by bolts. The protrusion 804 can slide inside the frame 805. The frame 805 is welded to the block 807 through the second connecting rod 806. The block 807 is connected to the slide rod 808 by threads. The slide rod 808 has a diameter of 8 mm and a chrome-plated surface. The slide rod 808 is slidably connected to the support plate 810 through a linear bearing. The support plate 810 is fixed to the inner wall of the shell 1 by bolts. The stirring rod 5 passes through the center of the support plate 810. The first spring 809 is sleeved on the slide rod 808. The elastic coefficient of the first spring 809 is 5N / mM. The two ends are fixed to the bottom of the support plate 810 and the top of the block 807 by slots respectively. The contact rod 811 is welded to the bottom of the block 807.

[0068] In actual use, after the cultivation is completed, the processor 14 starts the electric push rod 708 to push the plug plate 710 into the slot 715, the sleeve 702 stops rotating and moves upward along the thread 701; the sleeve 702 pushes the block 807 upward through the contact rod 811, the second connecting rod 806 drives the frame 805 and the protrusion 804 to slide, and the first connecting rod 803 drives the microporous filter 802 to flip around the annular plate 801 by 15°-20°, and the microcapsule is separated from the microporous filter 802 under gravity and saline flushing; after the discharge is completed, the electric push rod 708 is reset, the first spring 809 pushes the block 807 upward, and the microporous filter 802 returns to a horizontal state. The flip-type discharge design is set to prevent the microcapsules from blocking the filter pores, and the discharge is achieved in combination with saline flushing; the first spring 809 relies on the reset mechanism generated by its own elastic deformation to ensure the position accuracy of the microporous filter 802 and ensure the reliability of multiple uses.

[0069] In this embodiment, a trigger mechanism 7 is disposed inside the housing 1 , and includes a thread 701 , a sleeve 702 , an electric push rod 708 , a side plate 709 , a first limiting rod 711 , a plug plate 710 , a slot 715 , a second limiting rod 712 , and a second spring 713 . The sleeve 702 is fixed to the contact rod 811 by bolts, the thread 701 is processed on the stirring rod 5, the pitch is 5 mm, the sleeve 702 is connected to the thread 701 by a trapezoidal thread, the electric push rod 708 is fixed to the inner wall of the housing 1 by a bracket bolt, the output end of the electric push rod 708 is connected to the side plate 709 by a flange, the side plate 709 and the first limit rod 711 are slidably connected by a linear guide rail, the first limit rod 711 is welded to the plug plate 710, the plug plate 710 and the second limit rod 712 are connected by threads, the second limit rod 712 and the side plate 709 are slidably connected by a linear bearing, the second spring 713 is sleeved on the second limit rod 712, the elastic coefficient of the second spring 713 is 3N / mM, the two ends are respectively fixed to the side plate 709 and the plug plate 710 by a slot, the slot 715 is processed on the support plate 810, and the size is matched with the plug plate 710 clearance (clearance ≤0.1 mm).

[0070] In actual use, during the cultivation stage, the electric push rod 708 contracts to disengage the plug plate 710 from the slot 715, and the sleeve 702 rotates freely with the stirring rod 5; when the discharge stage starts, the electric push rod 708 extends to push the side plate 709 downward, and the plug plate 710 is inserted into the slot 715 under the action of the second spring 713, and the sleeve 702 stops rotating and presses down along the thread 701, triggering the action of the discharge mechanism 8; if the plug plate 710 is not aligned with the slot 715, the second spring 713 is compressed, and the stirring rod 5 automatically pops in and locks when it rotates to the aligned position of the slot 715. The elastic matching design of the plug plate 710 and the slot 715 realizes automatic alignment and locking to avoid mechanical jamming; the double limit rod structure ensures the precise movement trajectory of the plug plate 710 and improves the stability of the system.

[0071] In this embodiment, the bottom opening of the housing 1 is connected to a sealing plate 11, the bottom of the sealing plate 11 is connected to a discharge pipe 10, and the trigger mechanism 7 further includes an extension rod 703, a contact ring 704, a transmission rod 714, and a plug 716. The extension rod 703 is connected to the sleeve 702 by welding, a nylon roller is provided at the end of the extension rod 703, and the contact ring 704 can be pressed, the contact ring 704 and the transmission rod 714 are connected by threads, the transmission rod 714 passes through the center hole of the annular plate 801 and is welded to the plug 716, and the plug 716 is a conical rubber plug, which fits the conical sealing surface of the inner wall of the sealing plate 11.

[0072] In actual use, when the sleeve 702 is pressed down, the nylon roller at the end of the extension rod 703 abuts against the contact ring 704 and pushes the transmission rod 714 downward, the plug 716 is separated from the inlet of the discharge pipe 10, and the culture medium or microcapsule is discharged through the discharge pipe 10; when the sleeve 702 is reset, the third spring 706 pushes the transmission rod 714 upward, and the plug 716 reseals the discharge pipe 10. The seamless switching between the culture and discharge states is achieved by setting the linkage discharge control; the conical plugging structure ensures the sealing reliability and avoids liquid leakage during the culture stage.

[0073] In this embodiment, the trigger mechanism 7 further includes a limit sleeve 705, a stopper 707 and a third spring 706. The transmission rod 714 is slidably connected to the limit sleeve 705 through a linear bearing, the stopper 707 is welded to the end of the transmission rod 714, and the third spring 706 is sleeved on the transmission rod 714. The elastic coefficient of the third spring 706 is 8N / mM, and both ends are fixed to the limit sleeve 705 and the stopper 707 through slots.

[0074] In actual use, when the transmission rod 714 moves downward, the third spring 706 is stretched to store energy. When the sleeve 702 is reset, the third spring 706 releases elastic potential energy to push the transmission rod 714 and the plug 716 to reset quickly, ensuring that the plug 716 normally closes the opening position of the sealing plate 11.

[0075] In this embodiment, the top of the plug 716 is connected to a platform 12, and the bottom of the microporous filter 802 is connected to an inclined rod 13, and the bottom of the inclined rod 13 contacts the top of the platform 12. The platform 12 is a 316L stainless steel disc with a diameter of 5 cm and a Teflon coating on the surface; the inclined rod 13 is a 304 stainless steel rod with a diameter of 6 mm, and the contact surface of the end with the platform 12 is sprayed with a tungsten carbide wear-resistant coating.

[0076] In actual use, when the microporous filter 802 turns over, the inclined rod 13 rotates with the microporous filter 802 and presses down the platform 12, and the platform 12 drives the plug 716 to move down to open the discharge pipe 10; during the reset process of the microporous filter 802, the inclined rod 13 is separated from the platform 12, and the third spring 706 pushes the plug 716 to reseal the discharge pipe 10 and the sealing plate 11. The discharge and the microporous filter 802 are synchronously controlled through the mechanical linkage structure to ensure that there is no delay in the discharge process; and the contact surface of the inclined rod 13 and the platform 12 is coated with a wear-resistant coating to reduce long-term wear.

[0077] In general: after the user places the microcapsules or microspheres on the microporous filter 802 of the outer shell 1, the processor 14 controls all four first solenoid valves 305 to close and only opens one designated first solenoid valve 305, so that the culture medium in the first tank 307 passes through the pump body 303 in turn through the liquid outlet pipe 308 and the annular tube 301, and is finally evenly sprayed from the multiple nozzles 302 of the annular tube 301 into the outer shell 1 to complete the culture medium injection; after a single injection, pause for cell culture. During the culture, the oxygen sensor 15 monitors the dissolved oxygen content in real time. If the DO is lower than the set threshold, the processor 14 automatically triggers the oxygen injection mechanism 4 to work: the oxygen in the oxygen tank 401 is transported to the second cylinder 910 through the third solenoid valve 403 and the tube 402, and the oxygen is supplied to the housing 1 through the second air outlet 911 and the first air outlet 909 when corresponding. At the same time, the motor 2 drives the stirring rod 5 to stir intermittently (rotation speed 50rpm, 30 seconds each time) to improve the dissolved oxygen efficiency. It is worth noting that the motor 2 stirs the stirring rod 5 in a forward and reverse manner when driving the stirring rod 5 to rotate, and the trigger mechanism 7 will not work at this time. After the culture is completed, the motor 2 is started to drive the stirring rod 5 to rotate, and the side plate 709 and the plug plate 710 are driven downward by the electric push rod 708. When the plug plate 710 is not aligned with the slot 715, the second spring 713 is compressed. After alignment, the second spring 713 is released to insert the plug plate 710 into the slot 715. At this time, the sleeve 702 changes from rotating with the stirring rod 5 to linear motion; when the sleeve 702 moves downward, its extension rod 703 triggers the contact ring 704, and drives the plug 716 to move downward through the transmission rod 714 to discharge the culture medium (because the inner hole of the sealing plate 11 is smaller than the inner diameter of the discharge pipe 10 to achieve sealing), and the third spring 706 pushes the contact ring 704 to restore when resetting. In the subsequent cleaning stage, the processor 14 closes all the first solenoid valves 305 and opens the second solenoid valve 309. The 0.9% concentration of physiological saline in the first tank body 307 first passes through the main pipe 304, the pump body 303 and the liquid outlet pipe 308, etc., and finally sprays out from multiple nozzles 302, completing the cleaning of the entire pipeline. After the final differentiation is completed, the electric push rod 708 pushes the plug plate 710 down again to trigger the sleeve 702 to move up. When the contact rod 811 touches the block 807, the frame 805 and the second connecting rod 806 drive the protrusion 804 and the first connecting rod 803 to move up. At the same time, the microporous filter 802 flips and flushes the microcapsules with saline. The inclined rod 13 links the platform 12 and the plug 716 to move down to complete the automatic discharge of the microcapsules.

[0078] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells, characterized in that: The steps include: S1: Mix iPSCs with sodium alginate solution to form calcium alginate microspheres containing iPSCs; S2: culturing the iPSCs in the calcium alginate microspheres obtained in step S1 into cell clusters; S3: The cell cluster obtained in step S2 is subjected to stage-by-stage induction differentiation to obtain an endocrine cell cluster.

2. The method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells according to claim 1, characterized in that: Step S1 also includes: The calcium alginate microspheres are mixed with a film-forming solution, the calcium alginate microspheres after film formation are washed with a sodium chloride solution, mixed with a sodium alginate solution diluted ten times, and washed with a sodium chloride solution.

3. The pancreatic islet differentiation method of microencapsulated induced pluripotent stem cells according to claim 1 or 2, characterized in that: The weight average molecular weight of the sodium alginate solution is in the range of 100,000-500,000 Daltons, the ratio of guluronic acid to mannuronic acid in the sodium alginate molecule is (1:2)-(2:1), and the mass concentration range is 1%-3%.

4. The method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells according to claim 1 or 2, characterized in that: The seeding density of iPSC in sodium alginate solution ranges from (0.5-3)×10 6 cells / mL alginate solution.

5. The method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells according to claim 1 or 2, characterized in that: After the iPSC and the sodium alginate solution are evenly mixed, the sodium alginate mixture is passed through a high voltage electric field and dripped into the calcium chloride solidification solution for solidification, thereby obtaining calcium alginate microspheres containing iPSC. The concentration of the calcium chloride solidification solution is 50mM-200mM; the solidification time of the sodium alginate mixture is 10-30 minutes.

6. The method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells according to claim 2, characterized in that: The film-forming liquid is PLL, and the mass concentration percentage of the PLL solution is 0.05-0.2%; the time for mixing the calcium alginate microspheres and the PLL film-forming liquid to react is 5-15 minutes.

7. The method for pancreatic islet differentiation of microencapsulated induced pluripotent stem cells according to claim 1 or 2, characterized in that: The iPSC in the microspheres was cultured with iPSC culture medium to form cell clusters. The in vitro culture time range was 1-10 days. The definitive endoderm (DE) differentiation induction medium, pancreatic precursor cell (PP) differentiation induction medium and endocrine cell (EN) differentiation induction medium were used in sequence to induce differentiation of the iPSC in the calcium alginate microspheres or microcapsules.

8. A pancreatic islet differentiation device for microencapsulated induced pluripotent stem cells, characterized in that: The device is applied to the pancreatic islet differentiation method of microencapsulated induced pluripotent stem cells as claimed in claims 1 to 7, comprising a housing (1), a feeding mechanism (3), a motor (2) and a stirring rod (5); the output end of the motor (2) is connected to the stirring rod (5), and the stirring rod (5) is rotatably connected to the housing (1); The feeding mechanism (3) comprises a liquid outlet pipe (308), a pump body (303), a main pipe (304), four first solenoid valves (305), a second solenoid valve (309), a first tank body (307) and four second tank bodies (306); The liquid outlet pipe (308) is connected to the output end of the pump body (303), and the liquid outlet pipe (308) passes through the housing (1). The input end of the pump body (303) is connected to one end of the main pipe (304), and the other end of the main pipe (304) is connected to the first tank body (307) via the second solenoid valve (309). The main pipe (304) is connected to four first solenoid valves (305), and the four first solenoid valves (305) are respectively connected to four second tank bodies (306); The feeding mechanism (3) further comprises an annular tube (301) and a plurality of nozzles (302); the annular tube (301) is connected to the liquid outlet tube (308), and the plurality of nozzles (302) are in communication with the annular tube (301); The interior of the housing (1) is connected to an oxygen sensor (15), a monitoring end of the oxygen sensor (15) extends into the culture medium, and the housing (1) is provided with an oxygen injection mechanism (4); The oxygen injection mechanism (4) comprises an oxygen tank (401), a third solenoid valve (403) and a pipe body (402); The oxygen tank (401) is connected to the housing (1), the oxygen tank (401) is connected to the third solenoid valve (403), the third solenoid valve (403) is connected to the tube body (402), and the tube body (402) passes through the housing (1).

9. The pancreatic islet differentiation device of microencapsulated induced pluripotent stem cells according to claim 8, characterized in that: The control mechanism (9) further comprises a first gear (901), a second gear (902), a shaft (903), an inner ring (904), a plurality of protrusions (905), a plate (914), a rotating plate (915), a rod (916), a torsion spring (917), an outer ring (906), two first stoppers (913), a second stopper (912), a vertical cylinder (907), a first cylinder (908), a second cylinder (910), a first air outlet (909), and a second air outlet (911); The first gear (901) is connected to the first gear (901), the first gear (901) is meshed with the second gear (902), the second gear (902) is connected to the shaft (903), the shaft (903) is rotatably connected to the housing (1), the plate (914) is connected to the shaft (903), the plate (914) is connected to the rod (916), the rotating plate (915) is rotatably connected to the rod (916), the torsion spring (917) is sleeved on the rod (916), the two ends of the torsion spring (917) are respectively connected to the rod (916) and the rotating plate (915), the inner side of the inner ring (904) is connected to the plurality of protrusions (905), and the The inner ring (904) is connected to the vertical cylinder (907), the vertical cylinder (907) is connected to the first cylinder (908), the first cylinder (908) is rotatably connected to the second cylinder (910) via a bearing, the second air outlet (911) is machined on the first cylinder (908), the first air outlet (909) is machined on the second cylinder (910), the tube (402) passes through the second cylinder (910), the second stopper (912) is connected to the vertical cylinder (907), the two first stoppers (913) are connected to the inner side of the outer ring (906), the outer ring (906) is connected to the inner wall of the outer shell (1), and the second cylinder (910) is connected to the inner wall of the outer shell (1).

10. The pancreatic islet differentiation device of microencapsulated induced pluripotent stem cells according to claim 9, characterized in that: A discharge mechanism (8) is provided inside the housing (1); The discharge mechanism (8) comprises an annular plate (801), a microporous filter (802), a first connecting rod (803), a protrusion (804), a frame (805), a second connecting rod (806), a block (807), a sliding rod (808), a first spring (809), a support plate (810) and a contact rod (811); The annular plate (801) is connected to the inner wall of the housing (1); the annular plate (801) is rotatably connected to the microporous filter (802); the microporous filter (802) is rotatably connected to the first connecting rod (803); the first connecting rod (803) is connected to the protrusion (804); the protrusion (804) is slidably connected to the frame (805); the frame (805) is connected to the block (807) via the second connecting rod (806); the block (807) is rotatably connected to the first connecting rod (803); The first spring (809) is connected to the sliding rod (808), the sliding rod (808) is slidably connected to the support plate (810), the support plate (810) is connected to the inner wall of the housing (1), the stirring rod (5) passes through the center of the support plate (810), the first spring (809) is sleeved on the sliding rod (808), the two ends of the first spring (809) are respectively connected to the bottom of the support plate (810) and the top of the block (807), and the contact rod (811) is arranged below the block (807).

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