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Culture medium for amplifying human mesenchymal stem cell and amplification method of culture medium

A technology of high-quality stem cells and medium, applied in the field of stem cell culture, to achieve the effects of reducing expansion time, avoiding differentiation, and reducing costs

Inactive Publication Date: 2017-09-01
刘力伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to realize the effective clinical application of this method, the effective large-scale culture of the human mesenchymal stem cells and the maintenance of their stemness have become the main problems.

Method used

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  • Culture medium for amplifying human mesenchymal stem cell and amplification method of culture medium
  • Culture medium for amplifying human mesenchymal stem cell and amplification method of culture medium
  • Culture medium for amplifying human mesenchymal stem cell and amplification method of culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Isolation, purification and culture of human bone marrow-derived mesenchymal stem cells

[0055] 1. Materials and reagents

[0056] Lymphocyte separation medium (Ficoll) (Tianjin Haoyang Biotechnology Co., Ltd.); culture medium DMEM (Hyclone Company), human AB serum (central blood station); cell counting kit (CCK-8) (Japan Dojindo Company); death kit (Dojindo Japan); epidermal growth factor (EGF), platelet-derived growth factor (PDGF), dexamethasone (DEX), penicillin streptomycin (Pen / strep), insulin iron selenide transfer protein (ITS), Human serum albumin (HSA) was produced by Peprotech; fluorescently labeled mouse anti-human PE-CD29, FITC-CD34, PE-CD44, FITC-CD45 antibodies (eBioscience, USA); FC500 flow cytometer and corresponding software ( American Beckman Coulter Company).

[0057] 2. Culture medium components

[0058] 1) Stem cell culture medium

[0059] Calculated by 1 liter, the dosage of each component is as follows:

[0060] MCDB 50ml, insulin transferr...

Embodiment 2

[0069] Isolation and culture of human umbilical cord human mesenchymal stem cells

[0070] 1. Materials and reagents

[0071] Same as Example 1

[0072] 2. Culture medium components

[0073] 1) Stem cell culture medium

[0074] Calculated by 1 liter, the dosage of each component is as follows:

[0075] MCDB 50ml, insulin transferrin (ITS) 200μl, human serum albumin (HSA) 60μl, penicillin streptomycin (Pen / strep) 1ml, dexamethasone (DEX) 10μl, add DMEM medium to 1 liter.

[0076] 2) Expansion medium

[0077] Calculated by 1 liter, the dosage of each component is as follows:

[0078] MCDB 50ml, insulin transferrin (ITS) 200μl, human serum albumin (HSA) 60μl, penicillin streptomycin (Pen / strep) 1ml, dexamethasone (DEX) 10μl, linoleic acid (LA) 6ml, human Source AB type serum 5ml, platelet-derived growth factor (PDGF) 50μl, epidermal growth factor (EGF) 60μl, add DMEM medium to 1 liter.

[0079] 3. Culture method (adhesion method)

[0080] Including the following steps:

...

Embodiment 3

[0089] Isolation and culture of human umbilical cord human mesenchymal stem cells

[0090] 1. Materials and reagents

[0091] Same as Example 1

[0092] 2. Culture medium components

[0093] 1) Stem cell culture medium

[0094] Calculated by 1 liter, the dosage of each component is as follows:

[0095] MCDB 50ml, insulin transferrin (ITS) 200μl, human serum albumin (HSA) 60μl, penicillin streptomycin (Pen / strep) 1ml, dexamethasone (DEX) 10μl, add DMEM medium to 1 liter.

[0096] 2) Expansion medium

[0097] Calculated by 1 liter, the dosage of each component is as follows:

[0098] MCDB 50ml, insulin transferrin (ITS) 200μl, human serum albumin (HSA) 60μl, penicillin streptomycin (Pen / strep) 1ml, dexamethasone (DEX) 10μl, linoleic acid (LA) 6ml, human Source AB type serum 5ml, platelet-derived growth factor (PDGF) 50μl, epidermal growth factor (EGF) 60μl, add DMEM medium to 1 liter.

[0099] 3. Culture method (enzyme digestion method)

[0100] Including the following s...

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Abstract

The invention discloses a serum-free medium for expanding human mesenchymal stem cells, which is based on the medium for culturing stem cells, adding linoleic acid, human AB type serum, and platelet-derived growth factors. The invention also discloses a culture method for human mesenchymal stem cells. The serum-free medium of the present invention uses multiple cytokines in combination reasonably, which not only improves the purification effect of mesenchymal stem cells, but also shows that the morphology of human mesenchymal stem cells is still uniform even after 30 passages, thus ensuring The safety of human mesenchymal stem cells improves the expansion speed of cells, reduces the expansion time and reduces the cost.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a medium for expanding human mesenchymal stem cells, and also relates to a method for expanding human mesenchymal stem cells using the medium. Background technique [0002] Cell therapy has become the most active part in the field of biological therapy, and standardized cell therapy research has become an important part of modern medicine. Currently, the cell types commonly used in cell therapy in my country are adult-derived cells, such as immune cells, bone marrow stem cells, hematopoietic or mesenchymal stem cells derived from umbilical cord blood and umbilical cord. [0003] Mesenchymal stem cells (MSCs) have a wide range of sources, and are relatively easy to isolate, culture, expand and purify. They have low immunogenicity and no restrictions on moral and ethical issues, so they have huge applications in immunotherapy and gene therapy. prospect. Such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/30C12N2501/11C12N2501/135C12N2501/998
Inventor 刘力伟刘波
Owner 刘力伟
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