WO2022183740A1

WO2022183740A1 - Method for preparing pancreatic beta cell, and use thereof

Info

Publication number: WO2022183740A1
Application number: PCT/CN2021/124156
Authority: WO
Inventors: 杨子江; 王浩; 周围; 苏茵
Original assignee: 贝康医学科技有限公司
Priority date: 2021-03-05
Filing date: 2021-10-15
Publication date: 2022-09-09
Also published as: CN112980771B; CN112980771A

Abstract

The present invention relates to a method for preparing a differentiated pancreatic beta cell, and the use thereof. Specifically, the method comprises the following steps: (1) performing domesticated cultivation of pluripotent stem cells in a three-dimensional suspension manner; and (2) inducing the domesticated cells to differentiate into pancreatic beta cells. The present invention also relates to a method for preparing an alginate-polylysine-alginate (APA) microencapsulated artificial pancreas islet from the differentiated pancreatic beta cell. The method comprises preparing the microencapsulated artificial pancreas islet by means of using a sodium alginate solution. The present invention further relates to the use of the pancreatic beta cell and the artificial pancreas islet.

Description

A method for preparing pancreatic beta cells and its application

technical field

The invention belongs to the field of medical biotechnology, and in particular relates to a method for preparing pancreatic beta cells and its application.

Background technique

Diabetes is a disorder of the body's glucose metabolism caused by pancreatic islet dysfunction or failure. According to the latest statistics from the International Diabetes Federation (IDF), in 2019, more than 463 million people in the world have diabetes, and by 2045, 700 million people will have diabetes; China ranks first in the world with 116 million people with diabetes , is expected to reach 147 million by 2045. In 2019, about 4.2 million people (aged 20-79 years) died of diabetes or its complications worldwide, 1 person died of diabetes every 6 seconds, accounting for about 11.3% of global all-cause deaths.

Type 1 diabetes, which accounts for 5-10% of all diabetes, is caused by a complete loss of beta cell number and function. At present, the main treatment methods are long-term insulin injection therapy and islet transplantation. Insulin injection cannot cure diabetes and complications fundamentally. The new type of insulin pump also has the disadvantages of inconvenient portability, unstable blood sugar control and causing a lot of pain to patients. In addition, the extraction and purification process of islets in the islet transplantation strategy is very complicated. The lack of organ resources and how to efficiently and stably obtain islets that can meet the requirements of clinical transplantation are also the main challenges of islet transplantation. In view of the above problems, the development of stem cell therapy technology to restore the original missing pancreatic beta cells in the body is a better choice for the eradication of diabetes (Pagliuca FW et al., 2013).

At present, the methods for differentiating stem cells into beta cells mainly include: (1) Gene transfection method, which is to transfect multiple transcription factors PDX-1, NKX6.1, Ngn3, NeuroD, Pax4, etc. , which were induced to differentiate into insulin-secreting cells (Noguchi H et al., 2006). However, the gene transfection method has potential carcinogenic risk. It uses viral vectors to insert foreign genes into the cell genome, which may inactivate tumor suppressor genes. In addition, the insulin-secreting cells obtained by this method are immature and cannot respond well to glucose stimulation. (2) Cytokine induction method, which simulates the key steps in pancreatic development in vitro according to the development process of embryos to the pancreas, and induces the differentiation of stem cells into pancreatic beta cells or pancreatic progenitor cells (Pagliuca FW et al., 2014; Rezania A et al, 2014; Kroon E et al, 2008). However, this method has complicated steps and long differentiation time; and immature differentiation methods are prone to problems such as poor stability of the differentiation batch, low cell yield, and limited large-scale production. However, the development of a mature and stable cytokine-induced differentiation method can well overcome the above problems, which can also obtain stable, large-scale and mature functional pancreatic beta cells in vitro.

In addition, transplanting mature pancreatic beta cells into an allogeneic environment is often immune to the allogeneic rejection, thereby causing the transplanted beta cells to lose their original functions. At present, the main strategy to solve the problem of immune rejection during allogeneic transplantation is the use of combined immunosuppressive agents, but the immunosuppressive effect is often not ideal and faces the pain of lifelong medication. Recent strategies have shown the development of safe and effective immunoisolation tools to be a better option for addressing immune rejection during allogeneic transplantation (Omid Veiseh et al., 2015; Arturo J Vegas et al., 2016; Daniel G Anderson et al., 2016). The selection of the wrapping material used in the immunoisolation tool is the key to the success of transplantation. Due to the influence of different wrapping materials, the in vivo functional evaluation of differentiated beta cells is also quite different. Currently commonly used wrapping materials include: (1) natural materials. Lipids, polysaccharides and proteins; (2) semi-synthetic materials. Cellulose derivatives, etc.; (3) Synthetic materials. Both degradable and non-degradable materials are included. Many of these materials have serious fibrotic encapsulation and no immune isolation function in vivo, which seriously affects the activity and function of encapsulated cells. However, the development of an ideal wrapping material can solve the above-mentioned problems in the xenotransplantation process.

Therefore, pluripotent stem cell-derived pancreatic beta cells are expected to treat diabetes through cell replacement strategies, but how to better scale and stably obtain mature pancreatic beta cells in vitro and solve the problem of immune rejection after transplantation of allogeneic pancreatic beta cells has been is a research hotspot.

SUMMARY OF THE INVENTION

The present invention relates to a method for three-dimensional suspension directional differentiation of pluripotent stem cells (PSC, human embryonic stem cells or human induced pluripotent stem cells) into mature pancreatic beta cells, the method comprising the following steps:

(1) Three-dimensional suspension acclimation culture of pluripotent stem cells;

(2) Induce the acclimated cells to differentiate into pancreatic beta cells;

The step of the three-dimensional suspension acclimation culture of the pluripotent stem cells described in step (1) includes:

1) Y27632 pretreatment, preferably, the pluripotent stem cells cultured on the plane are replaced with the mTeSR1 medium containing 10 μM concentration of Y27632 2 hours before the suspension culture;

2) Use DMEM/F12 medium to wash and neutralize the pretreated cells and count the cells;

3) The pluripotent stem cells are stirred and cultured in mTeSR1+Y27632 medium, passaged 5-10 times in mTeSR1+Y27632 medium, half of the medium is replaced every day, and single cells and aggregated large cells in the culture supernatant are removed during passage. cell clumps;

Preferably, the seeding density of the pluripotent stem cells is 3-8*10 ⁵ /mL, and a disposable bioreactor (Disposable spinner flasks, Corning, 3152/3153) is used for three-dimensional stirring suspension culture, and the parameters are: 50- 120rpm rotation speed, constant temperature of 37°C, 5% CO ₂ , 100% humidity, the medium is mTeSR1 medium containing Y27632 at a concentration of 10 μM;

Preferably, the removal of large cell clumps is to use a 400 μm mesh to filter and remove cell clumps larger than 400 μm;

4) Detecting the acclimated pluripotent stem cells, if the cells grow in spheroid suspension and the ratio of Oct4+/SSEA4+ double positive cells is more than 95%, it is qualified for domestication;

The steps of cell differentiation and microencapsulation after the induction and domestication described in step (2) include:

The media used were:

S1 medium: MCDB131 basal medium+1:10000-100000 insulin-transferrin-seleno-ethanolamine (ITS-X)+1-5mM glutamine, necessary buffer salts (NaHCO ₃ ), antibiotics, antioxidants ( VC), glucose and serum albumin;

S2 medium: MCDB131 basal medium+ITS-X 1:10000-1:100000+1-5mM glutamine, necessary buffer salts (NaHCO ₃ ), antibiotics, antioxidants (VC), glucose and serum albumin;

S3/S4 medium: MCDB131 basal medium+ITS-X 1:50-1:500+1-5mM glutamine, necessary buffer salts (NaHCO ₃ ), antibiotics, antioxidants (VC), glucose and serum albumin protein;

S5 medium: MCDB131 basal medium+ITS-X 1:50-1:500+1-5mM glutamine+2-20μg/mL heparin sodium salt, necessary buffer salts (NaHCO ₃ ), antibiotics, antioxidants ( VC), glucose and serum albumin;

The specific method of inducing differentiation is as follows:

Use the pluripotent stem cells acclimated in step (1) as the starting seed cells for differentiation

1) Inoculate 0.3-0.7*10 ⁶ scattered three-dimensional suspension-acclimated pluripotent stem cells per ml of mTeSR1 medium (supplemented with 10 μM Y27632 in the medium), and after culturing for 24-72 hours, replace the medium on the first day and change the medium When the volume is reduced by 10-20%,

The medium on the first day is: S1 medium is added: 50-200ng/ml recombinant human activin A (Activin A), 10-100ng/mL recombinant human Wnt3a protein;

2) On the 2nd day, the medium was replaced on the 2nd day, and the volume of the medium remained unchanged during the medium change;

The medium on the 2nd day is: S1 medium is added: 50-200ng/ml Activin A;

3) The medium on the 4th day was replaced on the 4th and 6th respectively, and the volume of the medium did not change during the medium change;

The medium on the 4th day: S2 medium supplemented with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5μM transforming growth factor-βRI Kinase Inhibitor IV (TGF-βRI Kinase Inhibitor IV) ;

4) The medium on the 7th day was replaced on the 7th and 8th respectively, and the volume of the medium was unchanged during the liquid change;

The medium on the 7th day: S3/S4 medium supplemented with: 20-100ng/ml KGF, 0.1-0.5μM Sant1, 1-5μM retinoic acid (RA), 100-

500nM

1,1,4,4-tetra Phenyl-1,3-butadiene (TPB), 5-20 μM Y27632;

5) Change the medium on the 9th day on the 9th, 11th, and 13th respectively, and the volume of the medium does not change during the medium change;

The medium on the 9th day: S3/S4 medium supplemented with: 20-100ng/ml KGF, 0.1-0.5μM Sant1, 50-200nM RA, 10-100ng/mL EGF, 10-100ng/mL recombinant human noggin (NOG ), 5-20μM Y27632;

6) The medium on the 14th day was replaced on the 14th and the 16th day respectively, and the volume of the medium was unchanged during the liquid change;

The medium on the 14th day: S5 medium supplemented with: 0.1-0.5μM Sant1, 50-200nM RA, 0.5-2μM γ-Secretase Inhibitor XXI (γ-Secretase Inhibitor, XXI), 5-20μM RepSox, 0.5-2μM Triiodothyronine (L-3,3',5-Triiodothyronine, T3), 5-50ng/ml Recombinant Human Betacellulin Protein, 50-500nM LDN193189 hydrochloride (LDN193189 hydrochloride), 10μM Zinc sulfate;

7) Change the culture medium on the 18th day on the 18th and 20th respectively, and the volume of the culture medium does not change during the liquid change;

The medium on the 18th day was: S5 medium supplemented with: 10-50nM RA, 0.5-2μM XXI, 5-20μM RepSox, 0.5-2μM T3, 5-50ng/ml Betacellulin, 50-500nM LDN193189, 1mM N-cys ( Fmoc-N-Me-Cys(Trt)-OH);

8) On the 21st day, the differentiated cells were digested into single cells using TrypLE Express, and then re-inoculated into the reactor at a density of 0.5-2*10 ⁶ cells/ml, cultured at 50-120 rpm, and the parameters of the incubator were set to a constant temperature of 37 ℃, 5% CO ₂ and 100% humidity, from 21 to 35 days, replace the S3 medium every two days. During the culture, use a 10-50 μm reversible filter to collect the reaggregated normal cell clusters, and discard the unagglomerated cells. The supernatant, the differentiated pancreatic beta cells.

Optionally, in the method for inducing differentiation, any one or any combination of the following steps can also be included:

9) Before the above-mentioned differentiation step 3), enrichment and culture of differentiated CD177-positive groups can be performed, and the specific method is:

The differentiation stage cell mass was taken, digested into single cells, and then CD177-positive cell subsets were sorted, and the sorted cells were seeded at a density of 2-10×10 ⁵ /mL in the above-mentioned 4th day culture containing 10 μM Y27632 The base continues to differentiate;

10) Before the above-mentioned differentiation step 6), the enrichment and culture of GP2-positive groups can be carried out, and the specific method is:

Take the cell mass at the differentiation stage, digest it into single cells, and then sort out the GP2-positive cell subpopulation, and then inoculate the sorted cells at a density of 5-10×10 ⁵ /mL in the above-mentioned day 14 medium and proceed. differentiate;

11) Before the above-mentioned differentiation step 8), enrichment and culture of Procr-positive groups can be carried out, and the specific method is:

Take the cell mass at the differentiation stage, digest it into single cells, and then sort out the Procr-positive cell subsets. The enriched Proc-positive cells are passaged every 7-14 days. During each passage, the digested Procr-positive single cells The suspensions were supplemented with fresh human HUVEC cells at a ratio of 1:1; Procr-positive cells and human HUVEC cells were then mixed and re-inoculated at a ratio of 1:4-1:6 to make differentiated cells. The cell yield was significantly improved;

12) In the culture medium of the above differentiation step 8), 100ng/mL WNT4 can be added, which can drive the metabolic maturation necessary for glucose-stimulated insulin secretion of differentiated cells.

The present invention also relates to a method for preparing sodium alginate-polylysine-sodium alginate (APA) microencapsulated artificial islets from the differentiated pancreatic beta cells, the method comprising:

(1) Preparation of sodium alginate solution: the dissolved sodium alginate is filtered through PES sterile filtration devices of 0.8 μm, 0.45 μm and 0.22 μm in turn; the viscosity of the detected solution is 50-200cP, and it is stored at 4°C;

(2) Prepare microencapsulated artificial islets according to method 1 or method 2:

Method 1: Microfluidic method:

Mix 1%-3% sodium alginate solution with the pancreatic beta cells, 1 mL sodium alginate solution and 1-10*10 ⁶ cells;

After mixing, add one of the pipelines, and add 0.1-2g/L calcium chloride solution to the other pipeline;

Crosslinking into a gel through a 1-10cm collection tube, followed by calcification for 5-30 minutes;

Add 0.01-0.1% polylysine to react for 10-30 minutes;

Then add 0.1%-0.3% sodium alginate solution to react for 2-10 minutes;

Then add 10-100mM sodium citrate to react for 2-10 minutes to form APA microcapsules;

Method 2: High voltage electrostatic method:

After mixing, use a syringe with a 20-40G needle to inhale, and under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz, and flow rate of 100-1000μL/min, make it form a jet into 0.1-2g/L calcium chloride Cross-linking in solution to gel, followed by calcification for 5-30 minutes;

Add 0.01-0.1% polylysine to react for 10-30 minutes;

Then add 0.1%-0.3% sodium alginate solution to react for 2-10 minutes;

Then add 10-100mM sodium citrate and react for 2-10 minutes to form APA microcapsules.

The present invention also relates to the application of the pancreatic beta cells or artificial pancreatic islets in the preparation of medicines. The medicines are medicines for treating diabetes. Preferably, the diabetes is type I diabetes.

The present invention also relates to the application of the pancreatic beta cells or artificial pancreatic islets in the preparation of therapeutic active ingredients in cell therapy, wherein the cell therapy is a cell therapy for diabetic patients, preferably, the diabetes is Type 1 diabetes.

The present invention also relates to a method for inducing an endoderm progenitor cell line, the method comprising the steps of:

(1) Three-dimensional suspension acclimation culture of pluripotent stem cells;

(2) Induction of endoderm progenitor cell lines;

The three-dimensional suspension acclimation and culturing steps of the pluripotent stem cells in step (1) are the same as the relevant steps of the method for the three-dimensional suspension directional differentiation of the pluripotent stem cells into mature pancreatic beta cells;

The method for inducing the endoderm progenitor cell line described in step (2) is:

The medium on the 2nd day is: S1 medium is added: 50-200ng/ml Activin A;

4) When the proportion of SOX17 positive cells is >90% by flow cytometry, take the cell mass and digest it into single cells, and sort out CXCR4+/CD117+ double-positive cell subsets (common, differentiated to the 6th day, that is, CXCR4+/CD117+ Double positive cell subgroup sorting), this cell subgroup is committed endoderm progenitor cells;

5) inoculate the group of cells in a plate containing Matrigel, and use the directional endoderm progenitor cell culture and expansion medium to culture and expand;

The components of the directed endoderm progenitor cell culture and expansion medium are:

Add to S1 medium: 20-100ng/mL bone morphogenetic protein 4 (BMP4), 5-20ng/mL recombinant human basic fibroblast growth factor protein (bFGF), 5-20ng/mL recombinant human vascular endothelial growth factor Protein (VEGF), 5-20ng/mL recombinant human epidermal growth factor protein (EGF).

The beneficial effects of the present invention are:

The purpose of the present invention is to provide a method for obtaining mature, batch-stable, large-scale, three-dimensional suspension-directed differentiated beta cells in vitro, aiming at the defects of batch stability and poor in vivo function evaluation of the existing differentiated beta cells. At the same time, an ideal method for encapsulating and encapsulating differentiated beta cells is provided, so that the microencapsulated beta cells can function well in animals without the problems of immune rejection and fibrosis encapsulation. Specifically, it includes (1) three-dimensional large-scale suspension culture of pluripotent stem cells (including induced pluripotent stem cells and embryonic stem cells); (2) dynamic directional differentiation of three-dimensional suspension into mature pancreatic beta cells using compound combinations in vitro; (3) microbiology of beta cells Encapsulation and encapsulation; (4) functional evaluation of microencapsulated beta cells in vitro and in vivo.

Compared with the prior art, the method provided by the present invention has the following significant advantages: the method for PSC three-dimensional suspension culture provided by the present invention can successfully maintain the expression of PSC pluripotency gene, the cell proliferation is good, and the karyotype Stable, in particular, the planar cells need to be replaced with Y27632 fresh medium to pretreat cells for 2-4 hours before the 3D reactor. Appropriate initial cell seeding density, culture system, rotation speed, etc. can significantly increase the success rate of PSC three-dimensional suspension culture. The cell medium exchange method (half exchange medium) in the three-dimensional culture process, the list cell treatment method, the treatment method of large cell clumps larger than 400 μm, and the normal cell clump acquisition method not only saves costs, but also maintains cell viability, etc. . The combination of compounds used in each stage of the differentiation process can efficiently differentiate into the cell types of this stage, and the proportion of differentiated cells is significantly increased. In particular, the change of the pre-differentiation culture system, the preparation method and addition time of the composition, the method of changing the medium during the differentiation process, and the treatment method of the list cells on the differentiation process all have a good effect on promoting differentiation, and significantly increase the stability between different batches of differentiation. And differentiated beta cells are more mature and more functional. The improved sodium alginate microencapsulation packaged differentiated beta cells transplanted into animals can reverse hyperglycemia in diabetic mice in a short time (24h), and has a good immune isolation function. Complete mice maintain normoglycemia for a longer period of time.

Description of drawings

Figure 1. Stem cell pluripotency detection indicators and results during flat culture, 1A, microscopic photo; 1B, flow detection results.

Figure 2. Photographs of the results of environmental inspection after 3D culture of hPSCs cells.

Figure 3. The cell proliferation during the acclimation process. The multiplication ratio was between 2 and 4 times (Figure 3A), and the ratio of Oct4/SSEA4 double positive cells was above 99% (Figure 3B).

Figure 4. Morphology of cell mass after alginate-polylysine-sodium alginate (APA) encapsulation.

Figure 5. The results of in vivo functional evaluation of microencapsulated pancreatic beta cells transplanted into the peritoneal cavity of STZ-induced C57BL/6 mice.

Figure 6. qPCR detection results at various stages during the differentiation of pancreatic beta cells by the classical method and the expression trends of related markers at the transcriptional level.

Figure 7. The qPCR detection results of each stage in the process of obtaining pancreatic beta cells by the optimized differentiation method of the present invention and the expression trend of related markers at the transcription level.

Fig. 8 is the detection result of the properties of cell clusters in each differentiation stage of the optimized differentiation method of the present invention.

Figure 9. Schematic diagram of detection indicators at each stage in the differentiation process.

Detailed ways

Example 1. Planar culture, in vitro 3D culture and acclimation of hPSCs

1. Flat culture of hPSCs

The required reagent preparation method:

mTeSR1 pluripotent stem cell culture medium (STEMCELL, 85850);

Accutase digestive enzyme (STEMCELL, 07920);

Rho kinase inhibitor Y27632 (Abcam, ab120129): make up a 10 mM stock solution with DMSO, and the final concentration is 10 μM when used, that is, when used at 1000X;

mTeSR1+Y27632 medium: add 15μL of 10mM Y27632 to 15mL mTeSR1, mix well, store at 4°C when not in use, and use it up within two weeks after preparation.

hPSCs cells (clinical grade human embryonic stem cells or human induced pluripotent stem cells, sourced from Beijing Stem Cell Bank).

1. Recovery and culture of hPSCs

1.1. Matrigel-coated culture plate: Take out the 6-well plate, add 0.5-2 mL of Matrigel (Matrigel, Corning, 354277) to each well, gently shake the 6-well plate to make Matrigel completely cover the bottom of the dish, and place it in a 37°C incubator Incubate for 1h to 2h, take it out before the experiment and place it in a clean bench/biosafety cabinet to equilibrate at room temperature for 10-40min. If it is not used temporarily, it can be sealed with Parafilm and stored at 2-8°C and used within 1-2 weeks.

The quantity of a cryopreserved stem cell is about 1×10 ⁶ cells/mL, corresponding to 1 well of a 6-well plate;

1.2. First add 2-3 mL of mTeSR1 medium to a 15 mL centrifuge tube for later use.

1.3. Thawing: Immerse the cryopreservation tube taken out from the liquid nitrogen into warm water at 37°C and shake it quickly to thaw it within 1-2 minutes;

1.4. Centrifugation: After the freezing solution in the cryopreservation tube is thawed, add it dropwise to a 15mL centrifuge tube containing mTeSR1 medium, place the 15mL centrifuge tube in a low-speed centrifuge, and centrifuge at 800-1800rpm for 3min ;

1.5. Resuspension: After centrifugation, discard the supernatant and add 0.5-2 mL of mTeSR1+Y27632 cell culture medium to mix the stem cell pellet by pipetting and pipetting about 3 to 5 times.

1.6 Inoculation: After blowing and sucking evenly, discard the balanced Matrigel, add the homogeneously pipetted stem cell suspension to the coated 6-well plate, and complete the 2 mL culture system per well.

1.7 Culture: After inoculation, the 6-well plate can be placed under an inverted phase contrast microscope to observe the density of the seeded stem cells, and the 6-well plate can be gently shaken by a horizontal cross to make the cells evenly distributed. The cells were cultured in a constant temperature incubator at 37°C and 5% CO ₂ , and the adherence of cells was observed on the second day;

1.8 Change the medium: change the medium every 24-48h from the time of resuscitation.

During the flat culture process, the cell morphology was normal, and the stem cell pluripotency detection indicators and results during the flat culture process are shown in Figure 1; the results show that the hPSCs cultured in a flat surface by the above method can well maintain the stem cell clone shape and stemness-related gene expression. Very good; flow cytometry results showed that the ratio of Oct4+/SSEA4+ double positive cells was above 99%, indicating that the stemness was well maintained during the culture process; the ratio of SSEA1 cells was very low, and no cell differentiation occurred.

2. Passaging hPSCs for 3D culture

2.1 Add Y27632 pretreatment: 2-4h before the 3D reactor (Disposable spinner flasks, Corning, 3152/3153), the mTeSR1 mediμm pretreated cells containing Y27632 should be replaced;

2.2 Washing: Aspirate the original medium, slowly add 0.5-2mL DMEM/F12 to the wall and shake gently, and then aspirate DMEM/F12 along the edge of the petri dish; Digestion: Add 0.5-2mL/well Accutase to the 6-well plate Make it cover the bottom of the dish and place it in a 37°C incubator for 2-5min;

2.3 Neutralization: Add 1-4mL DMEM/F12 for neutralization, fan the bottom of the petri dish with a pipette, and gently pipette 3 to 5 times to detach the stem cell colonies at the bottom of the dish, and transfer them to a 15mL centrifuge tube;

2.4 Counting: Centrifuge at 800-1800rpm for 3-5min; discard the supernatant, pipet the cells with stem cell medium for 5-10 times, and take part of the cell suspension for counting;

2.5 Inoculation: According to the counting result, resuspend the density of 3-8*10^5/mL in mTeSR1+Y27632 medium, and then inoculate in Corning Spinner Flask, 40-300mL culture system. Spinner flasks were cultured on a nine-position magnetic stirrer at 50-120 rpm, and the parameters of the incubator were set to a constant temperature of 37°C, 5% CO ₂ and 100% humidity.

Figure 2

This culture remains unchanged during cell culture and differentiation. If the cell seeding density is not within this range, if the density is too small, the cells will not be able to form well, and the proliferation will be limited; if the density is too large, the cells will easily form into large clumps, the adhesion between the clumps will be serious, and the cell clumps will be If the diameter is too large, it will lead to hypoxia in the middle cells, lower cell viability and easy differentiation. If the rotational speed is not within this range, too much rotational speed will lead to failure to form a good cluster, too many single cells will reduce the cell viability, and dryness will be lost; The assembly leads to hypoxia in the middle cells, decreased cell viability and easy differentiation.

3. 3D culture acclimated hPSCs cells and functional detection

3.1 Before measuring the differentiation ability of hPSCs, ensure at least 5-10 generations of acclimation in the reactor. Half of the medium was changed every day, the large cell clumps larger than 400 μm were removed through a 400 μm mesh, and the single cells in the culture supernatant were added back to the reactor. Discard the single cells in the culture supernatant during passage, collect the normal pellet with a 37 μm reversible filter, disperse the cells with Accutase, and inoculate 0.3-0.7 million cells per ml of mTeSR1 medium supplemented with 5-20 μM Y27632 scattered cells. If the large cell clumps larger than 400 μm are not removed, the adhesion of the cell clumps will become more and more serious, and the large particle size of the cell clumps will lead to hypoxia in the middle cells, lower cell viability and easy differentiation. If the single cells in the culture supernatant are not added back to the reactor, cell proliferation will be restricted, which is not conducive to the formation of cell clusters.

3.2 Differentiate after 48-72 hours of culture. During this period, replace half of the mTeSR1 medium without Y27632 every day. Before differentiation, keep the diameter of the cell cluster within 300 μm. If the diameter of the cell cluster is larger than 300 μm, some cells in the cell cluster will be necrotic or spontaneous differentiation. Cell lines with different doubling times may require different seeding concentrations or times.

The cell proliferation during the acclimation process is shown in Figure 3: it can be seen that during the acclimation process, the pluripotent stem cells can proliferate well, and the proliferation multiple is between 2-4 times (Figure 3A). In the process of domestication, hPSCs can also express stemness-related genes well. Flow cytometry results showed that the ratio of Oct4+/SSEA4+ double positive cells was over 99% (Fig. 3B), which also indicated that the stemness of 3D pluripotent stem cells was maintained during the domestication process. very good.

Example 2. Differentiation of hPSCs into pancreatic beta cells

The names and formulations of the media used throughout the differentiation process are as follows,

S1 medium: MCDB131 basal medium (Thermofisher, 10372019) + 2-20 mM glucose (Glucose, Sigma, G7528-250G) + 1-5 g/L sodium bicarbonate (NaHCO ₃ , Sigma, S5761-500G) + 0.5-5 % Recombinant human serum albumin (Human serum albumin, HSA, Heyuan Bio, HYC002M01) + 1:10000-1:100000 Insulin Transferrin Selenium Ethanolamine (Insulin Transferrin Selenium Ethanolamine, ITS-X, Thermofisher, 51500056) +1-5mM glutamine substitute (GlutaMAX ^™ Supplement, Glutamax, Thermofisher, 35050061)+0.1-0.5mM vitamin C (L-Ascorbic acid, Vitamin C, Sigma, A4544-25G)+1% penicillin/streptomycin (Penicillin-Streptomycin, Pen/Strep, Thermofisher, 15140122).

S2 medium: MCDB131+2-20mM Glucose+1-3g/L _NaHCO3 +0.5-5%HSA+ITS-X 1:10000-1:100000+1-5mM Glutamax+0.1-0.5mM Vitamin C+1% Pen/Strep.

S3/S4 medium: MCDB131+2-20mM Glucose+1-3g/L _NaHCO3 +0.5-5%HSA+ITS-X 1:50-1:500+1-5mM Glutamax+0.1-0.5mM Vitamin C+ 1% Pen/Strep.

S5 medium: MCDB131+5-50mM Glucose+1-3g/L _NaHCO3 +0.5-5%HSA+ITS-X 1:50-1:500+1-5mM Glutamax+0.1-0.5mM Vitamin C+1% Pen/Strep+2-20μg/mL heparin sodium salt (Heparin sodium salt, Heparin, Sigma, H3149-500KU-9).

All of the above media were sterilized by filtration through a 0.22μm bottle top filter Filter.

And need to make up fresh medium before medium change (add small molecules and growth factors to basal medium in low light hood safety cabinet).

1. SC-β cell (pancreatic beta cell) differentiation

1. Classical method for differentiation and function detection of pancreatic beta cells

Using the method of Melton DA. (this method is cited from: doi.org/10.1016/j.cell.2014.09.040),

At each and final stage of differentiation, cells were taken separately for analysis for in vitro functional evaluation. The test results are as follows:

(1) S0 stage: hPSC, human pluripotent stem cells. Flow detection showed that the ratio of Oct4+/SSEA4+ double positive cells was normal, and the stemness was well maintained;

(2) S1 stage: DE, definitive endoderm cells. The proportion of SOX17 positive cells by flow cytometry was 82.9%, which did not reach more than 90%, and the differentiation efficiency was low;

(3) S2 stage: PGT, primitive gut tube cells. The proportion of HNF4a positive cells by flow cytometry was 47.9%, and the differentiation efficiency was low;

(4) S3 stage: PP1, early panic progenitor cells. The proportion of PDX1 positive cells by flow cytometry was 40.6%, and the differentiation efficiency was low;

(5) S4 stage: PP2, later panic progenitor cells. The proportion of PDX1+/NKX6.1+ double-positive cells by flow cytometry was only 8.62%, and the expression was low;

(6) S5 stage: EN, endocrine progenitor cells. The proportion of NKX6.1+/C-peptide double-positive cells by flow cytometry was only 10.1%, and the vast majority of C-peptide in the differentiation results were weakly positive;

(7) S6 stage: SC-βcells, stem cell-derivedβcells. The proportion of NKX6.1+/C-peptide double positive cells by flow cytometry was only 12.7%. The proportion of CHGA positive cells in endocrine cells was 47.71%, which was relatively low. Three consecutive rounds of glucose stimulation did not respond, and the secretion of insulin was very low when stimulated with high glucose.

(8) The qPCR detection results of each stage in the differentiation process using this method and the expression trend of related markers at the transcription level:

During the whole differentiation process, cells at different stages were taken for RNA extraction, and then qPCR was used to detect the expression trend of related genes throughout the differentiation process. It can be seen from Figure 6 that the relative expression levels of PDX1, NKX6.1 and Insulin obtained by this method are low, only several tens of times that of the initial cells. The expression of Oct4 did not drop to a low level until Stage 5, and there was a big safety issue.

2. Optimized method for pancreatic beta cell differentiation and functional assay

Pluripotent stem cells acclimated in a 3D reactor for 5-20 passages (passages of the 5-20 passages including the 3D acclimation process) were used as starting seed cells for differentiation.

2.1 Differentiation method

To initiate SC-β cell differentiation, seed 0.3-0.7 million dispersed hPSCs per ml of mTeSR1 medium supplemented with 10 μM Y27632. After culturing for 24-72 hours, replace the Day 1media. At this time, the 80-320mL culture system is converted into a 60-300mL differentiation system (the volume is reduced by 10-20%, and the amount of medium during the initial medium change is reduced by 10% compared to when the medium is not changed. -20%, and the culture volume will remain unchanged every time the medium is changed), this is the official start of differentiation, and the medium will be replaced at the following times in sequence (full medium change, single cells in the culture supernatant at the same differentiation stage) After centrifugation, it was added back to the reactor, and the single cells in the culture supernatant were discarded between stages). If the volume is not reduced at the onset of differentiation, it will result in very low cell yields at the later stages of differentiation. Vc, all factors and small molecule compounds used during differentiation were added on the same day. If added in advance, the efficiency of differentiation will be greatly reduced. And before the medium is replaced, the relevant medium needs to be freshly prepared, and the small molecule compounds and growth factors are added to the basal medium in a safety cabinet with a low light hood, and the whole process of changing is protected from light. If the operation is not protected from light, some small molecular compounds will be decomposed due to direct light from the light source, and finally the differentiation efficiency will be reduced.

0.3-0.7 million dispersed hPSCs were first seeded per ml of mTeSR1 medium supplemented with 10 μM Y27632. After culturing for 24-72 hours, replace the medium and add the corresponding cytokines. According to the culture process, the steps for replacing the medium and adding cytokines are as follows:

Day 1: S1+50-200ng/ml recombinant human activin A (Recombinant Human/Mouse/Rat Activin A Protein, Activin A, R&D systems, 338-AC-050/CF)+10-100ng/mL recombinant human Wnt3a protein (Recombinant Human Wnt-3a Protein, Wnt3a, R&D systems, 5036-WN-500). (The amount of medium during the initial medium change is reduced by 10%-20% compared to the time when the medium is not changed, and the culture volume is maintained with each medium change after that. Change)

Day 2: S1+50-200ng/ml Activin A.

Days 4, 6: S2+20-100ng/ml recombinant human keratinocyte growth factor protein (Recombinant Human KGF/FGF-7 Protein, KGF, R&D systems, 251-KG-050)+1-5 μM transforming growth factor-βRI Kinase Inhibitor IV (TGF-βRI Kinase Inhibitor IV, Sigma, 616454-2MG).

Days 7, 8: S3+20-100ng/ml KGF+0.1-0.5μM Sant1(Sigma,S4572-5MG)+1-5μM retinoic acid(RA,Sigma,R2625-50MG)+100-

500nM

1 ,1,4,4-Tetraphenyl-1,3-butadiene(1,1,4,4-Tetraphenyl-1,3-butadiene,TPB,Sigma,185213-5G)+5-20μM Y27632.

Days 9, 11, 13: S3+20-100ng/ml KGF+0.1-0.5μM Sant1+50-200nM RA+10-100ng/mL recombinant human epidermal growth factor protein (Recombinant Human EGF protein, EGF, R&D systems, 236-EG-200)+10-100ng/mL recombinant human Noggin Protein (Recombinant Human Noggin Protein, NOG, R&D systems, 6057-NG-025)+5-20μM Y27632.

Days 14, 16: S5+0.1-0.5μM Sant1+50-200nM RA+0.5-2μM γ-Secretase Inhibitor XXI (γ-Secretase Inhibitor XXI, Compound E, XXI, Millipore, 565790-500UG)+5-20μM RepSox (Alk5i II, Sigma, R0158-5MG) + 0.5-2 μM Triodethyronine (L-3, 3', 5-Triiodothyronine, T3, Millipore, 64245-250MG-M) + 5-50 ng/ml recombinant human β Cytokine (Recombinant Human Betacellulin Protein, Betacellulin, R&D systems, 261-CE-010) + 50-500nM LDN193189 hydrochloride (LDN193189 hydrochloride, LDN193189, Sigma, SML0559-5MG) + 10 μM zinc sulfate (ZnSO4, Sigma, Z0251) .

Days 18, 20: S5+10-50nM RA+0.5-2μM XXI+5-20μM RepSox+0.5-2μM T3+5-50ng/ml Betacellulin+50-500nM LDN193189+1mM N-cys(Fmoc-N-Me- Cys(Trt)-OH,Sigma,773069-1G).

Days 21-35 (change medium every two days): S3 medium.

On the 21st day of differentiation, the differentiated cells were digested into single cells using TrypLE Express, and then re-seeded into the reactor at a density of 0.5-2 million cells/ml, and cultured at 50-120 rpm. The parameters of the incubator were set to a constant temperature of 37 °C, 5% _CO2 and 100% humidity.

After that, the medium was changed every other day, and the re-aggregated normal clusters were collected using a 10-50 μm reversible filter, and the unagglomerated supernatant was discarded. At each and final stage of differentiation, cells were taken separately for analysis.

2.2 Detection indicators of each stage in the differentiation process of Stage0-Stage6:

At each and final stage of differentiation of the method of 2.1, cells were taken separately for analysis for in vitro functional evaluation. The test results are as follows:

(1) S0 stage. hPSC, human pluripotent stem cells. The test results are shown in Figure 8A;

The ratio of Oct4+/SSEA4+ double positive cells was more than 95% by flow cytometry, and the stemness was well maintained;

(2) S1 stage. DE,definitive endoderm cells. The test results are shown in Figure 8B;

The proportion of SOX17 positive cells reached 92.8% by flow cytometry, indicating that most of the cells differentiated into committed endoderm cells.

Alternatively, flow sorting can be performed at this stage to obtain committed endoderm progenitor cell lines. The main methods are:

At this stage of differentiation, the cell pellet was digested into single cells using TryPLE, and then the cells were labeled with CXCR4 and CD117, and the CXCR4+/CD117+ double-positive population was sorted by flow cytometry.

This population of cells is plated on Matrigel-containing plates and cultured using Committed Endoderm Progenitor Cell Culture and Expansion Medium.

S1media+20-100ng/mL bone morphogenetic protein 4 (Recombinant Human BMP-4 Protein, BMP4, R&D systems, 314-BP-050)+5-20ng/mL recombinant human basic fibroblast growth factor protein (Recombinant Human bFGF Protein,bFGF,R&D systems,233-FB-010)+5-20ng/mL recombinant human vascular endothelial growth factor protein (Recombinant Human VEGF 165 Protein,VEGF,R&D systems,293-VE-010)+5-20ng/ mL recombinant human epidermal growth factor protein (Recombinant Human EGF protein, EGF, R&D systems, 236-EG-200).

Committed endoderm progenitor cells obtained in culture can be used as the starting point for beta cell differentiation, which can further improve the stability of different differentiation batches and reduce the residual of stem/progenitor cells in the terminal beta cells of differentiation, which also significantly improves the in vivo transplantation. safety.

(3) S2 stage. PGT, primitive gut tube cells. The test results are shown in Figure 8C;

The proportion of HNF4a positive cells reached 98.4% by flow cytometry, indicating that almost all cells were differentiated into PGT cells, and the differentiation efficiency was very high.

(4) S3 stage. PP1, early panic progenitor cells. The test results are shown in Figure 8D;

The proportion of PDX1-positive cells reached 57.4% by flow cytometry, indicating that most of the cells entered the early stage of pancreatic progenitor cells and had a good differentiation effect.

(5) S4 stage. PP2, later pancreatic progenitor cells. The test results are shown in Figure 8E;

The ratio of PDX1+/NKX6.1+ double-positive cells was 27% by flow cytometry, indicating that a certain proportion of cells entered the next differentiation stage at this time, and the differentiation effect was better.

(6) S5 stage. EN,endocrine progenitor cells. The test results are shown in Figure 8F;

The proportion of NKX6.1+/C-peptide double positive cells reached 24% by flow cytometry, indicating that more than 20% of the cells had differentiated into SC-beta cells at this time.

(7) S6 stage. SC-βcells, stem cell-derivedβcells. The test results are shown in Figure 8G;

The proportion of NKX6.1+/C-peptide double positive cells by flow cytometry was more than 30%. Some cells expressed GCG (islet alpha cells) and SST (islet delta cells), and the proportion of endocrine cells CHGA positive cells was more than 90%.

There were three consecutive rounds of glucose response and the stimulation index was greater than 2, and the secretion of Insulin was greater than 1.0-5.0μIU/1000cells when stimulated by high glucose. Dithizone staining appears red.

(8) qPCR detection results at each stage during the differentiation process and the expression trend of related markers at the transcriptional level:

During the whole differentiation process, cells at different stages were taken for RNA extraction, and then qPCR was used to detect the expression trend of related genes throughout the differentiation process. It can be seen from Figure 7 that the relative expression levels of PDX1, NKX6.1 and Insulin obtained by this method are higher, especially the expression of Insulin is nearly 200,000 times the initial value. The expression of Oct4 drops to a lower level in Stage2, and the safety problem is greatly reduced.

Test conclusion:

Using the optimized method to induce hPSCs cells to differentiate into pancreatic beta cells needs to go through the following stages during the entire differentiation process. The detection indicators of each stage are as follows:

This value is the internal quality control standard. When each stage reaches the following value, the batch can be considered to be differentiated normally, the differentiation can proceed smoothly, and the function of the final pancreatic beta cells can be normal.

hPSCs cells: Octamer-binding transcription factor 4/stage-specific embryonic antigen 4 (Octamer-binding transcription factor 4/Stage-specific embryonic antigens 4, Oct4+/SSEA4+) double positive cells ratio is greater than 95%;

DE, committed endoderm: the proportion of SOX transcription factor family member 17 (Sex-determining region Y-box 17, Sox17+) positive cells is greater than 90%;

PGT, original gut tube: the proportion of hepatocyte nuclear factor 4a (Hepatocyte Nuclear Factor 4 alpha, HNF4a+) positive cells is greater than 80%;

PP1, early stage of pancreatic progenitor cells: the proportion of pancreatic and duodenal homeobox 1 (Pancreatic and duodenal homeobox 1, PDX1+) positive cells is greater than 60%;

PP2, late stage of pancreatic progenitor cells: PDX1+/homologous transcription factor NKX6.1 (homeobox transcription factor NK6 homeobox 1, NKX6.1+) double-positive cells ratio is greater than 25%;

EN, pancreatic endocrine progenitor cells: the proportion of NKX6.1+/C-peptide+ double positive cells is greater than 10%;

SC-beta cluster, stem cell-derived pancreatic beta cells: the ratio of NKX6.1+/C-peptide+ double positive cells is greater than 30%.

After the indicators at different stages reach the above-mentioned quality control standards, they can be well differentiated into functional beta cells.

2.3 Enrichment of cells including but not limited to CD177, CD117, CXCR4, GP2, Procr and other related markers at certain stages in the entire differentiation process of Stage0-Stage6 (for specific stages, see steps 9)-12) above (this group The enrichment is interspersed in the whole process of differentiation, which is equivalent to the optimization in the differentiation process. These enrichments can significantly improve the differentiation efficiency and are a selection method to improve the differentiation efficiency).

The enrichment can be determined according to the shape of the cells during the differentiation process. If the shape of the differentiated cells in the batch changes slightly, the function and yield of the differentiated cells can be further improved by enrichment:

(1) The specific enrichment and culture methods of the CXCR4+/CD117+ double-positive population are:

The cell clusters at the differentiation stage to be enriched were digested into single cells using TryPLE, and then the cells were labeled with CXCR4 and CD117, and CXCR4+/CD117+ double-positive clusters were sorted by flow cytometry. This population of cells is plated on Matrigel-containing plates and cultured using Committed Endoderm Progenitor Cell Culture and Expansion Medium.

The composition of endoderm progenitor cell culture and expansion medium is: S1media+BMP4(20-100ng/mL)+bFGF(5-20ng/mL)+VEGF(5-20ng/mL)+EGF(5-20ng/mL) mL).

Differentiation of the committed endoderm progenitor cells obtained in culture can further improve the stability of different differentiation batches and reduce the residual stem/progenitor cells in the terminal beta cells of differentiation, which significantly improves the safety.

(2) The specific enrichment and culture methods for CD177-positive groups are:

The cell clusters at the differentiation stage were digested into single cells using TryPLE, and then the cells were labeled with CD177, and CD177-positive clusters were selected by flow cytometry. The sorted cells were then seeded at a density of 2-10×10 ⁵ /mL in Stage1 medium containing 10 μM Y27632 for culture differentiation.

Cells subjected to the CD177+ enrichment program differentiated more uniformly in vitro into pancreatic progenitors and eventually into more functionally mature glucose-responsive beta cells than cells without this enrichment.

(3) Enrichment and culture of GP2-positive cell population, the specific methods are:

This method can significantly increase the ratio of differentiated cells PDX1+/NKX6.1+ double positive cells.

(4) The specific enrichment and culture methods of Procr-positive groups are:

The cell clusters at the differentiation stage were digested into single cells using TryPLE, and then the cells were labeled with Procr, and Procr-positive clusters were selected by flow cytometry.

Enriched Proc+ cells can be passaged every 7-14 days. During each passage, the digested Procr+ single-cell suspension is supplemented with fresh human HUVEC cells at a cell number ratio of 1:1.

Then mix Procr+ single cells and human HUVEC cells at a ratio of 1:4-1:6 to re-inoculate and culture. This method results in a significant increase in the yield of differentiated cells.

(5) WNT4 can be added in the last stage of differentiation, which can significantly drive metabolic maturation, and the results show that the response of differentiated beta cells to glucose can be significantly improved, and more insulin can be released.

Example 3. Improved APA microencapsulation of differentiated pancreatic beta cells

1. Preparation of improved ultrapure sodium alginate solution

The dissolved sodium alginate is filtered through 0.8μm, 0.45μm and 0.22μm PES sterile filtration devices in turn; the viscosity of sodium alginate is 50-200cP; the prepared sterile sodium alginate can be stored in a 4 degree refrigerator 1-4 weeks.

2. APA microencapsulated differentiated beta cells - microfluidic method or high voltage electrostatic method

2.1 Microfluidic method (the microfluidic board was purchased by Suzhou Wenhao Microfluidic Technology Co., Ltd., model WH-SP-01):

Mix 1%-3% sodium alginate solution with beta cells, 1mL sodium alginate corresponds to 1-10*10^6 cells;

Add 0.01-0.1% polylysine (Poly-L-lysine hydrobromide, Sigma, P7890-100MG) to react for 10-30 minutes;

Then add 0.1%-0.3% sodium alginate solution to react for 2-10 minutes;

2.2 High voltage electrostatic method:

Add 0.01-0.1% polylysine to react for 10-30 minutes;

Then add 0.1%-0.3% sodium alginate solution to react for 2-10 minutes;

The specific morphology of the wrapped cell mass is shown in Figure 4. Preferably, the particle size of the microcapsules is controlled at 250-750 μm.

Example 4. Functional evaluation of microencapsulated pancreatic beta cells (single transplantation functional evaluation)

Various compounds have been found to induce diabetes in animal models. The two most studied and routinely used compounds are streptozotocin (STZ) and alloxan (Alloxan, ALX), among which STZ is the most commonly induced. Both STZ and ALX are glucose analogs, and both act through the glucose transporter GLUT2 in beta cells, resulting in an almost complete destruction of beta cells in islets, resulting in a severe lack of insulin production in mice, hyperglycemia and weight loss, This reproduces the main symptoms of type I diabetes. The present invention causes C57BL/6 mice to produce type I diabetes symptoms by using STZ for drug induction.

1. Microencapsulated or naked pancreatic beta cells were transplanted into STZ-induced type I diabetic C57BL/6 mice subcutaneously for in vivo functional evaluation

1.1 Microencapsulated or naked pancreatic beta cell transplantation:

Transplanted mice were anesthetized by inhalation of 2% to 5% isoflurane (Isoflurane USP, Clipper Distribution). Subcutaneous transplantation is then performed: a small skin incision (0.3-0.5 cm) is made in the lower abdomen to form a subcutaneous "small pocket" in the left and right lower quadrants, microencapsulated beta cells or naked beta cells are mixed with 200-500 μL The beta-cell active Matrigel was mixed and transplanted into the subcutaneous "small pocket" of mice. The components of beta cell active Matrigel include: 10X Media(M) 199 basal medium (ThermoFisher, 11825015), L glutamine, fetal bovine serum, 5-10% sodium bicarbonate and type I collagen.

1.2 The transplantation of naked beta cells (unencapsulated beta cells) also needs to be combined with immunosuppressive agents. The specific scheme is:

Maintenance immunosuppression consisted of rapamycin (1-2 mg/kg intraperitoneally four times a day) starting on the day of transplantation for 5-10 days.

To deplete B cells, use 10F4 (provided by M. Cancro, Department of Pathology and Laboratory Medicine, University of Pennsylvania), a monoclonal antibody against mouse BLyS (50-200 μg intraperitoneally 15-25 d before transplantation, divided two injections, 24 hours apart). Beginning on day 10, 10F4 was also given gradually, with a gradually decreasing dose, from 20-100 μg per week in week 2 to 1-10 μg per week in week 8. On days 50-80, immunosuppressive dosing was discontinued.

1.3 Post-transplantation detection

The blood glucose level, glycosylated hemoglobin level, in vivo glucose stimulation and human C-peptide were detected. The transplanted APA microencapsulated beta cells or naked beta cells were taken for fixation and immunofluorescence was performed to identify relevant markers.

Alternatively, the islet graft site can be resected, with the aim of confirming that the islet graft is the only source of maintenance of euglycemia, isletectomy (skin) is performed on a group of islet recipients who have been receiving euglycemia for a long time, which leads to diabetes in 24-72 hours Rapid recurrence within. The same results showed that subcutaneously transplanted differentiated beta cells were functionally equivalent to microencapsulated beta cells after intraperitoneal transplantation (referring to a means of detecting that after the transplanted pancreatic beta cells were removed, the animals relapsed into diabetes, which means that the Transplanted pancreatic beta cells are functioning).

According to the above method, each mouse was transplanted with 1-10*10^6 cells, and an indwelling needle was used for intraperitoneal transplantation. After transplantation, the blood glucose level, glycosylated hemoglobin level, in vivo glucose stimulation and human C-peptide were detected.

The results show that microencapsulated beta cells can rapidly reverse hyperglycemia symptoms in diabetic mice, and this process takes 1-7 days, followed by maintaining normoglycemia for more than 30 days. During the transplantation period, the content of human C-peptide in the plasma of the transplanted mice was randomly detected, and the results showed that the content of human C-peptide was much higher than that of the control group, between 100-500 pM. The percentage of glycated hemoglobin HbA1C also decreased, reaching 6-12%. In vivo IPGTT experiments showed that microencapsulated beta cells could rapidly sense the rise of blood sugar and secrete sufficient amount of Insulin to maintain blood sugar stability. The transplanted APA microencapsulated beta cells were taken for fixation and immunofluorescence was performed to identify relevant markers. Microencapsulated beta cells can express beta cell-related Marker NKX6.1/C-peptide well, and the double-positive rate is greater than 30%. The specific evaluation results are shown in Figure 5.

Example 5. Functional evaluation of microencapsulated pancreatic beta cells (combined transplantation functional evaluation)

Microencapsulated or naked beta cells combined with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs) were transplanted into STZ-induced C57 mice for functional evaluation. The experimental steps are as follows:

1. After the cell mass of Stage5-Stage6 differentiation stage is digested into single cells with TryPLE digestion enzyme, the single cell suspension of mesenchymal stem cells (MSC) or endothelial progenitor cells (EPC) is mixed with differentiated cells in a ratio of 1:1. The single-cell suspension was mixed and cultured, and the medium used for the mixed culture was S3 differentiation basal medium, which was cultured on an orbital shaker in a low-adsorption 6-well plate.

2. The cell inoculation method of the animal experiment was the same as that in Example 4, the mixed cell inoculation density was 0.5-2*10^6/mL, the culture volume was 2-7mL/well, and the orbital shaker rotation speed was 70-120rpm. Single cells re-aggregate into clusters within 24-72 hours, and after 7-14 days of further maturation, they were transplanted into STZ-induced C57 mice subcutaneously or intraperitoneally for in vivo functional evaluation. Mesenchymal stem cells or endothelial progenitor cells and islet cells are co-aggregated and cultured to form three-dimensional cell clusters. Subcutaneous or intraperitoneal injection of a single dose of mixed islet cell clusters can provide long-term blood sugar control. Blood glucose was maintained within the normal range without the use of anti-immune rejection drugs or encapsulation systems. And in this process, the receptors can be exempted from the use of anti-immune rejection drugs or islet cell encapsulation systems (microencapsulation).

In this method, the mesenchymal stem cells are first transplanted at the transplant site, and the amount of the transplanted mesenchymal stem cells is 1-10*10^6 cells per the transplant site of each mouse. The main purpose is to build an angiogenesis environment at the transplant site, so that the transplanted beta cells can obtain nutrients and oxygen in the vascularized environment, which is more conducive to the survival and function of the transplanted beta cells. The results showed that the pre-transplanted mesenchymal stem cells were able to vascularize the transplanted part, and the vascularization time was 10-50 days. Subsequent transplantation of microencapsulated or naked beta cells was functionally equivalent to microencapsulated beta cells after intraperitoneal transplantation.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art to understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

Claims

A method for three-dimensional suspension directional differentiation of pluripotent stem cells into mature pancreatic beta cells, the method comprising the following steps:

(1) Three-dimensional suspension acclimation culture of pluripotent stem cells;

(2) Induce the acclimated cells to differentiate into pancreatic beta cells;

The pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.
The method according to claim 1, wherein the step of three-dimensional suspension acclimation culture of pluripotent stem cells in step (1) comprises:

1) Y27632 pretreatment, preferably, the pluripotent stem cells cultured on the plane are replaced with the mTeSR1 medium containing 10 μM concentration of Y27632 2 hours before the suspension culture;

2) Wash and neutralize pretreated cells with DMEM/F12 medium;

3) The pluripotent stem cells are shaken and cultured in mTeSR1+Y27632 medium, preferably, passaged 5-10 times in mTeSR1+Y27632 medium, half of the medium is replaced every day, and single cells and cells in the culture supernatant are removed during passage. Aggregated large cell clumps, preferably, the removal of large cell clumps is to filter and remove cell clumps larger than 400 μm using a 400 μm mesh;

More preferably, the seeding density of the pluripotent stem cells is 3-8*10 5 /mL, and the parameters of the three-dimensional stirring culture are: 50-120 rpm rotation speed, constant temperature of 37° C., 5% CO 2 , and 100% humidity. The base is mTeSR1 medium containing Y27632 at a concentration of 10 μM;

4) Detecting the acclimated pluripotent stem cells, if the cells grow in spheroid suspension and the ratio of Oct4+/SSEA4+ double positive cells is more than 95%, the domestication is qualified.
The method according to claim 1 or 2, wherein the step of inducing the acclimated cell differentiation and microencapsulation in step (2) comprises:

Using the acclimated pluripotent stem cells in step (1) as the initial seed cells for differentiation, the differentiation steps are:

1) Inoculate 0.3-0.7*10 6 scattered three-dimensional suspension-acclimated pluripotent stem cells in mTeSR1 medium (containing 10μM Y27632), after culturing for 24-72 hours, change the medium on the first day, and reduce the volume by 10- 20%,

The medium on the first day is: S1 medium is added: 50-200ng/ml recombinant human activin A (Activin A), 10-100ng/mL recombinant human Wnt3a protein;

2) On the 2nd day, replace the medium on the 2nd day;

The medium on the 2nd day is: S1 medium is added: 50-200ng/ml Activin A;

3) On the 4th to 6th day, replace the medium on the 4th day;

The medium on the 4th day: S2 medium supplemented with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5μM transforming growth factor-βRI Kinase Inhibitor IV (TGF-βRI Kinase Inhibitor IV) ;

4) On the 7th to 8th day, replace the medium on the 7th day;

The medium on the 7th day: S3/S4 medium supplemented with: 20-100ng/ml KGF, 0.1-0.5μM Sant1, 1-5μM retinoic acid (RA), 100-500nM 1,1,4,4-tetra Phenyl-1,3-butadiene (TPB), 5-20 μM Y27632;

5) On the 9th to 13th day, replace the medium on the 9th day;

The medium on the 9th day: S3/S4 medium supplemented with: 20-100ng/ml KGF, 0.1-0.5μM Sant1, 50-200nM RA, 10-100ng/mL EGF, 10-100ng/mL recombinant human noggin (NOG ), 5-20μM Y27632;

6) From the 14th to the 16th day, replace the medium on the 14th day;

The medium on the 14th day: S5 medium supplemented with: 0.1-0.5μM Sant1, 50-200nM RA, 0.5-2μM γ-Secretase Inhibitor XXI (γ-Secretase Inhibitor, XXI), 5-20μM RepSox, 0.5-2μM Triiodothyronine (L-3,3',5-Triiodothyronine, T3), 5-50ng/ml Recombinant Human Betacellulin Protein, 50-500nM LDN193189 hydrochloride (LDN193189 hydrochloride), 10μM Zinc sulfate;

7) On the 18th to 20th day, replace the medium on the 18th day;

The medium on the 18th day was: S5 medium supplemented with: 10-50nM RA, 0.5-2μM XXI, 5-20μM RepSox, 0.5-2μM T3, 5-50ng/ml Betacellulin, 50-500nM LDN193189, 1mM N-cys ( Fmoc-N-Me-Cys(Trt)-OH);

8) On the 21st day, the differentiated cells were digested into single cells and re-inoculated in the reactor for re-culturing. During the culture process, the reaggregated normal cell clusters were collected, and the unagglomerated supernatant was discarded, that is, the differentiated pancreatic beta. cell;

Preferably, the re-cultivation parameters are: 50-120 rpm rotation speed, constant temperature of 37 ° C, 5% CO 2 and 100% humidity, 21-35 days, replace the S3 medium every two days, use a 10-50 μm reversible filter to collect normal cell mass that will reaggregate;

Described each culture medium composition is:

S1 medium: MCDB131 basal medium+1:10000-100000 insulin-transferrin-seleno-ethanolamine (ITS-X)+1-5mM glutamine, necessary buffer salts (NaHCO 3 ), antibiotics, antioxidants ( VC), glucose and serum albumin;

S2 medium: MCDB131 basal medium+ITS-X 1:10000-1:100000+1-5mM glutamine, necessary buffer salts (NaHCO 3 ), antibiotics, antioxidants (VC), glucose and serum albumin;

S3/S4 medium: MCDB131 basal medium+ITS-X 1:50-1:500+1-5mM glutamine, necessary buffer salts (NaHCO 3 ), antibiotics, antioxidants (VC), glucose and serum albumin protein;

S5 medium: MCDB131 basal medium+ITS-X 1:50-1:500+1-5mM glutamine+2-20μg/mL heparin sodium salt, necessary buffer salts (NaHCO 3 ), antibiotics, antioxidants ( VC), glucose and serum albumin.
The method of claim 3, wherein:

2) On the 2nd day, the medium was replaced on the 2nd day, and the volume of the medium remained unchanged during the medium change;

3) The medium on the 4th day was replaced on the 4th and 6th respectively, and the volume of the medium did not change during the medium change;

4) Change the medium on the 7th day on the 7th and 8th respectively, and the volume of the medium remains unchanged during the medium change.

5) Change the medium on the 9th day on the 9th, 11th, and 13th respectively, and the volume of the medium does not change during the medium change;

6) The medium on the 14th day was replaced on the 14th and the 16th day respectively, and the volume of the medium was unchanged during the liquid change;

7) The medium on the 18th day was replaced on the 18th and 20th respectively, and the volume of the medium remained unchanged during the medium change.
The method according to claim 3 or 4, wherein, optionally, in the method for inducing differentiation, it further comprises any of the following steps or a combination of any of the steps:

9) Before the above differentiation step 3), enrichment and culture of differentiated CD177-positive groups are performed, and the specific method is:

The cell clusters differentiated in the previous step were taken, digested into single cells, and then the CD177-positive cell subsets were sorted, and then the sorted cells were seeded at a density of 2-10 × 10 5 /mL in the above-mentioned section containing 10 μM Y27632. Continue to differentiate in medium on day 4;

10) Before the above-mentioned differentiation step 6), enrichment and culture of GP2-positive groups are carried out, and the specific method is:

Take the cell mass differentiated in the previous step, digest it into single cells, and then sort out GP2-positive cell subsets, and then inoculate the sorted cells at a density of 5-10×10 5 /mL in the above-mentioned day 14 medium continue to differentiate;

11) before the above-mentioned differentiation step 8), carry out the enrichment and culture of Procr positive cell group, and the concrete method is:

Take the cell mass differentiated in the previous step, digest it into single cells, and then sort out Procr-positive cell subsets. The enriched Proc-positive cells are passaged every 7-14 days. During each passage, the digested Procr The positive single cell suspensions were supplemented with fresh human HUVEC cells, and the cell number ratio was 1:1; then, Procr-positive cells and human HUVEC cells were mixed and re-inoculated at a ratio of 1:4-1:6;

12) In the culture medium of the above differentiation step 8), 100ng/mL WNT4 was added to drive the metabolic maturation necessary for the glucose-stimulated insulin secretion of the differentiated cells.
A method for preparing sodium alginate-polylysine-sodium alginate (APA) microencapsulated artificial islets from the pancreatic beta cells obtained by the method described in any of claims 1-5, the method comprising:

(1) Prepare a solution with a viscosity of 50-200cP sodium alginate, preferably, the dissolved sodium alginate is filtered through PES sterile filtration devices of 0.8 μm, 0.45 μm and 0.22 μm in turn, and stored at 4°C;

(2) Prepare microencapsulated artificial islets according to method 1 or method 2:

Method 1: Microfluidic method:

Mix 1%-3% sodium alginate solution with the pancreatic beta cells, and 1 mL sodium alginate solution with 1-10*10 6 cells;

After mixing, add one of the pipelines, and add 0.1-2g/L calcium chloride solution to the other pipeline;

Crosslinking into a gel through a 1-10cm collection tube, followed by calcification for 5-30 minutes;

Add 0.01-0.1% polylysine to react for 10-30 minutes;

Then add 0.1%-0.3% sodium alginate solution to react for 2-10 minutes;

Then add 10-100mM sodium citrate to react for 2-10 minutes to form APA microcapsules;

Method 2: High voltage electrostatic method:

Mix 1%-3% sodium alginate solution with the pancreatic beta cells, and 1 mL sodium alginate solution with 1-10*10 6 cells;

After mixing, use a syringe with a 20-40G needle to inhale, and under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz, and flow rate of 100-1000μL/min, make it form a jet into 0.1-2g/L calcium chloride Cross-linking in solution to gel, followed by calcification for 5-30 minutes;

Add 0.01-0.1% polylysine to react for 10-30 minutes;

Then add 0.1%-0.3% sodium alginate solution to react for 2-10 minutes;

Then add 10-100mM sodium citrate and react for 2-10 minutes to form APA microcapsules.
Pancreatic beta cells prepared by any one of the methods of claims 1-5, or the application of artificial pancreatic islets prepared by the method of claim 7 in the preparation of medicines, the medicines are medicines for the treatment of diabetes, preferably, The diabetes is type I diabetes.
The application of the pancreatic beta cells prepared by the method according to any one of claims 1 to 5, or the artificial pancreatic islets prepared by the method according to claim 7, in the preparation of therapeutic active ingredients in cell therapy, the cell therapy For cell therapy for diabetic patients, preferably, the diabetes is type I diabetes.
A method for inducing an endoderm progenitor cell line, the method comprising the steps of:

(1) using the same steps as the method described in claim 1 to carry out three-dimensional suspension acclimation culture of pluripotent stem cells;

(2) Induction of endoderm progenitor cell lines;

The method for inducing the endoderm progenitor cell line described in step (2) is:

Use the pluripotent stem cells acclimated in step (1) as the starting seed cells for differentiation

1) Inoculate 0.3-0.7*10 6 scattered three-dimensional suspension-acclimated pluripotent stem cells per ml of mTeSR1 medium (supplemented with 10 μM Y27632 in the medium), and after culturing for 24-72 hours, replace the medium on the first day and change the medium When the volume is reduced by 10-20%,

The medium on the first day is: S1 medium is added: 50-200ng/ml recombinant human activin A (Activin A), 10-100ng/mL recombinant human Wnt3a protein;

2) On the 2nd day, the medium was replaced on the 2nd day, and the volume of the medium remained unchanged during the medium change;

The medium on the 2nd day is: S1 medium is added: 50-200ng/ml Activin A;

3) On the 4th and 6th day, the medium was replaced on the 4th day, and the volume of the medium was unchanged during the medium change;

The medium on the 4th day: S2 medium supplemented with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5μM transforming growth factor-βRI Kinase Inhibitor IV (TGF-βRI Kinase Inhibitor IV) ;

4) When the proportion of SOX17-positive cells was >90% in flow cytometry, the cell mass was digested into single cells, and the CXCR4+/CD117+ double-positive cell subgroup was sorted, and this cell subgroup was the directed endoderm progenitor cells;

5) inoculate the group of cells in a plate containing Matrigel, and use the directional endoderm progenitor cell culture and expansion medium to culture and expand;

The components of the directed endoderm progenitor cell culture and expansion medium are:

Add to S1 medium: 20-100ng/mL bone morphogenetic protein 4 (BMP4), 5-20ng/mL recombinant human basic fibroblast growth factor protein (bFGF), 5-20ng/mL recombinant human vascular endothelial growth factor Protein (VEGF), 5-20ng/mL recombinant human epidermal growth factor protein (EGF).