Bone-marrow mesenchymal stem cell culture medium and application thereof

A bone marrow mesenchymal and cell culture technology, applied in the field of bone marrow mesenchymal stem cell culture medium, can solve the problems of bMSC in general proliferation ability in vitro, limitation of clinical cell therapy, low self-renewal ability, etc. The effect of decreased sex and large nucleoplasmic ratio

Active Publication Date: 2019-11-19
海门生原干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, bMSCs are mesenchymal stem cells derived from bone marrow, with complex cell components and low self-renewal ability. At the same time, bMSCs have a general ability to proliferate in vitro, and their subsequent application in clinical cell therapy is also limited.

Method used

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  • Bone-marrow mesenchymal stem cell culture medium and application thereof
  • Bone-marrow mesenchymal stem cell culture medium and application thereof
  • Bone-marrow mesenchymal stem cell culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Analysis of conventional culture medium and bone marrow mesenchymal stem cell culture medium of the present invention for bone marrow mesenchymal stem cell culture

[0029] The control group adopts conventional culture medium, and the components of conventional culture medium are: 10% fetal bovine serum (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), low-sugar DMEM; Experimental group adopts bone marrow mesenchyme of the present invention See Table 1 for details of the stem cell culture medium and the medium composition of the experimental group.

[0030] Table 1 The medium composition of the experimental group

[0031]

[0032]

[0033] The two media were used for primary culture and subculture respectively.

[0034] 1. Primary culture:

[0035] For the bone marrow derived from clinical patients, wash it twice with PBS, use 4°C pre-cooled 0.16M Tris-NH4Cl to break red for 5 minutes, centrifuge at 1000 rpm for 5 min...

Embodiment 2

[0040] Example 2 Detection of bMSC-specific markers by flow cytometry

[0041] Take the bMSC cells in the logarithmic growth phase of the experimental group and the control group in Example 1 respectively, wash the cells three times with PBS, digest the cells with 0.05% Trypsin, stop the digestion of the medium and blow until a single cell suspension is obtained; about 10 5 ~10 6 According to the antibody instructions, add the corresponding detection amount of fluorescently labeled antibodies (CD34-FITC, CD45-FITC, CD106-FITC, CD44-FITC, HLA-1-FITC; CD90-FITC, CD29-PE,) and the corresponding isotype For the control antibody, mix well and place at room temperature in the dark for 30 minutes. Add 2ml of PBS, centrifuge at 1000g for 5min, discard the supernatant, and add 300μl of PBS to resuspend the cells. For flow cytometry detection, cells were acquired with Cell quest software, and the percentage of positive cells was analyzed. See Table 2 for analytical results. Cell sur...

Embodiment 3

[0045] The pluripotency gene expression identification (RT-PCR detection) of embodiment 3bMSC

[0046] 1. Extraction of total cellular RNA: using the TRIzol Total RNA Extraction Kit (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), respectively extract the bone marrow mesenchymal stem cells prepared in the experimental group and the control group in Example 1. For the total RNA at passage 7, the purity and quantity of the extracted RNA were measured by a spectrophotometer, and stored at -70°C.

[0047] 2. Synthesis of cDNA: 25 μl reaction system: 2 μg of cellular RNA sample, 3 μl of random primer or Oligo dT-Adaptor Primer, 10 μl of RNase Free ddH2O. 70°C, 10min. Quench on ice, 1 min. Centrifuge briefly. Add 1.5μl of 10mM dNTP, 0.5μl of RNase Inhibitor (25units), 1μl of M-MMLV Reverse Transcriptase (200units), 5μl of 5×RNA PCR Buffer, 4μl of RNase Free ddH2O, lightly flick, and put in a PCR machine at 37°C for 100min. After the reaction, mix well and store at ...

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Abstract

The invention provides a bone-marrow mesenchymal stem cell culture medium and application thereof, and belongs to the technical field of stem cells as well as cell culture and amplification. The bone-marrow mesenchymal stem cell culture medium consists of the following ingredients at the following concentrations: 52-53% by volume of Dulbecco modified MEM medium, 38-42% by volume of MCDB medium, 1XITS cell culture additives, 8-12 microgram/milliliter of linoleic acid, 40-60 microgram/milliliter of human serum albumin, 1X penicillin and streptomycin, 75-125 micromoles of L-ascorbic acid, 1-3% by volume of fetal bovine serum, 10-15 micromoles of dexamethasone, 12-18 ng/mL of platelet-derived growth factors, and 8-12 ng/mL of growth promoting factors. Being adopted, the bone-marrow mesenchymal stem cell culture medium provided by the invention is capable of increasing success rate of clonal culture of bone-marrow mesenchymal stem cells (bMSC); and moreover, the bone-marrow mesenchymal stem cell culture medium is capable of increasing passage times, ensuring relatively high cell purity and improving cell primitivity.

Description

technical field [0001] The invention relates to the technical field of stem cells and in vitro culture and expansion of cells, in particular to a culture medium for bone marrow mesenchymal stem cells and its application. Background technique [0002] The clinical treatment of stem cells requires suitable seed cells. Embryonic stem cells (ESCs), which are more researched at present, are the earliest pluripotent stem cells isolated. In vitro culture can maintain stable proliferation ability and have the potential to differentiate into multiple germ layers. However, embryonic stem cells are generally cells from heterologous tissues, and there is a risk of immune rejection. Although there are already ESCs obtained from adult cell nuclear transfer, which can avoid immune rejection, there is still a risk of teratoma formation in vivo, and there are many ethical controversies about ESCs. [0003] It has been reported many times that bone marrow contains many kinds of stem cells i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2500/36C12N2500/38C12N2501/135C12N2501/10C12N2501/39Y02A50/30
Inventor 刘厚奇
Owner 海门生原干细胞科技有限公司
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