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Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell

A mammalian and epithelial cell technology, applied in the fields of cell biology and toxicology, can solve problems such as the inability to establish normal cell lines that are immortalized without gene introduction, interference with cell physiological pathways, and immortalized cells that cannot maintain normal phenotypes.

Inactive Publication Date: 2015-06-10
SHENZHEN RES INST OF WUHAN UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many cell immortalization methods have been established, most of them introduce exogenous viruses and proto-oncogenes into target cells through virus introduction and gene transfection. In the internal physiological pathway, cells undergo unexpected changes. Compared with normal cells, their biological properties such as karyotype and serum-dependent type may change, and some even show the characteristics of tumor cells. The established immortalized cells cannot maintain its original normal phenotype
However, so far, it has not been possible to establish a normal cell line without gene transfer and immortalization as a research model in toxicology

Method used

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  • Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell
  • Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell
  • Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1 is used for the culture medium of the normal epithelial cell of isolation culture, subculture people or mammal

[0080] Its components include: a mixed medium of DMEM and Ham's F-12NUTRIENT MIX (GIBCO), the volume ratio is 3:1, while adding 5% fetal bovine serum (GIBCO), 2nM triiodothyronine (Sigma), 0.5% insulin-transferrin -selenium reagent (Life Technologies), 5 μg / ml transferrin (Life Technologies), 10 ng / mL epidermal growth factor (Sigma), 0.4 μg / mL hydrocortisone (Sigma), 1 nM cholera toxin (List Biological Labs, Campbell, CA ), 0.5 μg / mL amphotericin B (Fungizone; Bristol-Myers Squibb, Park Avenue NY), 40 μg / mL gentamicin (Gentacin; Schering-Plough, Kenilworth, NJ), 50 nM calpeptin (ENZO Life Sciences, Farmingdale, NY), 40ng / ml recombinant human IL-1RA (PeProTech), 3ug / ml recombinant human R-Spondin-1 (PeProTech).

Embodiment 2

[0081] Example 2 The Primary Isolation and Culture Method of Human or Mammalian Normal Epithelial Cells

[0082] 1) With the informed consent of the patient or patient guardian, collect normal tissue samples surgically removed from clinical patients; or strictly follow the animal experiment procedures and regulations of ARRIVE (Animal Research: Reporting of In Vivo Experiments), and collect normal tissue samples from mammals. tissue samples;

[0083] 2) Preparation of digestive solution: the above-mentioned medium containing collagenase / hyaluronidase (mixed solution with a final concentration of 1x) (the composition is a mixed medium of DMEM and Ham's F-12NUTRIENT MIX (GIBCO), and the volume ratio is 3:1, while adding 5% fetal bovine serum (GIBCO), 2nM triiodothyronine (Sigma), 0.5% insulin-transferrin-selenium reagent (Life Technologies), 5μg / ml transferrin (Life Technologies), 10ng / mL epidermal growth factor (Sigma ), 0.4 μg / mL hydrocortisone (Sigma), 1 nM cholera toxin (Li...

Embodiment 3

[0092] The subculture method of embodiment 3 people or mammalian normal epithelial cell

[0093] 1) When normal human or mammalian epithelial cells proliferate to 70-90% abundance, wash the cells twice with 1xPBS, and digest the monolayer cells with 0.05% trypsin / EDTA for 2-5 minutes.

[0094] 2) 10ml of complete DMEM neutralizes the digestion reaction.

[0095] 3) Centrifuge at 1000rmp for 5 minutes to remove the supernatant.

[0096] 4) In a certain ratio, such as 1:2:3, 1:4, 1:5, as long as the cells can adhere to the wall and grow normally), resuspend the cell pellet in 10ml of the medium in Example 1 for inoculation.

[0097] 5) If necessary, 1x10 6 Epithelial cells were resuspended in 1-2ml cell freezing solution (90% fetal bovine serum and 10% DMSO), and stored in liquid nitrogen for later use.

[0098] Normal epithelial cells derived from different tissues of human and mouse were isolated, cultured and subcultured according to the above method, and the morphology of...

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Abstract

The invention relates to a culture medium for a normal epithelial cell of a human or mammal, primary separation culture, subculture, 3D gas-liquid culture and 3D matrigel culture methods, the normal epithelial cell generated by using the culture medium and the culture methods and application of the normal epithelial cell to a toxicological evaluation system. The culture medium is prepared by mixing DMEM and Ham's F-12NUTRIENT MIX according to the volume ratio of 3:1 and also adding 4-6% of fetal calf serum, 1-3nM triiodothyronine, 0.4-0.65% of insulin-transferrin-selenium reagent, 4-6mu g / ml transferrin, 9-11ng / mL epidermal growth factors, 0.3-0.5mu g / mL hydrocortisone, 0.5-1.5nM cholera toxin, 0.4-0.6mu g / mL amphoterrible B, 35-45mu g / mL gentamicin, 45-55nM calpeptin, 35-45ng / ml recombinant human IL-1RA and 3mu g / ml recombinant human R-Spondin-1. The culture medium disclosed by the invention can be used for carrying out separation culture or subculture on the normal epithelial cell of the human or the mammal and any other various tissue source, rapidly proliferating the normal epithelial cell in vitro and establishing a cell line; and the normal epithelial cell is a normal diploid cell and is applied to the toxicological evaluation system of the human or the mammal.

Description

technical field [0001] The present invention relates to the field of cell biology and toxicology, in particular to culture medium, primary separation culture, subculture, air-liquid 3D culture and Matrigel 3D culture method of human or mammalian normal epithelial cells, as well as the culture medium, culture Methods Generated normal epithelial cells and their application in toxicology evaluation system. Background technique [0002] Environmental pollution has seriously endangered people's health, and respiratory diseases caused by it have also emerged continuously. Human epithelial cells are the first to be the main damage target. Organ-specifically differentiated epithelial cells perform important functions such as gas exchange in the lungs, filtration in the kidneys, detoxification and binding in the liver, insulin secretion by pancreatic islet cells, and skin resistance to harmful environments. Therefore, epithelial cell-based in vitro models have been used to investig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
Inventor 李晖李红亮刘红亚王玲
Owner SHENZHEN RES INST OF WUHAN UNIVERISTY
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