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Mesenchymal stem cell nutrient solution

A mesenchymal stem cell and culture medium technology, applied in the field of mesenchymal stem cell culture, can solve the problems of high cost, cell pollution, and complex formula of serum-free culture medium, and achieve the effect of high safety and simple formula

Active Publication Date: 2012-10-03
ASIA PACIFIC STEM CELL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. Serum from animals will be used in formulas containing serum, but the serum contains various unidentified substances and proteins, and cells are easily contaminated by animal viruses and bacteria identified by the scientific community;
[0006] 2. The existing serum-free culture medium uses blood products whose composition has not been fully determined to replace serum, and the risk of cell contamination is not lower than that using serum;
[0007] 3. The existing culture medium used for the growth of mesenchymal stem cells begins to differentiate after several generations of culture and loses the plasticity and medical efficacy of stem cells;
[0008] 4. The formula of the existing serum-free culture solution is very complicated, and the cost of materials required is higher than that of animal serum, which makes it very low in popularity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Wash 20cc adipose tissue 3 times with phosphate buffered saline, add collagenase (Type II, 50-100U / ml) to digest the tissue for one hour, centrifuge the digest and wash the resulting cells 3 times with phosphate buffered saline; mix the cells 7ml of culture solution was placed in a T-75 cell culture flask for cultivation. The components of the culture solution in this example were: alpha-MEM medium, B27 serum-free additive with a volume ratio of 1%, bFGF at 10 ng / ml, bFGF at 10 ng / ml EGF, 0.5mg / ml fetuin, 100units / ml each of penicillin and streptomycin, use the above culture medium, replace the culture medium once every 3 days, and detect when the cells grow to a T-75 cell culture flask. No less than one million adipose-derived mesenchymal stem cells were cultured.

Embodiment 2

[0023] Use an ethanol solution with a volume ratio of 70% to disinfect the oral cavity of the human body, and then use a 2mm diameter biopsy instrument to extract a tissue sample (not less than 2mmx2mmx2mm) from the human gum, wash it with phosphate buffer for 3 times, and add collagenase (Type II, 50-100U / ml) to digest the tissue for one hour, centrifuge the digest and wash the resulting cells 3 times with phosphate buffered saline; mix the gingival tissue cells with 10ml culture solution and put them in a T-75 cell culture bottle for cultivation. The components of the culture medium are: DMEM / F12 serum-free medium, B27 serum-free supplement with a volume ratio of 3%, bFGF at 50ng / ml, EGF at 50ng / ml, fetuin at 2mg / ml, and penicillin at 150units / ml each and streptomycin, use the above culture medium, replace the culture medium once every 3 days, when the cells grow to a T-75 cell culture flask, it is detected that no less than one million gingival tissue mesenchymal stem cells ...

Embodiment 3

[0026] Thaw adipose-derived mesenchymal stem cells (not less than 400,000 cells) to 37 degrees, wash the obtained cells twice with phosphate buffer, mix the cells with 20ml of culture medium and put them in a T-150 cell culture bottle for cultivation. This implementation The culture solution components of the example are: alpha-MEM medium, B27 serum-free supplement with a volume ratio of 2%, bFGF at 20ng / ml, EGF at 20ng / ml, fetuin at 1mg / ml, penicillin at 100units / ml each And streptomycin, using the above culture medium, replace the culture medium once every 3 days, when the cells grow to a T-75 cell culture flask, it is detected that no less than 3 million adipose-derived mesenchymal stem cells have been cultured.

[0027] Compared with the culture of adipose-derived mesenchymal stem cells using the serum-free medium formula of Dromard C et al., 2011, the cells will grow in suspension in the culture flask, while the mesenchymal stem cells of this example can be attached to the...

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Abstract

The invention provides a serum-free mesenchymal stem cell nutrient solution, which is simple in formula, low in cost and high in safety. The serum-free mesenchymal stem cell nutrient solution comprises a serum-free animal cell culture medium, a B27 serum-free additive, bFGFs (basic fibroblast growth factors) and EGFs (epidermal growth factors), wherein the nutrient solution also comprises fetuin and antibiotics. The nutrient solution does not contain animal serum, has higher safety and definite formula components, and is suitable for clinical treatment. Meanwhile, according to the serum-free mesenchymal stem cell nutrient solution, human fat stem cells can be normally attached to the surface of a culture vessel and grow, the antibiotics are added into the formula components, and the formula is simpler compared with that of the general serum-free nutrient solution. For the fat stem cells cultured according to the conventional formula, the cells began to differentiate and slowly grow after two generations. Compared with the fat stem cells cultured according to the conventional formula, the fat stem cells cultured according to the formula of the serum-free mesenchymal stem cell nutrient solution do not have the phenomena of differentiation and slow growth after being tested, so that the serum-free mesenchymal stem cell nutrient solution is extremely suitable for the growth of the fat stem cells and gum stem cells.

Description

technical field [0001] The invention relates to the culture technology of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are cells with high differentiation potential, which are different from ordinary somatic cells and have the ability to differentiate into various body tissues, such as bones, skin, heart, nerves, etc. Its application in regenerative medicine has been increasing in recent years. The source of cells is mainly extracted from human tissues and cultivated in vitro to obtain the required number of stem cells for treatment. However, the traditional culture medium used for in vitro proliferation of cells also contains 5% to 20% of animal serum, if the serum used contains incurable viruses or pathogens (such as mad cow disease prions), patients receiving cell therapy will be infected, and the consequences will be very serious. Since the main ingredient that stimulates cell growth in the culture medium is serum, scientists try to repla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 周向荣邓铭权杜洁华
Owner ASIA PACIFIC STEM CELL SCI
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