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Retinal pigment epithelium cell and preparation method and application thereof

A technology of retinal pigment and epithelial cells, applied in the field of retinal pigment epithelial cells and its preparation, can solve the problems that the number and quality of retinal pigment epithelial cells cannot meet the experimental requirements, waste the time of laboratory researchers and research and development funds, etc., and achieve large clinical and scientific research application potential, facilitate the operation and observation of cell state, and improve the effect of differentiation efficiency

Active Publication Date: 2019-07-23
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention provides a fast, efficient and simple method for obtaining retinal pigment epithelial cells from pluripotent stem cell differentiation and its application, and solves the problem of the method for preparing and cultivating retinal pigment epithelial cells and the reagents used by experimenters in the prior art Due to various limitations, the quantity and quality of the obtained retinal pigment epithelial cells cannot meet the experimental requirements, wasting the time of laboratory researchers and research and development funds, etc.

Method used

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  • Retinal pigment epithelium cell and preparation method and application thereof
  • Retinal pigment epithelium cell and preparation method and application thereof
  • Retinal pigment epithelium cell and preparation method and application thereof

Examples

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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 Image 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 inhib...

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Abstract

The invention relates to the field of cellular biology, in particular to a retinal pigment epithelial cell and a preparation method and application thereof. The retinal pigment epithelial cell expresses MITF and ZO-1. The preparation method of the retinal pigment epithelial cell comprises the following steps of S1: culturing human pluripotent stem cells; S2: differentiating the human pluripotent stem cells obtained in S1 into retinal pigment epithelial precursor cells; S3: differentiating the retinal pigment epithelial precursor cells obtained in S2 into immature retinal pigment epithelial cells; and S4: differentiating the immature retinal pigment epithelial cells obtained in S3 to obtain retinal pigment epithelial cells. The preparation method can differentiate pluripotent stem cells rapidly, efficiently and simply to obtain retinal pigment epithelial cells. In addition, no system containing serum or a serum substitute is used in the preparation method provided by the invention. Thedifferentiation efficiency is high and the differentiation effect is stable. The purification method is simple and the obtained cell has high purity.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a retinal pigment epithelial cell and its preparation method and application. Background technique [0002] Eye macular degeneration is a chronic eye disease that mostly occurs in the elderly over the age of 45. The older the age, the higher the incidence rate. Macular degeneration can be divided into two categories: wet and dry. Wet maculopathy, which accounts for 10% of the total incidence, is due to abnormal blood vessel growth under the retina, which ruptures and causes scar tissue to grow. Dry maculopathy, which accounts for 90% of the total incidence, is a kind of aging of the macula with age. It is manifested as the detachment of pigment epithelium or neuroepithelium into the vitreous body, forming drusen. Whether it is wet or dry macular degeneration, it will cause a type of single layer of cells located between the choroid and retina - the retinal pigment epithelium, to und...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079A61K35/30A61P27/02
CPCA61K35/30A61P27/02C12N5/0621
Inventor 焦璐琰俞君英张颖张东明
Owner 安徽中盛溯源生物科技有限公司
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