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Cultivation method of mesenchymal stem cell secretin

A technology of mesenchymal stem cells and culture methods, applied in the field of mesenchymal stem cell secretin culture, can solve the problems of high cost, protein concentration loss, etc., and achieve the effect of improving separation, reducing loss, reducing cost and required space

Inactive Publication Date: 2017-05-10
浙江译美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the prior art, mesenchymal stem cells need a lot of culture flasks and space for mesenchymal stem cells to attach during the culture process, which requires high cost; at the same time, the protein in secretin will be concentrated in a large amount Loss, and the product contains impurities such as high concentration of salt and cell fluid

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  • Cultivation method of mesenchymal stem cell secretin
  • Cultivation method of mesenchymal stem cell secretin
  • Cultivation method of mesenchymal stem cell secretin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Cultivate 100 million adipose-derived mesenchymal stem cells Secretin 1

[0044] Take 2 million mesenchymal stem cells and place them in a medium containing complete medium I, and place the medium at 37°C with a volume fraction of 5% CO 215 million adipose-derived mesenchymal stem cells were cultured overnight in a saturated humidity incubator; the adipose-derived mesenchymal stem cells were then transferred to a 4 liter bioreactor with 5 g of microcarriers and 600 ml of complete medium I device. The temperature inside the bioreactor is 37°C and contains 5% CO by volume 2 , and keep the humidity in a saturated state, and the speed is maintained at 50rpm. At the same time, add 100 milliliters of fresh complete medium I to the bioreactor every 24 hours, and continue cell culture until the number of adipose-derived mesenchymal stem cells reaches 100 million (about 8 days in total);

[0045] When the number of adipose-derived mesenchymal stem cells reached the...

Embodiment 2

[0050] Example 2: Culture 100 million adipose-derived mesenchymal stem cells secretin 2

[0051] The difference between the second embodiment and the first embodiment is that when the number of adipose-derived mesenchymal stem cells reaches the standard, the adipose-derived mesenchymal stem cells and the attached microcarriers are washed three times with PBS buffer containing double antibodies, and 5 1 liter of PBS buffer solution containing double antibodies and 1 liter of complete medium I, continue to cultivate, and collect the supernatant in the bioreactor after 72 hours; and the mass fraction of the primary filtrate accounted for by the PCH flocculant here is 0.5%; Furthermore, the formula of the complete medium I is 3% fetal bovine serum by volume fraction, 45% MCDB201 by volume fraction, 13 μg / L platelet-derived growth factor, 12 μg / L basic fibroblast growth factor, 15 μg / L DF12 medium with epidermal growth factor.

[0052] Therefore, the purity of secretin obtained fr...

Embodiment 3

[0053] Example three: culturing 100 million adipose-derived mesenchymal stem cells secretin three

[0054] The difference between the third embodiment and the first embodiment is that when the number of adipose-derived mesenchymal stem cells reaches the standard, the adipose-derived mesenchymal stem cells and the microcarriers attached to them are washed three times with PBS buffer containing double antibodies, and injected into the bioreactor for 4 1 liter of PBS buffer solution containing double antibodies and 1 liter of complete medium I, continue to cultivate, and collect the supernatant in the bioreactor after 72 hours; and the mass fraction of the primary filtrate accounted for by the PCH flocculant here is 0.4%; Furthermore, the formula of complete medium I is 2% fetal bovine serum by volume fraction, 40% MCDB201 by volume fraction, 10 μg / L platelet-derived growth factor, 10 μg / L basic fibroblast growth factor, 10 μg / L DF12 medium with epidermal growth factor.

[0055]...

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Abstract

A cultivation method of mesenchymal stem cell secretin comprises the following steps: S1, taking a mesenchymal stem cell and adding into a bioreactor for cultivation, obtaining a target amount, and washing mesenchymal stem cells in the bioreactor with a double-antibody-containing PBS buffer solution;S2, adding a complete medium I and the double-antibody-containing PBS buffer solution into the bioreactor to continue cultivation; S3, collecting supernate in the bioreactor, adding a PCH flocculating agent into the supernate, and precipitating protein in the supernate; S4,putting a protein precipitate obtained in S3 into a semi-permeable membrane, and using deionized water to repeatedly wash the protein precipitate in the semi-permeable membrane; S5, utilizing the deionized water to load a mesenchymal container with protein, and obtaining a protein liquid as the mesenchymal stem cell secretin. The cultivation method not only can effectively improve the space utilization rate and reduce the usage amount of a nutrient medium, but also improves the protein purity and reduces the loss rate.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a method for culturing mesenchymal stem cell secretin. Background technique [0002] Mesenchymal stem cells (MSC, mesenchymal stem cells) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development, and belong to pluripotent stem cells. Supporting and promoting stem cell engraftment, immune regulation and self-replication have attracted increasing attention. For example, mesenchymal stem cells can be differentiated into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells under specific induction conditions in vivo or in vitro, after continuous subculture and cryopreservation It still has multi-directional differentiation potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and disease. [0003] Moreover, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/36C07K1/34C07K1/30C12N5/0775C12R1/91
CPCC12P21/00C07K1/30C07K1/34C07K1/36C12N5/0662C12N2500/12C12N2501/11C12N2501/115C12N2501/135
Inventor 陈继冰吴振化
Owner 浙江译美生物科技有限公司
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