Fibroblast culture medium and preparation method thereof

A fibroblast and culture medium technology, applied in the direction of cell culture active agents, biochemical equipment and methods, animal cells, etc., can solve the problems affecting the safety of clinical application, the risk of animal-derived pathogens, and the difficulty in meeting actual needs, etc. Achieve the effect of stable passage, improve safety, and avoid risks

Active Publication Date: 2017-05-10
GUANGZHOU RAINHOME PHARM&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the current medium used to induce iPS cells into fibroblasts, such as MEF medium, has a low differentiation probability and is difficult to meet actual needs, and contains serum, which will bring the risk of animal-derived pathogens and affect clinical application. safety

Method used

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  • Fibroblast culture medium and preparation method thereof
  • Fibroblast culture medium and preparation method thereof
  • Fibroblast culture medium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Design the experimental group and the control group:

[0036] 1. Culture medium of the experimental group:

[0037] Experimental group culture medium comprises basal culture fluid and supplement, and described basal culture fluid is H-DMEM culture fluid and F12 culture fluid that volume ratio is 1:1, and total volume is 500mL; With the addition in described basal culture fluid Meter, the additive includes the following components:

[0038]

[0039]

[0040] Preparation method: add insulin 30mg, hydrocortisone 0.2mg, transferrin 50mg, sodium selenite 5μg, β-mercaptoethanol 0.05mmoL, NEAA 5mL, GlutaMAX 5mL, penicillin 50mg, Streptomycin 50mg, bFGF 7.5μg, E2 5nmoL, P4 0.5mmoL, 8-bromo-cAMP 0.5mmoL, human follicle stimulating hormone FSH 50nmoL, enough.

[0041] 2. Control medium

[0042] For the control group, 500 mL of the existing MEF culture medium was used, and 10 mL of FBS was added to it.

[0043] 2. Induction of iPS differentiation into fibroblasts in v...

Embodiment 2

[0057] In this example, a method for inducing fibroblasts by induced pluripotent stem cells, the method is the same as step 2 in Example 1, the difference is that the culture medium used includes basal culture fluid and additives, and the basal culture fluid is The H-DMEM culture fluid and the F12 culture fluid with a volume ratio of 1:2 have a total volume of 500 mL; in terms of the concentration in the basic culture fluid, the additives include the following components:

[0058] ingredient name working concentration NEAA 0.5%v / v GlutaMAX 2%v / v insulin 1μg / mL Hydrocortisone 1μg / mL Transferrin 150μg / mL Sodium Selenite 5ng / mL β-mercaptoethanol 0.2mmol / L penicillin 0.2mg / mL streptomycin 0.05mg / mL f 10ng / mL E2 5nmol / L P4 0.5mmol / L 8-Bromo-cAMP 0.5mmol / L FSH 150nmol / L

[0059] The identification method of culturing gained fibroblasts is the same as step 3 in embodiment 1, a...

Embodiment 3

[0061] In this example, a method for inducing fibroblasts by induced pluripotent stem cells, the method is the same as step 2 in Example 1, the difference is that the culture medium used includes basal culture fluid and additives, and the basal culture fluid is The H-DMEM culture fluid and the F12 culture fluid with a volume ratio of 1:0.5 have a total volume of 500 mL; in terms of the concentration in the basic culture fluid, the additives include the following components:

[0062] ingredient name working concentration NEAA 2%v / v GlutaMAX 0.5%v / v insulin 10μg / mL Hydrocortisone 0.1μg / mL Transferrin 50μg / mL Sodium Selenite 15ng / mL β-mercaptoethanol 0.05mmol / L penicillin 0.05mg / mL streptomycin 0.2mg / mL f 20ng / mL E2 15nmol / L P4 2mmol / L 8-Bromo-cAMP 2mmol / L FSH 50nmol / L

[0063] The identification method of culturing gained fibroblasts is the same as step 3 in embodiment ...

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Abstract

The invention relates to a culture medium by which induced pluripotent stem cells are induced into fibroblasts and a preparation method of the culture medium. The culture medium consists of a basic culture solution and an additive, wherein the basic culture solution is a mixture of an H-DMEM culture solution and an F12 culture solution at the volume ratio of 1: (0.5-2); and in terms of concentrations in the basic culture solution, the additive consists of the following components: nonessential amino acid, glutamine derivatives, insulin, hydrocortisone, transferrin, sodium selenite, fibroblast growth factors, estradiol, progestin, 8-bromo cyclophosphate and human follicle stimulating hormone. The culture medium, in comparison with the prior art, is higher than fibroblast differentiation probability; in addition, the culture medium can guarantee stable passage and improve cell propagation speed, so as to offer a fibroblast seed cell source for skin tissue engineering; and meanwhile, the application of serum is avoided in an entire course, so that risks caused by animal-derived pathogens are effectively avoided and the safety of clinical application is enhanced.

Description

technical field [0001] The invention relates to a cell culture medium, in particular to a fibroblast culture medium and a preparation method thereof. Background technique [0002] Skin tissue engineering is a research hotspot in recent years. However, related skin cells are all terminally differentiated cells, and it is difficult to perform large-scale culture and expansion in vitro. At the same time, the source of seed cells also hinders the development of skin tissue engineering. [0003] Due to the limited sources of autologous seed cells, rejection of allogeneic seed cells, and ethical issues with stem cells, urine cells are collected from urine and reprogrammed into induced pluripotent stem cells (ips cells), ips The cells can then be induced to become keratinocytes, fibroblasts, sweat gland cells, hair papilla cells, etc., which can solve the problem of the source of seeds for skin tissue engineering, and cells can be obtained from urine, which is convenient and quick...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0656C12N2500/25C12N2500/33C12N2500/40C12N2501/115C12N2501/31C12N2501/39C12N2501/392C12N2506/45
Inventor 车七石
Owner GUANGZHOU RAINHOME PHARM&TECH CO LTD
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