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Molecular assembly and application for adherent culture and proliferation of mesenchymal stem cells (MSC)

A technique for stem cell and adherent culture, applied in cell culture active agent, culture process, tissue culture and other directions, which can solve the problems affecting MSC growth, complex cell culture process, and changes in MSC adhesion.

Inactive Publication Date: 2018-07-17
北京三有利生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The addition of adhesion molecules to serum-free media will undoubtedly increase the cost of the media, while providing the adhesion matrix alone will complicate the cell culture process
In addition, due to the selection of culture flasks / dishes from different manufacturers or suppliers, the MSC adhesion will also change, affecting the growth of MSCs

Method used

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  • Molecular assembly and application for adherent culture and proliferation of mesenchymal stem cells (MSC)
  • Molecular assembly and application for adherent culture and proliferation of mesenchymal stem cells (MSC)
  • Molecular assembly and application for adherent culture and proliferation of mesenchymal stem cells (MSC)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1, in vitro adherent culture and expansion of human umbilical cord mesenchymal stem cells (MSC)

[0041] 1. Materials

[0042] Human umbilical cord MSCs: obtained from discarded umbilical cords of fetuses born in normal labor, using known methods to extract human umbilical cord MSCs (Ju XA, Chen J, Ding L, Li YZ, Xiao FJ, Li ZQ, Guo ZK. A slowly proliferating subpopulation in human umbilical cord mesenchymal stem cells in culture. InVitro Cell Dev Biol Anim. 2013;49(9):653-6).

[0043] Reagents: IMDM was purchased from Sigma. Fetal calf serum (FCS) was purchased from StemCell, Canada. The reagents for preparing serum-free culture were purchased from Sigma Company and were pure for culture. Human albumin is a recombinant protein purchased from North China Pharmaceutical Factory. Ascorbic acid was purchased from Sigma Company, product number is A4544. Thrombin was purchased from Sigma, Cat. No. T1063. TGF-β1 was purchased from Beijing Yiqiao Sino Biotechnolo...

Embodiment 2

[0061] Example 2, in vitro adherent culture expansion of human bone marrow mesenchymal stem cells (MSC)

[0062] 1. Materials

[0063] Human bone marrow MSCs: obtained from Beijing Sanli Biotechnology Co., Ltd., extracted from bone marrow by known methods (Ding L, Zhu H, Yang Y, Wang ZD, Zheng XL, Yan HM, Dong L, Zhang HH ,Han DM,Xue M,Liu J,Zhu L,Guo ZK,Wang HX.Functional mesenchymal stem cells remainpresent in bone marrow microenvironment of patients with leukemia post-allogeneic hematopoietic stem cell transplant.Leuk Lymphoma.2014;55(7): 1635-44.).

[0064] Reagent: Thiazolium Blue (MTT) was purchased from Sigma Company, the product number is M5655, dissolved in PBS, and the final concentration was 1 mg / mL. Other reagents are identical with embodiment 1.

[0065] The formula of serum-free medium is shown in Table 1.

[0066] 2. In vitro adherent culture and expansion of human bone marrow mesenchymal stem cells (MSCs)

[0067] 1) Cell collection

[0068] Collect the t...

Embodiment 3

[0079] Example 3, Cell Morphology of Human Bone Marrow Mesenchymal Stem Cells (MSC) Expanded in Vitro Adhesive Culture

[0080] 1. Materials

[0081] Human bone marrow MSCs were obtained from human heparinized anticoagulated bone marrow from healthy bone marrow donors. The extraction method can be found in literature (Ding L, Zhu H, Yang Y, Wang ZD, Zheng XL, Yan HM, Dong L, Zhang HH, Han DM, Xue M, LiuJ, Zhu L, Guo ZK, Wang HX. Functional mesenchymal stem cells remain present inbone marrow microenvironment of patients with leukemia post-allogeneicchematopoietic stem cell transplant.Leuk Lymphoma.2014;55(7):1635-44)

[0082] Reagents: human lymphocyte separation medium was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd. Other reagents are identical with embodiment 1.

[0083] The formula of serum-free medium is shown in Table 1.

[0084] 2. In vitro adherent culture and expansion of human bone marrow mesenchymal stem cells (MSCs)

[0085] 1) Isolat...

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Abstract

The invention discloses molecular assembly and application for adherent culture and proliferation of mesenchymal stem cells (MSC). The molecular assembly comprises thrombin, progesterone, a transforming growth factor beta1, ascorbic acid and hydrocortisone or hydrocortisone salt. The molecular composition is added into a basal culture medium of IMDM so as to obtain a culture medium for adherent culture and proliferation of the MSC. The culture medium in the invention is used for performing adherent culture and proliferation of the MSC, so that exogenous addition of lots of adhesion molecules is avoided, complex programs such as plate pre-paving and the like are avoided, the price is low, the action specificity is high, the adherent proliferation problem of the MSC is well solved, and the culture medium is applicable to culture of the MSC derived from human bone marrow and human umbilical cord and has wide application prospects.

Description

technical field [0001] The invention belongs to a cell culture method in the field of biotechnology, and in particular relates to a molecular combination for in vitro adherent culture and expansion of mesenchymal stem cells (MSC) and its application in in vitro culture and expansion of mesenchymal stem cells (MSC). application. Background technique [0002] Mesenchymal stem cells are also called mesenchymal stromal cells (MSCs) after in vitro culture and expansion, and are also called bone marrow stromal cells, adipose stem cells, and dental pulp stem cells according to their source. According to the definition of the International Society for Cell Therapy, MSCs are a group of fibroblast-like cells that express stem cell markers CD90, mesenchymal cell markers CD73, endothelial cell markers CD31, and hematopoietic cell markers CD34 and CD45. In addition, MSCs have osteogenic, chondrogenic and adipogenic abilities (Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N5/0663C12N5/0668C12N2500/38C12N2501/15C12N2501/30C12N2501/734
Inventor 郭子宽王恒湘
Owner 北京三有利生物技术有限公司