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
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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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