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Culture method for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells

A technology for pluripotent stem cells and directed differentiation, which is applied in the field of culturing the directional differentiation of induced pluripotent stem cells to prepare islet beta cells, and can solve the problems of imperfect function and low number of pancreatic islet beta cells.

Active Publication Date: 2021-06-18
SHANGHAI AISAER BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the existing problem that the number of pancreatic beta cells produced by the differentiation of pancreatic beta cells is small and the function is not perfect

Method used

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  • Culture method for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells
  • Culture method for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells
  • Culture method for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells

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Embodiment 1

[0038] A culture method for preparing pancreatic islet β cells through directed differentiation of induced pluripotent stem cells in this embodiment, the specific steps are as follows:

[0039] S100, preparing induced pluripotent cell spheres;

[0040] S200, primary differentiation, using culture medium A to perform directional differentiation culture on the induced pluripotent cell sphere to obtain definitive endoderm cells;

[0041] S300, secondary differentiation, using culture medium B to induce differentiation of definitive endoderm cells to obtain pancreatic precursor cells;

[0042] S400, third differentiation, using culture medium C to induce differentiation of pancreatic precursor cells to obtain pancreatic endocrine progenitor cells;

[0043] S500, four times of differentiation, using culture medium D to induce differentiation of pancreatic endocrine progenitor cells to obtain desired pancreatic islet β cells.

[0044] The preparation of induced pluripotent cell sp...

Embodiment 2

[0061] The content of this embodiment is mainly the preparation of islet β cell differentiation culture medium, specifically as follows:

[0062] The islet β cell differentiation culture medium is divided into four stages, stage 1 (DE induction medium composed of culture medium A), stage 2 (PP induction medium composed of culture medium B), and stage 3 (EN induction medium composed of culture medium C). culture medium) and stage four (islet β cell induction medium composed of culture medium D), the components of the culture medium are composed of basal medium (basal medium) and supplementary components (supplement), and the components and ratios are shown in Table 1- 4:

[0063] Table 1 Components and proportions of DE induction medium

[0064]

[0065]

[0066] Table 2 Components and proportions of PP induction medium

[0067]

[0068] Remarks: The addition of A83-01 can significantly improve the cell differentiation efficiency.

[0069] Table 3 Components and pro...

Embodiment 3

[0077] The main content of this example is to cultivate induced pluripotent stem cells by means of 3D suspension culture, as follows:

[0078] 1. Required reagents

[0079] The culture medium used for the culture of induced pluripotent stem cells (iPSCs) is mTeSR-1 complete medium, the digestive enzyme used for expansion and passage is EDTA, the reagent used for cell rinsing and balancing is DPBS, and the reagent used for promoting cell survival The inhibitor is Y27632.

[0080] 2. Training process

[0081] For iPSCs adherently cultured in a 100mm-dish, discard the culture medium and rinse with 2mL DPBS, add 4mL LEDTA digestive enzyme and place at 37°C for 3-4min, discard the digestive enzyme after contact and separation between cells, add 12mL containing final The mTeSR-1 complete medium with a concentration of 10 μM Y7632 was used to separate the cells into small cell pieces by pipetting, and each cell small piece was aggregated by 10-30 cells.

[0082] The isolated cell ...

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Abstract

The invention discloses a culture method for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells. The culture method comprises the following specific steps: S100, preparing induced multifunctional cell spheres; S200, performing primary differentiation, namely performing directional differentiation culture on the induced multifunctional cell spheres by using a culture solution A to obtain fixed endoderm cells; S300, performing secondary differentiation, namely performing induced differentiation on the fixed endoderm cells by using a culture solution B to obtain pancreatic precursor cells; S400, performing third differentiation, namely performing induced differentiation on the pancreatic precursor cells by using a culture solution C to obtain pancreatic endocrine progenitor cells; and S500, performing fourth differentiation, namely performing induced differentiation on the pancreatic endocrine progenitor cells by using a culture solution D to obtain the required pancreatic beta cells. The culture solutions have different proportions in different stages of cell culture, each component directionally plays a role in stages, and the induced multifunctional stem cells can be directionally differentiated and cultured in a short time by the method to obtain the pancreatic beta cells.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and specifically relates to a culture method for inducing pluripotent stem cells to differentiate and prepare pancreatic beta cells. Background technique [0002] Diabetes Mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and its main pathogenesis is the decreased secretion and utilization of insulin. At present, diabetes has become the third major disease that threatens human health and affects people's quality of life after tumors and cardiovascular and cerebrovascular diseases. According to the latest report of the World Health Organization (WHO) in 2016, 422 million people worldwide suffer from diabetes, and the number of diabetic patients in China exceeds 100 million, ranking first in the world. [0003] There are two types of diabetes: type 1 diabetes (T1D), which accounts for 5-10% of the number of diseases, is an autoimmune disease caused by the selective des...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/10
CPCC12N5/0678C12N5/0676C12N2506/45C12N2510/00C12N2501/727C12N2501/15C12N2501/415C12N2501/40C12N2501/11C12N2501/385C12N2501/117C12N2501/065C12N2501/42C12N2501/115C12N2501/395C12N2501/12
Inventor 高歌周安宇
Owner SHANGHAI AISAER BIOTECH CO LTD
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