CN113583938B - 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 PDFInfo
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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 cytoplasm content of the product and good application effect.
Description
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 method for fundamentally treating diabetes emerging in the century, but in the early stage, islet isolation and transplantation are mainly utilized, and an appropriate immunosuppression 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 a 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 material tissue of S1 includes one or both of placenta tissue or adipose tissue.
Preferably, the isolation culture of S1 comprises: cleaning raw material tissues, shearing, digesting by using digestive enzyme, centrifuging to obtain cell precipitates, cleaning, and inoculating to a cell culture medium for culture.
Preferably, the above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 45-60min.
Preferably, the centrifugation is carried out for 5-8min under the condition of 1200-1500 rpm; washing with PBS for 3-5 times.
Preferably, the cells are inoculated into a cell culture medium and then cultured under conditions of 37% by weight, 5% by weight, CO2, and 95% by weight saturation humidity.
Preferably, the culturing comprises: after inoculation, culturing for 3 days under the conditions of 37 and 5 percent of CO2 and 95 percent of saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80 to 90 percent, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell sediment, and suspending with the cell culture solution to obtain the mesenchymal stem cells.
Preferably, the S2 subculture comprises: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
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 10-15ng/mL; the concentration of the fibroblast growth factor is 10-15ng/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.10 4 /cm 2 。
Preferably, the passaged mesenchymal stem cell of S3 is a passaged cell of more than P3 generation.
Preferably, the induction medium for inducing differentiation in S3 is α -MEM medium, which includes betacellulin, EGF, nicotinamide, and human basic fibroblast growth factor.
Preferably, in the induction medium, the concentration of the beta-cell cytokine is 3-5 mug/L, the concentration of the EGF is 120-140pmol/L, the concentration of the nicotinamide is 15-18mmol/L, and the concentration of the human basic fibroblast growth factor is 7-10ug/L.
Preferably, said inducing of differentiation of S3 comprises: inoculating the subculture mesenchymal stem cells into a 6-hole ultralow 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 10 5 cells/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 the 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 a raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, carrying out induced differentiation on the obtained continuous mesenchymal stem cells to differentiate into islet-like cells.
S1, the raw material tissue comprises human placenta tissue.
S1, the separation culture comprises the following steps: 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 45min.
Centrifuging at 1200rpm for 5min; the washing was performed 3 times using PBS.
The cells were inoculated into the culture medium, and then cultured under conditions of 37, 5% CO2 and 95% saturation humidity.
The above culturing, comprising: after inoculation, culturing for 3 days under the conditions of 37 and 5 percent of CO2 and 95 percent of saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80 to 90 percent, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell sediment, and suspending with the cell culture solution to obtain the mesenchymal stem cells.
S2, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
The trypsin-EDTA solution contained trypsin at a concentration of 0.25% by mass and EDTA at a concentration of 0.02% by mass.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 10ng/mL; the concentration of the fibroblast growth factor is 10ng/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 10 4 /cm 2 。
S3, the passage mesenchymal stem cells are P3 passage cells.
S3, the induction medium used for inducing differentiation is an alpha-MEM (alpha-MEM) culture medium which comprises beta-cell hormone, EGF, nicotinamide and human basic fibroblast growth factor.
In the induction culture medium, the concentration of beta-cytomin is 3mu g/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 7ug/L.
S3, 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 10 5 cells/well.
Example 2 islet cells induced in vitro differentiation of stem cells form islet-like structures:
s1, separating and culturing a raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, carrying out induced differentiation on the obtained continuous mesenchymal stem cells to differentiate into islet-like cells.
S1, the raw material tissue comprises fat tissue.
S1, the separation culture comprises the following steps: 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 60min.
Centrifuging at 1500rpm for 8min; the washing was performed 5 times with PBS.
The cells were inoculated into the culture medium, and then cultured under conditions of 37, 5% CO2 and 95% saturation humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 and 5 percent of CO2 and 95 percent of saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80 to 90 percent, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell sediment, and suspending with the cell culture solution to obtain the mesenchymal stem cells.
S2, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
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 15ng/mL; the concentration of the fibroblast growth factor is 15ng/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 10 4 /cm 2 。
S3, the passage mesenchymal stem cells are P3 passage cells.
S3, the induction medium used for inducing differentiation is an alpha-MEM (alpha-MEM) culture medium which comprises beta-cell hormone, 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 10ug/L.
S3, 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 10 5 cells/well.
Example 3 islet cells induced in vitro differentiation of stem cells form islet-like structures:
s1, separating and culturing a 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 comprises placenta tissue.
S1, the separation culture comprises the following steps: cleaning raw material tissues, shearing, digesting by using digestive enzyme, centrifuging to obtain cell precipitates, cleaning, and inoculating to a cell culture medium for culture.
The above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 50min.
Centrifuging at 1400rpm for 6min; the washing was performed 4 times with PBS.
The cells were inoculated into the culture medium, and then cultured under conditions of 37, 5% CO2 and 95% saturation humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 and 5 percent of CO2 and 95 percent of saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80 to 90 percent, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell sediment, and suspending with the cell culture solution to obtain the mesenchymal stem cells.
S2, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
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 12ng/mL; the concentration of the fibroblast growth factor is 12ng/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 10 4 /cm 2 。
S3, the passage mesenchymal stem cells are P3 passage cells.
S3, the induction medium used for inducing differentiation is an alpha-MEM (alpha-MEM) culture medium which comprises beta-cell hormone, EGF, nicotinamide and human basic fibroblast growth factor.
In the induction culture medium, the concentration of beta-cytotoxin is 4 mu g/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 9ug/L.
S3, inducing differentiation, including: 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 10 5 cells/well.
Example 4 islet cells induced in vitro stem cell differentiation form islet-like structures:
s1, separating and culturing a 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 comprises placenta tissue.
S1, the separation culture comprises the following steps: cleaning raw material tissues, shearing, digesting by using digestive enzyme, centrifuging to obtain cell precipitates, cleaning, and inoculating to a cell culture medium for culture.
The above digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 50min.
Centrifuging at 1400rpm for 6min; the washing was performed 4 times with PBS.
The cells were inoculated into the culture medium, and then cultured under conditions of 37, 5% CO2 and 95% saturation humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 and 5 percent of CO2 and 95 percent of saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80 to 90 percent, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell sediment, and suspending with the cell culture solution to obtain the mesenchymal stem cells.
S2, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
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 12ng/mL; the concentration of the fibroblast growth factor is 12ng/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 10 4 /cm 2 。
S3, the passage mesenchymal stem cells are P3 passage cells.
S3, the induction medium used for inducing differentiation is an alpha-MEM (alpha-MEM) culture medium which comprises beta-cell hormone, 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 9ug/L.
S3, 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 10 5 cells/well.
S3, performing induction culture, namely removing the culture medium before replacing the induction culture medium for the first time, digesting the cells with a trypsin-EDTA solution for 25min, washing the cells with a PBS (phosphate buffer solution) for 4 times, adding 3ml of the 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 a 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 comprises placenta tissue.
S1, the separation culture comprises the following steps: 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 50min.
Centrifuging at 1400rpm for 6min; the washing was performed 4 times with PBS.
Inoculating the above strain into cell culture medium, and culturing under 37 deg.C, 5% CO2, and 95% saturated humidity conditions.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 percent (C), 5 percent (C) CO2 and 95 percent saturated humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80-90 percent, removing the culture solution, washing cells, digesting with pancreatin, then resuspending and diluting, centrifuging, taking cell sediment, and resuspending with a cell culture solution to obtain the mesenchymal stem cells.
S2, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
The trypsin-EDTA solution contained trypsin at a concentration of 0.25% by mass and EDTA at a concentration of 0.02% by mass.
The subculture medium comprises: alpha-MEM medium, platelet derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 12ng/mL; the concentration of the fibroblast growth factor is 12ng/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 10 4 /cm 2 。
S3, the passage mesenchymal stem cells are P3 passage cells.
S3 the induction culture medium used 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 by mass concentration.
S3, 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 a raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
and S3, carrying out induced differentiation on the obtained continuous mesenchymal stem cells to differentiate into islet-like cells.
S1, the raw material tissue comprises placenta tissue.
S1, the separation culture comprises the following steps: 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 50min.
Centrifuging at 1400rpm for 6min; the washing was performed 4 times with PBS.
The cells were inoculated into the culture medium, and then cultured under conditions of 37, 5% CO2 and 95% saturation humidity.
The above culturing includes: after inoculation, culturing for 3 days under the conditions of 37 and 5 percent of CO2 and 95 percent of saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80 to 90 percent, removing the culture solution, cleaning cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell sediment, and suspending with the cell culture solution to obtain the mesenchymal stem cells.
S2, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, culturing under the conditions of 37 and 5 percent of CO2 and 95 percent of saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80-90 percent, and subculturing according to a ratio of 1.
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 12ng/mL; the concentration of the fibroblast growth factor is 12ng/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 10 4 /cm 2 。
S3, the passage mesenchymal stem cells are P3 passage cells.
S3 the induction culture medium used 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 by mass concentration.
S3, inducing differentiation, comprising: collecting third generation placenta mesenchymal stem cells, digesting with trypsin and EDTA at mass concentration of 0.25% and 0.02% 5 And 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, an 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 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 staining solution, washing with PBS for 2 times, observing cell staining conditions, and taking a picture.
As a result: all examples and comparative examples are red in color after dithizone staining, and are positive reactions, and a control group is 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 concentration as 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 aggregates (50-100 um) are picked from each sample and placed into a 1.5mL centrifuge tube, PBS is used for washing for 2 times, 1mL sugar-free DMEM is added for pre-culture for 4h, then 400uL DMEM containing 6.0mmol/L glucose and 18mmol/L glucose is used for sequential culture for 2h, supernatant is collected, and the secretion amount of insulin under stimulation of glucose with different concentrations in the supernatant is detected by an ELISA method.
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 response according to the stimulation degree of glucose in the external environment, and the method is high in induction efficiency and cytoplasm quantity, 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 (7)
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 a raw material tissue to obtain mesenchymal stem cells;
s2, subculturing the obtained mesenchymal stem cells;
s3, inducing and differentiating the obtained continuous mesenchymal stem cells into islet-like cells;
s3, an induction culture medium used for inducing differentiation is an alpha-MEM culture medium, wherein the alpha-MEM culture medium comprises beta-cytomin, EGF, nicotinamide and human basic fibroblast growth factor;
s3, in the induction culture medium, the concentration of beta-cytomin is 3-5 mu 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 7-10ug/L;
s3, the passage mesenchymal stem cells are P3 passage cells;
s3, performing induction culture, namely removing a culture medium before replacing the induction culture medium for the first time, digesting cells for 20-30min by using a trypsin-EDTA solution, washing the cells for 3-5 times by using a PBS (phosphate buffer solution), adding 3ml of induction culture medium into each hole, continuing suspension induction, replacing the induction culture medium every 3 days, and keeping the total induction culture time for 5-7 days;
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 10-15ng/mL; the concentration of the fibroblast growth factor is 10-15ng/mL; the concentration of the ascorbic acid is 25-50 mug/mL.
2. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 1, wherein: s1, the raw material tissue comprises one or two of placenta tissue or fat tissue; s1, the separation culture comprises the following steps: 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, the digestive enzyme comprises collagenase type I or collagenase type II, and the digestion time is 45-60min; centrifuging at 1200-1500rpm for 5-8min; washing with PBS for 3-5 times.
4. The method of inducing differentiation of stem cells into islet cells to form islet-like structures in vitro of claim 3, wherein: s1, culturing, comprising: after inoculation at 37 ℃ with 5% CO 2 And culturing for 3 days under the condition of 95% saturation humidity, replacing half of the culture solution, culturing for 3 days, replacing the whole culture solution, continuously culturing until the cell fusion degree reaches 80% -90%, removing the culture solution, cleaning the cells, digesting with pancreatin, then suspending and diluting, centrifuging, taking cell precipitates, and suspending with the 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, subculturing comprises the following steps: adding the obtained mesenchymal stem cells into a subculture medium, and adding 5% CO at 37 deg.C 2 And culturing under the condition of 95% saturated humidity, digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80% -90%, and performing subculture according to the ratio of 1.
6. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 1, wherein: s2, subculturing, and inoculating on a plate with the diameter of 10cm, wherein the subculturing density is (1-2) multiplied by 10 4 /cm 2 。
7. The method of inducing islet cells of stem cell differentiation to form islet-like structures in vitro according to claim 1, wherein: s3, in the induction culture medium, the concentration of beta-cell cytokine is 3-5 mu 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 7-10ug/L; s3, inducing differentiation, including: 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.
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