Method for culturing mesenchymal stem cells through manual simulation of bone marrow microenvironment

A bone marrow mesenchymal, artificial simulation technology, applied in the field of cell engineering, can solve the problems of the expansion rate of unstable bone marrow mesenchymal stem cells, weak mechanical properties of the scaffold, poor cell activity, etc., to improve cell activity and proliferation rate, The effect of improving mechanical properties and expanding the contact area

Inactive Publication Date: 2016-02-10
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is that in the prior art, the method for cultivating bone marrow mesenchymal stem cells using a single three-dimensional scaffold causes low expansion rate of bone marrow mesenchymal stem cells and low cell v

Method used

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  • Method for culturing mesenchymal stem cells through manual simulation of bone marrow microenvironment

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preparation example Construction

[0040] 1, the preparation of collagen, comprises the following steps:

[0041] 1) Take fresh beef bones, remove muscle, fat, and remove fascia as much as possible; after repeated washing with three-distilled water, store in PE ziplock bags at -30°C;

[0042] 2) In the frozen state, use a scalpel to cut the beef bone into thin slices, and then mash it in a tissue masher;

[0043] 3) Weigh about 60 grams of crushed bovine bond, soak in 0.05% (w / v) chlorhexidine acetate for 5 minutes for disinfection, then wash with 0.9% normal saline for several times, filter with gauze, and wash away the residual acetic acid as much as possible Chlorhexidine;

[0044] 4) Digest the sterilized bovine bond in PBS digestion solution (pH=7.4) containing 0.25% trypsin at 37°C for 24 hours;

[0045] 5) After digestion, add a few drops of H 2 o 2 , soak for 10-15 minutes to remove residual trypsin; then wash repeatedly with three-distilled water to remove H 2 o 2 .

[0046] 6) Add a small amoun...

Embodiment 1

[0057] The method for cultivating bone marrow mesenchymal stem cells by artificially simulating the bone marrow microenvironment provided by the present invention comprises the following steps:

[0058] S1a, obtaining primary bone marrow mesenchymal stem cells;

[0059] Bone marrow samples were collected and anticoagulated with heparin. Add each sample to the Percoll separation medium with a density of 1.073g / ml at a ratio of 1:2, centrifuge at 2300rpm for 30min, take the middle mononuclear cells, add PBS buffer, centrifuge at 1000rpm for 10min, wash 3 times, discard The supernatant was used to obtain primary bone marrow mesenchymal stem cells.

[0060] S2a, subculture of primary bone marrow mesenchymal stem cells;

[0061] Adjust the bone marrow mesenchymal stem cells obtained in step S1a to 1×10 7 Individual / culture bottle (25ml) density, and add serum culture medium and cultivate, wherein, serum culture medium is to contain 10% fetal bovine serum, 10 -6 mol / L hydrocorti...

Embodiment 2

[0069] The difference from Example 1 is that in this example, the three-dimensional cell culture scaffold is a collagen-chitosan modified scaffold made of collagen, chitosan and glutaraldehyde;

[0070] In this embodiment, the process of culturing third-generation bone marrow mesenchymal stem cells in a three-dimensional cell culture scaffold includes the following steps:

[0071] S31b, preparation of three-dimensional cell culture scaffold:

[0072] Collagen is dissolved in the acetic acid solution of 3% (0.5M), stirs to form the collagen swelling solution that concentration is 0.5%; After mixing with the chitosan solution, dry heat cross-linking at 150°C in a vacuum oven (vacuum degree <0.2mbar) for 24 hours to obtain a collagen / chitosan scaffold; Soak in 0.25% (w / v) glutaraldehyde aqueous solution at a volume ratio of 1:1, and cross-link at 4°C for 12 hours. After cross-linking, fully rinse with triple distilled water for 10 minutes each time for 6 times to remove residual ...

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Abstract

The invention relates to a method for culturing mesenchymal stem cells through manual simulation of the bone marrow microenvironment. The method comprises the following steps: acquiring the mesenchymal stem cells, and carrying out culture in vitro in a culture medium by adopting a three-dimensional cell culture scaffold, wherein the three-dimensional cell culture scaffold is made of collagen and chitosan or lysine, or the three-dimensional cell culture scaffold is made of collagen, chitosan and glutaraldehyde. According to the method, by adopting chitosan or lysine, or adopting chitosan and glutaraldehyde to modify the collagen scaffold, the mechanical property of the collagen scaffold can be improved; a three-dimensional porous structure of the three-dimensional cell culture scaffold is adopted for maintaining the three-dimensional growing environment in the in-vitro culturing process of the mesenchymal stem cells, so that the bone marrow microenvironment suitable for the growth of the mesenchymal stem cells can be well simulated, and the problem that in the prior art, the single three-dimensional cell culture scaffold is adopted for culturing the mesenchymal stem cells, the mechanical property of the scaffold is poor, so that the cell proliferation rate is low, and the cell death rate is high, is solved.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for artificially simulating a bone marrow microenvironment for culturing bone marrow mesenchymal stem cells. Background technique [0002] Bone marrow mesenchymal stem cells (Mesenchymaistemcells, MSCs) are a type of cells in the bone marrow similar to fibroblasts. Cell and cardiomyocyte transformation. Autologous bone marrow mesenchymal stem cells also have the advantages of convenient extraction, non-immunogenicity, multi-directional differentiation potential, ethical requirements, and non-tumorigenicity, which are incomparable to other stem cells. Therefore, it is of far-reaching significance to explore the large-scale expansion of MSCs in vitro significance. [0003] At present, the in vitro expansion of bone marrow mesenchymal stem cells is mostly carried out by two-dimensional culture, and some are cultured by three-dimensional cell culture scaffolds, bu...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 曾宪卓鲁菲
Owner SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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