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A kind of culture medium and its application and preparation method

A culture medium and a complete medium technology, applied in the field of stem cell culture, can solve the problems of limited corneal tissue, short life cycle, and small number of cells, etc., and achieve a high probability of specific differentiation, convenient material collection, and low immunogenicity Effect

Active Publication Date: 2019-02-22
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study of corneal epithelium has enabled people to have a deeper understanding of the physiological and pathological characteristics of the cornea and corneal diseases; however, due to the short life cycle of the differentiated corneal epithelial cells grown in vitro, the number of cells obtained is small and the cost is high. Research on corneal tissue and construction of tissue engineered cornea

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  • A kind of culture medium and its application and preparation method
  • A kind of culture medium and its application and preparation method
  • A kind of culture medium and its application and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～12

[0042] The preparation of embodiment 1~12 culture medium

[0043] The culture medium was prepared as follows:

[0044] (1) Separation and extraction of New Zealand white rabbit corneal epithelial cells under sterile conditions, inoculated in 25cm 2 In cell culture flasks, at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator, change the medium for the first time 48 hours after the cells adhere to the wall, and change the medium every other day, and observe the growth of the cells under an inverted phase-contrast microscope every day. Corneal epithelial cell culture medium is 100U / mL penicillin and 100U / mL streptomycin, DMEM / F12 medium, 10% fetal bovine serum.

[0045] (2) When the cells in (1) reach 80%-90% confluence, digest and collect the cells with 0.25% trypsin-0.02% EDTA digestion solution, and adjust the cell density to 1×10 9 each / L, inoculated in a new 25cm 2 culture flask at 37°C, 5% CO 2, in a saturated humidity incubator. Corneal epithelial cell cultur...

Embodiment 13

[0054] Embodiment 13 directed differentiation test

[0055] The steps of directed differentiation test are as follows:

[0056] (1) hUC-MSCs were divided into control group and experimental group. Put the sterilized 18mm×18mm coverslip into a six-well plate, and prepare a prepared density of 5×10 4 Each / mL hUC-MSCs suspension was added to a six-well plate, 2 mL per well, and placed at 37 ° C, 5% CO 2 1. Cultivate in a saturated humidity incubator, change the medium for the first time in 24 hours, then change the medium every 2 days, and observe the cell growth every day under an inverted phase-contrast microscope.

[0057] Wherein, the experimental group adopts the culture medium of Examples 1-12 to culture hUC-MSCs; the control group adopts human mesenchymal stem cell complete medium to culture hUC-MSCs as a comparative example.

[0058] (2) After the cells were cultured for 7 days, the coverslips in the wells were taken out to detect the expression of CK12 by immunofluore...

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Abstract

The invention relates to the technical field of stem cell culture, in particular to a culture medium as well as application and a preparation method thereof. The culture medium consists of corneal epithelial cell culture supernatant and a corneal stroma. The culture medium provided by the invention can significantly improve the hUC-MSCs's rate of specific differentiation towards corneal epithelial cells.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a culture medium and its application and preparation method. Background technique [0002] Corneal disease is a common blinding eye disease with a high prevalence and difficult treatment. Allogeneic corneal transplantation is an effective way to restore vision due to corneal blindness, but corneal transplantation usually fails due to severe immune rejection, and suitable donor tissues are in short supply. Clinical studies have shown that culturing autologous limbal stem cells in vitro as seed cells for corneal epithelial repair has achieved good results. Limbal stem cells, like other stem cells, have the characteristics of low degree of differentiation, long cell cycle, high proliferation potential, self-renewal ability and stress proliferation, and studies have found that limbal stem cells cultured in vitro may lose part of their antigenicity, which is beneficial to c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 陈海佳王一飞葛啸虎戚康艺
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD