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Human serum-free culture medium and preparation method thereof

A serum-free medium, human-derived technology, applied in the field of human-derived serum-free medium and its preparation, to achieve the effect of improving safety and standardization

Active Publication Date: 2013-05-08
湘雅生物医药(湖州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is imperative to develop a human-derived serum-free medium. At present, there is no commercialized human-derived serum-free medium at home and abroad.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of human-derived serum-free medium

[0030] A. Preparation of iron-saturated human transferrin solution:

[0031] (1), first the FeCl of 16.22mg 3 Dissolve with 10ml of 1mM HCl to make a stock solution, then aliquot and store at -20°C to obtain No. 1 stock solution;

[0032] (2) Weigh 120 mg of human transferrin, dissolve it in 4.0 ml DMEM without adding sodium bicarbonate, then add 270 μl of No. 1 storage solution, and stir evenly to obtain an iron-saturated human transferrin solution;

[0033] B. Preparation of human cholesterol solution: Weigh 50 mg of human cholesterol powder, add 50 ml of DMEM without sodium bicarbonate, oscillate with an ultrasonic oscillator with a 2 cm diameter titanium probe, and oscillate at the maximum amplitude for 1 hour in a refrigerator at 4 ° C. Make it completely emulsified, filter and sterilize successively with 1.2 μm and 0.45 μm filter membranes to obtain a human cholesterol solution, which is stored at -30°C...

Embodiment 2

[0036] Example 2: Preparation of human-derived serum-free medium

[0037] A. Prepare iron-saturated human transferrin solution:

[0038] (1), first 27.30mg of FeCl 3 Dissolve with 20ml of 1mM HCl to prepare a stock solution, then aliquot and store at -20°C to obtain No. 1 stock solution;

[0039] (2) Weigh 200 mg of human transferrin, dissolve it in 10.0 ml of DMEM without adding sodium bicarbonate, then add 400 μl of No. 1 storage solution, and stir evenly to obtain an iron-saturated human transferrin solution;

[0040] B. Preparation of human cholesterol solution: Weigh 100 mg of human cholesterol powder, add 100 ml of DMEM without sodium bicarbonate, oscillate with an ultrasonic oscillator with a 2 cm diameter titanium probe, and oscillate at the maximum amplitude for 1 hour in a refrigerator at 4 ° C. Make it completely emulsified, filter and sterilize successively with 1.2 μm and 0.45 μm filter membranes to obtain a human cholesterol solution, which is stored at -30°C; ...

Embodiment 3

[0043] Example 3: Preparation of human-derived serum-free medium

[0044] A. Preparation of iron-saturated human transferrin solution:

[0045] (1), first 20mg of FeCl 3 Dissolve with 15ml of 1mM HCl to make a stock solution, then aliquot and store at -20°C to obtain No. 1 stock solution;

[0046] (2) Weigh 200 mg of human transferrin, dissolve it in 10.0 ml of DMEM without adding sodium bicarbonate, then add 400 μl of No. 1 storage solution, and stir evenly to obtain an iron-saturated human transferrin solution;

[0047] B. Preparation of human cholesterol solution: Weigh 100 mg of human cholesterol powder, add 100 ml of DMEM without sodium bicarbonate, oscillate with an ultrasonic oscillator with a 2 cm diameter titanium probe, and oscillate at the maximum amplitude for 1 hour in a refrigerator at 4 ° C. Make it completely emulsified, filter and sterilize successively with 1.2 μm and 0.45 μm filter membranes to obtain a human cholesterol solution, which is stored at -30°C;...

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Abstract

The invention discloses a human serum-free culture medium and a preparation method thereof. The human serum-free culture medium uses eleven raw materials, namely human serum albumin solution for treatment, human recombinant insulin solution, human transferrin solution, human cholesterol solution, human catalase solution, 2-mercaptoethanol solution, ascorbic acid solution, linoleic acid solution, ethanolamine solution, human vitronectin solution and L-glutamine solution; the added protein and lipid are both from the blood plasma, serum or tissue of human, the protein is pharmaceutical-grade orhighly purified human protein or human recombinant protein, the protein and lipid do not contains any animal component, the other components all meet the United States Pharmacopoeia or national standards; and the human serum-free culture medium is qualified through the cell culture test and is clinical, safe and reasonable human serum-free culture medium. The proliferation rate of the CIK cell cultured and inducted by the human serum-free culture medium is better than that of the CIK cell cultured and inducted by the culture medium with serum, the cell CD3+CD56+ percentage and the killing rate to the K562 leukemic cell are similar to that of the culture medium with serum. By adopting the human serum-free culture medium, the safety and standardization of cell therapy can be increased.

Description

technical field [0001] The invention relates to medical experiment supplies, in particular to a human-derived serum-free medium and a preparation method thereof. Background technique [0002] Cell culture media containing animal or human serum have been used since the establishment of tissue and cell culture techniques. In the 1970s, biologists were worried about the influence of uncertain substances in animal or human serum on cultured cells, especially the danger posed by pathogens in animal or human serum. With the further development of stem cell research, it is now possible to infuse or transplant in vitro cultured and expanded stem cells into the human body for cell replacement therapy, so it is very necessary to develop a human-derived serum-free medium. In 2007, we searched for and started research on human-derived broad-spectrum serum-free medium. [0003] At present, there is no formal production of human-derived serum-free medium at home and abroad. The serum-f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07C12N5/078C12N5/0789
Inventor 戴育成
Owner 湘雅生物医药(湖州)有限公司
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