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Human corneal endothelial cell culture medium and its preparation method and application

A technology of endothelial cells and corneal endothelium, which is applied in the field of human corneal endothelial cell culture medium and its preparation, can solve problems such as no obvious results, and achieve the effects of maintaining shape, inhibiting aging, and improving quantity and quality

Active Publication Date: 2011-12-28
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have confirmed that the conditioned medium of human corneal stromal cells contains basic fibroblast growth factor (bFGF) secreted by stromal cells, which can promote the proliferation of corneal endothelial cells. Cell growth factor (bFGF), epidermal growth factor (EGF) or nerve growth factor (NGF), etc. to promote the proliferation of corneal endothelial cells, but the long-term culture of corneal endothelial cells in the culture medium added with the above-mentioned long factors can maintain the shape and inhibit aging. No obvious effect on apoptosis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Analysis of the proliferation of human corneal endothelial cells by different concentrations of bovine corneal endothelial cell lysate by MTT method

[0027] (1) Human fetal corneal endothelial cells were seeded on a 96-well plate with the same number of starting cells (cell seeding density was 4-5×10 4 / ml), placed at 37°C, 5% CO 2 cultivated under conditions;

[0028] (2) After the cells adhere to the wall the next day, remove the original solution and replace it with D / F12 culture solution containing 0, 10, 20, 50, 100, 200 μg / ml protein bovine corneal endothelial cell lysate (10% fetal bovine serum), and each group had 6 replicate wells;

[0029] (3) After continuing to culture for 4-5 days, the proliferation of human embryonic corneal endothelial cells in each group was measured by MTT method.

[0030] The results showed that the bovine corneal endothelial cell lysate group with a protein content of 10-200 μg / ml was better than the group containing only fetal bo...

Embodiment 2

[0032] MTT assay to analyze the effect of different culture medium on promoting the proliferation of human corneal endothelial cells

[0033] (1) Human fetal corneal endothelial cells were seeded on a 96-well plate with the same number of starting cells (cell seeding density was 4-5×10 4 / ml), placed at 37°C, 5% CO2 cultivated under conditions;

[0034] (2) After the cells adhere to the wall the next day, remove the original solution and replace with D / F12 culture medium, bovine corneal endothelial cell lysate containing 50 μg / ml protein concentration, and bovine corneal epithelial cell lysate with 50 μg / ml protein concentration respectively , 50 μg / ml protein concentration of bovine corneal stromal cell lysate, human fetal corneal stromal cell conditioned culture medium and D / F12 culture medium of bovine corneal endothelial cell conditioned medium (the content of 10% fetal bovine Serum) 5 replicate wells were set up for each group;

[0035] (3) After continuing to culture f...

Embodiment 3

[0039] Observation of bovine corneal endothelial cell lysate on the maintenance of human corneal endothelial cell morphology under an inverted microscope

[0040] (1) Human fetal corneal endothelial cells were seeded on 24-well plates with the same number of starting cells, and placed at 37°C, 5% CO 2 cultivated under conditions;

[0041] (2) After the cells adhere to the wall the next day, remove the stock solution and replace it with D / F12 culture solution containing 0, 10, 20, 50, 100, 200 μg / ml protein bovine corneal endothelial cell lysate (10% fetal bovine serum), and each group had 3 replicate wells;

[0042] (3) Continuously observe and record the growth state of the cells under an inverted microscope.

[0043] The results showed that the bovine corneal endothelial cell lysate with a protein content of 10-200 μg / ml can well maintain the hexagonal endothelial morphology of fetal corneal endothelial cells, and the group without bovine corneal endothelial cell lysate is...

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Abstract

The invention relates to a human corneal endothelial cell culture medium, a preparation method and application thereof. The main active ingredients include bovine corneal endothelial cell lysate with a protein content of 10-200 μg / ml, fetal bovine serum with a content of 10%, 100 U / ml of penicillin and streptomycin, and the rest are supplemented with D / F12 basal culture medium. Method: First, culture bovine corneal endothelial cells in vitro; then prepare bovine corneal endothelial cell lysate, and finally prepare it in a sterile container. In the present invention, for the first time, bovine corneal endothelial cell lysate is made into cell culture liquid and applied to the in vitro culture of human corneal endothelial cells, which can promote the large-scale expansion of human corneal endothelial cells cultured in vitro, maintain the morphological characteristics of corneal endothelial cells, and inhibit cell aging And apoptosis, and more conducive to subculture of endothelial cells. The raw material bovine corneal endothelial cells are rich in sources and easy to culture in vitro, the preparation method of the culture solution is simple, easy to implement, beneficial to industrialization, and has broad application and development prospects.

Description

technical field [0001] The invention relates to a culture medium for culturing and expanding human corneal endothelial cells in vitro, in particular to a human corneal endothelial cell culture medium formulated with bovine corneal endothelial cell lysate as the main active ingredient and its preparation method and application. Background technique [0002] Corneal endothelial cell (CEC) is a single layer of cells located in the inner surface of the cornea. After its external attachment, the aqueous humor in the elastic membrane plays an important role in maintaining corneal transparency and visual function. When CEC dysfunction occurs (such as bullous keratopathy, Fuch corneal endothelial dystrophy, endothelial decompensation after intraocular surgery, etc.), CEC cannot regenerate and only rely on the expansion of surrounding normal endothelial cells to fill the lost cells and play a role in metabolism. When it exceeds its compensatory capacity, it will seriously affect norm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08
Inventor 史伟云高彦周庆军杨玲玲
Owner SHANDONG EYE INST