Special culture medium and culture method for long-term maintenance, propagation, and subcultring of human hepatocyte

A technology of subculture and culture medium, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems that liver cells cannot be used as a qualified cell model for liver disease research, complicated operations, and impossible to source liver cells.

Inactive Publication Date: 2015-12-16
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to many interfering factors (such as symbiotic culture), complicated operations (sandwich culture), or the inability to directly observe the morphological changes of cells (suspension culture and three-dimensional culture), it cannot meet the needs of liver disease research, so it is only partially used. To detect the hepatotoxicity of new drugs
However, even when used to detect hepatotoxicity of new drugs, the results obtained are only of relative significance and value due to the above-mentioned various defects
The bigger problem is that none of the above-mentioned existing methods for culturing human hepatocytes can proliferate and pass on
Therefore, the existing methods and the cultured hepatocytes not only cannot be used as qualified cell models for liver disease research, but also cannot provide qualified hepatocyte sources for the construction of bioartificial liver and hepatocyte transplantation

Method used

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  • Special culture medium and culture method for long-term maintenance, propagation, and subcultring of human hepatocyte
  • Special culture medium and culture method for long-term maintenance, propagation, and subcultring of human hepatocyte
  • Special culture medium and culture method for long-term maintenance, propagation, and subcultring of human hepatocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, preparation human hepatocyte culture medium

[0093] 1. Hepatocyte basal medium was prepared according to conventional methods, that is, the ingredients in Table 4 were added to DMEM / F12 (basic cell culture medium) (percentages are in v / v):

[0094] Table 4

[0095] N2

2.5％

B27

5％

Non-essential amino acids (Non-AA)

1％

Sodium pyruvate

1％

Penicillin and streptomycin mixture (100x)

1％

[0096] 2. On the basis of the basic medium for hepatocytes above, add the components with final concentrations as shown in Table 5 to obtain hepatocyte culture medium I-1.1 / 2, I-2.1 / 2 and hepatocyte culture medium I-3.1 respectively / 2 / 3 / 4.

[0097] table 5

[0098]

Embodiment 2

[0099] Example 2, long-term proliferation, subculture and maintenance of human hepatocytes

[0100]1. Hepatocyte Culture Medium Ⅰ-1.1, Hepatocyte Culture Medium Ⅰ-1.2 Culture of primary human hepatocytes

[0101] (1) Primary hepatocytes (separated from excised human liver tissue) were purchased from Lifetechnology Company.

[0102] (2) Prime the culture plate with matrigel (1:30) for 1 hour, then suspend the primary hepatocytes in the adherent medium (basic medium for hepatocytes supplemented with 10% serum), plate and store at 37°C Incubate for 1 hour;

[0103] (3) The culture medium in (2) was discarded, and the hepatocytes were cultured in hepatocyte medium I-1.1 and I-1.2 respectively.

[0104] Using hepatocyte culture medium Ⅰ-1.1 and Ⅰ-1.2, when cultured for 1, 14 and 20 days, the cell morphology was taken (the cells cultured without adding GSK3β inhibitor and TGFβ inhibitor were used as control), and the morphology diagram was as follows: figure 1 .

[0105] Depend ...

Embodiment 3

[0124] Embodiment 3, identification of liver cell characteristics

[0125] (1) Immunostaining

[0126] The immunostaining method is:

[0127] 1. Discard the cell culture medium, rinse with PBS once,

[0128] After fixing with 2.4% paraformaldehyde for 10 minutes, rinse with PBS for 5 minutes x 3 times,

[0129] 3. 10% goat serum blocking: room temperature, 60 minutes,

[0130] 4.0.2% Triton: 25-30min,

[0131] 5. Primary antibody (rabbit anti-ALB antibody, mouse anti-CYP3A antibody or rabbit anti-Asgpr antibody) at room temperature for 1 hour

[0132] or overnight at 4°C,

[0133] 6. Rinse with PBS for 5 minutes x 3 times,

[0134] 7. Secondary antibody (Cy3-labeled goat anti-rabbit antibody, FITC-labeled goat anti-mouse or goat anti-rabbit antibody) at room temperature

[0135] 45-60min,

[0136] 8. Wash with PBS, 5min×3 times,

[0137] 9. Seal the slides and take pictures with a fluorescence microscope.

[0138] The cells cultured in vitro for 84 days were immunost...

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Abstract

The invention discloses a special culture medium and culture method for long-term maintenance, propagation, and subcultring of human hepatocyte. The optimized culture medium for long-term culture of hepatocyte is disclosed for the first time. By using the culture medium, long-term propagation, subcultring and maintenance of the human hepatocyte can be performed, and forms and functions of the hepatocyte can be maintained. The culture medium is applied to culture hepatocyte, and the culture method is simple to operate and low in cost, and is safe and stable. The human hepatocyte obtained from culture, propagation and subcultring is applicable to clinical cell transplantation for treating liver diseases, is used for mechanism research related to liver diseases and for bioartificial liver construction, and is used as a hepatocyte source or a cell model for basic research relative to medicine hepatotoxicity, detection of medical efficacy and medical target, and the like. The culture medium and culture method has a bright application prospect.

Description

technical field [0001] The invention belongs to the fields of biology and medicine; more specifically, the invention relates to a special culture medium and a culture method for long-term maintenance, proliferation and subculture of human hepatocytes in vitro. Background technique [0002] According to the statistics of the World Health Organization, millions of people die of liver disease every year in the world. China is a big country with liver disease. There are 140 million hepatitis B and C virus carriers alone, accounting for 28% of the global total; there are 297,000 liver cancer patients, accounting for more than half (55%) of the global total. Therefore, a large number of qualified human hepatocytes are needed in liver disease research, new drug hepatotoxicity detection, bioartificial liver and hepatocyte transplantation. However, the lack of liver sources is a global problem, and the existing human hepatocyte culture methods all have the short time for conventiona...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 张培霖王红阳
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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