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Optimized culture method for primary rat hepatocytes

A technology of primary hepatocytes and a culture method is applied in the field of optimized culture of rat primary hepatocytes, which can solve the problems that the function of hepatocytes is not as good as that of normal hepatocytes, the yield is low, and the preparation is difficult, so as to achieve mass culture and compact arrangement. Effect

Inactive Publication Date: 2016-04-20
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the various cell materials currently constructed have their own advantages and disadvantages
For example, animal-derived liver cells have a wide range of sources and can be obtained in large quantities, but they carry animal-derived diseases, which are prone to immune rejection and immunopathogenicity of foreign proteins
Another type of hepatic cell line that can be obtained in large quantities is tumor-derived hepatic cell line, which can be proliferated in large quantities and cultured on a large scale, but has tumor characteristics, and the function of hepatocytes is not as good as that of normal hepatocytes, and there is a risk of tumorigenesis
Compared with the above two types, fetal liver cells are more ideal and have fewer defects, but their source is limited, and the conditions in the process of culture and preparation are very strictly controlled, the preparation is difficult and the yield is low, and it is not easy to obtain in large quantities.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] S1. Load primary rat hepatocytes on a microwell culture plate, centrifuge at 60 g for 11 minutes, remove excess cell suspension on the surface of the microwell culture plate, and culture in hepatocyte serum-free medium for 12 hours;

[0026] S2. After the culture is completed, collect the primary hepatocytes obtained in step S1 and transfect the primary cells with SV40Tag and human telomerase reverse transcriptase to obtain transformed cells;

[0027] S32, placing the obtained modified cells in a hepatocyte serum-free medium for microcarrier suspension culture;

[0028] The hepatocyte serum-free culture fluid comprises the following components:

[0029] Basal medium 500mL, insulin 0.5μg / mL, sodium selenite 6μg / L, epidermal growth factor 5ng / mL, hepatocyte growth factor 5ng / mL, fibronectin 0.3μg / mL, dexamethasone 0.3nmol / mL and Milk thistle extract 50mg / L;

[0030] The hepatocyte serum-free medium comprises the following components:

[0031] DMEM / F12 high-glucose medi...

Embodiment 2

[0033] S1. Load primary rat hepatocytes on a microwell culture plate, centrifuge at 260 g for 3 minutes, remove excess cell suspension on the surface of the microwell culture plate, and culture in hepatocyte serum-free medium for 48 hours;

[0034] S2. After the culture is completed, collect the primary hepatocytes obtained in step S1 and transfect the primary cells with SV40Tag and human telomerase reverse transcriptase to obtain transformed cells;

[0035] S32, placing the obtained modified cells in a hepatocyte serum-free medium for microcarrier suspension culture;

[0036] The hepatocyte serum-free culture fluid comprises the following components:

[0037] Basal medium 500mL, insulin 1.1μg / mL, sodium selenite 10μg / L, epidermal growth factor 110ng / mL, hepatocyte growth factor 110ng / mL, fibronectin 1.3μg / mL, dexamethasone 10.3nmol / mL and Milk thistle extract 110mg / L;

[0038] The hepatocyte serum-free medium comprises the following components:

[0039] DMEM / F12 high-gluco...

Embodiment 3

[0041] S1. Load primary rat hepatocytes on a microwell culture plate, centrifuge at 160 g for 7 minutes, remove excess cell suspension on the surface of the microwell culture plate, and culture in hepatocyte serum-free medium for 30 hours;

[0042] S2. After the culture is completed, collect the primary hepatocytes obtained in step S1 and transfect the primary cells with SV40Tag and human telomerase reverse transcriptase to obtain transformed cells;

[0043] S32, placing the obtained modified cells in a hepatocyte serum-free medium for microcarrier suspension culture;

[0044] The hepatocyte serum-free culture fluid comprises the following components:

[0045] Basal medium 500mL, insulin 0.8μg / mL, sodium selenite 8μg / L, epidermal growth factor 57.5ng / mL, hepatocyte growth factor 57.5ng / mL, fibronectin 0.8μg / mL, dexamethasone 5.3nmol / mL and milk thistle extract 80mg / L;

[0046] The hepatocyte serum-free medium comprises the following components:

[0047]DMEM / F12 high-glucos...

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Abstract

The invention discloses an optimized culture method for primary rat hepatocytes. The optimized culture method for the primary rat hepatocytes comprises the following steps: loading the primary rat hepatocytes on a micropore culture plate, centrifuging, and culturing in a hepatocyte serum-free culture solution; after culture, collecting primary hepatocytes, obtaining modified cells through transfection, putting the modified cells into a hepatocyte serum-free culture medium, and carrying out microcarrier suspension culture, wherein the hepatocyte serum-free culture solution comprises a basal culture medium, insulin, sodium selenite, an epidermal growth factor, a hepatocyte growth factor, fibronectin, dexamethasone and a milk thistle extract, and the hepatocyte serum-free culture medium comprises a high-glucose culture medium, epidermal regenerated fibroin fiber, dexamethasone, a nostoc sphaeroids kutz extract and the like. The optimized culture method disclosed by the invention has the advantages that primary hepatocytes are compactly planted by utilizing mechanics, the hepatocytes can be arranged compactly, close contact is formed, and high-density culture of the hepatocytes can be quickly formed; and a serum-free culture medium formula is adopted, so that the serum-free culture medium is more applicable to growth of the hepatocytes and function exertion, and mass culture of the hepatocytes in vitro is realized.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to an optimized culture method for primary rat hepatocytes. Background technique [0002] Liver failure is severe liver damage caused by a variety of factors, leading to serious impairment or decompensation of its synthesis, detoxification, excretion, and biotransformation functions, with coagulation dysfunction, jaundice, hepatic encephalopathy, and ascites as the main manifestations. A group of clinical syndromes. Among the current treatment methods for liver failure, the bioartificial liver system is the best choice. Biological artificial liver refers to the artificial liver support system composed of organs, tissues and cells of the same or different species of animals combined with special materials and devices, which can temporarily replace the liver function, thereby assisting treatment. [0003] Bioartificial livers include previous isolated liver perfusion, human-mammalian cro...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/071
CPCC12N5/067
Inventor 杨献光王棋文靳伟常翠芳朱琳和春翠
Owner HENAN NORMAL UNIV