Construction method for hepatic progenitor cell-like cell bank and cell lines prepared by method and applications of cell bank and cell lines

A construction method and progenitor cell technology, applied in the construction method and its prepared cell lines and application fields, can solve the problems of affecting the in vitro proliferation ability of mature hepatic parenchymal cells, the single source of donors, and the inability to carry out drug-specific toxicity research, etc. Achieving good in vitro proliferative ability

Active Publication Date: 2020-07-10
SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method first genetically modifies the hepatic parenchymal cells, and then carries out subsequent proliferation and differentiation cultures, which easily affects the in vitro proliferation ability of the obtained mature hepatic parenchymal cells, and the source of the donor is single, which makes it impossible to carry out drug-specific toxicity studies

Method used

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  • Construction method for hepatic progenitor cell-like cell bank and cell lines prepared by method and applications of cell bank and cell lines
  • Construction method for hepatic progenitor cell-like cell bank and cell lines prepared by method and applications of cell bank and cell lines
  • Construction method for hepatic progenitor cell-like cell bank and cell lines prepared by method and applications of cell bank and cell lines

Examples

Experimental program
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Embodiment 1

[0066] This example provides the first immortalized hepatic progenitor cell-like cell line, classified as 81.5, preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: C2019125, and the first immortalized hepatic progenitor cell-like cell line. A method for constructing an immortal hepatic progenitor-like cell line.

[0067] The coating solution in this embodiment is a mixed solution of HepX medium and Matrigel, and the volume ratio of the HepX medium to the Matrigel is 80:1.

[0068] When the culture device is a 6-well cell culture plate, add 250-350 microliters of coating solution to each culture well to perform the coating treatment in advance.

[0069] When the culture device is a 12-well cell culture plate, add 150-200 microliters of coating solution to each culture well to perform the coating treatment in advance.

[0070] When the culture vessel is a cell culture dish with a diameter of 6 cm, add 500-700 microliters of coating ...

Embodiment 2

[0098] This embodiment provides a method for constructing a second immortal hepatic progenitor cell-like cell line and a second hepatic progenitor cell-like cell line obtained by the method for constructing a second immortal hepatic progenitor cell-like cell line.

[0099] The difference between the construction method of the second immortalized hepatic progenitor cell line and the construction method of the first immortalized hepatic progenitor cell-like cell line in Example 1 is that:

[0100] In the step S1, after separating and treating the human liver hemangiomatoma tissue derived from the second donor, the obtained second primary human hepatocyte culture was transformed and cultured for 14 days to obtain the second liver Progenitor-like cell lines. The inoculation density of the second human primary hepatocyte culture is 5×10 4 pieces / square centimeter. The number of times of expansion culture is 2, specifically the first expansion culture and the second expansion cult...

Embodiment 3

[0102] This embodiment provides a method for constructing a third immortal hepatic progenitor cell-like cell line and a third hepatic progenitor cell-like cell line obtained by the method for constructing a third immortal hepatic progenitor cell-like cell line.

[0103] The difference between the construction method of the third immortal hepatic progenitor cell-like cell line and the construction method of the first immortal hepatic progenitor cell-like cell line in Example 1 is that:

[0104] In the step S1, after the human liver hemangioma tissue from the third donor is separated and treated, the obtained third human primary hepatocyte culture is subjected to the transformation culture to obtain the third hepatic progenitor Cell-like cell line.

[0105] In the step S2, after thawing the frozen culture to be multiplied, 0.5×10 4 Inoculate at an inoculation density of one per square centimeter.

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Abstract

The invention provides a construction method for a hepatic progenitor cell-like cell bank. The method includes the following steps: performing transformation culture on human primary hepatocyte cultures derived from different donors, performing cryopreservation treatment, performing propagation culture, performing primary passage treatment, performing virus infection, performing secondary passagetreatment, performing continuous screening culture and performing continuous passage culture. According to the construction method for the heterogeneous immortal hepatic progenitor cell-like cell bankprovided by the invention, the human primary hepatocyte cultures derived from the donors are firstly subjected to transformation culture before proliferation culture, so that the method is beneficialto giving good proliferation performance to the human primary hepatocyte cultures, and culture parameters are controlled in the subsequent stage, so that obtained immortal hepatic progenitor cell-like cell lines derived from the different donors have good in-vitro proliferation ability. The invention also provides an application of the liver progenitor cell-like cell bank and the cell lines obtained by the construction method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a hepatic progenitor cell-like cell bank, a cell line prepared therefrom and applications thereof. Background technique [0002] Liver failure is the end-stage manifestation of severe liver disease, and the mortality rate of patients can be as high as 50% to 90%. Among them, the problem of drug-induced liver injury caused by adverse drug reactions in patients is more prominent, accounting for more than 50% of acute liver failure cases, which leads to drug development failure or withdrawal after marketing. [0003] The majority of drug development failures or post-marketing withdrawals are due to patient-specific toxicity. In the prior art, liver cell immortalization is one of the important ways to solve the source of liver cells. Usually, primary liver cells or hepatocyte-like cells differentiated from induced stem cells are immortalized, and the obtained c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12Q1/02A61K35/407A61P1/16
CPCC12N15/86C12N5/0672G01N33/5014G01N33/5067A61K35/407A61P1/16C12N2740/15043C12N2800/107C12N2510/04C12N2503/02G01N2500/10C12N2740/16043C12N5/0671C12N2533/90C12N5/067C12N2513/00
Inventor 鄢和新
Owner SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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