Preparation method of retinal pigment epithelial cells
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A technique for retinal pigment and epithelial cells is applied in the field of preparation of retinal pigment epithelial cells, which can solve the problems of high cost, large batch difference, and many added factors.
Active Publication Date: 2021-11-26
GUANGXIU-GAOXIN LIFE SCI CO LTD HUNAN
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[0003] However, the "spontaneous differentiation method" has the disadvantage that the time and efficiency of RPE formation are uncontrollable. The method published in "Cell StemCell" is also slow in obtaining RPE, while the method of Dennies O Clegg needs to add more factors, and the cost High, and batch differences are large, the quality is difficult to control
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[0033] The method for preparing retinal pigment epithelial cells according to an embodiment of the present invention includes the following steps S1-S4:
[0034] S1, providing pluripotent stem cells;
[0035] S2, culturing the pluripotent stem cells to form embryoid bodies;
[0036] S3, using a medium containing Dorsomorphin and SB431542 to carry out suspension culture of the embryoid bodies;
[0037] S4. After the suspension culture, use the medium containing nicotine to carry out the adherent culture of the embryoid body for more than three weeks, and add Activin A to the medium for culture in the second to third weeks of the adherent culture to obtain the retina Pigment epithelium and retinal pigment epithelium precursors.
[0038] In the course of natural development, the human body will go through the stages of development from the fertilized egg to the inner, middle and outer germ layers. The retinal pigment epithelium develops from the ectoderm, and mainly undergoes ...
Embodiment 1
[0050] This embodiment starts from human embryonic stem cells, and the cell induction process is divided into three stages, such as figure 1 shown.
[0051] The basal medium used is D / F-EB medium but not limited thereto, and the following table is prepared:
[0052] Element Volume (mL) Knockout (KO) DMEM 30.8 Alien Free Knockout Serum Replacement 8 Glutamax I 0.4 2-Mercaptoethanol (70 μL diluted in 100 mL of KO DMEM) 0.4 non-essential amino acids 0.4
[0053] Stage 1: The feeder-free human embryonic stem cells used for induction were digested with Accumax digestion solution for about 5 minutes to form single cells, and the human embryonic stem cells were seeded into AggreWell well plates at a density of 1000 to 3000 cells and cultured in an incubator Leave overnight to form embryoid bodies (EBs). Our experimental results show that 1000-3000 cells / EB is the optimal planting density, less than 1000 cells / EB is not conducive to the ...
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Abstract
The invention relates to a preparation method of retinal pigment epithelial cells. The preparation method comprises the following steps: providing pluripotent stem cells; culturing the pluripotent stem cells to form embryoid bodies; using a culture medium containing Dorsomorphin and SB431542 for carrying out suspension culture on the embryoid body; after the suspension culture, a culture medium containing nicotine is used for carrying out adherent culture on the embryoid body for more than three weeks, Activin A is added into the culture medium for culture in the second week to the third week of the adherent culture, and the retinal pigment epithelial cells and the retinal pigment epithelial cell precursors are obtained. According to the preparation method disclosed by the invention, the culture medium needs fewer added components and is free of animal-derived components, the quantity of harvested cells is large, the induction efficiency is high, and a quality guarantee can be provided for the production of the retinal pigment epithelial cells.
Description
technical field [0001] The invention relates to the technical field of cell biology, in particular to a preparation method of retinal pigment epithelial cells. Background technique [0002] Currently, there are some methods for directing induction of human embryonic stem cells (hESC) into retinal pigment epithelium (Retinal pigment epithelial, RPE). This human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) is in the process of clinical research for the treatment of fundus diseases. The mainstream method for directional induction of human embryonic stem cells to differentiate into RPE for clinical research is the "spontaneous differentiation method". This method directly removes the factors that maintain the undifferentiation of human embryonic stem cells, allows them to spontaneously differentiate to obtain RPE cells, and then separates them. In addition to the "spontaneous differentiation method", the representative method is the method published in "Cel...
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