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Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change

A technology of retinal progenitor cells and retinal degeneration, applied in the field of stem cells, can solve problems such as abnormal integration and expression of viruses, unresolved exogenous vectors, cell ethics, immune rejection of teratomas, etc., and achieve the effect of improving visual function

Active Publication Date: 2017-01-11
HE EYE HOSPITAL SHENYANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some achievements have been made so far, the problem of exogenous vectors has not been resolved, and there is still a long way to go before clinical application
The cells commonly used for treatment mainly include: (1) Embryonic stem cells (ES): It has the ability to differentiate into a variety of cells, but this kind of cells has problems such as ethics, immune rejection and teratoma, and is not suitable for clinical application
(2) Induced pluripotent stem cells (iPS): Refers to the reprogramming of normal mature cells into pluripotent induced stem cells in vitro, which theoretically have the ability to differentiate into multiple directions, but it has problems such as virus integration and abnormal expression
However, the retinal progenitor cells used for injection should be of good quality. Unidentified retinal progenitor cells injected into the vitreous cavity have a high risk of treatment failure, and severe complications may occur
However, there is still no research showing which aspects should be identified to ensure that the obtained retinal progenitor cells can have a good therapeutic effect on retinal degenerative diseases

Method used

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  • Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change
  • Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change
  • Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 The cultivation of retinal progenitor cells

[0067]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, concentration 10ng / ml, basic growth factor bFGF, concentration 10ng / ml, L-glutamine, concentration 1:100) mix and count according to 1-2×10 5 / cm 2 Inoculate in low-adsorption T25 culture flasks, place at 37°C, 5% CO 2 Cultured in a constant temperature incubator, the next day the cells formed into spheres.

[0068] 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 the powder while adding For the remaining solvent, shake it with a vortex...

Embodiment 2

[0071] Example 2 Identification of Retinal Progenitor Cells

[0072] The vigorously proliferating fifth-generation retinal progenitor cells cultured in Example 1 were identified by flow cytometry: using TrypLE TM -Express digested retinal progenitor cells, resuspended cells in DPBS and counted according to 0.5×10 5 The amount of cells was evenly distributed to the flow detection tubes, and then the identification antibody (Nestin / Pax6 / / Ki67 / Chx10 / Sox2 / CD38 / HLA-DR) and the isotype control antibody were incubated at room temperature in the dark for 15min, and then added Centrifuge 1ml DMEM / F12 at 500-800rpm for 3min. Resuspended with 30μL DPBS for detection on the machine.

[0073] Identification antibodies and isotype control antibodies were purchased from BD and Abcam.

[0074] The identification criteria are: the expression of Nestin is greater than 90%; the expression of Pax6 is greater than 70%; the expression of Ki-67 is greater than 60%; the expression of Chx10 is grea...

Embodiment 3

[0077] Example 3 Preparation of Retinal Progenitor Cell Preparation

[0078] The retinal progenitor cells with the preservation number CGMCC NO.10419 were suspended in HBSS balanced salt solution, and the cell concentration was 4×10 7 individual / mL.

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Abstract

The invention relates to the technical field of stem cells, and especially relates to a method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change. The method comprises the following steps: culturing retinal progenitor cells to the logarithmic phase, and carrying out flow cytometry identification, wherein the retinal progenitor cells that meet the identification standards are capable of treating retinal degenerative change. The retinal progenitor cells identified by the provided method can maintain a good proliferation performance and stem cell characteristics and have a good curative effect when being applied to the treatment on retinal degenerative change. The experiment results show that after the retinal progenitor cells identified by the provided method are injected into the vitreous body cavities of RCS mice, the retinal outer nuclear layer cells are protected; the multifocal visual electrophysiology examination results show that the change of potential transmitted by retinal photoreceptor cells of the cell injection group is more prominent than the group, which is only injected with a HBSS buffer solution. The water maze experiment results also show that after injection, the visual performance of mice is improved.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a method and a kit for identifying retinal progenitor cells capable of treating retinal degeneration. Background technique [0002] Retinal degeneration is a type of retinal disease characterized by apoptosis and necrosis of retinal photoreceptor cells and pigment epithelial cells, mainly including atrophic age-related macular degeneration (Age-related macular degeneration, AMD), retinitis pigmentosa (Retinitis Pigmentosa, RP) and retinal atrophy. Retinal degenerative disease is one of the main blinding diseases at present. Most of them have a genetic predisposition. In the early stage of the disease, the retinal cells appear dysfunctional. As the disease progresses, the retinal neuroepithelium and pigment epithelium gradually undergo apoptosis and atrophy, and vision becomes irreversible. Descending, severe blindness. Because the pathogenesis is mainly cell apoptosis, there...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14C12N5/0797A61P27/02
Inventor 王卓实张明琦张东蕾张峰磊
Owner HE EYE HOSPITAL SHENYANG
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