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Medium and method for inducing differentiation of umbilical cord mesenchymal stem cells into corneal epithelial cells

A technology for corneal epithelial cells and amniotic membrane epithelial cells, applied in the field of stem cell culture, can solve problems such as difficulty in differentiation, and achieve the effects of reducing risks, widening sources, and increasing percentages

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

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Problems solved by technology

However, it is very difficult to induce hUC-MSCs to differentiate into corneal epithelial cells in vitro

Method used

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  • Medium and method for inducing differentiation of umbilical cord mesenchymal stem cells into corneal epithelial cells
  • Medium and method for inducing differentiation of umbilical cord mesenchymal stem cells into corneal epithelial cells
  • Medium and method for inducing differentiation of umbilical cord mesenchymal stem cells into corneal epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0062] Example 2 Preparation of Amnion Epithelial Cells

[0063] Human amniotic membrane was taken from healthy puerpera (serological examinations such as hepatitis B, hepatitis C, HIV, mycoplasma, and syphilis were all negative), and the amniotic membrane was rinsed twice in PBS containing 100 U / mL penicillin and 100 U / mL streptomycin. Cut into about 30-50cm 2 For large or small tissue pieces, add a sufficient amount of 0.25% trypsin digestion solution, digest in a 200R constant temperature oscillator at 37°C for 15-30min, take out every 10min and shake vigorously by hand, centrifuge at 2000rpm for 5min, and then add enough 2.5g / L collagenase Ⅱ+0.05g / L DNAs enzyme, digest at 37°C for 4h, centrifuge, then add keratinocyte serum-free medium (KFSM), 10-15ng / ml epidermal growth factor (EGF) for culture Liquid, pipetting evenly, with 1×10 7 cells / L were inoculated in a culture dish with a diameter of 10 cm, placed in a cell culture box with a volume fraction of 5% CO2, and cultu...

Embodiment 3

[0066] Take the 3rd-5th generation hUC-MSCs and the 2nd generation amniotic epithelial cells in the logarithmic phase of proliferation, inoculate them in the Transwell co-culture system with polylysine-treated sterile coverslips, and implant the amniotic membrane in the upper chamber Epithelial cells were seeded at a density of 4-6 x 10 5 / well, the lower chamber was implanted with hUC-MSCs, the seeding density was 1-1.5×10 5 / hole. Add 2.5ml containing 100U / mL penicillin and 100U / mL streptomycin to each well, the volume fraction is 55% LONZA human stem cell serum-free medium (Lonza Ultra CULTURE TM ), 45% keratinocyte serum-free medium (Keratinocyte serum-free medium, KFSM) culture solution, 5ng / ml epidermal growth factor (EGF), 5ug / ml insulin complete culture solution. Replace with new culture medium every 2-3 days. After 7 days of culture, the cell slides were taken out, and the immunohistochemical staining of cytokeratin AE1 and AE5 was carried out according to the inst...

Embodiment 4

[0068] Take the 3rd-5th generation hUC-MSCs and the 2nd generation amniotic epithelial cells in the logarithmic phase of proliferation, inoculate them in the Transwell co-culture system with polylysine-treated sterile coverslips, and implant the amniotic membrane in the upper chamber Epithelial cells were seeded at a density of 4-6 x 10 5 / well, the lower chamber was implanted with hUC-MSCs, the seeding density was 1-1.5×10 5 / hole. Add 2.5ml containing 100U / mL penicillin and 100U / mL streptomycin to each well, the volume fraction is 55% LONZA human stem cell serum-free medium (Lonza Ultra CULTURE TM ), 45% keratinocyte serum-free medium (Keratinocyte serum-free medium, KFSM) culture fluid, 10ng / ml epidermal growth factor (EGF), 10ug / ml insulin complete culture fluid. Replace with new culture medium every 2-3 days. After 7 days of culture, the cell slides were taken out, and the immunohistochemical staining of cytokeratin AE1 and AE5 was carried out according to the instruct...

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Abstract

The invention relates to the field of stem cell culture, in particular to a culture medium and method for inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells. An epidermal growth factor and insulin are added into a basic culture solution, so that the percentage of differentiation of hUC-MSCs to the corneal epithelial cells is increased. Moreover, the culture solution does not contain heterogenous serum, so that the risk is lowered. According to the method provided by the invention, the umbilical cord mesenchymal stem cells and amniotic epithelial cells are co-cultured, so that the differentiation percentage can be increased remarkably, and the required culture period is short. The adopted umbilical cord mesenchymal stem cells are derived from umbilical cords and placentas, so that the source is wide, and legal and ethical limitations are avoided.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a medium and a method for inducing differentiation of umbilical cord mesenchymal stem cells into corneal epithelial cells. Background technique [0002] The corneal epithelial cell layer is located on the outer surface of the cornea and consists of 4 to 5 layers of non-keratinizing squamous epithelial cells, which play an important role in maintaining ocular surface homeostasis and corneal transparency. Sound corneal epithelial cells are an important condition for the cornea to resist the damage of various harmful factors from the outside world. Limbal stem cells with sound structure and function are the main source of corneal epithelial cell regeneration. Various pathogenic factors such as ocular trauma, surgical trauma, inflammation, and drug toxicity can cause limbal damage, resulting in limbal stem cell dysfunction, resulting in epithelial cell regeneration. Deletion leads t...

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马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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