CN112980771A - Method for preparing pancreatic beta cells and application thereof - Google Patents
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- CN112980771A CN112980771A CN202110245407.0A CN202110245407A CN112980771A CN 112980771 A CN112980771 A CN 112980771A CN 202110245407 A CN202110245407 A CN 202110245407A CN 112980771 A CN112980771 A CN 112980771A
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Abstract
The invention relates to a method for preparing differentiated pancreatic beta cells and application thereof, and particularly relates to the method comprising the following steps of: (1) three-dimensional suspension domestication culture of pluripotent stem cells; (2) inducing the domesticated cells to differentiate 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 the artificial pancreatic 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 the hypofunction or failure of pancreatic islets leads to disturbance of glucose metabolism in the body. According to the latest statistics of the International Diabetes Federation (IDF), in 2019, more than 4.63 million people worldwide suffer from Diabetes, and by 2045, 7 million people suffer from Diabetes; china is the first to live in the world with 1.16 million diabetics, and 1.47 million diabetics are expected to be reached in 2045 years. In 2019, about 420 million people (20-79 years old) die from diabetes or its complications all over the world, and 1 person dies from diabetes every 6 seconds, accounting for about 11.3% of all death cause deaths all over the world.
The current method for differentiating stem cells into beta cells mainly comprises the following steps: (1) a gene transfection method comprises transfecting cells with a plurality of transcription factors PDX-1, NKX6.1, Ngn3, NeuroD, Pax4, etc., which control the development of an embryo into a pancreas, and inducing differentiation into insulin-secreting cells (Noguchi H, etc., 2006). However, the gene transfection method has a potential carcinogenic risk, and it is possible to inactivate the cancer suppressor gene by inserting a foreign gene into the genome of a cell using a viral vector. In addition, the insulin-secreting cells obtained by this method are not mature and do not respond well to glucose stimulation. (2) The cell factor induction method is a method for inducing stem cells to differentiate into pancreatic beta cells or pancreatic progenitor cells by simulating key steps in pancreatic development in vitro according to the development process from embryos to pancreas (Pagluca FW et al, 2014; Rezania A et al, 2014; Kroon E et al, 2008). However, the method has complicated steps and long differentiation time; and the immature differentiation method easily causes the problems of poor stability of differentiation batches, low cell yield, limited large-scale production and the like. However, the development of a mature and stable cytokine induced differentiation method can well overcome the 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 body environment, and are often subjected to immunological rejection by foreign bodies, so that the transplanted beta cells lose the original functions. The main strategy for solving the problem of immunological rejection in the process of allograft at present is the use of combined immunosuppressant, but the immunosuppressive effect is often not ideal and faces the pain of lifetime medication. Recent strategies have shown that the development of safe and effective immunoisolation tools is a better choice for solving the problem of immune rejection during xenotransplantation (Omid Veise et al, 2015; Arturo J Vegas et al, 2016; Daniel G Anderson et al, 2016). The selection of the coating material used in the immune isolation tool is the key for successful transplantation, and due to the influence of different coating materials, the difference of the in vivo function evaluation effect of the differentiated beta cells is obvious. Currently common wrapping materials include: (1) a natural material. Lipids, polysaccharides and proteins; (2) semi-synthetic materials. Cellulose derivatives and the like; (3) and (3) synthesizing the material. Including degradable and non-degradable materials. Many of them will have serious fibrosis wrapping and no immune isolation function in vivo, and will seriously affect the activity and function of the wrapped cells. However, the development of an ideal wrapping material can solve the problems involved in the xenograft transplantation process.
Therefore, pluripotent stem cell-derived pancreatic beta cells are expected to treat diabetes through a cell replacement strategy, but how to better scale stable in vitro mature pancreatic beta cells and solve the problem of immune rejection after transplantation of allogeneic pancreatic beta cells has been a research hotspot.
Disclosure of Invention
The invention relates to a method for the three-dimensional suspension directed differentiation of pluripotent stem cells (PSCs, human embryonic stem cells or human induced pluripotent stem cells) into mature pancreatic beta cells, which comprises 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 three-dimensional suspension acclimatization culture method of the pluripotent stem cells in the step (1) comprises the following steps:
1) y27632 pretreatment, preferably, the pluripotent stem cells cultured on a plane are subjected to liquid exchange 2 hours before suspension culture, and then are replaced with mTeSR1 medium containing Y27632 at a concentration of 10. mu.M;
2) washing and neutralizing the pretreated cells with DMEM/F12 medium and counting the cells;
3) culturing the pluripotent stem cells in mTeSR1+ Y27632 culture medium under stirring, carrying out passage 5-10 times in mTeSR1+ Y27632 culture medium, replacing half of the culture medium every day, and removing single cells and aggregated large cell masses in culture supernatant during passage;
preferably, the pluripotent stem cells are seeded at a density of 3 to 8 x 105mL, using a Disposable bioreactor (Disposable spinner flashes, Corning,3152/3153) for three-dimensional stirred suspension culture with the parameters: rotating at 50-120rpm, maintaining the temperature at 37 deg.C and 5% CO 2100% humidity, the medium is mTeSR1 medium with a concentration of 10 μ M of Y27632;
preferably, the removing of the large cell mass is to filter and remove the cell mass larger than 400 μm by 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 cells are qualified for domestication;
the step of inducing the domesticated cells to differentiate and microencapsulate in the step (2) comprises the following steps:
the media used were:
s1 medium: MCDB131 basic 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 medium: MCDB131 basal medium + ITS-X1: 10000-1:100000+1-5mM glutamine, necessary buffer salt (NaHCO)3) Antibiotics, antioxidants (VC), glucose and serum albumin;
S3/S4 Medium: MCDB131 basal Medium + ITS-X1: 50-1:500+1-5mM Glutamine, necessary buffer salts (NaHCO)3) Antibiotics, antioxidants (VC), glucose and serum albumin;
s5 medium: MCDB131 basal medium + ITS-X1: 50-1:500+1-5mM glutamine +2-20 μ g/mL heparin sodium salt, necessary buffer salt (NaHCO)3) Antibiotics, antioxidants (VC), glucose and serum albumin;
the induced differentiation method comprises the following steps:
using the pluripotent stem cells acclimatized in step (1) as starting seed cells for differentiation
1) 0.3-0.7 x 10 inoculations per ml mTeSR1 medium (10 μ M Y27632 added to the medium)6Culturing the scattered three-dimensional suspension domesticated pluripotent stem cells for 24-72 hours, then replacing the culture medium for the first day, reducing the volume by 10-20% when replacing the culture medium,
the culture medium for the first day is: s1 medium is added with: 50-200ng/mL recombinant human activin A (activin A), 10-100ng/mL recombinant human Wnt3a protein;
2) changing the culture medium of day 2 on day 2, wherein the volume of the culture medium is unchanged during liquid change;
the culture medium for the day 2 is: s1 medium is added with: 50-200ng/ml Activin A;
3) changing culture medium on day 4 and day 6 respectively, wherein the culture medium volume is unchanged during liquid change;
the culture medium for the 4 th day is: s2 medium is added with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5 μ M transforming growth factor- β RI Kinase Inhibitor IV (TGF- β RI Kinase Inhibitor IV);
4) changing culture medium on 7 th and 8 th days, wherein the volume of the culture medium is unchanged during liquid change;
the day 7 culture medium was: S3/S4 medium is added with: 20-100ng/ml KGF, 0.1-0.5. mu.M Sant1, 1-5. mu.M Retinoic Acid (RA), 100- 500nM 1,1,4,4-tetraphenyl-1,3-butadiene (TPB), 5-20. mu. M Y27632;
5) changing culture medium on 9 th, 11 th and 13 th days, wherein the volume of the culture medium is unchanged during liquid changing;
the culture medium on day 9 was: S3/S4 medium is added with: 20-100ng/mL KGF, 0.1-0.5. mu.M Sant1, 50-200nM RA, 10-100ng/mL EGF, 10-100ng/mL recombinant human Noggin (NOG), 5-20. mu. M Y27632;
6) changing the culture medium on day 14 and day 16 respectively, wherein the volume of the culture medium is unchanged during liquid change;
the culture medium for the 14 th day is: s5 medium is added with: 0.1-0.5 μ M Sant1, 50-200nM RA, 0.5-2 μ M gamma-Secretase Inhibitor XXI (gamma-Secretase Inhibitor XXI), 5-20 μ M Repsox, 0.5-2 μ M trispecific thyronine (L-3,3',5-Triiodothyronine, T3), 5-50ng/ml Recombinant Human β -cell (Recombinant Human Betacellulin Protein), 50-500nM LDN193189hydrochloride (LDN193189hydrochloride), 10 μ M zinc sulfate;
7) replacing the culture medium on the 18 th day and the 18 th day respectively, wherein the volume of the culture medium is unchanged when the culture medium is replaced;
the culture medium of day 18 was: s5 medium is added with: 10-50nM RA, 0.5-2. mu.M XXI, 5-20. mu.M Repsox, 0.5-2. mu. M T3, 5-50ng/ml Betacellulin, 50-500nM LDN193189, 1mM N-cys (Fmoc-N-Me-Cys (Trt) -OH);
8) on day 21, differentiated cells were digested into single cells using TrypLE Express, and then plated at 0.5-2 x 106Inoculating the cells/ml in the reactor again, culturing at 50-120rpm, and setting the parameters of the incubator at 37 deg.C and 5% CO2And changing the S3 culture medium every two days at the humidity of 100 percent for 21-35 days, collecting the reaggregated normal cell mass by using a 10-50 mu m reversible filter in the culture process, and discarding the supernatant without conglomeration to obtain the differentiated pancreatic beta cells.
Optionally, the method for inducing differentiation may further comprise any one or any combination of the following steps:
9) enrichment and culture of differentiated CD 177-positive populations may be performed prior to the differentiation step 3) above, by:
collecting cell mass at differentiation stage, digesting into single cells, sorting out CD 177-positive cell sub-population, and sorting out cells at 2-10 × 105The cells were inoculated at a density of/mL into the day 4 medium containing 10. mu. M Y27632 to continue differentiation;
10) before the differentiation step 6) described above, enrichment and culture of GP 2-positive populations can be carried out, in particular by:
collecting cell mass at differentiation stage, digesting into single cells, sorting GP2 positive cell subset, and sorting the sorted cells at 5-10 × 105The culture medium is inoculated in the 14 th day culture medium for continuous differentiation at the density of/mL;
11) before the differentiation step 8) above, enrichment and culture of the Procr-positive population can be carried out, in particular by:
taking a cell mass in a differentiation stage, digesting the cell mass into single cells, then sorting out Procr positive cell subsets, carrying out passage on the enriched Proc positive cells once every 7-14 days, and supplementing the digested Procr positive single cell suspension with fresh human HUVEC cells in the passage process of each time, wherein the cell ratio is 1: 1; then mixing Procr positive cells and human HUVEC cells, and inoculating and culturing again according to the proportion of 1:4-1:6, so that the yield of differentiated cells is obviously improved;
12) in the medium of the above differentiation step 8), 100ng/mLWNT4 can be added to drive the metabolic maturation necessary for glucose-stimulated insulin secretion by 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 a sodium alginate solution: filtering the dissolved sodium alginate by PES sterile filtering devices of 0.8 mu m, 0.45 mu m and 0.22 mu m in sequence; detecting the viscosity of the solution to be 50-200cP, and storing at 4 ℃;
(2) preparing microencapsulated artificial islets according to the method one or the method two:
the method comprises the following steps: a micro-fluidic method:
mixing 1% -3% sodium alginate solution with the pancreatic beta cells, mixing 1mL sodium alginate solution with 1-10 x 106Mixing cells;
after mixing, adding the mixture into one pipeline, 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 to react for 10-30 min;
then 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 microcapsules;
the second method comprises the following steps: high-voltage electrostatic method:
mixing 1% -3% sodium alginate solution with the pancreatic beta cells, mixing 1mL sodium alginate solution with 1-10 x 106Mixing cells;
sucking with a 20-40G syringe, forming jet flow under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz, and flow rate of 100-;
adding 0.01-0.1% polylysine to react for 10-30 min;
then adding 0.1-0.3% sodium alginate solution to react for 2-10 minutes;
adding 10-100mM sodium citrate, and reacting for 2-10 min to obtain APA microcapsule.
The invention also relates to application of the pancreatic beta cells or the artificial pancreatic islets in preparing a medicament, wherein the medicament is used for treating diabetes, and preferably, the diabetes is type I diabetes.
The invention also relates to the application of the pancreatic beta cells or the artificial islets of langerhans in preparing the active ingredients for treating cell therapy, wherein the cell therapy is cell therapy 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 step of three-dimensional suspension domestication culture of the pluripotent stem cells in the step (1) is the same as the related step of the method for directionally differentiating the pluripotent stem cells into mature pancreatic beta cells in three-dimensional suspension;
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 starting seed cells for differentiation
1) 0.3-0.7 x 10 inoculations per ml mTeSR1 medium (10 μ M Y27632 added to the medium)6Culturing the scattered three-dimensional suspension domesticated pluripotent stem cells for 24-72 hours, then replacing the culture medium for the first day, reducing the volume by 10-20% when replacing the culture medium,
the culture medium for the first day is: s1 medium is added with: 50-200ng/mL recombinant human activin A (activin A), 10-100ng/mL recombinant human Wnt3a protein;
2) changing the culture medium of day 2 on day 2, wherein the volume of the culture medium is unchanged during liquid change;
the culture medium for the day 2 is: s1 medium is added with: 50-200ng/ml Activin A;
3) changing culture medium on day 4 and day 6 respectively, wherein the culture medium volume is unchanged during liquid change;
the culture medium for the 4 th day is: s2 medium is added with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5 μ M transforming growth factor- β RI Kinase Inhibitor IV (TGF- β RI Kinase Inhibitor IV);
4) when the proportion of SOX17 positive cells is greater than 90%, digesting the cell mass into single cells, and sorting out a CXCR4+/CD117+ double-positive cell subset (common, differentiated to the 6 th day, namely sorting out the CXCR4+/CD117+ double-positive cell subset), wherein the cell subset is the directional endoderm progenitor cell;
5) inoculating the population of cells into a Matrigel-containing plate, and performing culture expansion using a directed endoderm progenitor cell culture and expansion medium;
the components of the culture and amplification medium for the directional endoderm progenitor cells are as follows:
s1 medium is added with: 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 invention has the beneficial effects that:
the invention aims to provide a method for obtaining mature and batch-stable large-scale three-dimensional suspension oriented 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 a wrapping material is provided, so that the microencapsulated beta cells can well function in an animal body and have no immune rejection and fibrosis wrapping problems. Specifically, the method comprises (1) three-dimensional large-scale suspension culture of pluripotent stem cells (including induced pluripotent stem cells and embryonic stem cells); (2) performing in-vitro three-dimensional suspension dynamic directional differentiation on the compound combination to obtain mature pancreatic beta cells; (3) microencapsulation and wrapping of beta cells; (4) in vivo and in vitro functional evaluation of microencapsulated beta cells.
Compared with the prior art, the method provided by the invention has the following remarkable advantages: the PSC three-dimensional suspension culture method provided by the invention can successfully maintain the expression of PSC pluripotency genes, the cell proliferation is good, the cell karyotype is stable, particularly, the planar cells need to be replaced by adding Y27632 fresh culture medium to pretreat the cells for 2-4h before being loaded in a 3D reactor, and the success rate of PSC three-dimensional suspension culture is remarkably increased by 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 list cell processing mode, the large cell mass processing mode with the cell mass larger than 400 mu m and the normal cell mass obtaining mode not only save the cost, but also can well maintain the survival rate of the cells and the like. The compound combination mode used in each stage in the differentiation process can be efficiently differentiated into the cell type of the stage, and the proportion of differentiated cells is obviously improved. Particularly, the culture system before differentiation is changed, the preparation method and the adding time of the composition, the liquid changing mode in the differentiation process, the list cell treatment mode in the differentiation process and the like have good differentiation promoting effect, the stability of differentiation between batches is obviously improved, and the differentiated beta cells are more mature and have stronger functions. The improved sodium alginate microencapsulated encapsulated differentiated beta cells can reverse hyperglycemia of diabetic mice in a short time (24h) in transplanted animals, has good immune isolation function, has less fibrosis encapsulation, and can maintain normal blood sugar in mice with complete immunity for a long time.
Drawings
FIG. 1 shows the stem cell pluripotency detection index and result in planar culture, 1A, and microscopic photograph; and 1B, flow detection results.
FIG. 2 is a photograph showing the results of environmental examination after 3D culture of hPSCs.
FIG. 3 shows cell proliferation during acclimation, wherein the proliferation rate was 2-4 times (FIG. 3A), and the ratio of Oct4/SSEA4 double positive cells was 99% or more (FIG. 3B).
FIG. 4 shows the cell mass morphology after sodium alginate-polylysine-sodium alginate (APA) encapsulation.
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 shows the qPCR detection results and the expression trend of the related markers at the transcription level at various stages during the differentiation of pancreatic beta cells by the classical method.
FIG. 7 shows 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 the related markers on the transcription level.
FIG. 8 shows the results of examining 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 detection indexes at various stages in the differentiation process.
Detailed Description
Example 1 planar culture, in vitro 3D culture and acclimatization of hPSCs cells
One, hPSCs cell plane culture
The preparation method of the required reagent comprises the following steps:
mTeSR1 pluripotent stem cell medium (STEMCELL, 85850);
accutase digestive enzyme (stemcel, 07920);
rho kinase inhibitor Y27632(Abcam, ab 120129): 10mM stock solution was prepared in DMSO, and the final concentration was 10. mu.M when used, i.e., 1000X;
mTeSR1+ Y27632 medium: 15 μ L of 10mM Y27632 was added to 15mL of mTeSR1, and the mixture was mixed, stored at 4 ℃ when not used, and used up to the optimum level within two weeks after compounding.
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, slightly shaking the 6-hole plate to enable the Matrigel to completely cover the bottom of the dish, placing the dish in an incubator at 37 ℃ for incubation for 1-2 h, taking out the dish before the experiment, and placing the dish in a clean bench/biological safety cabinet for balancing for 10-40min at room temperature. If not used temporarily, it can be sealed with Parafilm and stored at 2-8 deg.C and used within 1-2 weeks.
The number of the stem cells in a frozen state is 1X 106cells/mL or so, and correspondingly inoculating 1 hole of a 6-hole plate;
1.2, firstly adding 2-3 mL of mTeSR1 culture medium into a 15mL centrifuge tube for later use.
1.3, unfreezing: quickly immersing the freezing tube taken out from the liquid nitrogen into warm water at 37 ℃, and quickly shaking to quickly thaw the tube within 1-2 min;
1.4, centrifugation: after the frozen stock solution in the frozen stock tube is thawed, dropwise adding the frozen stock 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 1800rpm for 3min at 800-;
1.5, resuspension: and (3) after centrifugation, removing the supernatant, adding 0.5-2mL of mTeSR1+ Y27632 cell culture medium, and blowing and sucking stem cell sediment for about 3-5 times.
1.6 inoculation: and (3) after uniform blowing and sucking, discarding the balanced Matrigel, adding the stem cell suspension which is uniformly blown into the coated 6-hole plate, and completing a culture system with 2mL per hole.
1.7 culture: 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 to ensure that the cells are uniformly distributed. At 37 ℃ with 5% CO2Culturing in a constant-temperature incubator, and observing the cell adherence condition on the 2 nd day;
1.8 liquid changing: and changing the liquid once every 24-48h from the time of recovery.
In the planar culture process, the cell morphology is normal, and the stem cell pluripotency detection indexes and results 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 stem cell clone morphology, and the expression of the dry related genes is good; flow results show that the ratio of Oct4+/SSEA4+ double positive cells is above 99%, which indicates that the dryness is well maintained in the culture process; the proportion of SSEA1 cells was very low and no cell differentiation occurred.
Second, 3D culture of hPSCs by passage
2.1 pretreatment with Y27632: 2-4h before loading into a 3D reactor (dispersible spin flash, Corning,3152/3153), cells were pretreated by replacing mTeSR 1medi μm containing Y27632;
2.2, cleaning: absorbing the original culture medium, slowly adding 0.5-2mL of DMEM/F12 to the wall of the culture dish, gently shaking the culture dish, and absorbing the DMEM/F12 along the edge of the culture dish; digestion: adding 0.5-2 mL/well of Accutase into a 6-well plate to cover the bottom of the dish, and placing the dish in an incubator at 37 ℃ for 2-5 min;
2.3 neutralization: adding 1-4mL of DMEM/F12 for neutralization, blowing and beating the bottom of the culture dish in a fan shape by using a liquid transfer gun, gently blowing and beating 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 1800rpm for 3-5min at 800-; discarding the supernatant, blowing the cells for 5-10 times by using a stem cell culture medium, and taking part of cell suspension for counting;
2.5 inoculation: according to the counting result, 3-8 × 10^5/mL density is resuspended in mTeSR1+ Y27632 culture medium, and then inoculated in Corning Spinner flash, 40-300mL culture system. And placing Spinner flashes on a nine-point magnetic stirrer to culture at 50-120rpm, setting the parameters of the incubator at constant temperature of 37 ℃ and 5% CO2And 100% humidity.
The photograph showing the environmental test results of hPSCs after 3D culture is shown in FIG. 2
This culture mode is maintained during the cell culture and differentiation. If the cell inoculation density is not within the range, the too low density can cause the cells to be not well agglomerated, and the proliferation is limited; too high density can cause cell clusters to easily form large clusters, the clusters are seriously adhered, the large diameter of cell clusters can cause oxygen depletion of cells in the middle, the cell activity rate is reduced, and the cells are easy to differentiate. If the rotating speed is not in the range, the rotating speed is too high, so that the cells cannot be well agglomerated, the survival rate of the excessive single cells is reduced, and the cells are lost; the adhesion between cell clusters is serious due to the over-low rotating speed, the oxygen depletion of cells in the middle part is caused due to the over-large diameter of the cell clusters, the cell survival rate is reduced, and the cells are easy to differentiate.
Third, hPSCs cell cultured and domesticated by 3D and function detection
3.1 Prior to the determination of the differentiation capacity of hPSCs, at least 5-10 acclimations were ensured in the reactor. Half liquid change is carried out every day, large cell masses larger than 400 mu m are removed by a 400 mu m screen, and single cells in culture supernatant are added back to the reactor. Single cells in the culture supernatant were discarded at passage, normal clumps were collected with a 37 μm reversible filter, cells were dispersed with Accutase, and 0.3-0.7 million dispersed cells were seeded in mTeSR 1media supplemented with 5-20 μ M Y27632 per ml. If large cell aggregates larger than 400 μm are not removed, the cell aggregates are more and more seriously adhered, and the larger diameter of the cell aggregates can cause oxygen depletion of cells in the middle, reduce the cell viability and be easily differentiated. If the single cells in the culture supernatant are not added back to the reactor, the proliferation of the cells is limited, which is not favorable for the formation of cell clusters.
3.2 after 48-72 hours of culture, the culture medium of mTeSR1 without Y27632 is replaced by half every day, before differentiation, the diameter of the cell cluster is kept within 300 μm, if the diameter of the cell cluster is larger than 300 μm, partial cells in the cell mass are necrotized or spontaneously differentiated. Cell lines with different doubling times may require different seeding concentrations or times.
Cell proliferation during acclimation is shown in fig. 3: it can be seen that during the acclimation process, the pluripotent stem cells can be well proliferated, with the proliferation rate being 2-4 times (fig. 3A). hPSCs can well express genes related to the dryness in the acclimatization process, flow results show that the ratio of Oct4+/SSEA4+ double positive cells is more than 99% (figure 3B), and the dryness of the 3D pluripotent stem cells in the acclimatization process is well maintained.
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 basic medium (Thermofisiher, 10372019) +2-20mM Glucose (Glucose, Sigma, G7528-250G) +1-5G/L sodium bicarbonate (NaHCO)3Sigma, S5761-500G) + 0.5-5% recombinant Human serum albumin (Human serum albumin, HSA, Heyuanbio, HYC002M01) +1:10000-1:100000 insulin-transferrin-selenium-ethanolamine (insulin Transferrin Selenium ethanolamine, ITS-X, Thermofisiher, 51500056) +1-5mM glutamine substitute (GlutaMAX)TMSupplement, Glutamax, Thermofisiher, 35050061) +0.1-0.5mM Vitamin C (L-Ascorbic acid, Vitamin C, Sigma, A4544-25G) + 1% Penicillin/Streptomycin (penicilin-Streptomycin, Pen/Strep, Thermofisiher, 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-X1: 50-1:500+1-5mM Glutamax +0.1-0.5mM Vitamin C + 1% Pen/Strep + 2-20. mu.g/mL Heparin sodium salt (Heparin sodium salt, Heparin, Sigma, H3149-500KU-9).
All the above media were sterilized by 0.22 μm flask top Filter filtration.
And fresh media (small molecules and growth factors added to the basal media in a low-light-shade safety cabinet) needs to be prepared before media exchange.
First, SC-beta cell (pancreatic beta cell) differentiation
1. Classical method pancreatic beta cell differentiation and function detection
Using the method Melton DA. (cited in 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 assessment. The detection results are as follows:
(1) stage S0: hPSC, human pluratent cells. The proportion of Oct4+/SSEA4+ double positive cells is normal through flow detection, and the dryness is well maintained;
(2) stage S1: DE, definition endproduct cells. The flow detection shows that the ratio of SOX17 positive cells is 82.9 percent, which is not more than 90 percent, and the differentiation efficiency is low;
(3) stage S2: PGT, private gut tube cells. The proportion of HNF4a positive cells detected by flow is 47.9%, and the differentiation efficiency is low;
(4) stage S3: PP1, early commercial promoter cells. The proportion of PDX1 positive cells is 40.6 percent through flow detection, and the differentiation efficiency is low;
(5) stage S4: PP2, later commercial promoter cells. The proportion of PDX1+/NKX6.1+ double positive cells detected by flow detection is only 8.62%, and the expression is low;
(6) stage S5: EN, endicrine promoter cells. The flow detection NKX6.1+/C-peptide double positive cell ratio is only 10.1%, and the vast majority of C-peptide in the differentiation result is weakly positive expression;
(7) stage S6: SC-beta cells, stem-derived beta cells. The flow detection NKX6.1+/C-peptide double positive cell ratio is only 12.7%. The proportion of endocrine cells CHGA positive cells is 47.71%, which is relatively low. Three consecutive rounds of glucose stimulation were unresponsive and the amount of Insulin secreted was very low with high glucose stimulation.
(8) qPCR detection results of each stage in the differentiation process by using the method and the expression trend of related markers on the transcription level are as follows:
the RNA extraction is carried out on the cells at different stages in the whole differentiation process, and then the expression trend of related genes in the whole differentiation process is detected by using qPCR. As can be seen from FIG. 6, the relative expression levels of PDX1, NKX6.1 and Insulin obtained by the method are low, and are only dozens of times of that of the original cells. The expression of Oct4 did not drop to a low level until Stage5, and there was a great problem in safety.
2. Optimization method for pancreatic beta cell differentiation and function detection
Pluripotent stem cells that have been acclimated for 5 to 20 generations (the number of passages of the 3D acclimation process included in the number of 5 to 20 generations) in a 3D reactor are used as starting seed cells for differentiation.
2.1 differentiation method
To begin SC-beta cell differentiation, 0.3-0.7 million dispersed hPSCs were seeded in mTeSR 1media supplemented with 10. mu. M Y27632 per ml. After 24-72 hours of culture, Day 1media was replaced, at this time 80-320mL of the culture system was switched to 60-300mL of a differentiation system (volume was reduced by 10-20%, the amount of the culture medium used in the first liquid change was reduced by 10-20% compared to the amount used in the absence of the change, and then the culture volume was maintained for each liquid change), at this time, the formal start of differentiation was followed, and then the culture medium was replaced in the following order (liquid change all over, single cells in the culture supernatant at the same differentiation stage were centrifuged and added back to the reactor, and single cells in the culture supernatant at the crossover stage were discarded). If the volume is not reduced at the beginning of differentiation, it results in very low cell yield at the late stage of differentiation. Vc, all factors and small molecule compounds used during differentiation were added on the day. If added in advance, the efficiency of the differentiation is greatly reduced. And related culture media are required to be prepared fresh before the culture media are replaced, and the small molecular compounds and the growth factors are added into the basic culture media in a safety cabinet with a low light cover, and the whole replacement process is protected from light. If the operation is not performed in the dark, some small molecular compounds are decomposed by direct irradiation of a light source, and the differentiation efficiency is finally decreased.
First, 0.3-0.7 million dispersed hPSCs cells were seeded in mTeSR1 medium supplemented with 10. mu. M Y27632 per ml. After culturing for 24-72 hours, replacing the culture medium and adding corresponding cell factors, wherein the steps of replacing the culture medium and adding the cell factors according to the culture progress are as follows:
day 1: S1+50-200ng/mL Recombinant Human Activin A (Recombinant Human/Mouse/Rat Activin AProtein, Activin, 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 culture medium used in the first liquid change is reduced by 10% -20% compared with the amount used in the non-liquid change, and then the culture volume is maintained unchanged every liquid change)
Day 2:S1+50-200ng/ml ActivinA.
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 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 change 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 in a reactor at a density of 0.5-2 million cells/ml, cultured at 50-120rpm with the parameters of an incubator set at a constant temperature of 37 ℃ and 5% CO2And 100% humidity.
After this time, the fluid was changed every other day, the re-aggregated normal pellet was collected using a 10-50 μm reversible filter, and the non-aggregated supernatant was discarded. At each stage and the final stage of differentiation, cells were taken separately for analysis.
2.2 detection indexes of stages 0-6 in the differentiation process:
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 as follows:
(1) stage S0. hPSC, human pluratent cells. The detection results are shown in FIG. 8A;
the proportion of Oct4+/SSEA4+ double positive cells is larger than 95 percent through flow detection, and the dryness is well maintained;
(2) stage S1. DE, definition endproduct cells. The detection result is shown in FIG. 8B;
the flow detection shows that the proportion of SOX17 positive cells reaches 92.8 percent, which indicates that most of the cells are differentiated into directional endoderm cells.
In addition, this stage allows for flow sorting to obtain directed endoderm progenitor cell lines. The main method comprises the following steps:
differentiation at this stage, the cell pellet was depleted to single cells using TryPLE, and the cells were labeled with CXCR4 and CD117, and the double positive population CXCR4+/CD117+ was flow sorted.
The population of cells is seeded on Matrigel-containing plates and cultured using directed endoderm progenitor cell culture and expansion media.
Wherein the components of the culture medium for culturing and amplifying the directional endoderm progenitor cells are as follows:
s1 media +20-100ng/mL bone morphogenetic Protein 4 (recombined Human BMP-4Protein, BMP4, R & D systems,314-BP-050) +5-20ng/mL Recombinant Human basic fibroblast growth factor Protein (recombined Human bFGF Protein, bFGF, R & D systems,233-FB-010) +5-20ng/mL Recombinant Human vascular endothelial growth factor Protein (recombined Human VEGF 165Protein, VEGF, R & D systems,293-VE-010) +5-20ng/mL Recombinant Human epidermal growth factor Protein (recombined 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 differentiated terminal beta cells, and also obviously improve the safety in transplants.
(3) Stage S2. PGT, private gut tube cells. The detection result is shown in FIG. 8C;
the proportion of HNF4a positive cells reaches 98.4 percent through flow detection, which shows that almost all cells are differentiated into PGT cells, and the differentiation efficiency is very high.
(4) Stage S3. PP1, early commercial promoter cells. The detection result is shown in FIG. 8D;
the proportion of PDX1 positive cells reaches 57.4 percent through flow detection, which shows that most cells enter the early stage of pancreatic progenitor cells and have good differentiation effect.
(5) Stage S4. PP2, later commercial promoter cells. The detection result is shown in FIG. 8E;
the proportion of PDX1+/NKX6.1+ double positive cells detected by flow is 27%, which shows that the cells entering the next differentiation stage account for a certain proportion and have better differentiation effect.
(6) Stage S5. EN, endicrine promoter cells. The detection result is shown in FIG. 8F;
the flow detection of NKX6.1+/C-peptide double positive cells reaches 24%, which indicates that more than 20% of the cells are differentiated into SC-beta cells.
(7) Stage S6. SC-beta cells, stem-derived beta cells. The detection result is shown in FIG. 8G;
the proportion of NKX6.1+/C-peptide double positive cells detected by flow detection is more than 30 percent. Some cells express GCG (islet alpha cells) and SST (islet delta cells), and the proportion of endocrine cells CHGA positive cells is more than 90%.
There were three consecutive rounds of glucose response with stimulation index greater than 2 and Insulin secretion greater than 1.0-5.0. mu.IU/1000 cells at high glucose stimulation. The dithizone staining appeared red.
(8) qPCR detection results of each stage in the differentiation process and expression trend of related Marker on transcription level:
the RNA extraction is carried out on the cells at different stages in the whole differentiation process, and then the expression trend of related genes in the whole differentiation process is detected by using qPCR. As can be seen from FIG. 7, the relative expression levels of PDX1, NKX6.1 and Insulin obtained by the method are high, and especially the expression of Insulin is nearly 20 ten thousand times higher than the initial expression level. The expression of Oct4 is reduced to a lower level by the Stage2, and the safety problem is greatly reduced.
And (4) detection conclusion:
the whole differentiation process of inducing hPSCs to differentiate into pancreatic beta cells by using the optimization method needs to go through the following stages, and 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 differentiation of the batch can be considered to be normal, the differentiation can be smoothly carried out, and the finally obtained pancreatic beta cells can be normal in function.
hPSCs cells: the proportion of Octamer-binding transcription factor4/Stage-specific embryo antigen 4(Octamer-binding transcription factor4/Stage-specific embryo antigens 4, Oct4+/SSEA4+) double positive cells is more than 95%;
DE, directed endoderm: the proportion of SOX transcription factor family member 17(Sex-determining region Y-box 17, Sox17+) positive cells is more than 90 percent;
PGT, gastral tract: the proportion of Hepatocyte Nuclear Factor 4a (Hepatocyte Factor 4alpha, HNF4a +) positive cells is more than 80 percent;
PP1, pancreatic progenitor early stage: the proportion of Pancreatic duodenum homeobox-1 (Pancreatic and duodenal homeobox1, PDX1+) positive cells is more than 60 percent;
PP2, pancreatic progenitor late stage: the proportion of PDX1 +/homologous transcription factor NKX6.1(homeobox transcription factor NK6 homeobox1, NKX6.1+) double positive cells is more than 25%;
EN, pancreatic endocrine progenitor cells: the proportion of NKX6.1+/C-peptide + double positive cells is more than 10 percent;
SC-beta cluster, stem cell-derived pancreatic beta cells: the proportion of NKX6.1+/C-peptide + double positive cells is more than 30 percent.
After the indexes of different stages reach the quality control standard, the beta cells can be well differentiated into functional beta cells.
2.3 step of enriching the cells at certain stages (see the above 9-12) of the whole differentiation process of stages 0-6) including but not limited to CD177, CD117, CXCR4, GP2, Procr and other relevant markers (the enrichment is performed alternately in the whole differentiation process, which is equivalent to the optimization in the differentiation process, and the enrichment can obviously improve the differentiation efficiency and is a selection mode for improving the differentiation efficiency).
The enrichment can be judged whether to be executed according to the morphology of the cells in the differentiation process, if the morphology of the differentiated cells of the batch is slightly changed, the function and the yield of the differentiated cells can be further improved through the enrichment:
(1) the specific enrichment and culture method of CXCR4+/CD117+ double positive population is as follows:
the cell mass at the differentiation stage ready for enrichment was digested into single cells using TryPLE, and the cells were labeled with CXCR4 and CD117, and the double positive population CXCR4+/CD117+ was sorted by flow. The population of cells is seeded on Matrigel-containing plates and cultured using directed endoderm progenitor cell culture and expansion media.
Wherein the endoderm progenitor cell culture and amplification medium comprises the following components: s1 media + BMP4(20-100ng/mL) + bFGF (5-20ng/mL) + VEGF (5-20ng/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 and reduce the residue of stem/progenitor cells in differentiated terminal beta cells, thereby obviously improving the safety.
(2) Specific enrichment and culture methods for CD177 positive populations were:
the differentiation stage cell mass was digested into single cells using TryPLE, and the cells were labeled with CD177 and CD177 positive populations were sorted by flow. Then sorting out the cells at 2-10X 105The cells were cultured and differentiated by being inoculated in Stage1 medium containing 10. mu. M Y27632 at a density of/mL.
Cells subjected to the CD177+ enrichment procedure can differentiate more uniformly into pancreatic progenitor cells in vitro, and eventually into more functionally mature glucose-responsive beta cells, than cells not subjected to this enrichment.
(3) The GP2 positive cell population is enriched and cultured by the following specific method:
collecting cell mass at differentiation stage, digesting into single cells, sorting GP2 positive cell subset, and sorting the sorted cells at 5-10 × 105The culture medium is inoculated in the 14 th day culture medium for continuous differentiation at the density of/mL;
the method can obviously improve the proportion of the differentiated PDX1+/NKX6.1+ double positive cells.
(4) The specific enrichment and culture method of the Procr-positive population is:
the cell mass at the differentiation stage was digested into single cells using TryPLE, and the cells were labeled with Procr, and the Procr-positive population was sorted by flow sorting.
The enriched Proc + cells were passaged every 7-14 days, and during each passage the digested Procr + single cell suspension was supplemented with fresh human HUVEC cells at a cell ratio of 1: 1.
Then, the Procr + single cells and the human HUVEC cells were mixed and cultured again at a ratio of 1:4 to 1: 6. The method can remarkably improve the yield of differentiated cells.
(5) WNT4 was added at the last stage of differentiation, which clearly drives metabolic maturation, indicating that the response of differentiated beta cells to glucose can be significantly improved and more Insulin is released.
Example 3 modified APA microencapsulation of differentiated pancreatic beta cells
1. Preparation of improved ultrapure sodium alginate solution
Filtering the dissolved sodium alginate by PES sterile filtering devices of 0.8 mu m, 0.45 mu m and 0.22 mu m in sequence; the viscosity of the sodium alginate is 50-200 cP; the prepared sterile sodium alginate can be stored in a 4-degree refrigerator for 1-4 weeks.
2. Beta cell differentiated by APA microencapsulation-microfluidic method or high-voltage electrostatic method
2.1 microfluidic method (microfluidic plate available from Suzhou secretion microfluidic technologies GmbH, model WH-SP-01):
mixing 1% -3% sodium alginate solution with beta cells, wherein 1mL of sodium alginate corresponds to 1-10 x 10^6 cells;
after mixing, adding the mixture into one pipeline, 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, P7890-100MG) for reaction for 10-30 min;
then adding 0.1-0.3% sodium alginate solution to react for 2-10 minutes;
adding 10-100mM sodium citrate, and reacting for 2-10 min to obtain APA microcapsule.
2.2 high-voltage electrostatic method:
mixing 1% -3% sodium alginate solution with beta cells, wherein 1mL of sodium alginate corresponds to 1-10 x 10^6 cells;
sucking with a 20-40G syringe, forming jet flow under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz, and flow rate of 100-;
adding 0.01-0.1% polylysine to react for 10-30 min;
then adding 0.1-0.3% sodium alginate solution to react for 2-10 minutes;
adding 10-100mM sodium citrate, and reacting for 2-10 min to obtain APA microcapsule.
The morphology of the encapsulated cell mass is shown in FIG. 4. The particle size of the microcapsules is preferably controlled to be 250-750 μm.
Example 4 microencapsulated pancreatic beta cell function evaluation (Single transplant function evaluation)
It has now been found that a variety of compounds can induce diabetes in animal models. The two most studied and commonly used compounds are Streptozotocin (STZ) and Alloxan (ALX), respectively, with STZ induction being the most commonly used. STZ and ALX are both glucose analogs and both act through the glucose transporter GLUT2 in the beta cell, eventually leading to almost complete destruction of the beta cell in the islet, resulting in severe insulin deficiency in the mouse, exhibiting hyperglycemia and weight loss, thus reproducing the major symptoms of type I diabetes. The invention is induced by using STZ for medicine, so that C57BL/6 mice generate type I diabetes symptoms.
1. Microencapsulated or naked pancreatic beta cells implanted subcutaneously into STZ-induced type I diabetes C57BL/6 mice for in vivo functional assessment
1.1 microencapsulation or naked pancreatic beta cell transplantation:
the mice receiving the transplantation are anesthetized by inhalation of 2% to 5% Isoflurane (Isoflurane USP, Clipper Distribution). Then, subcutaneous transplantation is carried out: a small skin incision (0.3-0.5cm) was made in the lower abdomen to form a subcutaneous "pocket" in the left and right lower quadrants, and microencapsulated beta cells or naked beta cells were mixed with 200 and 500. mu.L of beta cell activated matrigel and transplanted into the subcutaneous "pocket" of the mouse. Wherein the beta cell active matrigel comprises the following components: 10X Media (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) needs to be combined with an immunosuppressant, and the specific scheme is as follows:
maintenance immunosuppression consisted of rapamycin (1-2mg/kg i.p., four times per day) for 5-10 days from the day of transplantation.
To eliminate B cells, 10F4 (supplied by university of pennsylvania pathology and laboratory medicine m. cancro), a monoclonal antibody against mouse BLyS was used (50-200 μ g injected intraperitoneally 15-25d prior to transplantation, in two injections, 24 hours apart). Starting on day 10, 10F4 was also administered gradually, with the dose gradually decreasing from 20-100 μ g per week on week 2 to 1-10 μ g per week on week 8. On days 50-80, immunosuppressive dosing was discontinued.
1.3 post-transplant detection
And detecting the blood sugar level of the mouse, the glycosylated hemoglobin level, the in vivo glucose stimulation, the human C peptide condition and the like. Fixing the transplanted APA microencapsulated beta cells or naked beta cells, then performing immunofluorescence, and identifying related markers.
In addition, islet transplantation sites can be resected in order to confirm that islet transplants are the only source of euglycemia, performing islet resection (skin) on a group of islet recipients who have long-term normoglycemia, which results in rapid recurrence of diabetes within 24-72 hours. The same results show that the subcutaneous transplanted differentiated beta cells have the same function as the microencapsulated beta cells after abdominal transplantation (which means a measure that the transplanted pancreatic beta cells are removed, and then the animals can relapse into diabetes, which means that the transplanted pancreatic beta cells have the function of doing the exercise).
According to the method, 1-10 x 10^6 cells are transplanted per mouse, an indwelling needle is used for abdominal cavity transplantation, and the blood sugar level, the glycosylated hemoglobin level, the in-vivo glucose stimulation, the 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 the symptoms of hyperglycemia in diabetic mice, which takes 1-7 days, and then maintain normal blood glucose for more than 30 days. The content of the human C peptide in the plasma of the transplanted mice is randomly detected in the transplantation period, and the result shows that the content of the human C peptide is far higher than that of the control group and is between 100 and 500 pM. The percentage of glycated hemoglobin HbA1C also decreased, reaching 6-12%. In vivo IPGTT experiments show that microencapsulated beta cells can rapidly sense the rise of blood sugar and secrete sufficient quantity of Insulin to maintain stable blood sugar. Fixing the transplanted APA microencapsulated beta cells, performing immunofluorescence, and identifying related markers. The microencapsulated beta cells can well express the related Marker NKX6.1/C-peptide of the beta cells, and the double positive rate is more than 30 percent. The specific evaluation results are shown in fig. 5.
Example 5 microencapsulated pancreatic beta cell function evaluation (Combined transplantation function evaluation)
Microencapsulated or naked beta cells in combination with Mesenchymal Stem Cells (MSC) or Endothelial Progenitor Cells (EPC) were transplanted into STZ-induced C57 mice for functional evaluation, the experimental steps were as follows:
1. after cell clusters at the Stage of differentiation of Stage5-Stage6 are digested into single cells by using TryPLE digestive enzyme, the single cell suspension of Mesenchymal Stem Cells (MSC) or Endothelial Progenitor Cells (EPC) and the differentiated single cell suspension are mixed and cultured according to the proportion of 1:1, the culture medium used for mixed culture is S3 differentiation basal culture medium, and the mixed culture medium is placed on a track shaker in a low-adsorption 6-pore plate for culture.
2. The cell inoculation method of animal experiments is the same as that of 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 70-120 rpm. The single cells are re-aggregated into clusters within 24-72h, and after further maturation for 7-14 days, the clusters are transplanted to STZ-induced C57 mice subcutaneously or intraperitoneally for in vivo functional evaluation. The mesenchymal stem cells or endothelial progenitor cells and the islet cells are subjected to co-aggregation culture to form a three-dimensional cell mass, the islet cell mass can be injected subcutaneously or intraperitoneally with a single dose to provide long-term blood sugar control for an organism, and blood sugar of type I diabetic mice can be kept within a normal range without using an anti-immune rejection drug or an encapsulation system. And the receptor can be protected from the use of anti-immune rejection drugs or islet cell encapsulation systems (microencapsulation) in the process.
In the method, transplantation of the mesenchymal stem cells is carried out at a transplantation position, and the quantity of the transplanted mesenchymal stem cells is 1-10 x 10^6 cells of the transplantation position of each mouse. The main purpose is to construct an angiogenesis environment of a transplantation part, so that transplanted beta cells can obtain nutrition and oxygen in a vascularization environment, and the survival and the function of the transplanted beta cells are facilitated. The results showed that the mesenchymal stem cells transplanted in advance were able to vascularize the transplanted part, and the generation of blood vessels was performed for 10 to 50 days. Subsequently transplanted microencapsulated or naked beta cells have the same function after intraperitoneal transplantation of the microencapsulated beta cells.
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.
Claims (9)
1. A method for the three-dimensional suspension directed differentiation of pluripotent stem cells into mature pancreatic beta cells, the method comprising the steps of:
(1) three-dimensional suspension domestication culture of pluripotent stem cells;
(2) inducing the domesticated cells to differentiate into pancreatic beta cells;
the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.
2. The method according to claim 1, wherein the step of three-dimensional suspension acclimatization culture of the pluripotent stem cells of step (1) comprises:
1) y27632 pretreatment, preferably, the pluripotent stem cells cultured on a plane are subjected to liquid exchange 2 hours before suspension culture, and then are replaced with mTeSR1 medium containing Y27632 at a concentration of 10. mu.M;
2) washing and neutralizing the pretreated cells by using DMEM/F12 culture medium;
3) shake culturing the pluripotent stem cells in mTeSR1+ Y27632 culture medium, preferably, passaging 5-10 times in mTeSR1+ Y27632 culture medium, replacing half of the culture medium every day, and removing single cells and aggregated large cell masses in culture supernatant during passaging, preferably, removing the large cell masses by filtering with 400 μm screen to remove cell masses larger than 400 μm;
more preferably, the pluripotent stem cells are seeded at a density of 3 to 8 x 105The parameters of three-dimensional stirring culture are as follows: rotating at 50-120rpm,Maintaining the temperature at 37 ℃, the CO2 at 5% and the humidity at 100%, wherein the culture medium is mTeSR1 culture medium containing Y27632 with the concentration of 10 mu M;
4) and 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 cells are qualified for domestication.
3. The method according to claim 1 or 2, wherein the step of inducing the acclimated cells to differentiate and microencapsulate in step (2) comprises:
using the pluripotent stem cells domesticated in the step (1) as starting seed cells for differentiation, wherein the differentiation steps are as follows:
1) mTeSR1 medium (containing 10. mu. M Y27632) was inoculated with 0.3-0.7 x 106Culturing the scattered three-dimensional suspension domesticated pluripotent stem cells for 24-72 hours, then replacing the culture medium for the first day, reducing the volume by 10-20% when replacing the culture medium,
the culture medium for the first day is: s1 medium is added with: 50-200ng/mL recombinant human activin A (activin A), 10-100ng/mL recombinant human Wnt3a protein;
2) changing culture medium on day 2;
the culture medium for the day 2 is: s1 medium is added with: 50-200ng/ml Activin A;
3) replacing the culture medium on day 4 to day 6;
the culture medium for the 4 th day is: s2 medium is added with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5 μ M transforming growth factor- β RI Kinase Inhibitor IV (TGF- β RI Kinase Inhibitor IV);
4) changing the culture medium on day 7 to day 8;
the day 7 culture medium was: S3/S4 medium is added with: 20-100ng/ml KGF, 0.1-0.5. mu.M Sant1, 1-5. mu.M Retinoic Acid (RA), 100-500nM 1,1,4,4-tetraphenyl-1,3-butadiene (TPB), 5-20. mu. M Y27632;
5) changing the culture medium on day 9 to day 13;
the culture medium on day 9 was: S3/S4 medium is added with: 20-100ng/mL KGF, 0.1-0.5. mu.M Sant1, 50-200nM RA, 10-100ng/mL EGF, 10-100ng/mL recombinant human Noggin (NOG), 5-20. mu. M Y27632;
6) replacing the culture medium on day 14 to day 16;
the culture medium for the 14 th day is: s5 medium is added with: 0.1-0.5 μ M Sant1, 50-200nM RA, 0.5-2 μ M gamma-Secretase Inhibitor XXI (gamma-Secretase Inhibitor XXI), 5-20 μ M Repsox, 0.5-2 μ M trispecific thyronine (L-3,3',5-Triiodothyronine, T3), 5-50ng/ml Recombinant Human β -cell (Recombinant Human Betacellulin Protein), 50-500nM LDN193189hydrochloride (LDN193189hydrochloride), 10 μ M zinc sulfate;
7) replacing the culture medium on day 18-20;
the culture medium of day 18 was: s5 medium is added with: 10-50nM RA, 0.5-2. mu.M XXI, 5-20. mu.M Repsox, 0.5-2. mu. M T3, 5-50ng/ml Betacellulin, 50-500nM LDN193189, 1mM N-cys (Fmoc-N-Me-Cys (Trt) -OH);
8) on day 21, the differentiated cells are digested into single cells and inoculated into the reactor again for culture, the reaggregated normal cell mass is collected in the culture process, and the supernatant without agglomeration is discarded, so that the differentiated pancreatic beta cells are obtained;
preferably, the parameters of the re-cultivation are: rotating at 50-120rpm, maintaining the temperature at 37 deg.C and 5% CO2And 100% humidity, changing the S3 culture medium every two days at 21-35 days, and collecting the normal cell mass to be re-aggregated by using a 10-50 μm reversible filter;
the components of each culture medium are as follows:
s1 medium: MCDB131 basic 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 medium: MCDB131 basal medium + ITS-X1: 10000-1:100000+1-5mM glutamine, necessary buffer salt (NaHCO)3) Antibiotics, antioxidants (VC), glucose and serum albumin;
S3/S4 Medium: MCDB131 basal Medium + ITS-X1: 50-1:500+1-5mM Glutamine, necessary buffer salts (NaHCO)3) Antibiotics, antioxidants (VC), glucose and serum albumin;
s5 medium: MCDB131Basal medium + ITS-X1: 50-1:500+1-5mM glutamine +2-20 μ g/mL heparin sodium salt, necessary buffer salt (NaHCO)3) Antibiotics, antioxidants (VC), glucose and serum albumin.
4. The method of claim 3,
2) changing the culture medium of day 2 on day 2, wherein the volume of the culture medium is unchanged during liquid change;
3) changing culture medium on day 4 and day 6 respectively, wherein the culture medium volume is unchanged during liquid change;
4) changing culture medium on 7 th and 8 th days, respectively, with the volume of the culture medium unchanged
5) Changing culture medium on 9 th, 11 th and 13 th days, wherein the volume of the culture medium is unchanged during liquid changing;
6) changing the culture medium on day 14 and day 16 respectively, wherein the volume of the culture medium is unchanged during liquid change;
7) the culture medium was changed on day 18 and day 20, respectively, and the volume of the culture medium was not changed at the time of medium change.
5. The method according to claim 3 or4, wherein optionally, the method for inducing differentiation further comprises any one or any combination of the following steps:
9) enrichment and culture of differentiated CD 177-positive populations is carried out before the differentiation step 3) described above, by:
collecting the cell mass differentiated in the previous step, digesting into single cells, sorting out CD 177-positive cell subsets, and sorting out the cells at 2-10 × 105The cells were inoculated at a density of/mL into the day 4 medium containing 10. mu. M Y27632 to continue differentiation;
10) before the differentiation step 6) described above, enrichment and culture of GP 2-positive populations were carried out by:
collecting the cell mass differentiated in the previous step, digesting into single cells, then sorting out GP2 positive cell subset, and sorting out cells in 5-10 × 105The culture medium is inoculated in the 14 th day culture medium for continuous differentiation at the density of/mL;
11) before the differentiation step 8), the enrichment and culture of the Procr-positive cell population are carried out by:
digesting the cell mass differentiated in the previous step into single cells, then sorting out Procr-positive cell subsets, carrying out passage on the enriched Proc-positive cells once every 7-14 days, and supplementing the digested Procr-positive single cell suspension with fresh human HUVEC cells in the process of passage each time, wherein the cell ratio is 1: 1; mixing Procr positive cells and human HUVEC cells, and inoculating and culturing at a ratio of 1:4-1: 6;
12) 100ng/mL WNT4 was added to the medium of differentiation step 8) above to drive the metabolic maturation necessary for glucose-stimulated insulin secretion by differentiated cells.
6. A method for preparing sodium alginate-polylysine-sodium alginate (APA) microencapsulated artificial islets from pancreatic beta cells made by the method of any one of claims 1-5, the method comprising:
(1) preparing a sodium alginate solution with the viscosity of 50-200cP, preferably, filtering the prepared and dissolved sodium alginate by a PES sterile filtering device with the diameters of 0.8 mu m, 0.45 mu m and 0.22 mu m in sequence, and storing at 4 ℃;
(2) preparing microencapsulated artificial islets according to the method one or the method two:
the method comprises the following steps: a micro-fluidic method:
mixing 1% -3% sodium alginate solution with the pancreatic beta cells, mixing 1mL sodium alginate solution with 1-10 x 106Mixing cells;
after mixing, adding the mixture into one pipeline, 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 to react for 10-30 min;
then 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 microcapsules;
the second method comprises the following steps: high-voltage electrostatic method:
mixing 1% -3% sodium alginate solution with the pancreatic beta cells, mixing 1mL sodium alginate solution with 1-10 x 106Mixing cells;
sucking with a 20-40G syringe, forming jet flow under the conditions of 5-20KV voltage, 1-10 pulse, 50-200Hz, and flow rate of 100-;
adding 0.01-0.1% polylysine to react for 10-30 min;
then adding 0.1-0.3% sodium alginate solution to react for 2-10 minutes;
adding 10-100mM sodium citrate, and reacting for 2-10 min to obtain APA microcapsule.
7. Pancreatic beta cells produced by the method of any one of claims 1-5, or artificial islets produced by the method of claim 7, for use in the manufacture of a medicament for the treatment of diabetes, preferably diabetes type i.
8. Use of pancreatic beta cells prepared by the method of any one of claims 1-5 or artificial islets prepared by the method of claim 7 for the preparation of a therapeutically active ingredient in cell therapy for diabetic patients, preferably diabetes type i.
9. A method of inducing an endodermal progenitor cell lineage, the method comprising the steps of:
(1) performing three-dimensional suspension acclimatization culture of pluripotent stem cells using the same steps as the method of claim 1;
(2) induction of endodermal progenitor cell lines;
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 starting seed cells for differentiation
1) 0.3-0.7 x 10 inoculations per ml mTeSR1 medium (10 μ M Y27632 added to the medium)6Culturing the scattered three-dimensional suspension domesticated pluripotent stem cells for 24-72 hours, then replacing the culture medium for the first day, reducing the volume by 10-20% when replacing the culture medium,
the culture medium for the first day is: s1 medium is added with: 50-200ng/mL recombinant human activin A (activin A), 10-100ng/mL recombinant human Wnt3a protein;
2) changing the culture medium of day 2 on day 2, wherein the volume of the culture medium is unchanged during liquid change;
the culture medium for the day 2 is: s1 medium is added with: 50-200ng/ml Activin A;
3) replacing the culture medium on the 4 th day on the 4 th and 6 th days respectively, wherein the volume of the culture medium is unchanged when the culture medium is replaced;
the culture medium for the 4 th day is: s2 medium is added with: 20-100ng/ml recombinant human keratinocyte growth factor protein (KGF), 1-5 μ M transforming growth factor- β RI Kinase Inhibitor IV (TGF- β RI Kinase Inhibitor IV);
4) when the proportion of SOX17 positive cells is over 90 percent through flow detection, digesting the cell mass into single cells, and separating a CXCR4+/CD117+ double positive cell subset, wherein the cell subset is a directional endoderm progenitor cell;
5) inoculating the population of cells into a Matrigel-containing plate, and performing culture expansion using a directed endoderm progenitor cell culture and expansion medium;
the components of the culture and amplification medium for the directional endoderm progenitor cells are as follows:
s1 medium is added with: 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).
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CN114958718A (en) * | 2022-05-20 | 2022-08-30 | 呈诺再生医学科技(北京)有限公司 | Method for inducing cells with high FOXA2 expression |
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