Method for forming pancreatic islet like structure by external evoked stem cell differentiation pancreatic islet cell

A stem cell differentiation and islet cell technology, applied in the field of inducing the formation of islet-like structures, can solve the problems of low islet-like structure formation rate, incompleteness, and no use of extracellular matrix, and achieves high islet-like structure formation rate, simple operation, uniform size effect

Inactive Publication Date: 2008-03-12
CHINA JAPAN FRIENDSHIP HOSPITAL
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Problems solved by technology

[0005] In previous studies, some researchers used the method of culturing in Matrigel gel to promote the aggregation of insulin-secreting cells, but this method has several shortcomings: first, the formation rate of islet-like structures is low, and the size of the formed islet-like structures Different; secondly, the envelope of the islet-like structure formed is inconspicuous or incomplete
However, at present, there is no report on the use of extracellular matrix in the method of inducing insulin-secreting cells to form islet-like structures in vitro, and there is no report on the induction of complete envelopes outside the islet-like structures.

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  • Method for forming pancreatic islet like structure by external evoked stem cell differentiation pancreatic islet cell

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Embodiment 1

[0024] A method for inducing stem cell-differentiated islet cells to form islet-like structures in vitro, comprising the following steps: expanding the second-generation human embryonic pancreatic stem cells to T75 flasks and digesting them with 0.2% trypsin, counting, and then dividing the human embryos Pancreatic stem cells were inoculated with 120,000 cells in 3ml medium and cultured in a sterile culture tube treated with silane; after the culture tube was treated with silane, the cells would not be able to attach to the wall of the culture tube and would be suspended in the culture medium. Suspension culture medium uses M199 medium containing 20% ​​heat-inactivated fetal bovine serum, and contains 4.5g / L of glucose, 1mmol / L of Ca 2+ , 0.01mmol / L adenosine triphosphate, 2mmol / L glutamine, 100U / ml penicillin and 100U / ml streptomycin; in 95% air, 5% CO 2 , In a humid environment at 37°C, the embryonic pancreatic stem cells were cultured in this suspension culture condition fo...

Embodiment 2

[0026] A method for inducing stem cell-differentiated islet cells to form islet-like structures in vitro, comprising the following steps: expanding the third-generation human embryonic pancreatic stem cells to T75 flasks after they are substantially confluent, digesting with 0.2% trypsin, counting, and then dividing the human embryos Pancreatic stem cells were inoculated with 1 million cells in 3ml medium and cultured in a sterile culture tube treated with silane; after the culture tube was treated with silane, the cells would not be able to attach to the wall of the culture tube and would be suspended in the culture medium. Suspension culture medium uses M199 medium containing 20% ​​heat-inactivated fetal bovine serum, and contains 4.5g / L of glucose, 2mmol / L of Ca 2+ , 2mmol / L adenosine triphosphate, 2mmol / L glutamine, 100U / ml penicillin and 100U / ml streptomycin; in 95% air, 5% CO 2 1. In a humid environment at 37°C, the embryonic pancreatic stem cells were cultured in this s...

Embodiment 3

[0028] After the third-generation human embryonic pancreatic stem cells were amplified to the confluence of the T75 flask, they were digested with 0.2% trypsin and counted, and then the human embryonic pancreatic stem cells were inoculated with 3ml of medium and cultured at a density of 500,000 cells in a silane-treated Bacteria culture tube; after the culture tube is treated with silane, the cells will not be able to attach to the wall of the culture tube and will be suspended in the medium. The medium for this suspension culture uses M199 medium containing 20% ​​heat-inactivated fetal bovine serum. And contain 4.5g / L of glucose, 1.5mmol / L of Ca 2+ , 1mmol / L adenosine triphosphate, 2mmol / L glutamine, 100U / ml penicillin and 100U / ml streptomycin; in 95% air, 5% CO 2 In a humid environment at 37°C, the embryonic pancreatic stem cells were cultured in this suspension culture condition for 30 hours to form islet-like structures.

[0029] The human embryonic pancreatic stem cells ...

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Abstract

A method for forming langerhans' islet like structure by inlet cells differentiated from dry cells by extracorporal induction. The inlet cells differentiated from dry cells are placed in a culture dish that has been treated by silane, the cells are induced to form langerhans' islet like structure by suspension culturing way with a cell culture medium containing 20% fetal bovine serum, out-cell substrate, calcium ion of certain concentration and ATP. The invention has the following advantages: 1. the method is simple, the induction time is short, the forming rate of langerhans' islet like structure is high; 2. the formed langerhans' islet like structure is very much similar with natural langerhans' islet, and can be steadily positioned in sinus hepaticus by transplanting through a portal; 3. the formed langerhans' islet like structure has an obvious diolame, and substrate component very similar to natural langerhans' islet. The langerhans' islet like structure formed by the method can meet the quality for transplanting langerhans' islet.

Description

technical field [0001] The invention relates to a technical method for inducing the formation of islet-like structures, in particular to a method for inducing stem cell-differentiated islet cells to form islet-like structures in vitro. Background technique [0002] Diabetes is one of the diseases that seriously endanger human health. The current common method for treating diabetes is to inject insulin. Insulin therapy can improve the patient's glucose metabolism disorder to a certain extent, but it cannot effectively prevent or reverse the microangiopathy and its complications caused by diabetes. Therefore, finding new means of diabetes treatment has always been a frontier topic of medical research. Pancreatic islet transplantation is currently an effective method for the treatment of insulin-dependent diabetes, and experimental data show that islet transplantation can not only correct the disorder of glucose metabolism, but also prevent or reverse the microangiopathy of di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/071
Inventor 蔡寒青娄晋宁许世清门秀丽张文健
Owner CHINA JAPAN FRIENDSHIP HOSPITAL
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