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Method for differentiating and inducing stem cells into hepatocytes

A stem cell differentiation and hepatocyte technology, applied in the field of biomedicine, can solve the problems of weak function of hepatocytes, lack of cells in stem cells, difficulty in harvesting a large number of cells, etc., and achieve the effect of low cost and stable culture environment

Pending Publication Date: 2021-08-03
弗元(上海)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, stem cells in the monolayer adherent state lack the three-dimensional and extensive connections between cells, and the function of the differentiated hepatocytes is weak, and the differentiation is limited to the area of ​​the culture plate, so it is difficult to harvest a large number of cells, which is not suitable for the large-scale preparation of hepatocytes.

Method used

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  • Method for differentiating and inducing stem cells into hepatocytes
  • Method for differentiating and inducing stem cells into hepatocytes
  • Method for differentiating and inducing stem cells into hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Human embryonic stem cell (HESCS) recovery

[0070] MATRIGEL (original solution) was taken in advance, placed in ice, then diluted 100 times with pre-cooled DMEM / F12 medium, add 1 mL to join the six-well plate, gently shake well, full hole The bottom of the plate. The six-hole plate is placed in a 37 ° C incubator (about 30 minutes), spare. MTESR1 human embryonic stem cell culture medium was taken from 4 ° C refrigerator, and the room temperature was placed for about 10 minutes, and 75% ethanol was transferred into the ultra-clean station. The H9 cells are removed from the liquid nitrogen freeze box, rapidly transferred into a 37 ° C water bath, gently shake, melted to the frozen contained small ice 即 即, 75% ethanol is transferred into the ultra-clean station. Carefully transfer cells to 15 ml of centrifuge tube, and approvressed 5 ml of MTESR1 medium from slow until 5 minutes, 1000 rock, centrifuged for 5 minutes, carefully discard the supernatant, adding the M...

Embodiment 2

[0071] Example 2, cultivation, passage, and freezing of human embryonic stem cells

[0072] The cell state should be observed before replacing fresh mtessr1 medium daily, if a large amount of dead cells are found to float the medium (poor in the cell state of the rigidity), the medium is discarded, and the preheating DMEM / F12 is easily washed once, discard DMEM / F12, add fresh MTESR1 medium, which is placed in a 37 ° C incubator to continue culture. The cells grow from about 3 to 5 days to 80% conventional, and the human embryonic stem cell clone is obvious, and the edge is smooth.

[0073] The cell passage, according to the experimental need to be used in advance by Matrigel package by the culture plate (1: 5 to 1:10 ratio), with the pre-package by Matrigel, shake well in a 37 ° C incubation. The cells were washed twice with DMEM / F12, and 1 mLACCutase digestive solution was added per well, and was placed in a 37 ° C incubator for 3 to 5 minutes. Invert the microscope every 2...

Embodiment 3

[0075] Example III, human embryonic stem cell differentiation into a liver spheroid (Liver spheroid)

[0076] In this example, the type endoderm-induced medium is purchased from Fuyuan (Shanghai) Biotechnology Co., Ltd., the item number RC200123.

[0077] (1) Try cells were cultured in a stereotype induced endoderm induced in Activin A and CHIR99021.

[0078] Divided human embryonic stem cells grown to about 90% of the converged human embryonic stem cells were induced. Human embryonic stem cells grown to approximately 90% were placed twice with preheating RPMI1640 base medium, and the added endoderm-induced medium containing 100 ng / ml Activin A and 1 to 2 μm CHIR99021 was added, and liquidated for a total of 3 days. On day 4, the cells were laminated twice with AdvancedDMEM / F12 medium, and the amount of intuntase digestive fluid digestion was added 3 to 5 minutes, collecting cells, 1000 rpm for 5 minutes, abandoned, collecting cells.

[0079] (2) The cells obtained in the Adva...

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Abstract

The invention discloses a method for differentiating and inducing stem cells into hepatocytes. In the application, the method comprises the following steps: step (1), culturing stem cells in a first culture medium added with Activin A and a Wnt signaling pathway agonist, and collecting the cells; and (2) culturing the cells obtained in the step (1) in a second culture medium added with BMP2 and FGF4, and collecting the cells, and (3) culturing the cells obtained in the step (2) in a third culture medium added with HGF and KGF, and collecting the cells, and (4) culturing the cells obtained in the step (3) in a fourth culture medium added with Vc, EGF and insulin, and collecting the cells to obtain the hepatocytes. According to the method for differentiating and inducing the stem cells into the hepatocytes, provided by the invention, the stem cells can be successfully induced to be differentiated into the hepatocytes by adding a combination of specific cell factors and small molecular compounds.

Description

Technical field [0001] Embodiments of the present invention relate to biopharmaceutical areas, and more particularly to stem cell differentiation induction as hepatocytes. Background technique [0002] Getting hepatocytes is the key to constructing artificial liver, pre-puzzle drug metabolism and hepatotoxic assessment, and liver regenerative medical seed cell size production, however, the human source of hepatocytes is limited by the number of donors, weak in vitro amplification capacity and easy Defects such as defective loss function are difficult to meet the increasing demand for hepatocytes. [0003] Multi-capable stem cells have an infinite proliferation and differentiation of human hepatocytes. In the prior art, multi-capable cell differentiation induces hepatocytes, mainly for single-layer postgraduate growth. However, the stem cells in single-layer adhesive state lack of cellular linkage, and the differentiated hepatocyte function is weak, and the differentiation is limi...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2506/02C12N2506/45C12N2501/415C12N2501/155C12N2501/119C12N2501/117C12N2501/11C12N2501/33C12N2500/38
Inventor 董永聘陈勇冯荣桑伟建
Owner 弗元(上海)生物科技有限公司
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