CN113583938A - Method for forming islet-like structure by islet cells differentiated by in vitro induced stem cells - Google Patents

Method for forming islet-like structure by islet cells differentiated by in vitro induced stem cells Download PDF

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CN113583938A
CN113583938A CN202110776877.XA CN202110776877A CN113583938A CN 113583938 A CN113583938 A CN 113583938A CN 202110776877 A CN202110776877 A CN 202110776877A CN 113583938 A CN113583938 A CN 113583938A
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cells
islet
stem cells
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concentration
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CN113583938B (en
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顾军
顾帅
兰丹
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Pluripotent Stem Cell Regeneration Medical Technology Guangzhou Co ltd
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Pluripotent Stem Cell Regeneration Medical Technology Guangzhou Co ltd
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Abstract

The invention discloses a method for forming an islet-like structure by islet cells differentiated by stem cells induced in vitro, which relates to the technical field of biology and comprises the following steps: separating and culturing the raw material tissue to obtain mesenchymal stem cells; subculturing the obtained mesenchymal stem cells; and inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells. The method adopts a specific induction culture medium and the steps of pretreatment and induction culture, can quickly and efficiently induce the islet cells differentiated from the stem cells in vitro to form the islet-like structure, and has the advantages of high induction rate, high product cytoplasm amount and good application effect.

Description

Method for forming islet-like structure by islet cells differentiated by in vitro induced stem cells
Technical Field
The invention relates to the technical field of biology, in particular to a method for forming an islet-like structure by islet cells differentiated by stem cells induced in vitro.
Background
Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. The chronic hyperglycemia results in chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves.
Islet transplantation is a fundamental method for treating diabetes emerging in this century, but in the early stage, islet isolation and transplantation are mainly utilized, and an appropriate immunosuppressive scheme is combined, so that the scheme is extremely dependent on donors, and large-scale application cannot be realized. The application of stem cell differentiation in the medical field is gradually paid attention and primarily developed by people in recent years, and researches begin to aim at preparing islet cells through stem cell differentiation. In the technology, the important part is the part for inducing the stem cells to differentiate into the islet-like cells, the part mostly takes the mesenchymal stem cells as raw materials, the mesenchymal stem cells are multipotent stem cells with high self-renewal and multidirectional differentiation potential in mesoderm, widely exist in various tissues of the whole body, and can be differentiated into islet beta cells or insulin-producing cells under certain conditions. However, the existing operation step generally has the problems of cell growth inhibition, more induction factors, long induction period, various residual factors and the like, reduces the integral induction rate, and leaves adverse effects on subsequent application of products.
Disclosure of Invention
In order to solve the problems of low induction rate, negative influence on product quality and the like existing in the existing method, the invention provides a novel method for forming an islet-like structure by islet cells capable of rapidly and efficiently inducing stem cell differentiation in vitro, the method is high in induction rate and high in product quality, and the specific scheme is as follows:
a method for inducing islet cells differentiated from stem cells to form islet-like structures in vitro, comprising the steps of:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
Preferably, the raw tissue of S1 includes one or both of placenta tissue or adipose tissue.
Preferably, the isolation culture of S1, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
Preferably, the digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 45-60 min.
Preferably, the centrifugation is carried out under the conditions of 1200-1500rpm for 5-8 min; washing with PBS for 3-5 times.
Preferably, the cells are inoculated into a cell culture medium and cultured under conditions of 37, 5% CO2 at a temperature of 95% saturated humidity.
Preferably, the culturing comprises: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
Preferably, the subculturing of S2 comprises: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
Preferably, the trypsin-EDTA solution comprises trypsin at a mass concentration of 0.25% and EDTA at a mass concentration of 0.02%.
Preferably, the subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 10-15 ng/mL; the concentration of the fibroblast growth factor is 10-15 ng/mL; the concentration of the ascorbic acid is 25-50 mug/mL.
Preferably, the subculture is performed on a plate having a diameter of 10cm at a subculture density of (1-2). times.104/cm2。
Preferably, the passaged mesenchymal stem cell of S3 is a passaged cell of more than P3 generations.
Preferably, the induction medium used for inducing differentiation in S3 is an α -MEM medium, which includes betacellulin, EGF, nicotinamide, and human basic fibroblast growth factor.
Preferably, in the induction medium, the concentration of beta-cell factor is 3-5 μ g/L, the concentration of EGF is 120-140pmol/L, the concentration of nicotinamide is 15-18mmol/L, and the concentration of human basic fibroblast growth factor is 7-10 ug/L.
Preferably, the inducing differentiation of S3 comprises: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 5-9 days.
Preferably, the seeding density is 1.5-2X 105cells/well.
Preferably, the induction culture of S3, before the first replacement of the induction culture medium, removing the culture medium, digesting the cells with trypsin-EDTA solution for 20-30min, washing the cells with PBS buffer solution for 3-5 times, adding 3ml of induction culture medium to each well, continuing suspension induction, replacing the induction culture medium every 3 days, and the total induction culture time is 5-7 days.
Advantageous effects
The invention has the beneficial effects that:
the method for forming the islet-like structure by the islet cells differentiated by the in vitro induced stem cells adopts a specific induction culture medium and the steps of pretreatment and induction culture, can quickly and efficiently form the islet-like structure by the islet cells differentiated by the in vitro induced stem cells, and has the advantages of high induction rate, high product cytoplasm amount and good application effect.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The following examples and comparative examples are parallel runs, with the same processing steps and parameters, unless otherwise indicated.
Example 1 in vitro induction of stem cell differentiated islet cells to form islet-like structures:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
S1 the raw material tissue includes human placenta tissue.
S1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
The above digestive enzyme comprises collagenase type I, and digestion time is 45 min.
Centrifuging at 1200rpm for 5 min; the washing was performed 3 times using PBS.
The cells were inoculated into a cell culture medium and cultured under conditions of 37 ℃ and 5% CO2 at 95% saturated humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
S2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
The trypsin-EDTA solution comprises trypsin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 10 ng/mL; the concentration of the fibroblast growth factor is 10 ng/mL; the concentration of the ascorbic acid is 25 mug/mL.
The subculture was carried out on a plate having a diameter of 10cm at a subculture density of 1X 104/cm2。
The subculture mesenchymal stem cells of S3 are P3 generation subcultured cells.
S3, wherein the basic culture medium is alpha-MEM culture medium, and comprises beta-cell factor, EGF, nicotinamide, and human basic fibroblast growth factor.
In the induction culture medium, the concentration of beta-cell element is 3 mug/L, the concentration of EGF is 120pmol/L, the concentration of nicotinamide is 15mmol/L, and the concentration of human basic fibroblast growth factor is 7 ug/L.
S3, the inducing differentiation comprising: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 5 days.
The inoculation density is 1.5X 105cells/well.
Example 2 in vitro induction of stem cell differentiated islet cells to form islet-like structures:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
S1 the raw tissue includes adipose tissue.
S1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
The above digestive enzyme comprises collagenase type II, and the digestion time is 60 min.
Centrifuging at 1500rpm for 8 min; the washing was performed 5 times with PBS.
The cells were inoculated into a cell culture medium and cultured under conditions of 37 ℃ and 5% CO2 at 95% saturated humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
S2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
The trypsin-EDTA solution comprises trypsin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 15 ng/mL; the concentration of the fibroblast growth factor is 15 ng/mL; the concentration of the ascorbic acid is 50 mug/mL.
The subculture was carried out on a plate having a diameter of 10cm at a subculture density of 2X 104/cm2。
The subculture mesenchymal stem cells of S3 are P3 generation subcultured cells.
S3, wherein the basic culture medium is alpha-MEM culture medium, and comprises beta-cell factor, EGF, nicotinamide, and human basic fibroblast growth factor.
In the induction culture medium, the concentration of beta-cell element is 5 mug/L, the concentration of EGF is 140pmol/L, the concentration of nicotinamide is 18mmol/L, and the concentration of human basic fibroblast growth factor is 10 ug/L.
S3, the inducing differentiation comprising: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 9 days.
The inoculation density is 2X 105cells/well.
Example 3 in vitro induction of stem cell differentiated islet cells to form islet-like structures:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
The raw material tissue of S1 includes placenta tissue.
S1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
The above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 50 min.
Centrifuging at 1400rpm for 6 min; the washing was performed 4 times with PBS.
The cells were inoculated into a cell culture medium and cultured under conditions of 37 ℃ and 5% CO2 at 95% saturated humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
S2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
The trypsin-EDTA solution comprises trypsin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 12 ng/mL; the concentration of the fibroblast growth factor is 12 ng/mL; the concentration of the ascorbic acid is 40 mug/mL.
The subculture was carried out on a plate having a diameter of 10cm and the subculture density was 1.5X 104/cm2。
The subculture mesenchymal stem cells of S3 are P3 generation subcultured cells.
S3, wherein the basic culture medium is alpha-MEM culture medium, and comprises beta-cell factor, EGF, nicotinamide, and human basic fibroblast growth factor.
In the induction culture medium, the concentration of beta-cell element is 4 mug/L, the concentration of EGF is 130pmol/L, the concentration of nicotinamide is 16mmol/L, and the concentration of human basic fibroblast growth factor is 9 ug/L.
S3, the inducing differentiation comprising: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 7 days.
The inoculation density is 1.7X 105cells/well.
Example 4 islet cells induced in vitro stem cell differentiation form islet-like structures:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
The raw material tissue of S1 includes placenta tissue.
S1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
The above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 50 min.
Centrifuging at 1400rpm for 6 min; the washing was performed 4 times with PBS.
The cells were inoculated into a cell culture medium and cultured under conditions of 37 ℃ and 5% CO2 at 95% saturated humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
S2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
The trypsin-EDTA solution comprises trypsin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 12 ng/mL; the concentration of the fibroblast growth factor is 12 ng/mL; the concentration of the ascorbic acid is 40 mug/mL.
The subculture was carried out on a plate having a diameter of 10cm and the subculture density was 1.5X 104/cm2。
The subculture mesenchymal stem cells of S3 are P3 generation subcultured cells.
S3, wherein the basic culture medium is alpha-MEM culture medium, and comprises beta-cell factor, EGF, nicotinamide, and human basic fibroblast growth factor.
In the induction culture medium, the concentration of beta-cell element is 4 mug/L, the concentration of EGF is 130pmol/L, the concentration of nicotinamide is 16mmol/L, and the concentration of human basic fibroblast growth factor is 9 ug/L.
S3, the inducing differentiation comprising: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 7 days.
The inoculation density is 1.7X 105cells/well.
S3, removing the culture medium before replacing the induction culture medium for the first time, digesting the cells with trypsin-EDTA solution for 25min, washing the cells with PBS buffer solution for 4 times, adding 3ml of induction culture medium into each hole, continuing suspension induction, and replacing the induction culture medium every 3 days, wherein the total induction culture time is 5-7 days.
Comparative example 1 islet cells induced in vitro stem cell differentiation form islet-like structures:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
The raw material tissue of S1 includes placenta tissue.
S1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
The above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 50 min.
Centrifuging at 1400rpm for 6 min; the washing was performed 4 times with PBS.
The cells were inoculated into a cell culture medium and cultured under conditions of 37 ℃ and 5% CO2 at 95% saturated humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
S2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
The trypsin-EDTA solution comprises trypsin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 12 ng/mL; the concentration of the fibroblast growth factor is 12 ng/mL; the concentration of the ascorbic acid is 40 mug/mL.
The subculture was carried out on a plate having a diameter of 10cm and the subculture density was 1.5X 104/cm2。
The subculture mesenchymal stem cells of S3 are P3 generation subcultured cells.
S3 the induction medium for inducing differentiation comprises 0.1mmol/L beta-mercaptoethanol, 5ug/L human basic fibroblast growth factor, 10mmol/L dexamethasone, and alpha-MEM containing 10% FBS.
S3, the inducing differentiation comprising: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 7 days.
Comparative example 2 islet cells induced in vitro stem cell differentiation form islet-like structures:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
The raw material tissue of S1 includes placenta tissue.
S1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
The above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 50 min.
Centrifuging at 1400rpm for 6 min; the washing was performed 4 times with PBS.
The cells were inoculated into a cell culture medium and cultured under conditions of 37 ℃ and 5% CO2 at 95% saturated humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 at the temperature and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging to take out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
S2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 at the temperature of 5% and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
The trypsin-EDTA solution comprises trypsin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 12 ng/mL; the concentration of the fibroblast growth factor is 12 ng/mL; the concentration of the ascorbic acid is 40 mug/mL.
The subculture was carried out on a plate having a diameter of 10cm and the subculture density was 1.5X 104/cm2。
The subculture mesenchymal stem cells of S3 are P3 generation subcultured cells.
S3 the induction medium for inducing differentiation comprises 0.1mmol/L beta-mercaptoethanol, 5ug/L human basic fibroblast growth factor, 10mmol/L dexamethasone, and alpha-MEM containing 10% FBS.
S3, the inducing differentiation comprising: taking the third generation placenta mesenchymal stem cells with the mass concentration of 0.2Cells were digested with 5% trypsin + 0.02% EDTA at 2X 105And performing density inoculation on the cells in a 6-plate, removing the old culture medium after 24 hours when the cells are fully filled with 60%, washing the cells for 3 times by using PBS buffer solution, adding 3mL of induction culture medium into each hole, removing the old culture medium after 2 days of induction culture, continuously adding 3mL of induction culture medium into each hole for culture, and repeating the steps for 14 days of induction culture.
The differentiation results of the islet-like cells obtained in the above examples and comparative examples were identified:
setting a control group: the differences from example 3 are: in step S3, α -MEM medium containing 10% by mass of FBS was used in place of the induction medium.
And (4) microscopic observation: the non-induced placenta mesenchymal stem cells grow in a long fusiform adherent manner, and the induced cells become gradually round and gather into clusters.
(1) And (3) carrying out dithizone dyeing reaction: taking islet-like cells obtained after induction culture, removing the islet-like cells from an original culture medium, washing with PBS for 3 times, adding 1ml of LPBS and 50uL of dithizone working solution respectively, incubating at 37 ℃ for 10min, removing a staining solution, washing with PBS for 2 times, observing the cell staining condition, and taking a picture.
As a result: all examples and comparative examples were red in color after dithizone staining, and were positive reactions, while the control group was negative;
(2) chemiluminescence immunoassay method for detecting insulin level: collecting cell culture supernatant after induction culture, and detecting the content of insulin.
As a result: the control group detected insulin concentrations at zero. Examples 1 to 4 were 413mU/L, 426mU/L, 429mU/L, 449mU/L, comparative example 1 was 361mU/L, and comparative example 2 was 313mU/L, respectively. Therefore, the induction culture medium and the induction culture method can obviously improve the induction efficiency.
(3) Glucose stimulation experiment: 50 islet-like cell masses (50-100um) are picked from each sample, the islet-like cell masses are placed in a 1.5mL centrifuge tube, washed with PBS for 2 times, added with 1mL sugar-free DMEM for pre-culture for 4h, then sequentially cultured with 400uL DMEM containing 6.0mmol/L glucose and 18mmol/L glucose for 2h, and the supernatant is collected and the secretion of insulin under stimulation of glucose with different concentrations in the supernatant is detected by ELISA.
As a result: insulin was hardly detectable in the control cell supernatant. All the samples of the examples and the comparative examples have insulin secretion under the condition of low sugar, and the insulin secretion amount is obviously increased under the condition of high sugar, after 18mmol/L glucose DMEM is cultured for 2 hours, the insulin secretion amount is 1.17 times of that of the example 1, the insulin secretion amount of the example 4 is 1.21 times of that of the example 3, the insulin secretion amount of the example 3 is 1.83 times of that of the comparative example 1, and the insulin secretion amount of the example 3 is 2.76 times of that of the comparative example 2. Therefore, the islet-like cells induced by the method provided by the invention can give corresponding responses according to the stimulation degree of glucose in the external environment, and the method is high in induction efficiency and high in cytoplasm amount and is more suitable for practical application.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Claims (10)

1. A method for inducing islet cells differentiated from stem cells in vitro to form an islet-like structure, comprising: the method comprises the following steps:
s1, separating and culturing the raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells.
2. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 1, wherein: s1 wherein the raw tissue includes one or both of placenta tissue and adipose tissue; s1, the isolation culture, comprising: cleaning raw material tissue, cutting, digesting with digestive enzyme, centrifuging to obtain cell precipitate, cleaning, and inoculating to cell culture medium.
3. The method of inducing islet cells of stem cell differentiation in vitro to form islet-like structures of claim 2, wherein: s1, digesting the mixture for 45-60min by using collagenase I or collagenase II; the centrifugation is carried out for 5-8min under the conditions of 1200 and 1500 rpm; washing with PBS for 3-5 times.
4. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 3, wherein: s1, comprising: after inoculation, culturing for 3 days under the conditions of 37 ℃, 5% CO2 and 95% saturation humidity, replacing half of culture solution, culturing for 3 days, replacing all the culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking out cell precipitates, and suspending with cell culture solution to obtain the mesenchymal stem cells.
5. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 1, wherein: s2 the subculturing includes: adding the obtained mesenchymal stem cells into a subculture medium, culturing at 37 ℃, 5% CO2 and 95% saturated humidity, digesting with a trypsin-EDTA solution when the cell fusion degree reaches 80-90%, and subculturing according to a ratio of 1:4 after digestion is finished.
6. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 5, wherein: s2 the subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 10-15 ng/mL; the concentration of the fibroblast growth factor is 10-15 ng/mL; the concentration of the ascorbic acid is 25-50 mug/mL.
7. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 6, wherein: s2, inoculating on a plate with a diameter of 10cm, and subculturing at a density of (1-2). times.104/cm2。
8. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 1, wherein: s3, wherein the basic culture medium is alpha-MEM culture medium, and comprises beta-cell factor, EGF, nicotinamide, and human basic fibroblast growth factor.
9. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 8, wherein: s3, the concentration of beta-cell factor is 3-5 mug/L, the concentration of EGF is 120-140pmol/L, the concentration of nicotinamide is 15-18mmol/L, and the concentration of human basic fibroblast growth factor is 7-10 ug/L; s3, the inducing differentiation comprising: inoculating the subculture mesenchymal stem cells into a 6-hole ultra-low adsorption culture plate, adding 3ml of induction culture medium into each hole, performing suspension induction, and replacing the induction culture medium every 3 days for 5-9 days.
10. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 9, wherein: s3, removing the culture medium before replacing the induction culture medium for the first time, digesting the cells with trypsin-EDTA solution for 20-30min, washing the cells with PBS buffer solution for 3-5 times, adding 3ml of induction culture medium into each hole, continuing suspension induction, replacing the induction culture medium every 3 days, and the total induction culture time is 5-7 days.
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