Establishment method for immortal bone mesenchymal stem cell line of Chang-Bai piglet

A technology for bone marrow mesenchyme and establishment of methods, applied in biochemical equipment and methods, cells modified by introducing foreign genetic material, botanical equipment and methods, etc., can solve the problems of lack of porcine bMSCs cell lines, growth stagnation, and limited biological scientific research and other issues

Active Publication Date: 2015-11-04
SOUTH CHINA AGRI UNIV
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Problems solved by technology

However, the ability of adult cells to proliferate in vitro is limited, and bMSCs will enter a state of growth stagnation after a certain period of proliferation in vitro, which limits the large-scale expansion in vitro and the study of their biological properties.
[0004] At present, there is still a lack of porcine bMSCs cell lines available in the market, which also limits the progress of research to some extent

Method used

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  • Establishment method for immortal bone mesenchymal stem cell line of Chang-Bai piglet
  • Establishment method for immortal bone mesenchymal stem cell line of Chang-Bai piglet
  • Establishment method for immortal bone mesenchymal stem cell line of Chang-Bai piglet

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Embodiment 1

[0029] see figure 1 , is a roadmap of a method for establishing an immortal line of Landrace piglet bone marrow mesenchymal stem cells of the present invention, comprising the following specific steps:

[0030] 1. Isolation and culture of porcine bone marrow mesenchymal stem cells

[0031] 1) Take femurs of 3-day-old Landrace male piglets (such as figure 2 A) and tibia, soaked in 75% ethanol for disinfection and degreasing;

[0032] 2) Use bone forceps to remove both ends, keep only the femoral body, use a 20mL disposable syringe to draw sterile PBS solution containing 20U / mL heparin sodium, and flush out the bone marrow cells;

[0033] 3) Collect the washed liquid, mix well, and centrifuge at 402g for 3min;

[0034] 4) Discard the supernatant, resuspend with new PBS solution, and pass through a 500-mesh cell sieve to remove bone fragments and blood clots;

[0035] 5) Collect the liquid and add it to the sterile lymphatic separation liquid prepared in advance at a ratio o...

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Abstract

The invention discloses an establishment method for an immortal bone mesenchymal stem cell line of a Chang-Bai piglet. The method comprises the following steps: taking bone marrow cells of a Chang-Bai boar piglet, culturing the bone marrow cells with a nutrient solution for 20 to 28 h, then replacing a fresh nutrient solution, continuing culture and carrying out passage until F3-generation Chang-Bai piglet bMSCs with uniform cellular morphology is obtained; infecting the F3-generation Chang-Bai piglet bMSCs with a lentivirus liquid carrying a Large T gene and then carrying out cell screening and passage until a cell strain stably expressing Egfp is obtained; and subjecting the cell strain stably expressing Egfp to passage for more than 50 generations and carrying out cytobiological analysis and detection so as to obtain the immortal bone marrow mesenchymal stem cell line of the Chang-Bai piglet. According to the invention, a system capable of supporting stable culture of porcine bMSCs (bone mesenchymal stem cells) is obtained; rapid and stable propagation of the cells in vitro is promoted and biological characteristics of the original generation of the cells are maintained; it is proved that immortal bMSCs of the Chang-Bai piglet are obtained; and in-depth research on biological properties of the porcine bMSCs can be carried out and the bMSCs can be applied to research and development of drugs or vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a culture solution for establishing an immortal line of Landrace piglet bone marrow mesenchymal stem cells and a method and application of using the culture solution to establish an immortal line of Landrace piglet bone marrow mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells with multi-lineage differentiation, which originate from mesoderm and are widely distributed in various tissues and organs. Under different induction conditions, MSCs can generate fat, bone, cartilage, muscle, skin, blood vessel, nerve and liver cells. In addition, MSCs also show the characteristics of easy isolation and culture in vitro, strong proliferation and differentiation ability, and low immunogenicity, so they are considered to be an ideal cell resource for cell therapy. Studies have shown that there are antigens expr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/28A61K39/00A61P43/00
Inventor 张守全白银山冯美莹卫恒习李莉
Owner SOUTH CHINA AGRI UNIV
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