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

A technology of pluripotent stem cells and directed differentiation, applied in the field of induced pluripotent stem cells directed differentiation to prepare the culture medium of islet β cells, which can solve the problems of small number of islet β cells and imperfect functions

Pending Publication Date: 2021-06-15
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 solution for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells
  • Culture solution for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells
  • Culture solution for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells

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Experimental program
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Effect test

Embodiment 1

[0033] An inducible multi-capable cell directional differentiation of the present embodiment, the culture solution, culture fluid comprising AdvancedDMEM / F12 medium, glucose, sodium bicarbonate, human serum albumin (HSA), N2, B27, glutamine , Vitamin C, JNK inhibitor, Hedgehog passage antagonist, EGF signal factor, FGF signal factor, TGF-β inhibitor, Wnt signaling pathway activator, NOTCH signaling path inhibitor, small molecule compound, and trace elements, the culture solution Different ratios in different stages of cell culture. Among them, AdvancedDMEM / F12 medium serves as a base medium, which adds nutrient components including glucose (0.4-1 g / l), sodium bicarbonate (1.23 g / l), HSA (1 ~ 5g / L), N2 (1 ×), B27 (1 ×), glutamine (1 ×), vitamin C (0.1 to 0.5 mm).

[0034] The JNK inhibitor is CC-930, the Hedgehog passage antagonist is N-acetyl cysteine ​​or cycloamine, and the EGF signal factor is EGF, and the FGF signal factor is FGF10, and the TGF-β super family factor ...

Embodiment 2

[0061] The content of the present embodiment is mainly formulated by islet beta cell differentiation culture fluid, as follows:

[0062] Islet β cell differentiation culture liquid is divided into four stages, stage (DE-induced medium composed of culture fluid A), stage 2 (PP inducing medium composed of culture fluid B), stage three (Composed of culture C) induced Medium and stage 4 (islet beta cell inducing medium of culture fluid D), the culture liquid components are composed of basal culture components (Basal Medium) and add components, components and ratios are as follows 1 ~ 4:

[0063] Table 1 DE induced by the composition and proportion of the medium

[0064]

[0065]

[0066] Table 2 ingredients and ratios of PP induced medium

[0067]

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

[0069] Table 3 EN induced by the composition and proportion of the medium

[0070]

[0071]

[0072] Remarks: The addition of AT7519...

Embodiment 3

[0078] The main content of this embodiment is to cultivate induced multi-functional stem cells using 3D suspension culture, as follows:

[0079] First, the required reagents

[0080] The culture solution used in induced multifunctional stem cells (IPSCs) is MTESR-1 fully medium, and the reagent used by the amplification of the passage is EDTA, cellular lubrication, and balance used by DPBS, promoting cell survival. The inhibitor is Y27632.

[0081] Second, the training process

[0082] After the wall of 10 mm-Dish is discarded into the medium and the 4ML DPBs was placed 3 to 4 minutes from 37 ° C for 37 ° C, and the digestive enzyme was discarded after the cells were discarded. The concentration of 10 μm Y7632 mTESR-1 fully medium and cells were blown into cell small pieces, and each cell small piece was gathered from 10 to 30 cells.

[0083] The separated cell small pieces were transferred to an ultra-low adsorbed 6-well plate, cultured at 37 ° C, 5% oxygen conditions, cultured 2...

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Abstract

The invention relates to a culture solution for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells, the culture solution comprises an AdvancedDMEM / F12 culture medium, glucose, sodium bicarbonate, human serum albumin (HSA), N2, B27, glutamine, vitamin C, a JNK inhibitor, a hedgehog pathway antagonist, EGF signal factors, FGF signal factors, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecule compound and trace elements, the ratios of the culture solution in different stages of cell culture are different, all the components play roles directionally in stages, and the pluripotent stem cells can be efficiently induced to be directionally differentiated into the pancreatic beta cells.

Description

Technical field [0001] The present invention belongs to the field of cellular engineering, in particular, a culture solution induces multidocondected cell directional differentiation prepared byouting beta cells. Background technique [0002] Diabetesmellitus, DM is a metabolic disorder disease with blood glucose is clinical characteristics, and its main pathogenesis is reduced by insulin secretion and its utilization obstacles. At present, diabetes have become a third largest threat to human health and affect people's quality of life. The latest report of the World Health Organization (WHO) shows that there have been 422 million people in the world with diabetes, while Chinese diabetic patients have more than 100 million people, ranking the world. [0003] There are two types of diabetes: a type of diabetes (T1D), accounting for 5 to 10% of the number of diseases, is an autoimmune disease caused by islet beta cells; secondary diabetes (T2D), accounting for the number of diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0676C12N2506/45C12N2500/34C12N2500/12C12N2501/998C12N2500/32C12N2500/38C12N2501/405C12N2501/11C12N2501/119C12N2501/15C12N2501/415C12N2501/42C12N2501/41C12N2501/16C12N2501/117C12N2501/727C12N2501/395C12N2501/50Y02A50/30
Inventor 高歌周安宇
Owner SHANGHAI AISAER BIOTECH CO LTD
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