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Culture method for retina progenitor cells and culture medium thereof

A retinal progenitor cell, cell culture technology, applied in animal cells, nervous system cells, vertebrate cells, etc., can solve problems such as plasma loss, pain caused by patients, physical diseases of patients, etc., to prevent premature aging or differentiation, Increase the number of growth and reproduction, break through the effect of small cell yield

Active Publication Date: 2012-10-24
HE EYE HOSPITAL SHENYANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fibrin is derived from the patient's own plasma, and the collection process causes pain to the patient, and may cause physical illness in the patient due to a large loss of plasma
The use of allogeneic or animal-derived fibrin to culture cells will cause contamination of foreign substances, and the cost of the experiment is high, and it is difficult to meet the wide application of clinical and scientific research.
The traditional culture of retinal progenitor cells with serum-free medium tends to lead to low cell yield, and fetal bovine serum medium cannot meet the clinical needs due to the heterogeneous components

Method used

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  • Culture method for retina progenitor cells and culture medium thereof
  • Culture method for retina progenitor cells and culture medium thereof
  • Culture method for retina progenitor cells and culture medium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: culture method of the present invention carries out retinal progenitor cell culture

[0041] Sphere-forming culture of retinal progenitor cells: After the tissue is sterilized, take the retinal tissue, digest it with enzymes, centrifuge at 1000r for 3 minutes, take the precipitate, and use DMEM / F12 basal medium (add N-2 supplement, the concentration is 1:100, Epidermal growth factor EGF, the concentration is 10ng / ml, basic growth factor bFGF, the concentration is 10ng / ml, L-glutamine, the concentration is 1:100) mix and count the sediment, according to 1-2×10 5 / cm 2 Inoculate in low-adsorption T25 culture flasks at 37°C, 5% CO 2 Cultured in a constant temperature incubator, the next day the cells formed into spheres.

[0042] Preparation of sodium alginate gel: Use deionized water as the solvent of sodium alginate, first weigh the sodium alginate powder, slowly add the powder into a centrifuge tube containing a small amount of solvent (20ml), and add th...

Embodiment 2

[0045] Embodiment 2: MTT assay cell viability and proliferation rate

[0046] Referring to the experimental method described in Example 1, the retinal progenitor cells in the logarithmic growth phase of the third generation were taken, and a single cell suspension was prepared, and 750 cells / well were inoculated in the culture medium prepared in Example 1 and covered with sodium alginate gel. The MTT test was carried out every day within 6 days in a 96-well culture plate. At the same time, a traditional culture method was established as a control, that is, retinal progenitor cells were cultured in a suspension or adherent manner, and the medium was 10%-20% serum medium.

[0047] Add 10μl MTT (1g / L) to each well of cells and continue to culture for 4h. The culture medium was removed by centrifugation, the cells were lysed by adding dimethyl sulfoxide, and the OD value was measured at a wavelength of 570 nm. The wells without MTT were used as the background, and the OD values ...

Embodiment 3

[0049] Example 3: Cell cycle detection by flow cytometry

[0050] Take 1×10 retinal progenitor cells cultured by the method described in Example 1 6 One, adding propidium iodide PI dye, staining for 20min, adding PBS and centrifuging to wash, and flow cytometry to detect the cycle of the cells. Cell cycle detection uses PI (propidium iodide) staining to detect the cell cycle, which can be combined with DNA and RNA in cells. After RNA is digested with RNA inhibitors, the fluorescence intensity of PI bound to DNA detected by flow cytometry It directly reflects the amount of DNA in the cell.

[0051] figure 2 and image 3 The cells tested are the 10th passage cells, from figure 2 It can be seen that the cells cultivated by the method of this patent are still in a vigorous proliferative phase, and there is no sign of aging; image 3 The results of flow cytometry identification proved that the retinal progenitor cells cultured at the 10th passage in vitro expressed the retin...

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Abstract

The invention relates to the bio-technical field, and discloses a cultural method for retina progenitor cells and a culture medium of the retina progenitor cells. According to the cultural method for cells disclosed by the invention, anchorage-dependent cells or suspension cells grow in an extracellular matrix with a certain three-dimensional space, so that so as to the growth velocity of cells is prevented effectively, cells are prevented from being aged or differentiated, the gene expression of the cells is not changed, high-quality and high-density cell reproduction is guaranteed and limitations of low yield of cells, poor quality of cells and the like in the traditional cultural method can be broken, so that the cultural method has a wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell culture method and culture medium. Background technique [0002] Retinal degenerative diseases are the leading causes of blindness in the world today. The main feature of such diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) is the death of retinal photoreceptor cells. Since human retinal cells are nerve cells and have no ability to regenerate, the most effective treatment currently is to replace or repair damaged retinal photoreceptor cells. [0003] In the past few decades, the application of stem / progenitor cell transplantation to induce tissue reconstruction and functional regeneration in the field of medical regeneration has attracted widespread interest, especially in the field of retinal regeneration, which has achieved many exciting results. In 2004, scientists found that after transplanting retinal progenitor cells into the eyes o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0797
Inventor 王卓实张明琦徐玲
Owner HE EYE HOSPITAL SHENYANG
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