Serum-free medium for porcine hepatocytes and preparation method of serum-free medium

A serum-free medium and cell technology, applied in cell culture medium, cell culture active agents, biochemical equipment and methods, etc. Characteristics and biological functions, maintenance of growth state, effect of good growth state

Inactive Publication Date: 2017-08-15
SHAOGUAN COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some researchers have used other serum-free media for related research, but these serum-free media contain animal-derived components and cannot be used for suspension culture of porcine primary liver cells, which is difficult to meet the needs of scientific research and clinical applications
Other commercial hepatocyte serum-free media have many problems, such as the secret formula of the media, high price, and uncertain preparation methods, which make it difficult to popularize and apply in the field of serum-free culture of hepatocytes in vitro
Although some domestic patent applications disclose some serum-free hepatocyte culture medium, the developed serum-free hepatocyte culture medium can be applicable to the bioartificial liver support system, but it is difficult to be used or suitable for the cultivation of porcine hepatocyte serum-free. The research and application of the latter cannot be satisfied, so it is urgent to develop a new serum-free medium for porcine hepatocytes

Method used

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  • Serum-free medium for porcine hepatocytes and preparation method of serum-free medium
  • Serum-free medium for porcine hepatocytes and preparation method of serum-free medium

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preparation example Construction

[0030] The preparation method of pig hepatocyte serum-free medium of the present invention comprises the following steps:

[0031] S1: In a sterile environment, prepare the mother solution of each component according to the content of each component.

[0032] Specifically, in step S1, the preparation method of the mother liquor of each component is as follows:

[0033] Dissolve bovine insulin, human transferrin, sodium selenite and ethanolamine in water to prepare a solution containing 10 μg / mL bovine insulin, 5.5 μg / mL human transferrin, 2.9×10 -8 The mother liquor of M sodium selenite and 2 μ g / mL ethanolamine;

[0034] Dissolving dexamethasone dry powder in absolute ethanol to prepare a dexamethasone ethanol solution with a concentration of 1 mg / mL, and then diluting it with basal culture medium to a concentration of 0.1 mg / mL to obtain a mother solution of dexamethasone;

[0035] Fibronectin is dissolved in PBS buffer solution, is formulated into the fibronectin stock so...

Embodiment 1

[0046] A serum-free culture medium for pig hepatocytes of the present invention, which is added with the following components in the basal culture medium: bovine insulin 10.00 mg / L, human transferrin 5.50 mg / L, sodium selenite 5.02 μg / L, ethanolamine 2.00mg / L, dexamethasone 196.26μg / L, fibronectin 0.5mg / L, vitamin C 10.00mg / L, hepatocyte growth factor 60.00μg / L, epidermal growth factor 80.00μg / L, glucagon 2.00 mg / L and bovine serum albumin 1.50g / L.

[0047] The basal culture solution is Williams' Medium E basal culture solution, more preferably Williams' Medium E basal culture solution containing penicillin and streptomycin. The basal culture solution can effectively inhibit bacterial growth and avoid cell contamination.

[0048] A preparation method of pig hepatocyte serum-free medium of the present invention comprises the following steps:

[0049] Step S1: In a sterile environment, prepare the mother liquor of each component according to the content of each component. spe...

Embodiment 2

[0062] The porcine hepatocyte serum-free medium of the present invention is used for the cultivation of porcine primary hepatocytes, and its operation is as follows.

[0063] After the piglet liver cells were separated and counted, the cell suspension was diluted to 3×10 with the adherent culture medium. 5 cells / mL, inoculate 2 mL in a 6-well plate, then inoculate 100 μL in a 96-well plate, place at 37°C, 5% CO 2 Cultured in a cell incubator with saturated humidity. After 4 hours of cell attachment, the medium was completely replaced with the growth medium, which was a basal medium containing 5 wt% neonatal bovine serum and 100 IU / mL double antibody, to remove dead cells and tissue debris, and continued to cultivate for 20 hours. Then use the serum-free medium of the present invention to change the whole amount of the medium. The serum-free medium is: the following components are added to the basal culture medium: 10 μg / mL bovine insulin, 5.5 μg / mL human transferrin, 2.9×10 ...

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Abstract

The invention relates to a serum-free medium for porcine hepatocytes and a preparation method of the serum-free medium. According to the serum-free medium for the porcine hepatocytes, a basic culture solution is added with the following components: 10.00mg/L of bovine insulin, 5.50mg/L of human transferrin, 5.02mg/L of sodium selenite, 2.00mg/L of ethanol amine, 196.26mug/L of dexamethasone, 0.5mg/L of fibronectin, 10.00mg/L of vitamin C, 60.00mug/L of hepatocyte growth factors, 80.00mug/L of epidermal growth factors, 2.00mg/L of glucagon and 1.50g/L of bovine serum albumin. The serum-free medium for the porcine hepatocytes is capable of being used for culturing porcine primary hepatocytes, the porcine primary hepatocytes are very equivalent to that cultured by a serum-containing medium in form and vigor and the growth condition is obviously good; the original biological characteristics and biological functions of the porcine hepatocytes can be well kept; the negative effects caused by a traditional serum-containing medium can be avoided; the condition requirements of normal growth and division and proliferation of the hepatocytes can be met.

Description

technical field [0001] The invention relates to the technical field of animal cell culture, in particular to a serum-free culture medium for pig hepatocytes and a preparation method thereof. Background technique [0002] The liver is an important organ for human and animal bodies to store glycogen, regulate metabolism, deoxidize, secrete active factors, and biotransform, and it is also the main site for drug metabolism and detoxification. Liver cells are the main executors of the above-mentioned biological functions of liver tissue. For this reason, many in vitro experiments often use primary human or livestock hepatocytes as research models to develop the physiological regulation function of liver cells, metabolic diseases, and pharmacology. , Molecular mechanisms of pathology and toxicology, establishment of virus-infected cell models, and research in the fields of physical and chemical factors and toxic effects. The in vitro culture of primary hepatocytes is the first st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0037C12N5/067C12N2500/25C12N2500/38C12N2500/46C12N2500/90C12N2501/11C12N2501/12C12N2501/335C12N2501/855C12N2501/998
Inventor 唐胜球江青艳董小英朱晓彤宾艳芳周桂炫方心灵
Owner SHAOGUAN COLLEGE
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