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Serum-free complete medium for mesenchymal stem cell

A complete medium and mesenchymal stem cell technology, applied in the field of serum-free complete medium for mesenchymal stem cells, can solve the problems of cytotoxicity, instability between serum batches, and a large number of heterologous proteins, so as to promote cell proliferation, Supports long-term subculture and avoids the effect of instability between serum batches

Inactive Publication Date: 2012-08-15
四川全组生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the objectives of the present invention is to solve the deficiencies of the prior art, provide a serum-free complete medium for in vitro culture of human mesenchymal stem cells, overcome the instability between batches of serum containing serum medium, cytotoxicity and a large number of heterogeneous Protein and other defects

Method used

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  • Serum-free complete medium for mesenchymal stem cell
  • Serum-free complete medium for mesenchymal stem cell
  • Serum-free complete medium for mesenchymal stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Preparation of 1L serum-free complete medium for mesenchymal stem cells: 1 bag of DMEM / F12 dry powder (packing: 10 bags*1L / box) with product model number 12400-024, 7 mg of recombinant human insulin, 12 g of human serum albumin, human Mix 1 mg transferrin, 25 mg cholesterol, 0.185 mg sodium selenite, 4 mg ferric nitrate, and 50 mg dextran, add 800 ml water for injection, stir to dissolve, adjust the pH to 7.1 with 0.5 mol / L hydrochloric acid, and dilute to 1 L. After mixing, use a 0.22um filter membrane to filter and sterilize, and store at 4°C.

[0019] BMSCs (Bone Marrow Mesenchymal Stem Cells) Culture in Vitro: Take 10ml of bone marrow from volunteers in the hospital, place an equal volume on 1.073g / ml lymphocyte separation medium, centrifuge at 2250rpm for 30min, take cloudy mononuclear cell layer, wash with normal saline 3 times; centrifuge at 2000rpm, 1800rpm, and 1600rpm in turn to remove the supernatant, and centrifuge for five minutes each time; resuspend the c...

Embodiment 2

[0030] Preparation of 1L serum-free complete medium for mesenchymal stem cells: 1 bag of DMEM / F12 dry powder (package: 10 bags*1L / box) with the product model Gibco 884400, 2 mg of recombinant human insulin, 30 g of human serum albumin, human transfection Mix ferritin 10mg, cholesterol 10mg, sodium selenite 0.05mg, ferric nitrate 10mg, dextran 10mg, add 800ml water for injection, stir to dissolve, adjust the pH to 7.1±0.1 with 0.5mol / L hydrochloric acid, and dilute to 1L , mix well, filter and sterilize with a 0.22um filter membrane, and store at 4°C.

[0031] BMSCs (Bone Marrow Mesenchymal Stem Cells) Culture in Vitro: Take 10ml of bone marrow from volunteers in the hospital, place an equal volume on 1.073g / ml lymphocyte separation medium, centrifuge at 2250rpm for 30min, take cloudy mononuclear cell layer, wash with normal saline 3 times; then centrifuge at 2000rpm, 1800rpm, 1600rpm to remove the supernatant, each time for five minutes, resuspend the cells obtained after remo...

Embodiment 3

[0042] Preparation of 1L serum-free complete medium for mesenchymal stem cells: 1 bag of DMEM / F12 dry powder (packing: 10 bags*1L / box) with product model number 12400-024, 10 mg of recombinant human insulin, 1 g of human serum albumin, human Mix 1 mg transferrin, 50 mg cholesterol, 0.2 mg sodium selenite, 1 mg ferric nitrate, and 100 mg dextran, add 800 ml water for injection, stir to dissolve, adjust the pH to 7.1±0.1 with 0.5 mol / L hydrochloric acid, and dilute to 1L, after mixing, filter and sterilize with a 0.22um filter membrane, and store at 4°C.

[0043] BMSCs (Bone Marrow Mesenchymal Stem Cells) Culture in Vitro: Take 10ml of bone marrow from volunteers in the hospital, place an equal volume on 1.073g / ml lymphocyte separation medium, centrifuge at 2250rpm for 30min, take cloudy mononuclear cell layer, wash with normal saline 3 times, then centrifuge at 2000rpm, 1800rpm, 1600rpm to remove the supernatant, each time for five minutes; resuspend the supernatant-removed cel...

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Abstract

The invention discloses a serum-free complete medium for mesenchymal stem cell, consisting of a basal medium and an additive component, wherein the basal medium is DMEM / F12 (dulbecco modified eagle medium), and the additive component consists of recombinant human insulin, human serum albumin, human transferrin, cholesterol, sodium selenite, ferric nitrate and glucan. The complete medium is free from serum, so that the defects that the serum-containing medium is unstable in serum batches, high in cell toxicity, abundant in heterologous protein and the like can be greatly avoided, and a good tool can be provided for the basic research and the clinic treatment of the mesenchymal stem cell; and the invention further achieves the effect which is approximate or equal to the serum-containing complete medium and the other serum-free complete mediums in the aspect of cell proliferation, so that the long-term subcultring of the cell can be supported.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a serum-free complete medium for mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are adult pluripotent stem cells derived from mesoderm, which can be isolated from bone marrow, periosteum, synovium, muscle, fat and other tissues, and have the potential to differentiate into various types of mature cells. Throughout life, tissues and organs need mesenchymal stem cells to continuously replenish and renew whether they are in a stable state or stimulated by environmental changes. Therefore, mesenchymal stem cells become ideal seed cells for cell engineering and tissue engineering. The number of mesenchymal stem cells is small, and it is difficult to meet the needs of clinical treatment and research. Therefore, it is necessary to massively expand mesenchymal stem cells in vitro. [0003] At present, a certain proportion of animal serum is added to the conventi...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 张琼戴成祥邓涛
Owner 四川全组生命科技有限公司
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