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.
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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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