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Liver oval cell immortalization culture medium as well as preparation method and application thereof

A technology of hepatocyte growth factor and culture medium is applied in the field of immortalization medium of hepatic oval cells and preparation thereof, and can solve the problem of difficulty in immortalizing and culturing isolated hepatic oval cells of mice and the like.

Pending Publication Date: 2021-11-02
成都以邦医药科技有限公司
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  • Abstract
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Problems solved by technology

[0004] The present invention intends to provide a liver oval cell immortalization medium to solve the technical problem that it is difficult to immortalize isolated mouse liver oval cells

Method used

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  • Liver oval cell immortalization culture medium as well as preparation method and application thereof
  • Liver oval cell immortalization culture medium as well as preparation method and application thereof
  • Liver oval cell immortalization culture medium as well as preparation method and application thereof

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Experimental program
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Embodiment 1

[0025] Embodiment 1: immortalization medium and cell culture

[0026] In this scheme, the basic components of the immortalization medium are DMEM / F12 (1:1) medium and fetal bovine serum, wherein, DMEM / F12 (1:1) medium is composed of DMEM medium and F12 medium , the volume ratio of DMEM medium and F12 medium is 1:1. A variety of additive factors are added to the basic ingredients, including hepatocyte growth factor, epidermal growth factor, leukemia inhibitory factor, double antibody, L-glutamine and fetal bovine serum. The contents of hepatocyte growth factor, epidermal growth factor, leukemia inhibitory factor, double antibody, L-glutamine and fetal bovine serum in the immortalization medium were 10μg / L, 20μg / L, 10μg / L, 100U / ml , 2mmol / L and 10% (volume percentage). Among them, the double antibody is penicillin and streptomycin, and both double antibody and fetal bovine serum are substances often used in the process of cell culture to avoid contamination of the culture syst...

Embodiment 2

[0048] Embodiment 2: Separation and acquisition of primary HOC

[0049] The primary HOC was prepared by the following method: 8-week-old male C57BL / 6 mice were added to the drinking water with 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydro-collidine), and fed continuously for 2 weeks to construct DDC mouse model, the mice were sacrificed on the 14th day for detection. figure 2 The pathological conditions of the liver after the establishment of the DDC mouse model are shown. After 2 weeks of induction, the DDC mice developed obvious cholestatic cholangitis. Immunohistochemical staining showed that oval cells aggregated and proliferated around the portal vein in the portal area of ​​the liver and expressed CK19 and ALB , indicating that the model was built successfully.

[0050] The DDC model mice were anesthetized with isoflurane, opened the abdomen and exposed the hepatic portal vein, placed a heparinized venous indwelling needle and fixed it; washed the intrahepatic blood with ...

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Abstract

The invention relates to the technical field of cancer cell culture, in particular to a liver oval cell immortalization culture medium and a preparation method and application thereof. The liver oval cell immortalization culture medium comprises basic components and additive factors, the additive factors comprise a hepatocyte growth factor, an epidermal growth factor and a leukemia inhibition factor. The liver cell growth factor, the epidermal growth factor and the leukemia inhibition factor are combined for use, so that the immortalization process of the mouse liver oval cells can be effectively promoted, the liver oval cells can at least maintain the cell proliferation activity for more than 8 weeks under the in-vitro condition, cell differentiation is avoided, and the cells are round or oval. According to the scheme, the culture medium can enable the hepatic oval cells to have the characteristic of repeated passage, can be applied to practical operation of maintaining the proliferative activity of the hepatic oval cells and inhibiting differentiation of the hepatic oval cells, and can provide more cell resources for researchers.

Description

technical field [0001] The invention relates to the technical field of cancer cell culture, in particular to a hepatic oval cell immortalization medium and a preparation method and application thereof. Background technique [0002] Cells derived from normal tissues can divide and grow under in vitro culture conditions, but after a limited number of passages, they stop proliferating, senescence and die. Some cells can escape from the crisis of proliferation and senescence spontaneously or affected by external factors, and thus have the ability to proliferate indefinitely. This process is called cell immortalization. Immortalized cells can provide a stable and uniform source of cells with consistent traits, and can reduce material costs. Immortalized cells are ideal models for studying cell proliferation, differentiation, apoptosis, aging, etc. in vitro. At this stage, there are already a large number of commercial immortalized cells, namely standard cell lines, which create...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2501/18C12N2501/235C12N2501/11
Inventor 邱焕迪郑利孙媛媛潘聪何林烨
Owner 成都以邦医药科技有限公司
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