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Culture method of marrow dedifferentiated mesenchymal stem cell

A bone marrow mesenchymal and mesenchymal stem cell technology, applied in the field of culture of human bone marrow dedifferentiated mesenchymal stem cells, can solve the problems of high experimental cost, long time period, and complicated experimental process, and achieve low experimental cost and short experimental cycle , Experimental operation is simple and convenient

Inactive Publication Date: 2015-02-11
吉林奥唯姿生物医学工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have problems such as long experimental operation period, extremely complicated experimental process, and high experimental cost.
In addition, although Chan et al. have studied that mouse De-MSCs induced by neuron-inducing factors have stronger neural differentiation ability, but this method is only specific for neuron induction. A frugal and feasible culture method to induce bone marrow dedifferentiated mesenchymal stem cells with high directional differentiation efficiency and high survival rate

Method used

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  • Culture method of marrow dedifferentiated mesenchymal stem cell
  • Culture method of marrow dedifferentiated mesenchymal stem cell
  • Culture method of marrow dedifferentiated mesenchymal stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Induced and cultured osteogenic dedifferentiated mesenchymal stem cells and their biological characteristics analysis experiments

[0067] 1. Materials

[0068] 1.1 Reagents

[0069] 1640 complete medium: Hyclone (US); fetal bovine serum, trypsin, penicillin and streptomycin: Gibco (US); PBS: solarbio (US); DMSO, trypsin, dexamethasone, β-glycerol phosphate , 2-Phosphate-L-VitC, were purchased from Sigma Company (USA), all antibodies involved in the test were purchased from eBioscience Company (USA), cell culture dishes and 48, 96-well plates: Corning (USA), cck8 kit ( Dojindo Japan). The rest of the reagents were of domestic analytical grade.

[0070] 1.2. Instruments

[0071] CO 2 Incubator, Thermo (USA); ultra-clean workbench (Suzhou Purification Equipment Factory); biological safety cabinet (Beijing Wuzhou Technology Development Co., Ltd.); high-pressure steam sterilizer, constant temperature oven (Babajin Experimental Equipment Co., Ltd.); enzyme I...

Embodiment 2

[0096] Example 2: In vitro osteogenic potential analysis experiment of induced cultured dedifferentiated mesenchymal stem cells

[0097] 1. Materials

[0098] 1.1 Reagents

[0099] 1640 medium: Hyclone (USA); fetal bovine serum, trypsin, penicillin and streptomycin: Gibco (USA); PBS: solarbio (USA); DMSO, trypsin, dexamethasone, β-glycerol phosphate, 2-Phosphate-L-VitC and Alizarin Red were purchased from Sigma (USA), all antibodies involved in the test were purchased from eBioscience (USA), cell culture dishes and 48, 96-well plates: Corning (USA), cck8 reagent Kit (Dojindo Japan), reverse transcription reagents and qPCR reagents were purchased from Takara Company (Japan), ALP kit (Nanjing Jitian Company). The rest of the reagents were of domestic analytical grade.

[0100] 1.2 Instruments

[0101] CO 2 Incubator, Thermo (USA); ultra-clean workbench: Suzhou Purification Equipment Factory; biological safety cabinet: Beijing Wuzhou Technology Development Co., Ltd.; high-pr...

Embodiment 3

[0154] Example 3: In vivo osteogenic potential analysis experiment of induced cultured dedifferentiated mesenchymal stem cells

[0155] 1. Materials

[0156] 1.1 Reagents

[0157] 1640 medium: Hyclone (USA); fetal bovine serum, trypsin, penicillin and streptomycin: Gibco (USA); PBS: solarbio (USA); DMSO, trypsin, dexamethasone, β-glycerol phosphate, 2-Phosphate-L-VitC was purchased from Sigma (USA); cell culture dishes and 48 and 96-well plates: Corning (USA); Trizol, reverse transcription reagents and qPCR reagents were purchased from Takara Company (Japan); ALP reagents Box (Nanjing Jitian Company); collagen module (gifted by Professor Dai Jianwu, Chinese Academy of Sciences); BCA protein quantification kit, RIPA lysate, PMSF: Beyond Institute of Biotechnology (Shanghai). The rest of the reagents were of domestic analytical grade.

[0158] 1.2 Instruments

[0159] CO 2 Cell incubator, Thermo (USA); paraffin slicer: Leica (Germany); ultra-clean bench: Suzhou Purification...

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Abstract

The invention discloses a cultivation method of marrow dedifferentiated mesenchymal stem cell. The method comprises the following steps: carrying out Ficcol isolated culture on bone marrow to obtain mesenchymal stem cell, when the cell is in logarithmic growth phase, abandoning a mesenchymal stem cell culture medium until the cell density achieves 70-80%, replacing as an osteogenic induction culture medium to culture for 3-7 days, abandoning the induction culture medium, cleaning for three times by using PBS, cleanly eliminating the culture medium residual liquid, replacing the cell in the mesenchymal stem cell culture medium, replacing new culture medium per 2-3 days, when the cell overspread a culture dish, performing normal digestion passage treatment, and trypsinizing, wherein the digestion time is not over 1 min, further culturing after the cell passage till to the 12th-14th day. Compared with the traditional bone marrow mesenchymal stem cell, the method for acquiring the dedifferentiated mesenchymal stem cell by replacing the culture medium has improved propagation efficiency and the osteogenesis dedifferentiation capability is enhanced.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing human bone marrow dedifferentiated mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) [0003] Derived from the mesoderm and ectoderm in the early stage of development, it was first discovered in the bone marrow as non-hematopoietic stem cells by Friendenstein et al. Because of its ability to differentiate into mesenchymal tissue cells, it is called mesenchymal stem cells. Later, it was isolated from many tissues, including fat, synovium, cartilage, periosteum, placenta and cord blood. Mesenchymal stem cells can be differentiated into endoderm, mesoderm and ectoderm cells under suitable conditions, and can be easily isolated and cultured. With its unique low immunogenicity, multi-directional differentiation and self-renewal functions, mesenchymal stem cells have been widely recognized and widely used in tissue enginee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 施勤杨惠林张慧
Owner 吉林奥唯姿生物医学工程有限公司
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