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Serum-free epithelial cell culture fluid

A technology of epithelial cells and culture medium, applied in the field of biomedicine, can solve the problems of aging, easy apoptosis of cells, slow growth of epithelial cells, etc., and achieve the goal of regulating cell proliferation and differentiation, synergistically inhibiting cell apoptosis, and avoiding proliferation inhibition Effect

Active Publication Date: 2017-11-03
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The embodiment of the present invention provides a serum-free epithelial cell culture medium, aiming at the slow growth of epithelial cells and the decline of original stem cell characteristics under the current serum-free culture system , Cells are prone to apoptosis, aging phenomenon, and difficult to expand in large quantities. Based on the basal medium, the present invention provides a serum-free medium system that can enable high-density proliferation of epithelial cells, reducing or Inhibit the cytotoxic damage produced in the process of cell culture, inhibit cell apoptosis, delay cell aging, and make it expand more times in vitro

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Examples

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Embodiment 1

[0033] An embodiment of the present invention provides a serum-free epithelial cell culture solution, which specifically includes basal culture solution, essential supplementary factors, cell growth factors, adhesion factors, hormones, cell proliferation and differentiation regulators, and apoptosis death inhibitors.

[0034] Among them, the essential supplement factors are insulin and transferrin with a concentration of 5-20 μg / ml; the cell growth factors are epidermal growth factor EGF, nerve growth factor NGF, insulin growth factor IGF-I, Transforming growth factor TGFβ and bovine pituitary extract (BPE); adherence factor is fibronectin with a concentration of 5-50ng / ml; hormone is hydrocortisone with a concentration of 1-10μM; cell proliferation and differentiation regulator is a concentration of both 1-5μM retinoic acid, glycolic acid and CaCl2; the apoptosis inhibitor is a combination of P53 inhibitor Pifithrin-α, antioxidant resveratrol and flavin adenine dinucleotide F...

Embodiment 2

[0055] Synergistic inhibition of apoptosis by p53 inhibitor Pifithrin-α, antioxidant resveratrol and flavin adenine dinucleotide (FAD).

[0056] Experimental operation method:

[0057] First, based on the basal medium MCDB153 and essential supplementary factors, cell growth factors, adherence factors, hormones, cell proliferation and differentiation regulators, and apoptosis inhibitors, prepare three types of medium: (1) Add only natural extracts The antioxidant resveratrol; (2) only add flavin adenine dinucleotide FAD (3) only add P53 inhibitor Pifithrin-α, (4) add P53 inhibitor Pifithrin-α, natural extract anti Oxidants resveratrol and flavin adenine dinucleotide (FAD).

[0058]Oral mucosal epithelial cells of passage P1 were resuscitated, inoculated into 25ml culture flasks containing these four mediums, and then placed in a 37°C, 5% CO2 incubator for culture, and the culture medium was replaced every 2 days. When the oral mucosal epithelial cells were 80% to 90% confluen...

Embodiment 3

[0061] Oral mucosal epithelial cells were cultured with serum-free medium MCDB153 and the serum-free medium provided in the examples of the present invention, and a comparative study of cell apoptosis rate during subculture:

[0062] Experimental operation method:

[0063] Under sterile conditions, recover the frozen P2 oral mucosal epithelial cells, and then inoculate them in 25ml culture flasks of serum-free medium MCDB153 and the culture medium provided in the examples of the present invention, and place them at 37°C with 5% Cultured in a CO2 incubator, the culture medium was replaced every 2 days. When the oral mucosal epithelial cells were 80%-90% confluent, they were digested to form a cell suspension, and then inoculated in a 6-well plate at a cell density of 6E4 cells / well, and cultured in a 5% CO2 incubator at 37°C. Culture medium was changed once a day. The apoptosis rate of the cells was analyzed by flow cytometry on the 6th day of culture.

[0064] The results s...

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Abstract

An embodiment of the invention provides a serum-free epithelial cell culture fluid, and relates to the field of biomedicine. The serum-free epithelial cell culture fluid comprises a basic culture fluid, necessary added factors, growth factors, anchoring factors, hormones, regulatory factors for cell proliferation and differentiation and an apoptosis inhibitor. By adding a P53 inhibitor, a naturally-extracted antioxygen resveratrol and flavin adenine dinucleotide (FAD) to inhibit apoptosis coordinately, apoptosis during cell culturing can be inhibited, and by adding the regulatory factors for cell proliferation and differentiation, presenility of cells is avoided, so that the serum-free epithelial cell culture fluid is applicable to large-scale multiplication culture of the cells.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a serum-free epithelial cell culture solution. Background technique [0002] Direct contact or exposure of the chemical to the eyes can cause irritation and local toxicity. Therefore, ocular surface irritation risk assessment is a much-needed item in hazard assessment and risk avoidance procedures. The evaluation of the potential risk of eye irritation of new compounds in traditional methods mainly relies on in vitro rabbit experiments, which is time-consuming, labor-intensive and costly. Therefore, finding rapid and economical compound screening methods has become an urgent need in this field, and has inspired the establishment of a large number of in vitro alternative methods for ocular surface irritation risk assessment, such as bovine corneal opacity and permeability testing, chicken embryo The allantoic membrane test, etc., the above-mentioned techniques can achieve in vitro eye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625C12N5/0632C12N2500/40C12N2500/35C12N2500/24C12N2500/30C12N2500/14C12N2501/105C12N2501/11C12N2501/13C12N2501/10C12N2500/84C12N2501/33C12N2501/998C12N2501/195
Inventor 张晓娥卢永波李潇吴迪
Owner GUANGDONG BOXI BIO TECH CO LTD
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