Method for amplifying autologous bone marrow mesenchymal stem cells

A bone marrow mesenchymal and stem cell technology, applied in bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of unable to meet clinical needs in time, high autologous serum concentration, application limitations, etc. Instable between times, reducing the amount of serum, beneficial to clinical application

Inactive Publication Date: 2010-06-30
GENERAL HOSPITAL OF PLA
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AI Technical Summary

Problems solved by technology

However, due to the large amount of serum required for mass culture of autologous MSCs, more autologous serum or plasma needs to be collected, which limits its application.
[0004] In order to make up for the defects of the above-mentioned prior art, those skilled in the art propose to use autologous serum homologous to bone marrow mesenchymal stem cells for expansion and culture, but the concentration of autologous serum required by the prior art is relatively large, generally about 10%. To ensure the normal culture and growth of bone marrow mesenchymal stem cells
Due to the high concentration and large dosage, it is necessary to extract more plasma from the patient before culturing to achieve the required dosage, which has caused certain harm to the health of the patient.
In addition, the prior art of bone marrow mesenchymal stem cells takes a long time to culture, and it usually takes about 30 days to meet the demand, so it cannot meet the clinical needs in time and delay the disease.

Method used

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  • Method for amplifying autologous bone marrow mesenchymal stem cells
  • Method for amplifying autologous bone marrow mesenchymal stem cells
  • Method for amplifying autologous bone marrow mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Autologous Bone Marrow Mesenchymal Stem Cell Culture

[0038] (1) Preparation of 1000ml autologous bone marrow mesenchymal stem cell medium

[0039] Weigh 10g of DMEM low sugar, dissolve it in 950ml of ultrapure water, add 3.7g of sodium bicarbonate, 100,000 U of gentamycin, 5ug of basic fibroblast growth factor, 5mg of insulin, 5ug of transferrin, 5g of white Egg white, 10ug sodium selenite, 10mg hydrocortisone, stir well, adjust pH to 7.1, add water to 1000ml, sterilize by 0.22μ filter membrane, store at 4°C for 1-3 months.

[0040] (2) Isolation, culture and expansion of human bone marrow mesenchymal stem cells

[0041] Aseptically extract about 5ml of bone marrow, transfer it into a 50ml centrifuge tube containing 1ml (500u / ml heparin) normal saline, dilute with an equal amount of normal saline containing 20u / ml heparin, and slowly trickle along the tube wall to superimpose an equal volume of human The lymphocyte separation medium was centrifuged at 2000...

Embodiment 2

[0047] Example 2 Detection of cell growth performance

[0048] (1) Cell growth curve

[0049] Take the 2nd and 3rd generation cultured cells, prepare single cell suspension, count and adjust the cell concentration to 5×10 3 / ml, seeded in a 24-well cell culture plate, digested the cells in 3 wells of the well plate with trypsin after 24 hours and counted, took 3 wells continuously every day, and stopped counting until the cells were full of the wells, and counted as time (d) The abscissa is the abscissa, and the cell number is the ordinate, and the growth curve is drawn.

[0050] The result is as figure 2 As shown, there was no obvious change in the number of cells on the 1st and 2nd day after inoculation, and the number of cells increased a lot from the 3rd day, and reached the peak on the 6th day, and the growth rate of the cells slowed down thereafter.

[0051] (2) Phenotype detection

[0052] Take 1ml of P3 generation cell suspension (1×10 6 cells / ml), add 2ml of PBS...

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Abstract

The invention relates to a method for amplifying autologous bone marrow mesenchymal stem cells, in particular to the purpose of finding out the optimal environment for cell growth by utilizing the compound of culture media and a little of autoserum as well as the purpose of strictly controlling the cell fusion degree in the method so as to achieve the purpose of fast culturing the bone marrow mesenchymal stem cells in the end; and the use of the autoserum remedies the defects of fetal calf serum. The culture time of the bone marrow mesenchymal stem cells is greatly shortened, thereby being beneficial to clinical application. Simultaneously, the method is used for culturing the bone marrow mesenchymal stem cells, and 1.5-2*107 mesenchymal stem cells can be obtained through amplification innearly twenty days by extracting 5 ml bone marrow. The dosage of blood serum is greatly reduced, thereby reducing the quantity of blood collected from patients. The obtained bone marrow mesenchymal stem cells have the characteristics of the stem cells in the aspects of cell morphology, growth cycle, phenotype, inducing differentiation capacity, and the like.

Description

technical field [0001] The invention relates to a method for culturing and expanding bone marrow mesenchymal stem cells, in particular to an expansion method for culturing bone marrow mesenchymal stem cells using autologous serum. Background technique [0002] Mesenchymal stem cells (MSCs) are widely distributed in various tissues of the human body, including bone marrow, fat, placenta, and skeletal muscle, among which bone marrow mesenchymal stem cells are considered to be the most accessible and abundant MSCs bank. Human bone marrow mesenchymal stem cells have the characteristics of multidirectional differentiation potential, self-renewal, homing to diseased tissues and immune regulation, and have the following advantages: ①Good proliferation ability in vivo and in vitro, easy to cultivate and regulate, After several generations of amplification, it can still maintain its multi-directional differentiation potential; ②The survival rate after transplantation is high, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 赵亚力郝好杰韩为东司艺玲伍志强
Owner GENERAL HOSPITAL OF PLA
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