Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Dissociation and Optimal Cryopreservation Stage Determination

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03:06 min

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November 3rd, 2023

DOI :

10.3791/200823-v

November 3rd, 2023

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Xinyue Bai*¹^,²^,³^,⁴^,⁵^,⁶^,⁷, Ting Zhang*¹^,²^,³^,⁴^,⁵^,⁶^,⁷, Xinyue Zhu¹^,²^,³^,⁴^,⁵^,⁶^,⁷, Xianyu Huang¹^,²^,³^,⁴^,⁵^,⁶^,⁷, Haiyun Liu¹^,²^,³^,⁴^,⁵^,⁶^,⁷, Xiaoyan Ding⁸, Mei Jiang¹^,²^,³^,⁴^,⁵^,⁶^,⁷, Xiaodong Sun¹^,²^,³^,⁴^,⁵^,⁶^,⁷

¹Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, ²National Clinical Research Center for Eye Diseases, ³Shanghai Clinical Research Center for Eye Diseases, ⁴Shanghai Key Clinical Specialty, ⁵Shanghai Key Laboratory of Ocular Fundus Diseases, ⁶Shanghai Engineering Center for Visual Science and Photomedicine, ⁷Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, ⁸Stem Cell Core Facility, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences

* Diese Autoren haben gleichermaßen beigetragen

Transkript

To begin, dilute a vial of thawed cold basement membrane matrix with 12 milliliters of DM EM F12. Mix the suspension well. Add one cover slip into each well of a 24-well plate.

Then, pipette 250 microliters of the dilute matrix solution into each well. Discard the culture medium of the cultured hESC derived retinal pigment epithelial cell plates. Wash the plates two times with one milliliter of preheated PBS per well.

Next, add 0.5 milliliters of the cell dissociation reagent to the wells of the culture plates, and incubate them at 37 degrees Celsius for 15 minutes to initiate digestion. After incubation, observe the cells under the microscope to confirm the end of digestion. Now with a one milliliter pipette, gently pipette the cell suspension up and down 10 times.

Add preheated culture medium to dilute the suspension. Then, centrifuge it at 250 G for three minutes at room temperature. Pour out the supernatant from the centrifuge tube.

Then, re-suspend the cell pellet in two milliliters of the culture medium. Use a pipette to mix the cells 10 to 15 times. Filter the cell suspension through a 40 micrometer cell strainer.

Next, count the retinal pigment epithelial cells. Aspirate the coating solution before plating. Then, add one milliliter of the culture medium into each well and seed the cells on the cover slips.

After EDU incorporation and staining, capture the images of five random fields with a fluorescence microscope. Calculate the percentage of EDU positive cells. Then, plot the corresponding proportion time curves to generate a growth curve.

Lastly, determine the freezing window from the exponential phase for each cell line. The retinal pigment epithelial cells initially lost their characteristic hexagonal morphology, but later regained it in the exponential phase of growth. When the cells were cultured for an additional week, cell proliferation decreased.

EDU proliferation assay confirmed that P2 day five cells exhibited a higher proliferation rate, whereas P2 day 11 cells had entered the deceleration phase.

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