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Cell culture substrate and application and use method thereof

A technology for cell culture and culture substrate, applied in the field of stem cell culture, can solve problems such as difficulty in differentiation, and achieve the effects of cheap materials, convenient material acquisition and good biocompatibility

Active Publication Date: 2015-11-25
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Cell culture substrate and application and use method thereof
  • Cell culture substrate and application and use method thereof
  • Cell culture substrate and application and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0066] The preparation method of collagen film is:

[0067] Dissolve 60mg-240mg collagen in 20mL acetic acid solution, stir to dissolve, transfer to a 60mm Petri dish and place it in a refrigerator at 4°C until the air bubbles are removed. Add 0.5g / L glutaraldehyde to the solution, and put it in a 4°C refrigerator for cross-linking for 24h. Put the petri dish into a drying oven at 50°C for 48 hours, take it out, soak it in 56g / L NaOH solution for 30 minutes, take out the film, rinse it with distilled water to obtain a collagen film.

[0068] The prepared collagen film is light yellow in color, transparent and flexible, and can be trimmed into any shape at will. Due to the cross-linking of glutaraldehyde, the strength is also enhanced.

[0069] Collagen membranes need to be soaked in ethanol and then soaked in medium before cell culture or induction of differentiation. Specifically, the collagen film was first soaked in ethanol solution for 24 hours, and then soaked in cultu...

Embodiment 1

[0077] Culture medium: containing 100U / mL penicillin and 100U / mL streptomycin, the volume fraction is 55% LONZA human stem cell serum-free medium (LonzaUltraCULTURE TM ), 45% keratinocyte serum-free medium (Keratinocyteserum-freemedium, KFSM) culture medium, 10ng / ml epidermal growth factor (EGF), 10ug / ml insulin.

[0078] Select hUC-MSCs of passage 3-5 in the logarithmic phase of proliferation, and adjust the concentration to 3×10 5 / ml was inoculated on 3g / L collagen membrane, each group of membrane was inoculated with 0.5mL of cell suspension, and 6 samples were set up in each group. Carefully pipette the cell-laden membrane into a 6-well plate. Incubate under standard environment, and then carefully add 2mL culture medium after 2h to continue culturing. Replace with new culture medium every 2-3 days. After 7 days of culture, the cells were collected for cell smear, immunohistochemical staining of cytokeratin AE1 and AE5 was performed according to the instructions of the i...

Embodiment 2

[0080] Culture medium: containing 100U / mL penicillin and 100U / mL streptomycin, the volume fraction is 55% LONZA human stem cell serum-free medium (LonzaUltraCULTURE TM ), 45% keratinocyte serum-free medium (Keratinocyteserum-freemedium, KFSM) culture medium, 10ng / ml epidermal growth factor (EGF), 10ug / ml insulin.

[0081] Select hUC-MSCs of passage 3-5 in the logarithmic phase of proliferation, and adjust the concentration to 3×10 5 / ml was inoculated on 6g / L collagen membrane, each group of membrane was inoculated with 0.5mL cell suspension, and each group set up 3 samples. Carefully transfer the cell-laden membrane to a 6-well plate in 5% CO 2 , 37°C, humidity 95% and incubate after 2 hours, carefully add 2mL culture medium to continue the cultivation. Replace with new culture medium every 2-3 days. After 7 days of culture, epithelioid cells crawled out of the cell colonies, adhered to the wall, proliferated rapidly, and gradually grew in colonies. Cells were collected f...

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Abstract

The invention relates to the field of stem cell culture, in particular to a cell culture substrate and an application and a use method thereof. The cell culture substrate comprises a collagen membrane and a culture liquid. The cell culture substrate has the advantages that the collagen membrane is used as a carrier and is matched with the effective culture liquid, so the differentiation of hUC-MSCs (human umbilical cord mesenchymal stem cells) to cornea epithelial cells is promoted; after being proofed by experiments, when the hUC-MSCs are inoculated to the collagen membrane and are induced for seven days by the culture liquid, the differentiation efficiency of hUC-MSCs can reach 32%, but the differentiation efficiency is only 9.54% when the collagen membrane is not used as the carrier for inducing, so the collagen membrane can obviously improve the differentiation efficiency of the hUC-MSCs to the cornea cell.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a cell culture substrate and its application and usage method. Background technique [0002] Sound corneal epithelial cells are an important condition for the cornea to resist damage from various external harmful factors, and limbal stem cells with sound structure and function are the main source of corneal epithelial cell regeneration. A variety of pathogenic factors, such as: ocular trauma, surgical trauma, inflammation, and drug toxicity can cause corneal limbal damage, resulting in limbal stem cell dysfunction and loss of epithelial cells, resulting in increased risk of corneal infection, perforation, and neovascularization . Therefore, the problem of obtaining a large number of epithelial cells for corneal repair through in vitro culture needs to be solved urgently. Clinical studies have shown that culturing autologous limbal stem cells in vitro as seed cells for corneal e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/071
Inventor 陈海佳王一飞葛啸虎戚康艺
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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