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Supplement for cell cultivation media

A technology of nutrient additives and culture medium, applied in cell culture medium, animal cells, culture process, etc., can solve the problems of research quality impact, complex composition, existence of pollutants, etc.

Active Publication Date: 2012-08-01
陈嘉有 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In science, serum is rich in proteins, growth factors, hormones, nutrients and metabolites, lipids, minerals and inhibitors. Its composition is complex and there is a possibility of pollutants, which often affect the quality of research.

Method used

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  • Supplement for cell cultivation media
  • Supplement for cell cultivation media
  • Supplement for cell cultivation media

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of porcine blood blood preparation:

[0031] 1. After the pigs are bled by the carotid artery, the blood is put into a centrifuge tube containing anticoagulant, and stored in an ice bath for processing in the laboratory.

[0032] 2. Centrifuge the collected blood three times at 100-5000g for 5-10 minutes at room temperature and take out the supernatant (first centrifugation at 100-500g for 5-10 minutes, second centrifugation at 100-500g for 5-10 minutes, third centrifugation Centrifuge at 1000-5000g for 5-10 minutes to concentrate).

[0033] 3. Add different concentrations of collagen, thrombin and calcium chloride formula combination, the concentration of Co (+) added collagen is 1000-5000 μg / ml, Co (-) is collagen 10-800 μg / ml, T (+ ) is added thrombin 5~50U / ml, T(-) is thrombin 0.1~2.0U / ml, Ca(+) is calcium chloride 5~30%, Ca(-) is calcium chloride 0.1~4.0 % (Table 1), then shake and activate at 50-100rpm in a water bath at 15 to 45°C for 10 to 60 minut...

Embodiment 2

[0038] Growth factor content detection:

[0039] 1. Use the R&D system DG100 growth factor ELISA analysis kit to analyze the content of P-BP growth factor, and calculate the IGF content of P-BP according to the standard curve.

[0040]2. Use the CUSABIO CSE-E06800p growth factor ELISA analysis kit to analyze the content of P-BP growth factor, and calculate the KGF content of P-BP according to the standard curve.

[0041] 3. Use the R&D system DHD00B growth factor ELISA analysis kit to analyze the content of P-BP growth factor, and calculate the PDGF content of P-BP according to the standard curve.

[0042] 4. Use the R&D system MB100B growth factor ELISA analysis kit to analyze the content of P-BP growth factor, and calculate the TGF-β content of P-BP according to the standard curve.

[0043] 5. The results are shown in Table 2. After P-BP is activated, the secretion of all growth factors is significantly increased (p<0.05), and the secretion of TGF-β, PDGF, and KGF is higher...

Embodiment 3

[0047] Optimal Activation Formula Design:

[0048] The results of analyzing the content of growth factors with Design-Expert software were used to calculate the influence of each activator on the release of each growth factor, and expressed by analysis of variance (ANOVA) values ​​(Table 3), A is collagen, B is thrombin, C For calcium chloride, the ANOVA index shows the effect of the activator on the release of growth factors, and a larger value indicates that the activator can cause platelets to release more growth factors. Among them, the activation of collagen and calcium chloride will increase the release of TGF-β and PDGF, and calcium chloride will increase the secretion of KGF. The results refer to RSM (response surface methodology) and cube (Cube) benefit diagram ( Figure 1 to Figure 8 ), and finally Co-T+Ca-, Co+T-Ca- and Co+T+Ca- are the best possible formulations.

[0049] Table 3 is analyzed by Design-Expert software, and each activator is to the exponent of growt...

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Abstract

A method of preparing a supplement for cell cultivation media, which comprises concentrating porcine blood by centrifugation and obtaining supernatant, adding agonist for activating platelets into the supernatant to obtain activated supernatant, and sterilizing the activated supernatant is presented. A supplement for cell cultivation media, which is made by the above method, a cell cultivation media comprising the supplement, and a use of the cell cultivation media in culturing or treating cells in tissue engineering or regenerative medicine is also presented.

Description

technical field [0001] The present invention relates to a novel cell culture medium additive. Background technique [0002] In recent years, stem cells have become one of the most important studies in tissue engineering. They can be differentiated into various tissue cells for regenerative medicine, such as bone cells, nerve cells, cardiomyocytes, and islet cells. However, the most clinical and tissue applications The bottleneck is that it cannot be quickly cultured in vitro to the amount required for treatment, so that it is difficult to be widely used in medical treatment. [0003] Fetal bovine serum (FBS) is commonly used as a cell culture medium additive in many researches. Fetal bovine serum is a serum obtained from the blood of unborn fetal bovine after centrifugation to remove red blood cells. Generally, 5-20% fetal bovine serum is added to the culture medium to promote cell proliferation. The use of fetal bovine serum as a medium supplement has always been a contro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N2502/115C12N2500/84C12N5/0037C12N5/0667
Inventor 林咏凯尤伟勋
Owner 陈嘉有
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