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Mycer immortalized hepatocyte

A liver cell and fusion protein technology, applied in the field of liver cells, can solve the problems of differences between species, unpredictable in vivo toxicity, and low expression of markers

Inactive Publication Date: 2007-09-26
RENEURON LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] A number of bioanalytical systems have been used to evaluate the various isoforms of cytochrome P450 enzymes, but they all have a major disadvantage in that none of them can predict in vivo toxicity
The main disadvantage of existing assays is that only a very limited number of liver enzymes can be studied at one time, limiting the similarity between the assay and the environment of the liver itself 1，2
However, it is currently not possible to upregulate all or most hepatocyte enzymes to "normal" levels by adding substances to the culture medium or changing culture conditions 10
Some hepatic cell lines are also available from other species including rats and primates 11，12 , but these cell lines also have issues with lower expression of markers, and there are also issues with species differences

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Materials and methods

[0047] All chemicals were purchased from Sigma Corporation unless otherwise indicated.

[0048] Tissue culture

[0049] All cells were grown in a Millennium incubator (Jencon) at 37°C, 5% CO 2 grow under conditions. Cells were cultured on a regular basis with three medium changes per week. Subculture was performed when 90% confluency was reached.

[0050] culture medium

[0051] Eight media are used during tissue culture. Human fetal liver cells and clones were cultured on all of these media. These media were filter sterilized using 1 liter filter units (Nalgene) and prewarmed to 37°C before use. These eight media are referred to as media A, B, C, D, E, F, G, and H, respectively, and have the following compositions:

[0052] Medium A: arginine-free Williams E standard medium (Gibco), 2.2g / L sodium bicarbonate, 10% dialyzed fetal bovine serum (dFBS) (Gibco 26400-044), 2mM L-glutamine , 100 nM insulin, 100 IU / ml penicillin-streptomycin (Gi...

Embodiment 2

[0099] Materials and methods

[0100] All chemicals were purchased from Sigma (Dorset, UK) unless otherwise indicated.

[0101] Tissue culture

[0102] cells at 37°C, 5% CO 2 grown in a millennium incubator. The medium was changed three times a week, and the cells were passaged when the confluency was about 90%.

[0103] culture medium

[0104] The following kinds of media were used in the culture of liver tissues and clones. The medium was filter-sterilized using a 1 L filter unit (Nalgene, NY, USA) and pre-warmed to 37°C before use.

[0105] Medium A: Dulbecco's modified eagle medium (DMEM) (Invitrogen, Paisley, UK), 10% fetal bovine serum (FBS) (Hyclone Perbio Science, UT, USA), gentamicin (50mg / ml), L - Glutamine (2mM) and Epidermal Growth Factor (EGF) (20ng / ml).

[0106] Medium B: arginine-free Williams E standard medium (Invitrogen, Paisley, UK): DMEM (Invitrogen, Paisley, UK) (1:1), 10% FBS, EGF (20 ng / ml).

[0107] Medium C: DMEM (+Glutamax) (Invitrogen, Paisle...

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PUM

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Abstract

The invention relates to the production of hepatocyte cell lines useful in toxicology screens or therapy. A mammalian hepatocyte comprises, as a single polypeptide, a fusion protein comprising a c-myc protein and an oestrogen receptor, or functional fragments thereof.

Description

technical field [0001] The present invention relates to conditionally immortalized hepatocytes that can be scaled up for clinical and commercial use. Background technique [0002] The liver is responsible for the detoxification of drugs and toxins in the body. The most common type of cell in the liver is the hepatocyte; each hepatocyte performs the functions required by the entire organ. Since drugs are metabolized in the liver, Absorption, Distribution, Metabolism, Excretion and Toxicity Testing (ADME / Tox) of drug candidates is usually performed in liver tissue to obtain their potential toxicity in vivo. [0003] The traditional ADME / Tox test is performed in the late stage of new drug development, but the number of new drugs that fail the toxicity test accounts for about 30%. It is therefore highly desirable to be able to develop high-throughput ADME / Tox screens that can be applied early in drug development, thereby saving time and money. [0004] Many in vitro ADME / Tox ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/10C07K14/705C07K14/82C12N5/071
CPCC12N5/067C12N2510/04C07K2319/81C07K2319/00C07K14/82C07K14/70567A61P1/16A61P29/00A61P35/00
Inventor D·约翰逊J·辛丹P·戈尔德法布A·斯万伯格
Owner RENEURON LTD
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