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Preparation and amplification culture method of human multipotential stem cell sourced human retinal pigment epithelium cells

A technology of human pluripotent stem cells and retinal pigment, applied in the field of stem cells, can solve the problems of lack of RPE cells restricting development, and achieve the effects of low cost, high yield and simple technology

Pending Publication Date: 2018-11-02
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Retinal pigment epithelial cell transplantation is one of the most promising means of restoring eyesight, but the lack of RPE cells restricts the development of this treatment

Method used

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  • Preparation and amplification culture method of human multipotential stem cell sourced human retinal pigment epithelium cells
  • Preparation and amplification culture method of human multipotential stem cell sourced human retinal pigment epithelium cells
  • Preparation and amplification culture method of human multipotential stem cell sourced human retinal pigment epithelium cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Expansion and culture of human pluripotent stem cells

[0041] Three human pluripotent stem cell (hPSC) lines were used for the study. BC1-GFP-hiPSC and BC1-hiPSC were gifts from friends, and the other hiPSC was purchased from American Life Technology Company ( Episomal hiPSC Line, A18945). All cells were seeded on 6-well culture plates coated with extracellular matrix MatriGel (Corning, 354277), and expanded and cultured with mTeSR1. When the cell confluency reaches 80%-90%, it is digested with 0.5mM EDTA (Life, 15575-038), and subcultured at 1:8 to 1:12. Observed under an inverted microscope, the cells were in sheet form, growing like colonies, and the cells in the colonies were tightly arranged with unclear borders ( figure 1 ), thereby expanding human pluripotent stem cells (hPSCs).

Embodiment 2

[0042] Example 2: Induction of differentiation of hPSCs into retinal cells including pigment epithelial cells

[0043] Referring to the reported method (reference: Xiufeng Zhong, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from humaniPSCs. Nat Commun. 2014 Jun 10; 5:4047), hPSCs were induced to differentiate into retinal cells including RPE cells. The day when hPSCs start to differentiate (that is, the hPSC expansion medium is replaced with differentiation medium or embryoid bodies are prepared) is set as the "0" day of differentiation.

Embodiment 3

[0044] Example 3: Obtaining and culturing of hPSC-derived 3D-RPE spheres

[0045] The retinal cells at the 4th week of hPSC-induced differentiation included RPE cells, and the neural retina (NR) and RPE with slight bulge, ring, and strong refraction could be recognized under the microscope. RPE can grow around NR, or it can grow individually or in sheets ( figure 2 ). Use a self-made tungsten needle or a 1mL syringe to stir up NR and its nearby RPE, and transfer it to a low-adsorption petri dish for suspension culture. The culture medium is RPE cell culture medium. Its formula is: every 100mL cell culture medium contains 10mL fetal bovine serum (Gibco, 10099-141), 2mL 50×B27 nerve cell growth supplement (Gibco, 12587-010), 1mL 100× penicillin-streptomycin mixed solution (Gibco, 15240), 1 mL of 100× non-essential amino acids (Gibco, 11140-050), 1 mL of 100× glutamine (Gibco, 35050-061) and 0.1 mL of 1000× taurine (Sigma, T-0652), the balance being DMEM / F12 (3:1) medium, th...

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Abstract

The invention discloses a preparation and amplification culture method of human multipotential stem cell sourced human retinal pigment epithelium cells. The method comprises the following steps of collecting a 3D-RPE sphere from the human multipotential stem cell sources; performing mechanical separation, removing non-RPE cells or agglomerates containing no pigments; remaining RPE cell sheets containing the pigments; performing enzymolysis digestion on the pigment-containing RPE cell sheets to obtain RPE unicellular suspension; thus obtaining the human multipotential stem cell sourced human retinal pigment epithelium cells. The features of the human RPE cells prepared by the method are similar to the features of human embryo sourced RPE cells; the typical RPE cell form features are realized; the normal physiological functions are shown. The human RPE cells prepared by the method can realize the subculture; the mass amplification is realized; the seed cells are provided for the study and treatment of retinal diseases; benefits are brought to blind patients; the problem of important defects of limited retinal pigment epithelium cells and RPE transplanting donor lack in the prior artcan be solved; great significance is realized.

Description

Technical field: [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for preparing and expanding human retinal pigment epithelial cells derived from human pluripotent stem cells. Background technique: [0002] Retinal pigment epithelium (RPE, retinal pigment epithelium) is located outside the retinal neuroepithelium, providing nutrition for the latter and participating in phototransduction reactions. Its degeneration, death or abnormal function is an important cause of retinal degeneration eye disease. Retinal pigment epithelial cell transplantation is one of the most promising means of restoring eyesight, but the lack of RPE cells restricts the development of this treatment. Before the rise of stem cell technology, the source of RPE cells was limited to the isolation of early aborted embryos or eyeballs donated by volunteers. Recent studies have shown that human pluripotent stem cells (hPSC), including human embryonic s...

Claims

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Application Information

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IPC IPC(8): C12N5/079
CPCC12N5/0621C12N2500/30C12N2500/32C12N2501/998C12N2509/00C12N5/062C12N2501/13C12N2501/999C12N2506/45C12N2513/00C12N2533/90C12N2500/00C12N2502/45C12N2509/10
Inventor 钟秀风葛坚刘胜旭彭福华
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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