Screening and culturing method of retinal precursor cells
A technology of precursor cells and culture methods, applied in the field of screening and culture of retinal precursor cells, can solve the problems of small total amount of single cells, difficult expansion, poor state of positive cells, etc.
Pending Publication Date: 2022-01-18
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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Problems solved by technology
At present, the enrichment of this group of retinal precursor cells from organoids adopts mechanical separation plus digestive enzymes to separate single cells --- surface antibody staining --- cell screen --- machine sorting --- positive cell inoculation (whole The process takes about 3-4 hours), the main problems are as follows: 1. Digestion of retinal organoid nerve tissue is difficult, and the viability of isolated cells is low; 2. The total amount of single cells sorted immediately after digestion is small, and the positive cells enriched The number of cells is small; 3. It takes a long time from digestion to staining and sorting, and the positive cells obtained in the final sorting are in poor condition, which leads to difficulties in amplification
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Embodiment 1
[0028] Screening and culture of retinal precursor cells
[0029] (1) Take the retinal organoids derived from hESC, mechanically separate the retinal nerve layer with a 1ml empty needle under a microscope, put the retinal nerve layer into the improved digestive solution at 37°C for 30 minutes, and digest the undigested powder after 15 minutes The neural layer was replaced with another tube of fresh improved digestion solution, shaken several times every 3 minutes, blown lightly, and then adhered to the wall for 2 days; the improved digestion solution was TrypLE digestive enzyme containing 0.125% trypsin.
[0030] (2) Take out the adherent cultured cells in step (1), add only TrypLE digestive enzyme for 3-5 minutes, and sort ckit by flow cytometry + ssea4 - collect cells;
[0031] (3) The cells collected in step (2) are cultured with improved N2B27 neural stem cell medium; the improved N2B27 neural stem cell medium components include DMEM / F12 and Neurobasal medium 1:1 mixed, a...
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Abstract
The invention relates to a screening and culturing method of retinal precursor cells, and belongs to the technical field of biology. According to the screening method, the TrypLE digestive juice is improved, and sorting is performed after direct adherent culture, so that the cell activity is improved, the cell death rate is reduced, and the sorting activity rate is improved from 40-60% to 95% or above. The separated cells are more stable in cell amplification in the improved N2B27 neural stem cell culture medium, the amplification generations are improved, the amplification generations can be stably improved from original P5 to about P10, and the activity of the retinal stem cells is kept.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening and culturing retinal precursor cells. Background technique [0002] Retinal degenerative disease (RD) is a severe blinding eye disease characterized by the loss of retinal pigment epithelial cells and retinal neurons. There is currently no effective treatment for neuronal death caused by retinal degeneration, and stem cell transplantation is a promising therapeutic strategy for retinal degeneration. [0003] Retinal progenitor cells (RPCs) are a class of stem cells with therapeutic potential. In the past, human retinal precursor cells were mostly enriched from the retina of aborted embryos, and the sources were limited and involved ethical issues. Therefore, it is urgent to develop new sources of therapeutic cells. Human retinal organoids are three-dimensional retinal tissues induced by human pluripotent stem cells, which are highly similar to in...
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IPC IPC(8): C12N5/0797
CPCC12N5/0621C12N2509/00C12N2501/115
Inventor 曾玉晓邹婷徐海伟段平
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV