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Myocardial cell induction and growth medium

A technology of growth medium and cardiomyocytes, applied in the direction of cell culture active agent, culture process, tissue culture, etc., can solve the problems of residual additive components, excessive additives, unstable differentiation of cardiomyocytes, etc.

Pending Publication Date: 2020-08-07
上海中医大生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing cardiomyocyte differentiation and culture techniques often add fetal bovine serum, bovine serum albumin or B27 additives (B-27 TM Supplement, minus insulin (ThermoFisher, A1895601)) is achieved, and these additives are different between different batches, resulting in the instability of cardiomyocyte differentiation
In

Method used

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  • Myocardial cell induction and growth medium
  • Myocardial cell induction and growth medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] Example 1, Cell pretreatment

[0176] Pretreat the petri dishes with substrates such as Laminin (10ng / ml) and Fibronectin (10ng / ml) and place them in a cell incubator (temperature 37 degrees, humidity 95%, CO2 concentration 5%) for 2 hours. Press DYR-0-100 cell DYR0100 cell (Shanghai Cell Bank of Chinese Academy of Sciences) according to 0.2E+5~0.6E+5 / cm 2 The density is planted on the pretreated petri dish. It usually grows to 3-4 days, the cell density reaches 3.5E+5~4.0E+5 / cm 2 At that time, the method of this patent can be used for cardiomyocyte production.

Embodiment 2

[0177] Example 2. Induction of cell differentiation

[0178] After the cells reach the density, wash the cells twice with PBS to remove residual medium and dead cells. From the start of differentiation (Day 0, D0) to the seventh day (Day 7, D7), use differentiation medium (IS) to culture the cells ; And, in D0-D2, add 3-12uM CHIR-99021; in D2-D4, add 2.5-10uM IWR1. Generally, some beating cardiomyocytes can be seen on D7.

[0179] The differentiation medium components are: basal medium RPMI 1640 Medium (ThermoFisher), plus 0.25g / L albumin (Sigma) and 0.1g / L ascorbic acid (Sigma).

Embodiment 3

[0180] Example 3. Cardiomyocyte growth

[0181] After D7, the cells were transferred to growth medium (LCS), and LCS was used for long-term culture. Generally, cardiomyocytes can be seen beating in sheets on D10-D14.

[0182] The specific components of the growth medium are: basic medium DMEM / F12 (ThermoFisher), supplemented with 0.5g / L transferrin (Sigma), 0.1g / L ascorbic acid (Sigma), 0.5mg / L sodium selenite (Sigma) , 1% Lipid Concentrate (Chemically Defined Lipid Concentrate, ThermoFisher), 0.5g / L insulin (Sigma).

[0183] The yield of cardiomyocytes cultured in different containers is shown in Table 1.

[0184] Table 1

[0185]

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PUM

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Abstract

The invention relates to the technical field of biology, relates to a myocardial cell induction and growth culture medium, and discloses a serum-free production method for stably and economically producing myocardial cells. A whole set of system is divided into an induced differentiation system (IS) and a long-term culture system (LCS). The induced differentiation system comprises amino acid, vitamin, inorganic salt, glucose, glutathione, HEPES, phenol red, recombinant human albumin and ascorbic acid; and the long-term culture system comprises amino acid, vitamin, inorganic salt, glucose, glutathione, HEPES, phenol red, ascorbic acid, transferrin, insulin, sodium selenite and lipid concentrate. The whole system avoids the components of a conventional unstable mixture additive, improves thestability of myocardial cell differentiation and culture, is low in additive price, and is suitable for large-scale myocardial cell production.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a culture medium for inducing and growing cardiomyocytes, and a production method and application thereof. Background technique [0002] Human cardiomyocytes have broad application prospects in drug evaluation and cell therapy. Due to the extremely weak proliferation ability of cardiomyocytes and the difficulty in obtaining human heart tissue, the differentiation of human pluripotent stem cells to obtain cardiomyocytes has become the main way to obtain human cardiomyocytes. [0003] The study of cardiomyocyte differentiation has been very in-depth. By manipulating the WNT signaling pathway, human pluripotent stem cells can be successfully induced to differentiate into cardiomyocytes, but the differentiation stability of cardiomyocytes varies greatly in different systems. Existing cardiomyocyte differentiation and culture technologies often add fetal bovine serum, bovine serum albumin or B2...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2500/32C12N2500/38C12N2500/05C12N2501/998C12N2500/34
Inventor 王志敏虞修简黄薇
Owner 上海中医大生物科技有限公司
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