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Stem cell amplifying culture medium and stem cell culture method

A technology for stem cells and culture medium, applied in the fields of bioengineering and biomedicine, can solve the problems of inconsistent amount of use, low stem cell expansion ratio, and low purity of hematopoietic stem cells, and achieves an improved expression rate and obvious cell expansion effect. Effect

Inactive Publication Date: 2019-12-31
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used cytokines in research include stem cell factor, monocyte chemoattractant, and interleukin-1β, but their amplification factor for stem cells is low, and the amount used is not uniform, and the purity of hematopoietic stem cells is not high

Method used

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  • Stem cell amplifying culture medium and stem cell culture method
  • Stem cell amplifying culture medium and stem cell culture method
  • Stem cell amplifying culture medium and stem cell culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0068] 1. Preparation of culture medium for expanding stem cells

[0069] The cytokines stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, and StemRegenin 1 were added to the serum-free StemSpan SFEMII basal medium, and the final concentrations of the above cytokines were respectively 90ng / mL , 20ng / mL, 100ng / mL, 20ng / mL, and 1μM culture medium for expanding stem cells, and put them at 4°C for later use.

[0070] 2. Cell culture

[0071] Seed CD34+ hematopoietic stem cells in a 35mm cell culture dish at a seeding density of 1×10 5 cells / mL, add 2 mL of the above-mentioned expanded stem cell culture medium, at 37 °C, 5% CO 2 Cultivate under culture conditions, and add 500 μL of fresh culture medium for expanding stem cells every 2 days according to the state of cell culture.

Embodiment 2

[0073] 1. Preparation of culture medium for expanding stem cells

[0074] The cytokines stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, and StemRegenin 1 were added to the serum-free StemSpan SFEMII basal medium to obtain a final concentration of 100 ng / mL containing the above cytokines, respectively. , 20ng / mL, 90ng / mL, 20ng / mL, and 1μM culture medium for expanding stem cells, and put them at 4°C for later use.

[0075] 2. Cell culture

[0076] Seed CD34+ hematopoietic stem cells in a 35mm cell culture dish at a seeding density of 1×10 5 cells / mL, add 2 mL of the above-mentioned expanded stem cell culture medium, at 37 °C, 5% CO 2 Cultivate under culture conditions, and add 500 μL of fresh culture medium for expanding stem cells every 2 days according to the state of cell culture.

Embodiment 3

[0078] 1. Preparation of culture medium for expanding stem cells

[0079] The cytokines stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, and StemRegenin 1 were added to the serum-free StemSpan SFEMII basal medium to obtain a final concentration of 100 ng / mL containing the above cytokines, respectively. , 30ng / mL, 100ng / mL, 20ng / mL, and 1μM medium for expanding stem cells, and put them at 4°C for later use.

[0080] 2. Cell culture

[0081] Seed CD34+ hematopoietic stem cells in a 35mm cell culture dish at a seeding density of 1×10 5 cells / mL, add 2 mL of the above-mentioned expanded stem cell culture medium, at 37 °C, 5% CO 2 Cultivate under culture conditions, and add 500 μL of fresh culture medium for expanding stem cells every 2 days according to the state of cell culture.

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Abstract

The invention relates to a stem cell amplifying culture medium and a stem cell culture method. The ingredients of the stem cell amplifying culture medium comprise a basic culture medium, a stem cell factor, thrombopoietin, an FMS like tyrosine kinase 3 ligand and interleukin-6, wherein the final concentration of the stem cell factor is 90ng / mL-110ng / mL, the final concentration of the thrombopoietin is 10ng / mL-30ng / mL, the final concentration of the FMS like tyrosine kinase 3 ligand is 90ng / mL-110ng / mL, and the final concentration of the interleukin-6 is 10ng / mL-30ng / mL. When the stem cell amplifying culture medium is used for culturing cord blood hematopoietic stem cells, obvious cell amplifying effects can be presented, CD34+ on cell surfaces is also notably increased, during cell amplification, the cell state is stable, the multi-directional differentiation capacity is maintained, and the culture medium is an economic efficient culture system.

Description

technical field [0001] The invention belongs to the technical fields of bioengineering and biomedicine, and in particular relates to a stem cell expansion medium and a method for culturing stem cells. Background technique [0002] Hematopoietic stem cells are a group of cells with self-renewal and multi-directional differentiation potential. While maintaining the relative stability of stem cells, they can proliferate and differentiate into various lines of cells in the hematopoietic system. Hematopoietic stem cell transplantation is currently the most effective way to treat these diseases. Umbilical cord blood is another important source of hematopoietic stem cells besides bone marrow and peripheral blood stem cells. The chance of virus contamination is small, and the incidence of immune rejection after transplantation is lower than that of bone marrow transplantation from adult donors. The HLA-matched stem cells needed for transplantation can be provided at any time by est...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/125C12N2501/145C12N2501/2306C12N2501/26C12N2501/50
Inventor 孙筱放何丽娜程怡刘能青谢英俊
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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