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Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells

A serum-free medium, bone marrow mesenchymal technology, applied in the direction of bone/connective tissue cells, tissue culture, animal cells, etc., can solve residual animal serum, fail to achieve serum-free culture, and cannot promote bone marrow mesenchymal stem cell adhesion. wall and other problems to achieve the effect of reducing residues, maintaining multi-directional differentiation potential, and maintaining growth

Active Publication Date: 2009-04-22
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these serum-free media can promote bone marrow mesenchymal stem cell attachment
When cultured in vitro, bone marrow mesenchymal stem cells need to be completely adhered to the wall in a serum-containing medium, and then replaced with a serum-free medium for culture, which will cause a certain amount of animal serum to remain in the medium. It is said that the true serum-free culture has not been achieved, and it may still have a great risk in clinical application

Method used

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  • Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells
  • Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells
  • Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells

Examples

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Embodiment 1

[0044] Based on α-MEM, its components are as follows:

[0045] α-MEM 10.2g / L

[0046] Basic fibroblast growth factor 10ng / mL

[0047] Epidermal Growth Factor 10ng / mL

[0048] Bovine serum albumin 2mg / mL

[0049] Bovine insulin 10μg / mL

[0050] Transferrin 5.5μg / mL

[0051] Ethanolamine 0.02mM

[0052] Putrescine 200μM

[0053] Vitamin C 100μM

[0054] Hydrocortisone 10μg / mL

[0055] Dexamethasone 10nM

[0056] FeSO 4 ·7H 2 O 0.834mg / mL

[0057] CuSO 4 ·5H 2 O 0.0025mg / mL

[0058] ZnSO 4 ·7H 2 O 0.863mg / mL

[0059] Mercaptoethanol 0.05mM

[0060] Sodium Selenite 0.01mM

[0061] Dissolve the above components in three-distilled water or ultrapure water at room temperature, stir and dissolve, which is the serum-free medium mentioned in the present invention; after being sterilized by a 0.2 μm filter membrane, it can be used for bone marrow mesenchyme Stem cell culture and expansion in vitro.

Embodiment 2

[0063] Dissolve type I rat tail collagen in 0.02N acetic acid solution with a collagen concentration of 50 mg / mL. Dilute this collagen solution 10 times, filter and sterilize through a 0.2 μm filter membrane, and then use 100 μg / cm 2 Spread on a sterile petri dish, air-dry at room temperature in a sterile environment, rinse the pre-coated collagen dish twice with PBS, and the dish can be used for serum-free culture of bone marrow mesenchymal stem cells.

Embodiment 3

[0065] One-month-old New Zealand white rabbits were killed by air embolism, dissected, and the femur, tibia, ulna, and radius were separated, and soaked in 75% ethanol for 5 minutes. Rinse with PBS 3 to 5 times, cut the bones with bone pliers, transfer to two centrifuge tubes, add 15 to 30 mL of PBS, oscillate, and take the supernatant. Centrifuge at 1000rpm for 5 minutes and discard the supernatant. Add 30mL PBS, mix well, centrifuge at 1000rpm for 5 minutes, and discard the supernatant. Add 10 mL of PBS and mix well, filter through a 150-mesh sieve, collect the filtrate, centrifuge, and resuspend in serum-free α-MEM medium. The bone marrow extract was slowly added to an equal volume of Ficoll separation solution along the wall of the centrifuge tube, and centrifuged horizontally at 3000 rpm for 30 minutes. Use a pointed pipette to absorb the middle mist layer, add PBS and wash by centrifugation. Cells were suspended in α-MEM+10% FBS culture medium, and counted by trypan b...

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Abstract

The present invention belongs to the field of biotechnology, and discloses a serum-free culture medium with specific chemical compositions for in vitro culture and amplification of bone marrow mesenchymal stem cells. By adding insulin, transferrin, ethanolamine, sodium selenite, growth factors, adherent factors, hormone, putrescine, inorganic salt, vitamin, albumin and antioxidant into a basic culture medium, the bone marrow mesenchymal stem cells can attach to the culture medium under a serum free condition, so the in vitro culture and amplification are realized, the potential of multi-directional differentiation is maintained, and the amplified cells can be induced to be osteoblast and lipocyte in vitro. The serum-free culture medium has the advantages that the clinic level cell products for human produced by the serum free culture medium can effectively avoid the potential risk of producing cell products by serum culture medium. The drawing appended is a photo of the confluence of the bone marrow mesenchymal stem cells cultured by the serum-free culture medium.

Description

【Technical field】 [0001] The invention relates to a cell culture medium in the field of biotechnology, in particular to a serum-free culture medium for culturing and expanding bone marrow mesenchymal stem cells in vitro. 【Background technique】 [0002] Bone marrow mesenchymal stem cells (MSCs) are the main component of the bone marrow stroma, providing structural and functional support for the hematopoietic function of the bone marrow. Current studies have shown that bone marrow mesenchymal stem cells can promote the reconstruction of the hematopoietic system, have effective immune regulation functions, can inhibit the proliferation of T-lymphocytes in vitro, delay immune rejection after tissue transplantation, and can also be used for prevention and treatment. Treatment of graft-versus-host disease (GVHD) arising from autologous stem cell transplantation (ASCT), avoidance of immune rejection in organ transplantation, and treatment of autoimmune diseases such as rheumatoid a...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/08C12N5/0775
Inventor 周燕吴伟谭文松
Owner EAST CHINA UNIV OF SCI & TECH
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