Retinal pigment epithelial cell and its preparation method and application
A retinal pigment and epithelial cell technology, applied in the field of retinal pigment epithelial cells and their preparation, can solve the problems of wasting laboratory researchers' time and R&D funds, and the quantity and quality of retinal pigment epithelial cells cannot meet experimental requirements, and achieve large clinical results. and scientific research application potential, facilitate the manipulation and observation of cell status, and improve the effect of differentiation efficiency
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Embodiment 1
[0060] This embodiment discloses a method for preparing retinal pigment epithelial cells, the design flow chart is as follows figure 1 As shown, it specifically includes the following steps:
[0061] 1. D0: Culture of pluripotent stem cells
[0062] The pluripotent stem cells are prepared by the method disclosed in the patent CN108085299A, and then the pluripotent stem cells are cultured, and when the polymerization degree reaches 70-80%, they are digested into complete single cells with TrypLE (pancreatin) or Accutase (cell digestion solution). Cells were resuspended in pluripotent stem cell maintenance medium and seeded on Matrigel plates at 37°C, 5% CO 2 Concentration, cultivated in an incubator with saturated humidity.
[0063] The details and optimization of the above D0 experiment operation are as follows:
[0064] The pluripotent stem cells used in the experiment have undergone strict pluripotency verification (expressing various pluripotency markers, and can form te...
Embodiment 2
[0086] In this embodiment, the retinal pigment epithelial cells (RPE) obtained in Example 1 are detected, including the following methods:
[0087] Method 1: using RT-QPCR detection, hPSC cells were used as the control group, and the retinal pigment epithelial cells (RPE) obtained in Example 1 were used as the experimental group, and it was proved that the obtained retinal pigment epithelial cells were OTX2+ / MITF+ / RPE65+ / BEST1+ / ZO- 1+ / MERTK+ / CRALBP+ / PEDF+, see details image 3 shown.
[0088] Among them, OTX2 is an indicator of the front end of neural tube development; MITF, RPE65, and BEST1 are indicators of mature retinal pigment epithelial cells; ZO-1 is an indicator of cell tight junctions (mature marker); MERTK is an indicator of phagocytosis; CRALBP is a visual substance An indicator of recycling protein; PEDF is an indicator of secretory function. The kit used for RNA extraction is RNAprepPure Cell / Bacteria Kit, the manufacturer is TIANGEN, and the product number is D...
Embodiment 3
[0094] This example studies the role of small molecules in the differentiation of pluripotent stem cells into retinal pigment epithelial precursor cells.
[0095] In this example, it was found that on days 0-5 of culture of pluripotent stem cells, the addition of specific mature differentiation medium and combined small molecules can accelerate the differentiation of pluripotent stem cells into retinal pigment epithelial precursor cells, and the effect is stable.
[0096] After adding the specific mature differentiation medium and combined small molecules, the specific indicators of retinal pigment epithelial precursor cells were detected by RT-QPCR on day 0, day 3 and day 5, and the results are shown in Figure 6 (Pax6 is a pan-nervous indicator; OTX2 is a front-end indicator of neural differentiation, which can eventually differentiate into eye regions; TLL1, LHX2, TYR and RAX are eye-specific indicators).
[0097] The small molecules used include: BMP inhibitors, Nodal inhi...
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