CN112980771B - Method for preparing pancreatic beta cells and application thereof - Google Patents
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Abstract
The invention relates to a method for preparing differentiated pancreatic beta cells and application thereof, in particular to the method comprising the following steps: (1) three-dimensional suspension domestication culture of pluripotent stem cells; (2) inducing the differentiation of the acclimatized cells into pancreatic beta cells. The invention also relates to a method for preparing sodium alginate-polylysine-sodium alginate (APA) microencapsulated artificial islets from the differentiated pancreatic beta cells, which comprises the following steps: microencapsulated artificial islets were prepared using sodium alginate solution. The invention also relates to application of the pancreatic beta cells and artificial islets.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a method for preparing pancreatic beta cells and application thereof.
Background
Diabetes is a disease in which islet function is reduced or depleted, resulting in a disorder of the body's glycometabolism. According to the latest statistics of the international diabetes consortium (International Diabetes Federation, IDF), in 2019, more than 4.63 million people worldwide had diabetes mellitus, by 2045 7 million people would have diabetes mellitus; china was first in the world with 1.16 million people diabetic patients, and it is expected that 1.47 million people will be reached by 2045 years. In 2019, about 420 people (20-79 years old) die worldwide from diabetes or complications thereof, and 1 person dies from diabetes every 6 seconds, accounting for about 11.3% of all deaths worldwide.
Type 1 diabetes accounts for 5-10% of the total number of diabetes mellitus, and is caused by complete loss of beta cell number and function. At present, the treatment means mainly comprise long-term insulin injection treatment and islet transplantation, and the diabetes and complications cannot be cured by insulin injection at all; the novel insulin pump also has the disadvantages of inconvenient carrying, unstable blood sugar control and great pain for patients. In addition, the islet extraction and purification process in the islet transplantation strategy is very complex, the lack of organ resources and how to obtain the islets which can meet the clinical transplantation requirements efficiently and stably are also the main challenges of islet transplantation. In response to the above problems, the development of stem cell therapy techniques to restore the originally deleted pancreatic beta cells in the body is a better option to radically treat diabetes (Pagliuca FW et al, 2013).
The current methods for stem cell differentiation into beta cells mainly include: (1) Gene transfection is a method in which a plurality of transcription factors PDX-1, NKX6.1, ngn3, neuroD, pax4, etc., which control embryo development toward pancreas, are transfected into cells and induced to differentiate into insulin-secreting cells (Noguchi H et al, 2006). However, the gene transfection method has a potential oncogenic risk that the insertion of foreign genes into the cell genome using viral vectors may inactivate cancer suppressor genes. In addition, the insulin-secreting cells obtained by this method are not mature and do not respond well to glucose stimulation. (2) The cytokine induction method, which is a key step in simulating pancreatic development in vitro according to embryo to pancreatic development process, induces and differentiates 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, the method has complex steps and long differentiation time; moreover, the immature differentiation method is easy to bring the problems of poor stability of differentiation batches, low cell yield, limited mass production and the like. However, the development of a method for inducing differentiation of mature and stable cytokines can well overcome the above problems, and stable, large-scale and mature functional pancreatic beta cells can be obtained in vitro.
In addition, mature pancreatic beta cells are transplanted into a foreign environment and often undergo foreign immune rejection, so that the transplanted beta cells lose the original functions. The main strategy for solving the problem of immune rejection in the process of allograft transplantation is to use a combined immunosuppressant, but the immunosuppression effect is often less than ideal and suffers from drug administration for life. Recent strategies have shown that the development of safe and effective immunoisolation tools is a better option to address the problem of immune rejection during allograft 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 immune isolation tool is the key to success of transplantation, and the difference of the in-vivo function evaluation effect of the beta cells obtained by differentiation is obvious due to the influence of different wrapping materials. Wrapping materials commonly used today include: (1) natural materials. Lipids, polysaccharides and proteins; (2) semi-synthetic materials. Cellulose derivatives and the like; (3) synthetic material. Including degradable and non-degradable materials. The problems of serious fibrosis encapsulation, no immune isolation function and the like can occur in a plurality of material implants, and the activity and the function of encapsulated cells are seriously influenced. However, the development of an ideal packaging material would solve the problems associated with the xenograft procedures described above.
Therefore, multipotent stem cell-derived pancreatic beta cells are expected to treat diabetes by cell replacement strategies, but how to better scale up and stabilize mature pancreatic beta cells obtained in vitro and solve the problem of immune rejection after allogeneic pancreatic beta cell transplantation has been a research hotspot.
Disclosure of Invention
The 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, comprising the following steps:
(1) Three-dimensional suspension domestication culture of pluripotent stem cells;
(2) Inducing the domesticated cells to differentiate into pancreatic beta cells;
the step of three-dimensional suspension domestication culture of the pluripotent stem cells in the step (1) comprises the following steps:
1) Y27632 pretreatment, preferably, the planar culture of pluripotent stem cells is replaced 2 hours before suspension culture with mTESR1 medium containing Y27632 at 10 μm concentration;
2) The neutralization pretreatment cells were washed with DMEM/F12 medium and counted;
3) Culturing the pluripotent stem cells in a mTESR1+Y27632 culture medium under stirring, performing passage on the mTESR1+Y27632 culture medium for 5-10 times, changing half of the culture medium every day, and removing single cells and aggregated large cell aggregates in a culture supernatant during passage;
Preferably, the pluripotent stem cells have an seeding density of 3-8 x 10 5 Three-dimensional stirred suspension culture was performed using a disposable bioreactor (Disposable spinner flasks, corning, 3152/3153) with parameters: 50-120rpm, constant temperature 37 ℃ and 5% CO 2 100% humidity, wherein the culture medium is mTESR1 culture medium containing Y27632 with the concentration of 10 mu M;
preferably, the removing of the large cell mass is removing of cell mass larger than 400 μm by filtration using a 400 μm screen;
4) Detecting the domesticated pluripotent stem cells, wherein the cells grow in a spherical suspension manner, and the proportion of Oct4+/SSEA4+ double-positive cells is more than 95%, namely the domestication is qualified;
the step of inducing acclimatized cells and microencapsulating in step (2) comprises:
the culture medium used was:
s1 culture medium: MCDB131 basal medium +1:10000-100000 insulin-transferrin-selenium-ethanolamine (ITS-X) +1-5mM glutamine, necessary buffer salt (NaHCO) 3 ) Antibiotics, antioxidants (VC), glucose, and serum albumin;
s2 culture medium: MCDB131 basal medium+ITS-X1:10000-1:100000+1-5 mM glutamine, necessary buffer salts (NaHCO 3 ) Antibiotics, antioxidants (VC), glucose, and serum albumin;
S3/S4 medium: MCDB131 basal medium+ITS-X1:50-1:500+1-5 mM glutamine, necessary buffer salts (NaHCO 3 ) Antibiotics, antioxidants (VC), glucose, and serum albumin;
s5 culture medium: MCDB131 basal medium +ITS-X1:50-1:500+1-5 mM glutamine+2-20. Mu.g/mL heparin sodium salt, necessary buffer salt (NaHCO 3 ) Antibiotics, antioxidants (VC), glucose, and serum albumin;
the induced differentiation method specifically comprises the following steps:
using the pluripotent stem cells acclimatized in step (1) as differentiated starting seed cells
1) Inoculating 0.3-0.7×10/ml mTESR1 culture medium (10 μ M Y27632 added to the culture medium) 6 The scattered three-dimensional suspension domesticated pluripotent stem cells are cultured for 24 to 72 hours, the first day culture medium is replaced, the volume is reduced by 10 to 20 percent when the liquid is replaced,
the first day medium was: s1, adding the culture medium: 50-200ng/mL recombinant human Activin A (Activin A), 10-100ng/mL recombinant human Wnt3a protein;
2) The culture medium on the 2 nd day is replaced on the 2 nd day, and the volume of the culture medium is unchanged when the liquid is replaced;
the 2 nd culture medium is: s1, adding the culture medium: 50-200ng/ml Activin A;
3) Changing the culture medium on the 4 th day and the 6 th day respectively, wherein the volume of the culture medium is unchanged during liquid changing;
the culture medium on day 4 is: s2, adding the culture medium: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5. Mu.M transforming growth factor-. Beta.RI kinase inhibitor IV (TGF-. Beta. RI Kinase Inhibitor IV);
4) Changing the culture medium on the 7 th day and the 8 th day respectively, wherein the volume of the culture medium is unchanged during liquid changing;
the 7 th medium was: S3/S4 medium: 20-100ng/ml KGF, 0.1-0.5. Mu.M Sant1, 1-5. Mu.M Retinoic Acid (RA), 100-500nM 1, 4-tetraphenyl-1, 3-butadiene (TPB), 5-20. Mu. M Y27632;
5) Changing the culture medium on 9 th day, the culture medium on 11 th day and the culture medium on 13 th day respectively, wherein the volume of the culture medium is unchanged during liquid changing;
the 9 th medium was: S3/S4 medium: 20-100ng/mL KGF, 0.1-0.5 mu M Sant1, 50-200nM RA, 10-100ng/mL EGF, 10-100ng/mL recombinant human NOG (NOG), 5-20 mu M Y27632;
6) The 14 th culture medium is respectively replaced on the 14 th day and the 16 th day, and the volume of the culture medium is unchanged when the liquid is replaced;
the culture medium on day 14 is: s5, adding the culture medium: 0.1-0.5. Mu.M Sant1, 50-200nM RA, 0.5-2. Mu.M gamma. -secretase inhibitor XXI (gamma. -Secretase Inhibitor, XXI), 5-20. Mu.M RepSox, 0.5-2. Mu.M trisaccharide thyronine (L-3, 3', 5-triodothiron, T3), 5-50ng/ml recombinant human beta-cytokine (Recombinant Human Betacellulin Protein), 50-500nM LDN193189hydrochloride (LDN 193189 hydrochloride), 10. Mu.M zinc sulfate;
7) Changing the culture medium on the 18 th day and the culture medium on the 20 th day respectively, wherein the volume of the culture medium is unchanged during liquid changing;
The 18 th medium was: s5, adding the culture medium: 10-50nM RA, 0.5-2 μM XXI, 5-20 μM RepSox, 0.5-2 μ M T3, 5-50ng/ml Betacelllin, 50-500nM LDN193189, 1mM N-Cys (Fmoc-N-Me-Cys (Trt) -OH);
8) On day 21, differentiated cells were digested to single cells using TrypLE Express, then following 0.5-2 x 10 6 The cells/ml density is re-inoculated in a reactor, and the culture is carried out at the rotating speed of 50-120rpm, the parameters of the incubator are set to be constant temperature of 37 ℃ and 5 percent CO 2 And 100% humidity, and replacing the S3 culture medium every two days when the humidity is 21-35 days, collecting the normal cell mass which is reaggregated by using a 10-50 mu m reversible filter in the culture process, and discarding the supernatant which is not aggregated to obtain the differentiated pancreatic beta cells.
Optionally, in the method of inducing differentiation, any one or any combination of the following steps may be further included:
9) Enrichment and culture of the differentiated CD 177-positive population can be performed before the differentiation step 3), and the specific method is as follows:
taking cell mass at differentiation stage, digesting into single cells, separating CD177 positive cell subgroup, and separating the separated cells at a ratio of 2-10×10 5 The differentiation was continued in the medium containing 10. Mu. M Y27632 on day 4 at a density of/mL;
10 Before the differentiation step 6), enrichment and culture of GP 2-positive populations can be performed, in particular by:
taking cell mass at differentiation stage, digesting into single cells, separating GP2 positive cell subgroup, and then subjectingSorting cells to 5-10X 10 5 Density inoculation of/mL continued differentiation in day 14 medium;
11 Before the differentiation step 8), enrichment and culture of Procr positive populations can be performed, in particular by:
taking cell mass at the differentiation stage, digesting the cell mass into single cells, then separating out a Procr positive cell subgroup, carrying out passage on the enriched Proc positive cells every 7-14 days, supplementing the digested Procr positive single cell suspension with fresh human HUVEC cells in the process of passage, wherein the cell number ratio is 1:1; then mixing the Procr positive cells and the human HUVEC cells, and re-inoculating and culturing the two cells in a ratio of 1:4-1:6, so that the yield of differentiated cells is obviously improved;
12 100ng/mLWNT4 may be added to the medium of the above-described differentiation step 8) to drive metabolic maturation necessary for glucose-stimulated insulin secretion in the differentiated cells.
The invention also relates to a method for preparing sodium alginate-polylysine-sodium alginate (APA) microencapsulated artificial islets from said differentiated pancreatic beta cells, said method comprising:
(1) Preparing sodium alginate solution: the dissolved sodium alginate is filtered by a PES sterile filter device of 0.8 μm, 0.45 μm and 0.22 μm in sequence; detecting the viscosity of the solution to be 50-200cP, and preserving at 4 ℃;
(2) Preparing microencapsulated artificial islets according to a first method or a second method:
the method comprises the following steps: microfluidic method:
mixing 1% -3% sodium alginate solution with the pancreatic beta cells, and mixing 1mL sodium alginate solution with 1-10 x 10 6 Mixing cells;
adding one of the pipelines after mixing, and adding 0.1-2g/L calcium chloride solution into the other pipeline;
crosslinking into gel in a collecting tube of 1-10cm, and calcification for 5-30 min;
adding 0.01-0.1% polylysine for reaction for 10-30 min;
adding 0.1% -0.3% sodium alginate solution to react for 2-10 minutes;
adding 10-100mM sodium citrate to react for 2-10 minutes to form APA microcapsule;
the second method is as follows: high voltage electrostatic process:
mixing 1% -3% sodium alginate solution with the pancreatic beta cells, and mixing 1mL sodium alginate solution with 1-10 x 10 6 Mixing cells;
sucking the mixture by using a syringe with a 20-40G needle after mixing, allowing the mixture to form jet flow into 0.1-2G/L calcium chloride solution to crosslink into gel under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz and flow speed of 100-1000 mu L/min, and then calcification for 5-30 minutes;
Adding 0.01-0.1% polylysine for reaction for 10-30 min;
adding 0.1% -0.3% sodium alginate solution to react for 2-10 minutes;
and adding 10-100mM sodium citrate to react for 2-10 minutes to form the APA microcapsule.
The invention also relates to application of the pancreatic beta cells or artificial islets in preparation of medicines, wherein the medicines are medicines for treating diabetes, and preferably the diabetes is type I diabetes.
The invention also relates to application of the pancreatic beta cells or artificial islets in preparing a therapeutically active ingredient in a cytotherapeutic procedure, wherein the cytotherapeutic procedure is a cytotherapeutic procedure aiming at diabetics, and preferably, the diabetes is type I diabetes.
The invention also relates to a method of inducing an endodermal progenitor cell line, the method comprising the steps of:
(1) Three-dimensional suspension domestication culture of pluripotent stem cells;
(2) Induction of endodermal progenitor cell lines;
the three-dimensional suspension domestication culture step of the pluripotent stem cells in the step (1) is the same as the related steps of the method for three-dimensional suspension directional differentiation of the pluripotent stem cells into mature pancreatic beta cells;
the method for inducing the endoderm progenitor cell line in the step (2) comprises the following steps:
Using the pluripotent stem cells acclimatized in step (1) as differentiated starting seed cells
1) Inoculating 0.3-0.7×10/ml mTESR1 culture medium (10 μ M Y27632 added to the culture medium) 6 The scattered three-dimensional suspension domesticated pluripotent stem cells are cultured for 24 to 72 hours, the first day culture medium is replaced, the volume is reduced by 10 to 20 percent when the liquid is replaced,
the first day medium was: s1, adding the culture medium: 50-200ng/mL recombinant human Activin A (Activin A), 10-100ng/mL recombinant human Wnt3a protein;
2) The culture medium on the 2 nd day is replaced on the 2 nd day, and the volume of the culture medium is unchanged when the liquid is replaced;
the 2 nd culture medium is: s1, adding the culture medium: 50-200ng/ml Activin A;
3) Changing the culture medium on the 4 th day and the 6 th day respectively, wherein the volume of the culture medium is unchanged during liquid changing;
the culture medium on day 4 is: s2, adding the culture medium: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5. Mu.M transforming growth factor-. Beta.RI kinase inhibitor IV (TGF-. Beta. RI Kinase Inhibitor IV);
4) When the flow detection SOX17 positive cell proportion is more than 90%, cell clusters are taken to be single cells, and cell subsets with CXCR4+/CD117+ double positive are separated (commonly, differentiation is carried out until the 6 th day, namely, the cell subsets with CXCR4+/CD117+ double positive can be separated), and the cell subsets are directional endodermal progenitor cells;
5) Inoculating the population of cells in a Matrigel-containing plate, and culturing and expanding the population of cells by using a directional endoderm progenitor cell culture and expansion medium;
the culture and amplification medium for the directional endoderm progenitor cells comprises the following components:
s1, adding the culture medium: 20-100ng/mL of bone morphogenetic protein 4 (BMP 4), 5-20ng/mL of recombinant human basic fibroblast growth factor protein (bFGF), 5-20ng/mL of recombinant human vascular endothelial growth factor protein (VEGF), and 5-20ng/mL of recombinant human epidermal growth factor protein (EGF).
The invention has the beneficial effects that:
the invention aims at providing a method for obtaining mature and batch-stable large-scale three-dimensional suspension directional differentiated beta cells in vitro aiming at the defects of poor batch stability and in-vivo function evaluation of the existing differentiated beta cells. Meanwhile, an ideal method for microencapsulating and wrapping differentiated beta cells by using the wrapping material is provided, so that the microencapsulated beta cells can well function in an animal body and have no problems of immune rejection and fibrosis wrapping. Specifically comprises (1) three-dimensional large-scale suspension culture of pluripotent stem cells (including induced pluripotent stem cells and embryonic stem cells); (2) In vitro three-dimensional suspension dynamic directional differentiation into mature pancreatic beta cells using a combination of compounds; (3) microencapsulated encapsulation of beta cells; (4) evaluation of in vitro and in vivo functions of microencapsulated beta cells.
Compared with the prior art, the method provided by the invention has the remarkable advantages of: the PSC three-dimensional suspension culture method provided by the invention can successfully maintain the expression of PSC multipotency genes, has good cell proliferation and stable cell nucleus type, and particularly needs to replace a fresh culture medium added with Y27632 for pretreating cells for 2-4 hours before a planar cell is put into a 3D reactor, so that the success rate of PSC three-dimensional suspension culture is obviously increased due to proper initial cell inoculation density, culture system, rotating speed and the like. The cell liquid changing mode (half liquid changing) in the three-dimensional culture process, the cell processing mode of the upper list, the large cell mass processing mode of more than 400 mu m and the normal cell mass acquisition mode not only save the cost, but also can well maintain the cell viability and the like. The compound combination mode used in each stage in the differentiation process can efficiently differentiate into the cell type in the stage, and the proportion of differentiated cells is obviously improved. Especially, the culture system before differentiation is changed, the preparation method and the addition time of the composition have good differentiation promoting effects such as liquid changing mode, cell treatment mode of the upper differentiation list and the like in the differentiation process, the stability among various differentiation batches is obviously improved, and the differentiated beta cells are more mature and have stronger functions. The improved sodium alginate microencapsulation package differentiated beta cells transplanted into animals can reverse hyperglycemia of diabetic mice in a short time (24 h), have a good immune isolation function, have less fibrosis package, and can maintain normal blood sugar in the immunized mice for a long time.
Drawings
FIG. 1, 1A, microscopic photographs of the index and results of the multipotency detection of stem cells in the planar culture process; 1B, streaming detection results.
FIG. 2 is a photograph showing the result of the environmental test of hPSCs after 3D culture.
FIG. 3 shows the proliferation of cells in the acclimation process, the proliferation multiple is 2-4 times (FIG. 3A), and the ratio of Oct4/SSEA4 double positive cells is more than 99% (FIG. 3B).
FIG. 4, morphology of the cell mass after encapsulation with sodium alginate-polylysine-sodium alginate (APA).
FIG. 5 results of in vivo functional assessment of microencapsulated pancreatic beta cells transplanted into the abdominal cavity of STZ-induced C57BL/6 mice.
FIG. 6, qPCR assay results at various stages during differentiation of pancreatic beta cells by classical methods and expression trends of related markers at the transcriptional level.
FIG. 7 shows the expression trend of related markers at the transcription level by qPCR detection results of various stages in the pancreatic beta cell process obtained by the optimized differentiation method of the present invention.
FIG. 8 shows the results of measurement of the properties of cell clusters at each differentiation stage in the optimized differentiation method of the present invention.
FIG. 9 is a schematic diagram showing the detection index at each stage in the differentiation process.
Detailed Description
EXAMPLE 1 planar culture, in vitro 3D culture and acclimatization of hPSCs cells
1. hPSCs cell plane culture
The preparation method of the required reagent comprises the following steps:
mTeSR1 pluripotent stem cell medium (stemell, 85850);
ackutase digestive enzyme (STEMCELL, 07920);
rho kinase inhibitor Y27632 (Abcam, ab 120129): the stock solution was prepared with DMSO at a final concentration of 10. Mu.M for use, i.e., 1000X;
mtesr1+y27632 medium: 15mL of mTESR1 is added with 15 mu L of 10mM Y27632, and the mixture is uniformly mixed and stored at 4 ℃ when not used, and the mixture is optimally used within two weeks after preparation.
hPSCs (clinical grade human embryonic stem cells or human induced pluripotent stem cells, from Beijing stem cell bank).
1. hPSCs resuscitation and culture
1.1, matrigel coated plates: taking out the 6-hole plate, adding 0.5-2mL Matrigel (Matrigel, corning, 354277) into each hole, gently shaking the 6-hole plate to enable the Matrigel to completely cover the bottom of the dish, placing the dish in a 37 ℃ incubator for incubation for 1-2 h, taking out the dish before experiment, and placing the dish in an ultra-clean workbench/biological safety cabinet for balancing for 10-40min at room temperature. If temporarily unused, the container can be stored at 2-8deg.C after being sealed by Parafilm and used within 1-2 weeks.
The number of one frozen stem cell is 1×10 6 About cells/mL, 1 hole of 6 hole plates is correspondingly inoculated;
1.2, adding 2-3 mL of mTESR1 culture medium into a 15mL centrifuge tube for standby.
1.3, thawing: quickly immersing the freezing tube taken out of the liquid nitrogen into warm water at 37 ℃ and quickly shaking to quickly defrost the tube within 1-2 min;
1.4, centrifuging: after the frozen solution in the frozen storage tube is frozen, dropwise adding the frozen storage solution into a 15mL centrifuge tube containing mTESR1 culture medium, placing the 15mL centrifuge tube into a low-speed centrifuge for proportioning balance, and centrifuging at 800-1800rpm for 3min;
1.5, resuspension: and (3) adding 0.5-2mL mTeSR1+Y27632 cell culture medium into the supernatant after centrifugation to perform blowing-suction mixing on the stem cell sediment, and performing blowing-suction for about 3-5 times.
1.6 inoculation: after the pipetting is uniform, the equilibrated Matrigel is discarded, the pipetting uniform stem cell suspension is added to the coated 6-well plate and 2mL of culture system per well is filled.
1.7 culturing: the inoculated 6-hole plate can be placed under an inverted phase contrast microscope to observe the density of the inoculated stem cells, and the 6-hole plate is gently shaken horizontally by a cross so that the cells are uniformly distributed. And placed at 37℃in 5% CO 2 Culturing in a constant temperature incubator, and observing the cell adhesion condition on the 2 nd day;
1.8 liquid change: the liquid is changed every 24-48h from the time of resuscitating.
In the planar culture process, the cell morphology is normal, and the stem cell pluripotency detection index and the result in the planar culture process are shown in figure 1; the result shows that the hPSCs subjected to planar culture by the method can well maintain the clone form of stem cells, and the dry related genes are well expressed; the flow result shows that the ratio of Oct4+/SSEA4+ double positive cells is more than 99%, which indicates that the dryness is maintained well in the culture process; the SSEA1 cells were very low in proportion and no differentiation occurred.
2. hPSCs passage for 3D culture
2.1 adding Y27632 pretreatment: 2-4h before the 3D reactor (Disposable spinner flasks, corning, 3152/3153) the mTESR1 media μm pretreatment cells containing Y27632 were replaced;
2.2 cleaning: sucking the original culture medium, slowly adding 0.5-2mL of DMEM/F12 to the wall, slightly shaking, and then sucking the DMEM/F12 along the edge of the culture dish; digestion: adding 0.5-2 mL/hole Ackutase into a 6-hole plate to cover the bottom of the dish, and placing the dish in a 37 ℃ incubator for 2-5 min;
2.3 neutralization: adding 1-4mL of DMEM/F12 for neutralization, blowing the bottom of the culture dish by a sector of a liquid-transferring gun, gently blowing for 3-5 times to enable stem cell colonies at the bottom of the dish to fall off, and transferring the stem cell colonies into a 15mL centrifuge tube;
2.4 count: centrifuging at 800-1800rpm for 3-5min; discarding the supernatant, blowing the cells with a stem cell culture medium for 5-10 times, and taking part of cell suspension for counting;
2.5 inoculation: according to the counting result, 3-8 x 10≡5/mL density is resuspended in mTESR1+Y27632 medium, then inoculated in Corning Spinner Flask, 40-300mL culture system. Culturing the Spinner discs on a nine-site magnetic stirrer at 50-120rpm, and setting parameters of an incubator to constant temperature of 37 ℃ and 5% CO 2 And 100% humidity.
The photograph of the result of the environmental test of hPSCs after 3D culture is shown in FIG. 2
This mode of culture remains unchanged during cell culture and differentiation. If the cell inoculation density is not within the range, the cell cannot be well agglomerated due to the too small density, and proliferation is limited; the excessive density can cause that the cell clusters are easy to form large clusters, the adhesion between the clusters is serious, the particle size of the cell clusters is bigger, the hypoxia of the cells in the middle part can be caused, the cell activity rate is reduced, and the cell clusters are easy to differentiate. If the rotating speed is not in the range, the too large rotating speed can lead to poor agglomeration, the excessive cell activity rate of single cells is reduced, and the dryness is lost; too small a rotational speed can result in serious adhesion between cell clusters, and too large a cell cluster particle size can result in hypoxia of cells in the middle part, and the cell activity is reduced and differentiation is easy.
3. hPSCs cell domesticated by 3D culture and function detection
3.1 ensuring acclimation in the reactor for at least 5-10 passages before measuring the differentiation ability of hPSCs cells. Half-changing liquid is carried out every day, large cell aggregates larger than 400 mu m are removed through a 400 mu m screen, and single cells in culture supernatant are added back into the reactor. The single cells in the culture supernatant were discarded at passage, normal pellet was collected with 37 μm reversible filter, cells were dispersed with Accutase, and 0.3-0.7 million dispersed cells were inoculated in mTeSR1 medium added with 5-20 μ M Y27632 per ml. If the large cell mass larger than 400 μm is not removed, the cell mass is adhered more and more seriously, and further the cell aggregate size is bigger, so that the cell hypoxia in the middle part is caused, the cell activity is reduced, and the cell is easy to differentiate. If the single cells in the culture supernatant are not returned to the reactor, cell proliferation is limited, which is disadvantageous for cell mass formation.
3.2 differentiation is carried out after 48-72 hours of culture, during which half of the mTESR1 medium without Y27632 is replaced every day, before differentiation, the cell cluster diameter is kept within 300 mu m, and if the cell cluster diameter is larger than 300 mu m, partial cell necrosis or spontaneous differentiation of the cell cluster can be caused. Cell lines with different doubling times may require different seeding concentrations or times.
The proliferation of cells during acclimation is shown in FIG. 3: it can be seen that the pluripotent stem cells proliferated well during acclimation, with a proliferation factor between 2-4 fold (fig. 3A). hPSCs can well express the genes related to the dryness in the domestication process, and the flow result shows that the ratio of Oct4+/SSEA4+ double positive cells is more than 99% (figure 3B), which also shows that the 3D pluripotent stem cells can well maintain the dryness in the domestication process.
Example 2 differentiation of hPSCs into pancreatic beta cells
The names and formulas of the culture medium used in the whole differentiation process are as follows,
s1 culture medium: MCDB131 basal medium (thermo filter, 10372019) +2-20mM Glucose (Glucose, sigma, G7528-250G) +1-5G/L sodium bicarbonate (NaHCO) 3 Sigma, S5761-500G) +0.5-5% recombinant human serum albumin (Human serum albumin, HSA, gramineae, HYC002M 01) +1:10000-1:100000 insulin-transferrin-selenium-ethanolamine (InsulinTransferrinSeleniumEthanolamine, ITS-X, thermofiser, 51500056) +1-5mM glutamine substitutes (GlutaMAX) TM Supplement, glutamax, thermofiser, 35050061) +0.1-0.5mM Vitamin C (L-Ascorbic acid, vitamin C, sigma, A4544-25G) +1% Penicillin/Streptomycin (Penicillin-Streptomycin, pen/Strep, thermofiser, 15140122).
S2 culture medium: MCDB131+2-20mM glucose+1-3g/L NaHCO 3 +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 NaHCO 3 +0.5-5%HSA+ITS-X 1:50-1:500+1-5mM Glutamax+0.1-0.5mM Vitamin C+1%Pen/Strep.
S5 culture medium: MCDB131+5-50mM glucose+1-3g/L NaHCO 3 +0.5-5% HSA+ITS-X1:50-1:500+1-5 mM Glutamax+0.1-0.5mM Vitamin C+1%Pen/strep+2-20. Mu.g/mL Heparin sodium salt (Heparin sodium salt, heparin, sigma, H3149-500 KU-9).
All the above media were sterilized by filtration through 0.22. Mu. m bottle top filter Filter.
And fresh media (small molecules and growth factors are added to the basal medium in a low-light-hood safety cabinet) needs to be configured before media replacement.
1. Differentiation of SC-beta cells (pancreatic beta cells)
1. Classical method pancreatic beta cell differentiation and functional detection
Using the method of Melton DA. (which is cited in: doi.org/10.1016/j.cell.2014.09.040),
cells were taken separately for analysis for in vitro functional assessment at each and final stages of differentiation. The detection results are shown below:
(1) S0 phase: hPSC, human pluripotent stem cells. The proportion of Oct4+/SSEA4+ double-positive cells is normal in the flow detection, and the dryness is maintained better;
(2) S1,: DE, definitive endoderm cells. The proportion of SOX17 positive cells detected by the flow detection is 82.9%, the proportion is less than 90%, and the differentiation efficiency is low;
(3) S2, stage: PGT, primitive gut tube cells. The proportion of HNF4a positive cells detected by the flow is 47.9%, and the differentiation efficiency is low;
(4) S3, stage: PP1, early pancreatic progenitor cells. The proportion of PDX1 positive cells detected by the flow is 40.6%, and the differentiation efficiency is low;
(5) S4, stage: PP2, later pancreatic progenitor cells. The ratio of PDX1+/NKX6.1+ double positive cells in the flow detection is only 8.62%, and the expression is low;
(6) S5, stage: EN, endocrine progenitor cells. The ratio of the flow detection NKX6.1+/C-peptide double-positive cells is only 10.1%, and the majority of C-peptide in the differentiation result is weakly positive expression;
(7) S6, stage: SC-. Beta.cells, stem cell-derived-. Beta.cells. The proportion of NKX6.1+/C-peptide double-positive cells in the flow assay is only 12.7%. The CHGA positive cell fraction of endocrine cells was 47.71% and relatively low. Glucose stimulation for three consecutive rounds was not responsive and the secretion of ins was low at high glucose stimulation.
(8) qPCR detection results of various stages in the differentiation process by using the method and expression trend of related markers on the transcriptional level:
The cells at different stages are taken for RNA extraction in the whole differentiation process, and then qPCR is used for detecting the expression trend of related genes in the whole differentiation process. As can be seen from FIG. 6, the relative expression levels of PDX1, NKX6.1 and ins obtained by this method were low, only tens of times that of the original cells. The Oct4 expression does not drop to a low level until Stage5, which presents a significant safety problem.
2. Pancreatic beta cell differentiation and function detection by optimizing method
Pluripotent stem cells acclimatized in a 3D reactor for 5-20 passages (the passage number of the 3D acclimation process is included in the 5-20 passages) are used as differentiated starting seed cells.
2.1 differentiation method
At the beginning of SC-beta cell differentiation, 0.3-0.7 million dispersed hPSCs cells were inoculated into mTESR1 medium added with 10. Mu. M Y27632 per ml. After 24-72 hours of culture, day 1media was changed, at this time 80-320mL of culture system was changed to 60-300mL of differentiation system (volume was reduced by 10-20%, medium usage at the time of the first liquid change was reduced by 10% -20% compared with that at the time of no liquid change, and then culture volume was maintained at each liquid change) at this time of formal start of differentiation, and then medium change was sequentially performed according to the following times (full liquid change, single cells in culture supernatants at the same differentiation stage were centrifuged and then fed back to the reactor, and single cells in culture supernatants at the time of the cross-stage were discarded). If the volume is not reduced at the beginning of differentiation, it results in very low cell yields at the end of differentiation. Vc, all factors and small molecule compounds used in the differentiation process are added on the same day. If added in advance, the efficiency of differentiation is greatly reduced. And the related culture medium is required to be freshly prepared before the culture medium is replaced, and the small molecular compound and the growth factor are added into the basic culture medium in a safety cabinet with a low light cover, so that the whole replacement process is protected from light. If the light shielding operation is not performed, a part of the small molecular compounds are decomposed by direct irradiation with a light source, and the differentiation efficiency is reduced.
First 0.3-0.7 million dispersed hPSCs cells were inoculated in mTESR1 medium supplemented with 10. Mu. M Y27632 per ml. After culturing for 24-72 hours, the culture medium is replaced and the corresponding cytokines are added, and according to the culture process, the steps of replacing the culture medium and adding the cytokines are as follows:
day 1 S1+50-200ng/mL recombinant human Activin A (Recombinant Human/Mouse/Rat Activin AProtein, activinA, 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), (10% -20% less medium than without changing the fluid for the first fluid change, and then the culture volume is maintained for each fluid change)
Day 2:S1+50-200ng/ml ActivinA.
Days 4, 6:S2+20-100ng/ml recombinant human keratinocyte growth factor protein (Recombinant Human KGF/FGF-7Protein,KGF,R&D systems,251-KG-050) +1-5. Mu.M transforming growth factor-. Beta.RI kinase inhibitor IV (TGF-. Beta. RI Kinase Inhibitor IV, sigma,616454-2 MG).
Days 7, 8:S3+20-100ng/ml KGF+0.1-0.5. Mu.M Sant1 (Sigma, S4572-5 MG) +1-5. Mu.M Retinoic acid (RA, sigma, R2625-50 MG) +100-500nM 1, 4-Tetraphenyl-1,3-butadiene (1, 4-Tetraphenyl-1,3-butadiene, TPB, sigma, 185213-5G) +5-20. Mu. M Y27632.
Days 9, 11, 13:S3+20-100NG/mL KGF+0.1-0.5. Mu.M 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 (Recombinant Human Noggin Protein, NOG, R & D systems, 6057-NG-025) +5-20. Mu. M Y27632.
Days 14, 16:S5+0.1-0.5. Mu.M Sant1+50-200nM RA+0.5-2. Mu.M gamma. -secretase inhibitor XXI (gamma-Secretase Inhibitor XXI, compound E, XXI, millipore,565790-500 UG) +5-20. Mu.M RepSox (Alk 5i II, sigma, R0158-5 MG) +0.5-2. Mu.M trisaccharide (L-3, 3', 5-trisaccharine, T3, millipore,64245-250 MG-M) +5-50ng/ml recombinant human beta-cytokine (Recombinant Human Betacellulin Protein, betacelloulin, R & D systems,261-CE 010) +50-500nM LDN193189hydrochloride (LDN 193189 hydrogel, LDN193189 MG, SML 0559-5) +10. Mu.M zinc sulfate (ZnSO 4, Z51).
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 (medium changed every two Days) S3 medium.
At 21 days of differentiation, 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 million cells/ml, and cultured at a rotation speed of 50-120rpm, with parameters of the incubator set to constant temperature of 37℃and 5% CO 2 And 100% humidity.
Thereafter, the solution was changed every other day, and the normal pellet was collected by re-aggregation using a 10-50 μm reversible filter, and the supernatant of the non-aggregation was discarded. Cells were taken separately for analysis at each and final stages of differentiation.
2.2 detection index of each Stage in the differentiation process of Stage0-Stage 6:
at each and final stage of differentiation of the method of 2.1, cells were taken separately for analysis for in vitro functional assessment. The detection results are shown below:
(1) S0 stage. hPSC, human pluripotent stem cells. The detection result is shown in FIG. 8A;
the ratio of Oct4+/SSEA4+ double positive cells in the flow detection is more than 95%, and the dryness is maintained better;
(2) S1 stage. DE, definitive endoderm cells. The detection result is shown in FIG. 8B;
the flow test shows that the proportion of SOX17 positive cells reaches 92.8%, which indicates that most cells differentiate into the definitive endoderm cells.
In addition, this stage can be followed by flow sorting to obtain directional endodermal progenitor cell lines. The main method is as follows:
at this stage of differentiation, the cell mass was taken and digested to single cells using TryPLE, and then the cells were labeled with CXCR4 and CD117, and cxcr4+/cd117+ double positive populations were sorted by flow-through.
The population of cells was seeded into Matrigel-containing plates and cultured using directional endodermal progenitor cell culture and expansion medium.
Wherein the composition of the culture and expansion medium of the directional endoderm progenitor cells is as follows:
s1 media+20-100ng/mL bone morphogenic protein 4 (Recombinant Human BMP-4Protein,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 165Protein,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).
The directional endoderm progenitor cells obtained by culture can be used as the starting point of beta cell differentiation, which can further improve the stability of different differentiation batches and reduce the residue of stem/progenitor cells in beta cells at the differentiation terminal, and the safety in the transplanted body is also obviously improved.
(3) S2, a stage. PGT, primitive gut tube cells. The detection result is shown in FIG. 8C;
the proportion of HNF4a positive cells reaches 98.4% in the flow detection, which shows that almost all cells are differentiated into PGT cells, and the differentiation efficiency is very high.
(4) S3, stage. PP1, early pancreatic progenitor cells. The detection result is shown in FIG. 8D;
the proportion of PDX1 positive cells reaches 57.4% by flow detection, which shows that most cells enter the early stage of pancreatic progenitor cells, and the differentiation effect is good.
(5) And S4, a stage. PP2, later pancreatic progenitor cells. The detection result is shown in FIG. 8E;
the ratio of PDX1+/NKX6.1+ biscationic cells is 27% by flow detection, which shows that the cells entering the next differentiation stage account for a certain ratio at the moment, and the differentiation effect is better.
(6) S5, a stage. EN, endocrine progenitor cells. The detection result is shown in FIG. 8F;
flow detection of the NKX6.1+/C-peptide double positive cell proportion reached 24%, indicating that more than 20% of cells were differentiated into SC-beta cells at this time.
(7) S6, a stage. SC-. Beta.cells, stem cell-derived-. Beta.cells. The detection result is shown in FIG. 8G;
the proportion of NKX6.1+/C-peptide double-positive cells in the flow detection is more than 30%. Some cells express GCG (islet alpha cells) and SST (islet delta cells), and the proportion of endocrine cell CHGA positive cells is greater than 90%.
Has glucose response of three successive rounds and stimulation index of more than 2, and the secretion amount of Insulin is more than 1.0-5.0 mu IU/1000cells when high sugar is stimulated. Dithizone staining appears red.
(8) qPCR detection results at each stage in the differentiation process and expression trend of related markers on the transcriptional level:
the cells at different stages are taken for RNA extraction in the whole differentiation process, and then qPCR is used for detecting the expression trend of related genes in the whole differentiation process. As can be seen from FIG. 7, the relative expression levels of PDX1, NKX6.1 and ins obtained by this method are high, in particular the expression of ins is approximately 20 ten thousand times that of the original ones. The expression of Oct4 is reduced to a lower level in Stage2, and the safety problem is greatly reduced.
Detection conclusion:
the whole differentiation process of inducing hPSCs to differentiate into pancreatic beta cells by using the optimization method needs to undergo the following stages, and the detection indexes of each stage are as follows:
the value is an internal quality control standard, and when each stage reaches the following value, the batch can be considered to be normal in differentiation, the differentiation can be smoothly carried out, and finally the functions of the obtained pancreatic beta cells can be normal.
hPSCs cells: octamer binds to transcription factor4/Stage specific embryonic antigen 4 (Octamer-binding transcription factor4/Stage-specific embryonic antigens 4, octamer +/ssea4+) double positive cell ratio greater than 95%;
DE, directional endoderm: SOX transcription factor family member 17 (Sex-determining region Y-box 17, sox17+) positive cell proportion is greater than 90%;
PGT, raw intestinal tube: the proportion of positive cells of the hepatocyte nuclear factor 4a (Hepatocyte Nuclear Factor alpha, HNF4a+) is more than 80 percent;
PP1, early stage of pancreatic progenitor cells: the proportion of pancreatic duodenal homology frame-1 (Pancreatic and duodenal homeobox, pdx1+) positive cells is greater than 60%;
PP2, late stage of pancreatic progenitor cells: PDX1+/homologous transcription factor NKX6.1 (homeobox transcription factor NK Homeobox1, NKX 6.1+) double positive cell proportion is greater than 25%;
EN, pancreatic endocrine progenitor cells: the NKX6.1+/C-peptide+ double positive cell ratio is more than 10%;
SC-beta cluster, stem cell-derived pancreatic beta cells: the NKX6.1+/C-peptide+ double positive cell ratio is greater than 30%.
After indexes at different stages reach the quality control standard, the beta cells can be well differentiated into functional beta cells.
2.3 enrichment of cells at certain stages throughout the differentiation process of Stage0-Stage6 (see steps 9-12 above for specific stages) including, but not limited to, CD177, CD117, CXCR4, GP2, procr, etc. related markers (this set of enrichments is interspersed throughout the differentiation process, corresponding to optimisation during differentiation), which enrichment can significantly increase differentiation efficiency, is an alternative to increase differentiation efficiency).
The enrichment can judge whether to execute according to the cell morphology in the differentiation process, and if the batch of differentiated cells has slightly changed morphology, the functions and the yield of the differentiated cells can be further improved through the enrichment:
(1) Specific enrichment and culture methods for cxcr4+/cd117+ double positive populations are:
cell clusters at the differentiation stage, ready for enrichment, were digested to single cells using TryPLE, and then the cells were labeled with CXCR4 and CD117, and cxcr4+/cd117+ double positive populations were sorted by flow-sorting. The population of cells was seeded into Matrigel-containing plates and cultured using directional endodermal progenitor cell culture and expansion medium.
Wherein the components of the endoderm progenitor cell culture and expansion medium are as follows: s1 media+BMP4 (20-100 ng/mL) +bFGF (5-20 ng/mL) +VEGF (5-20 ng/mL) +EGF (5-20 ng/mL).
The differentiation of the directional endoderm progenitor cells obtained by culture can further improve the stability of different differentiation batches, reduce the residue of stem/progenitor cells in the beta cells at the differentiation terminal, and obviously improve the safety.
(2) Specific enrichment and culture methods for CD177 positive populations are:
cell clusters at the differentiation stage were taken and digested to single cells using TryPLE, and then the cells were labeled with CD177, and CD177 positive populations were flow-sorted. The sorted cells were then used to select 2-10X 10 cells 5 The culture was performed by inoculating with a density of 10. Mu. M Y27632 in Stage1 medium.
Cells subjected to the cd177+ enrichment procedure differentiate more uniformly in vitro into pancreatic progenitor cells and eventually into more functionally mature glucose-responsive beta cells than cells not subjected to the enrichment.
(3) Enrichment and culture of GP2 positive cell populations is performed by:
taking cell mass at differentiation stage, digesting into single cells, separating GP2 positive cell subgroup, and separating cells at 5-10X10 5 Density inoculation of/mL continued differentiation in day 14 medium;
the method can obviously increase the proportion of the differentiated cells PDX1+/NKX6.1+ double positive cells.
(4) Specific enrichment and culture methods for procar positive populations are:
cell clusters at the differentiation stage were taken and digested to single cells using TryPLE, and then the cells were labeled with Procr, and Procr positive populations were sorted by flow-through.
The enriched proc+ cells can be passaged every 7-14 days, and during each passaging the digested procr+ single cell suspension is supplemented with fresh human HUVEC cells at a cell number ratio of 1:1.
The two Procr+ single cells and human HUVEC cells were then mixed and re-inoculated at a ratio of 1:4-1:6. The method can remarkably improve the yield of the differentiated cells.
(5) WNT4 can be added in the last stage of differentiation, metabolic maturation can be obviously driven, and the result shows that the response of differentiated beta cells to glucose can be obviously improved, and more ins are released.
EXAMPLE 3 improved APA microencapsulation of differentiated pancreatic beta cells
1. Improved preparation of ultrapure sodium alginate solution
The dissolved sodium alginate is filtered by a PES sterile filter device of 0.8 μm, 0.45 μm and 0.22 μm in sequence; the viscosity of the sodium alginate is 50-200cP; the prepared sterile sodium alginate can be stored in a 4-degree refrigerator for 1-4 weeks.
2. APA microencapsulated differentiated beta cells-microfluidic method or high-voltage electrostatic method
2.1 microfluidic method (microfluidic plate purchased from WH-SP-01 model, inc. of microfluidic technology Co., ltd.):
mixing 1% -3% sodium alginate solution with beta cells, wherein 1mL sodium alginate corresponds to 1-10 x 10-6 cells;
adding one of the pipelines after mixing, and adding 0.1-2g/L calcium chloride solution into the other pipeline;
crosslinking into gel in a collecting tube of 1-10cm, and calcification for 5-30 min;
adding 0.01-0.1% polylysine (Poly-L-lysine hydrobromide, sigma, P7890-100 MG) for reaction for 10-30 min;
adding 0.1% -0.3% sodium alginate solution to react for 2-10 minutes;
and adding 10-100mM sodium citrate to react for 2-10 minutes to form the APA microcapsule.
2.2 high voltage electrostatic method:
mixing 1% -3% sodium alginate solution with beta cells, wherein 1mL sodium alginate corresponds to 1-10 x 10-6 cells;
sucking the mixture by using a syringe with a 20-40G needle after mixing, allowing the mixture to form jet flow into 0.1-2G/L calcium chloride solution to crosslink into gel under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz and flow speed of 100-1000 mu L/min, and then calcification for 5-30 minutes;
Adding 0.01-0.1% polylysine for reaction for 10-30 min;
adding 0.1% -0.3% sodium alginate solution to react for 2-10 minutes;
and adding 10-100mM sodium citrate to react for 2-10 minutes to form the APA microcapsule.
The morphology of the specifically packed cell mass is shown in FIG. 4. Preferably, the particle size of the microcapsules is controlled to 250-750 μm.
EXAMPLE 4 microencapsulated pancreatic beta cell function evaluation (Single transplantation function evaluation)
It has now been found that a variety of compounds can induce diabetes in animal models. The two most studied and conventionally used compounds are Streptozotocin (STZ) and tetraoxapyrimidine (ALX), respectively, of which again induction with STZ is most common. STZ and ALX are both glucose analogs, both acting through the glucose transporter GLUT2 in the beta cells, ultimately resulting in almost complete destruction of the beta cells in the islets, rendering the mice severely deficient in insulin production, exhibiting hyperglycemia and weight loss, reproducing the principal symptoms of type I diabetes. The invention causes C57BL/6 mice to produce type I diabetes symptoms by using STZ for drug induction.
1. In vivo functional assessment of microencapsulated or naked pancreatic beta cells transplanted subcutaneously into STZ-induced type I diabetes C57BL/6 mice
1.1 microencapsulated or naked pancreatic beta cell transplantation:
mice that received the transplant 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) was made in the lower abdomen to form a subcutaneous "small pocket" in the lower left and right quadrants, and microencapsulated or naked beta cells were mixed with 200-500. Mu.L of beta cell-activated matrigel and transplanted into the subcutaneous "small pocket" in mice. Wherein the components of the beta cell active matrigel comprise: 10 Xmedia (M) 199 basal medium (ThermoFisher, 11825015), L-glutamine, fetal bovine serum, 5-10% sodium bicarbonate, and type I collagen.
1.2 transplantation of naked beta cells (non-microencapsulated beta cells) also requires the use of a combination of immunosuppressive agents, specifically:
maintenance immunosuppression consisted of rapamycin (1-2 mg/kg intraperitoneally four times per day) for 5-10d from the day of transplantation.
For B cell removal, 10F4 (supplied by pennsylvania pathology and laboratory medical laboratory m.cancro), a monoclonal antibody against mouse BLyS (15-25 d intraperitoneal injection of 50-200 μg, two injections at 24 hour intervals) was used. From day 10, 10F4 was also given gradually, with a dose decreasing gradually from 20-100 μg per week at week 2 to 1-10 μg per week at week 8. On days 50-80, immunosuppressive dosing was discontinued.
1.3 post-implantation testing
Detecting the blood sugar level, glycosylated hemoglobin level, in vivo glucose stimulation, human C peptide condition, etc. And fixing the transplanted APA microencapsulated beta cells or bare beta cells, performing immunofluorescence, and performing identification of related markers.
In addition, islet graft sites can be resected in order to confirm that islet grafts are the only source of maintenance of euglycemia, a group of long-term euglycemic islet recipients undergo a pancreatectomy (skin), which results in rapid recurrence of diabetes within 24-72 hours. The same results show that the differentiated beta cells transplanted subcutaneously are functionally equivalent to microencapsulated beta cells after peritoneal transplantation (a detection means, i.e. transplanted pancreatic beta cells are removed and the animals relapse diabetes, which means that transplanted pancreatic beta cells are functioning).
According to the above method, the transplanted amount of each mouse is 1-10 x6 cells, the abdominal cavity transplantation is performed by using an indwelling needle, and the blood glucose level, glycosylated hemoglobin level, in vivo glucose stimulation, human C-peptide condition and the like of the mouse are detected after the transplantation.
The results show that: microencapsulated beta cells can rapidly reverse hyperglycemic symptoms in diabetic mice, which takes 1-7 days, followed by maintenance of normoglycemia for more than 30 days. The content of human C peptide in the plasma of the transplanted mice was randomly detected during the transplantation period, and the result shows that the content of human C peptide is far higher than that of the control group and is between 100 and 500 pM. The percentage content of glycosylated hemoglobin HbA1C is also reduced to 6-12%. In vivo IPGTT experiments showed that microencapsulated beta cells were able to rapidly sense the rise in blood glucose, secreting sufficient amounts of instrin to maintain blood glucose stable. And fixing the transplanted APA microencapsulated beta cells, performing immunofluorescence, and performing identification of related markers. The microencapsulated beta cells can well express the Marker NKX6.1/C-peptide related to the beta cells, and the double positive rate is more than 30%. The specific evaluation results are shown in fig. 5.
EXAMPLE 5 microencapsulated pancreatic beta cell function assessment (Combined transplantation function assessment)
Microencapsulated or naked beta cells are transplanted into STZ-induced C57 mice in combination with Mesenchymal Stem Cells (MSC) or Endothelial Progenitor Cells (EPC) for functional evaluation, and the experimental steps are as follows:
1. after cell clusters of Stage5-Stage6 differentiation Stage are digested into single cells by TryPLE digestive enzyme, single cell suspension of Mesenchymal Stem Cells (MSC) or Endothelial Progenitor Cells (EPC) is mixed with the differentiated single cell suspension according to the ratio of 1:1, and the culture medium used for the mixed culture is S3 differentiation basal medium, and the mixed culture is placed on an orbital shaker in a low-adsorption 6-well plate for culture.
2. The cell inoculation method for animal experiments is the same as in example 4, the mixed cell inoculation density is 0.5-2 x 10≡6/mL, the culture volume is 2-7 mL/hole, and the rotation speed of the orbital shaker is 70-120rpm. Single cells are reagglomerated within 24-72h, and after 7-14 days of further maturation, transplanted into STZ-induced C57 mice for in vivo functional evaluation subcutaneously or intraperitoneally. The mesenchymal stem cells or the endothelial progenitor cells and the islet cells are aggregated and cultured together to form a three-dimensional cell mass, and the single-dose mixed islet cell mass can be injected subcutaneously or intraperitoneally to provide long-time blood sugar control for the organism, so that the blood sugar of the type I diabetes mice can be kept within a normal range without using an anti-immune rejection drug or an encapsulation system. And the receptor is protected from the use of anti-immune rejection drugs or islet cell encapsulation systems (microencapsulation) during this process.
In the method, mesenchymal stem cells are transplanted at a transplantation position, wherein the amount of the transplanted mesenchymal stem cells is 1-10 x 6 cells at the transplantation position of each mouse. The main purpose is to construct an angiogenic environment of the transplanted site, so that the transplanted beta cells can obtain nutrition and oxygen in the vascularized environment, and the survival and the functions of the transplanted beta cells are facilitated. The results show that the pre-transplanted mesenchymal stem cells can vascularize the transplanted part, and the time for generating the blood vessel is 10-50 days. The microencapsulated or naked beta cells that are subsequently transplanted are functionally equivalent to those after the abdominal transplantation of the microencapsulated beta cells.
Finally, it should be noted that the above embodiments are only for helping the person skilled in the art to understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
Claims (8)
1. A method for three-dimensional suspension directed differentiation of pluripotent stem cells into mature pancreatic beta cells, comprising the steps of:
(1) Three-dimensional suspension domestication culture of pluripotent stem cells; detecting multipotent stem cells after domestication, wherein the cells grow in a spherical suspension and Oct4 + /SSEA4 + The proportion of the double positive cells is more than 95 percent, which is qualified for domestication; the pluripotent stem cells are human induced pluripotent stem cells;
(2) Inducing the domesticated cells to differentiate into pancreatic beta cells; the differentiation steps are as follows:
1) Inoculating 0.3-0.7X10 to mTESR1 culture medium containing 10mu M Y27632 6 The scattered three-dimensional suspension domesticated pluripotent stem cells are cultured for 24 to 72 hours, the first day culture medium is replaced, the volume is reduced by 10 to 20 percent when the liquid is replaced,
the first day medium was: adding 50-200ng/mL of Activin A and 10-100ng/mL of Wnt3a into the S1 culture medium;
2) Day 2 medium was changed on day 2;
the 2 nd culture medium is: s1, adding 50-200ng/ml of Activin A into a culture medium;
3) Changing the culture medium on the 4 th day from the 4 th day to the 6 th day;
the culture medium on day 4 is: 20-100ng/ml KGF and 1-5 mu M TGF-beta RI Kinase Inhibitor IV are added into the S2 culture medium;
4) Changing the culture medium on the 7 th day from the 7 th day to the 8 th day;
the 7 th medium was: 20-100ng/ml KGF, 0.1-0.5 mu M Sant1, 1-5 mu M retinoic acid, 100-500nM1, 4-tetraphenyl-1, 3-butadiene and 5-20 mu M Y27632 are added into the S3/S4 culture medium;
5) 9 th day medium is replaced from 9 th day to 13 th day;
the 9 th medium was: 20-100ng/mL KGF, 0.1-0.5 mu M Sant1, 50-200nM retinoic acid, 10-100ng/mL EGF, 10-100ng/mL recombinant human noggin and 5-20 mu M Y27632 are added to the S3/S4 medium;
6) Changing the culture medium on 14 th day from 14 th day to 16 th day;
The culture medium on day 14 is: 0.1-0.5. Mu.M Sant1, 50-200nM retinoic acid, 0.5-2. Mu.M gamma. -secretase inhibitor XXI, 5-20. Mu.M RepSox, 0.5-2. Mu.M tricin thyronine, 5-50ng/ml recombinant human beta-cytokine, 50-500nM LDN193189 hydrochloride and 10. Mu.M zinc sulfate were added to the S5 medium;
7) Changing the culture medium on the 18 th day from the 18 th day to the 20 th day;
the 18 th medium was: 10-50nM retinoic acid, 0.5-2. Mu.M gamma. -secretase inhibitor XXI, 5-20. Mu.M RepSox, 0.5-2. Mu.M classical thyronine, 5-50ng/ml recombinant human betacellulin, 50-500nM LDN193189 and 1mM Fmoc-N-Me-Cys (Trt) -OH were added to the S5 medium;
8) On day 21, the differentiated cells are digested into single cells and re-inoculated into a reactor for re-culture, the normal cell clusters which are re-aggregated are collected in the culture process, and the supernatant which is not aggregated is discarded, so that the differentiated pancreatic beta cells are obtained;
the culture mediums are as follows:
s1 culture medium: MCDB131 basal medium +1:10000-100000 insulin-transferrin-selenium-ethanolamine +1-5
mMGlutamate+1-5 g/L sodium bicarbonate+1% penicillin/streptomycin+0.1-0.5 mM vitamin C+2-20mM glucose+0.5-5% recombinant human serum albumin;
s2 culture medium: MCDB131 basal medium +1:10000-1:100000 insulin-transferrin-selenium-ethanolamine +1-5mM Glutamax +1-3g/L sodium bicarbonate +0.1-0.5mM vitamin C +1% penicillin/streptomycin +2-20mM glucose +0.5-5% recombinant human serum albumin;
S3/S4 medium: MCDB131 basal medium +1:50-1:500 insulin-transferrin-selenium-ethanolamine +1-5mM Glutamax +1-3g/L sodium bicarbonate +0.1-0.5mM vitamin C +1% penicillin/streptomycin +2-20mM glucose +0.5-5% recombinant human serum albumin;
s5 culture medium: MCDB131 basal medium +1:50-1:500 insulin-transferrin-selenium-ethanolamine +1-5mM Glutamax +2-20 μg/mL heparin sodium salt +1-3g/L sodium bicarbonate +0.1-0.5mM vitamin C +1% penicillin/streptomycin +5-50mM glucose +0.5-5% recombinant human serum albumin.
2. The method of claim 1, wherein upon the step of lysing the differentiated cells to single cells and re-seeding the reactor for re-culturing, the parameters of the re-culturing are: 50-120rpm, constant temperature 37 ℃,5% CO 2 And 100% humidity, replacing the S3 medium every two days at 21-35 days, collecting the reaggregation with 10-50 μm reversible filterNormal cell mass.
3. The method of claim 1, wherein the step of three-dimensional suspension acclimation culture of pluripotent stem cells of step (1) comprises:
1) Pretreatment: 2 hours before suspension culture, replacing the planar culture pluripotent stem cells with mTESR1 culture medium containing Y27632 with the concentration of 10 mu M;
2) Washing the neutralized pretreated cells with DMEM/F12 medium;
3) Passaging in mTESR1+Y27632 medium for 5-10 times, changing half of the medium every day, and removing single cells and aggregated large cell mass in culture supernatant during passaging.
4. The method of claim 3, wherein the step of,
the inoculation density of the pluripotent stem cells is 3-8 multiplied by 10 5 /mL,
The parameters of three-dimensional stirring culture are as follows: 50-120rpm, constant temperature 37 ℃ and 5% CO 2 100% humidity, the culture medium is mTESR1 culture medium containing Y27632 with the concentration of 10 mu M.
5. The method of claim 1, wherein in the step of inducing the differentiation of the acclimatized cells into pancreatic beta cells,
in the step 2), the 2 nd culture medium is replaced on the 2 nd day, and the volume of the culture medium is unchanged during liquid replacement;
in the step 3), the culture medium on the 4 th day is replaced on the 4 th day and the culture medium on the 6 th day respectively, and the volume of the culture medium is unchanged during liquid replacement;
in the step 4), the culture medium on the 7 th day is replaced on the 7 th day and the culture medium on the 8 th day respectively, and the volume of the culture medium is unchanged during liquid replacement;
in the step 5), the culture medium on the 9 th day is replaced on the 9 th day, the 11 th day and the 13 th day respectively, and the volume of the culture medium is unchanged during liquid replacement;
in the step 6), the 14 th culture medium is respectively replaced on the 14 th day and the 16 th day, and the volume of the culture medium is unchanged when the liquid is replaced;
In the step 7), the 18 th culture medium is replaced on the 18 th day and the 20 th day respectively, and the volume of the culture medium is unchanged when the liquid is replaced.
6. The method of claim 1, wherein in the step of inducing the differentiation of the acclimatized cells into pancreatic beta cells, further comprising any one or a combination of any of the following steps:
9) Enrichment and culture of differentiated CD 177-positive populations is performed prior to the differentiation step 3) above, in particular by:
taking the cell mass differentiated in the previous step, digesting into single cells, separating CD177 positive cell subgroup, and separating the cells at a ratio of 2-10×10 5 The differentiation was continued in the medium containing 10. Mu. M Y27632 on day 4 at a density of/mL;
10 Before the differentiation step 6), the enrichment and culture of GP2 positive groups is carried out by the following specific methods:
taking the cell mass differentiated in the previous step, digesting into single cells, separating GP2 positive cell subgroup, and separating cells by 5-10×10 5 Density inoculation of/mL continued differentiation in day 14 medium;
11 Before the differentiation step 8), enrichment and culture of the Procr positive cell population are carried out by the following specific methods:
taking the cell mass differentiated in the previous step, digesting the cell mass into single cells, then separating out a Procr positive cell subgroup, carrying out passage on the enriched Proc positive cells every 7-14 days, supplementing the digested Procr positive single cell suspension with fresh human HUVEC cells in the process of each passage, wherein the cell number ratio is 1:1; then mixing the Procr positive cells and the human HUVEC cells, and re-inoculating and culturing the two cells in a ratio of 1:4-1:6;
12 100ng/mL WNT4 was added to the medium of the above differentiation step 8) to drive metabolic maturation necessary for glucose-stimulated insulin secretion in the differentiated cells.
7. Use of the method according to any one of claims 1-6 for the preparation of a medicament for the treatment of diabetes.
8. The use according to claim 7, wherein the diabetes is type i diabetes.
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