Human fibroblast serum-free medium and preparation method and method for obtaining human fibroblast serum-free conditioned medium

A human fibroblast, serum-free culture medium technology, applied in the field of skin repair, can solve the problem of human fibroblast unsafe, and achieve the effect of promoting and protecting cell activity, good cell activity and protecting integrity

Active Publication Date: 2021-05-14
西安艾尔菲生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a serum-free medium for human fibroblasts, which solves the unsafe factors in the prior art for culturing human fibroblasts containing serum

Method used

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  • Human fibroblast serum-free medium and preparation method and method for obtaining human fibroblast serum-free conditioned medium
  • Human fibroblast serum-free medium and preparation method and method for obtaining human fibroblast serum-free conditioned medium
  • Human fibroblast serum-free medium and preparation method and method for obtaining human fibroblast serum-free conditioned medium

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Experimental program
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preparation example Construction

[0063] A preparation method for human fibroblast serum-free medium, comprising the following specific steps:

[0064] Step 1: Prepare serum-free culture supernatant concentrate

[0065] Human fibroblasts are inoculated in serum-free medium for culture, when the cell confluence is 80-90%, collect the supernatant of human fibroblasts; filter, concentrate by ultrafiltration, add water to determine the total protein, and obtain serum-free culture supernatant clear concentrate;

[0066] Step 2: Prepare Growth Factor Concentrate

[0067] Recombinant human epidermal growth factor (rh-EGF), recombinant human basic fibroblast growth factor, recombinant human transforming growth factor β1, recombinant platelet-derived growth factor-C, recombinant human insulin-like growth factor-1 five growth factors Prepare a 50ug / mL concentrated solution A, and freeze it under sterile conditions;

[0068] Step 3: Preparation of human fibroblast serum-free medium

[0069] Dissolving the vitamin com...

Embodiment 1

[0086] Prepare a kind of human fibroblast serum-free medium, its method is as follows:

[0087] Step 1: Prepare serum-free culture supernatant concentrate

[0088] Human fibroblasts at 5×10 3 / cm 2 Inoculate in serum-free medium for culture, collect the culture supernatant when the cell confluence is 80%, and obtain a supernatant with a volume of 6.5 L, filter the 6.5 L supernatant through a plate filter, and filter the membrane pore size The microfiltrate is concentrated by ultrafiltration using a cellulose acetate ultrafiltration membrane with a molecular weight cut-off of 3KDa. The area of ​​a single membrane of the ultrafiltration membrane is 0.1m 2 , a total of 4 membrane packs; the tangential flow process is used for concentration, the liquid at the permeate end is discarded, the microfiltrate is concentrated to 1 / 15 of the volume of the original microfiltrate, and the concentration is stopped, and the liquid in the ultrafiltration system is emptied to obtain a concent...

Embodiment 2

[0096] Prepare a kind of human fibroblast serum-free medium, its method is as follows:

[0097] Step 1: Prepare serum-free culture supernatant concentrate

[0098] Human fibroblasts in 6×10 3 / cm 2 Inoculate in serum-free medium for culture, collect the culture supernatant when the cell confluence is 85%, obtain a supernatant with a volume of 20L, filter the 20L supernatant through a plate filter, and filter the filter membrane with a pore size of 0.45 μm. The microfiltrate is concentrated by ultrafiltration using a cellulose acetate ultrafiltration membrane with a molecular weight cut-off of 3KDa. The area of ​​a single membrane of the ultrafiltration membrane is 0.1m 2 , a total of 6 membrane packs, using the tangential flow process for concentration, the liquid at the permeate end is discarded, the microfiltrate is concentrated to 1 / 17 of the volume of the original microfiltrate, and the concentration is stopped, and the liquid in the ultrafiltration system is emptied to...

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Abstract

A serum-free medium for human fibroblasts, including basal medium and serum-free culture supernatant concentrate, also includes the following components and their concentrations: the vitamin composition is 5-100ng / mL, and the grape polyphenols is 50-200μg / mL, recombinant human epidermal growth factor 1-50 ng / mL, recombinant human basic fibroblast growth factor 50-200 ng / mL, recombinant human transforming growth factor β1 1-50 ng / mL, recombinant platelet-derived growth factor -C is 1-10ng / mL, recombinant human insulin-like growth factor-1 concentration is 1-10ng / mL, β-mercaptoethanol is 50-200μM, human albumin concentration is 1-100μg / mL, glutathione The concentration is 1-100μg / mL, the astragalus polysaccharide is 1-50μg / mL, the hyaluronic acid is 0.1-5μg / mL, and the sodium bicarbonate is 1.0-3.7g / L. Human fibroblasts are cultivated by using serum-free medium instead of traditional medium containing animal serum, which avoids unsafe factors such as contamination of animal-derived components and human immune response caused by foreign proteins.

Description

technical field [0001] The invention belongs to the technical field of skin restoration, and relates to a human fibroblast serum-free medium, a preparation method of a human fibroblast serum-free medium, and a method for obtaining a human fibroblast serum-free culture medium. Background technique [0002] Human fibroblasts are the main cells that constitute the dermis of the skin, and their normal proliferation and differentiation maintain the normal structure and physiological functions of the skin. Cell growth factors, collagen, stress proteins and other extracellular proteins can be secreted by autocrine or paracrine, and the nutrients secreted by these cells can synergistically act on the skin, stimulating hyaluronic acid, collagen, elasticity in skin tissue Protein regeneration, repair damaged cells, and promote wound healing. [0003] In recent years, the in vitro expansion of human fibroblasts is carried out in a culture medium supplemented with fetal bovine serum or...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077A61K8/99A61Q19/00A61Q19/08
CPCA61K8/99A61Q19/00A61Q19/08C12N5/0656C12N2500/12C12N2500/30C12N2500/34C12N2500/38C12N2500/44C12N2500/90C12N2501/105C12N2501/11C12N2501/115C12N2501/135C12N2501/15
Inventor 王静王海江胡春梅肖萍
Owner 西安艾尔菲生物科技有限公司
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