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Preparation method of purified and amplified human mesenchymal stem cells

A bone marrow mesenchymal and mesenchymal stem cell technology, applied in the field of cell culture and preparation, can solve the problems of limiting the application of autologous serum, complicated serum components, pollution, etc., and achieve good clinical application value and potential effect

Active Publication Date: 2017-12-01
SHANGHAI LIFE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MSCs can grow and passage normally in the presence of fetal bovine serum, but the composition of serum is relatively complex, containing both nutrients that promote cell growth and low levels of substances that inhibit cell growth
Serum is also potentially contaminated by viruses and mycoplasma, and the performance of different batches of serum varies significantly
Autologous serum can also be used instead of fetal bovine serum, but a large amount of autologous serum is required to expand MSCs, which limits the application of autologous serum in MSCs culture
Although many studies have reported the use of serum-free medium, MSCs proliferate slowly under serum-free conditions, and there is a possibility of loss of differentiation ability

Method used

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  • Preparation method of purified and amplified human mesenchymal stem cells
  • Preparation method of purified and amplified human mesenchymal stem cells
  • Preparation method of purified and amplified human mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] (1) Isolation of mesenchymal stem cells from bone marrow

[0055] Using antibody-based negative isolation, the steps are as follows:

[0056] 1. Add 500μl of antibody mixture directly to 10ml of bone marrow in the ratio of 1ml:50μl, mix well, and place at room temperature for 20 minutes.

[0057] 2. Add 15 ml of lymphocyte separation solution to a 50 ml centrifuge tube.

[0058] 3. Mix 20 ml of sorting buffer (normal saline containing 2% human serum albumin and 1 mM EDTA) with bone marrow, and then add it to the lymphocyte splitting solution.

[0059] 4. Turn off the deceleration valve of the centrifuge and centrifuge at 300g for 25 minutes.

[0060] 5. Collect the MSCs layer under the plasma layer and transfer to a new centrifuge tube; add 30 ml of sorting buffer and centrifuge at 300 g for 10 minutes.

[0061] 6. Pour out the supernatant, add 30 ml of sorting buffer, centrifuge at 300 g for 10 minutes, and discard the supernatant again.

[0062] 7. Resuspend MSCs in culture medium...

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Abstract

The invention relates to a preparation method of purified and amplified human mesenchymal stem cells. The method comprises the following steps: (1) collecting mesenchymal stem cells by adopting an antibody-based negative sorting method; and (2) amplifying the mesenchymal stem cells by adopting a serum-free culture medium. The preparation method is superior to a conventional separation method, and the serum-free culture medium provided by the method is remarkably better than a conventional serum culture medium in the fields of safety and amplifying capability and has excellent application prospect.

Description

Technical field [0001] The invention belongs to the field of cell culture and preparation, and particularly relates to a preparation method for purifying and expanding human bone marrow mesenchymal stem cells. Background technique [0002] Mesenchymal Stem Cells (MSCs) are a branch of stem cells. They are a type of cells with self-replication and multidirectional differentiation capabilities. They are widely present in a variety of tissues, such as bone marrow, umbilical cord blood, umbilical cord tissue, and placental tissue And adipose tissue. Mesenchymal stem cells have three notable characteristics: 1. Mesenchymal stem cells cultured in vitro are adherent growth; 2. Mesenchymal stem cells highly express CD73, CD90 and CD105, but do not express CD31, CD34, CD45, HLA- Markers such as DR, CD14, CD19 and CD11b; 3. Under suitable stimulating factors, mesenchymal stem cells can differentiate into cells of various tissues such as osteoblasts, adipocytes and nerve cells. [0003] Bon...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2500/25C12N2500/32C12N2500/38C12N2500/40C12N2500/46C12N2500/90C12N2501/11C12N2501/115C12N2501/135C12N2501/15C12N2501/39C12N2501/734C12N2501/999C12N2533/52
Inventor 李新峰
Owner SHANGHAI LIFE SCI & TECH CO LTD
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