Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Our lab aims to better understand the molecular mechanism of retinal diseases with a focus on unmet medical needs of patients. We aim to develop effective treatments such as stem cell-based therapies for them. Cryopreservation of stem cell-derived RP cells is an important step for RP replacement therapy.

Currently, the cells are frozen at various stages, resulting in highly variable survival rates for sowing. This is needed to determine the optimal cell staging for freezing these cells. Our protocol provides a simple, efficient, and inexpensive ways of freezing RP cells by determining the exponential phase of RP cells for freezing.

It's less dependent of differentiation methods or cell lines. To begin, dilute a vial of thawed cold basement membrane matrix with 12 milliliters of DMEM/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 250G for three minutes at room temperature.

Pour out the supernatant from the centrifuged tube. Then resuspend 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. To begin, dissociate the hESC-derived retinal pigment epithelial cells.

Prepare the cell suspension and then count the cells. Now, centrifuge the cells at 250G for three minutes at room temperature. Pour out the supernatant.

Then resuspend the cell pellet in cryo-preservation medium. Next, transfer one milliliter of the cell suspension to a 1.2-milliliter cryogenic vial. Place the cryogenic vials in a freezing container and freeze overnight at minus 80 degrees Celsius.

Transfer the vials to liquid nitrogen for long-term storage. To thaw the frozen vials, first heat the culture medium in metallic beads heated to 37 degrees Celsius. Pre-fill 10 milliliters of the warmed culture medium into a 15-milliliter tube.

Now, remove the cryogenic vials from the liquid nitrogen and place them in an automated thawing system for rapid thawing. Drop 0.5 to one milliliter of the preheated culture medium into the cryogenic vial to ensure gradual cell adaptation. Then pipette 1.5 to two milliliters of the cell suspension to the 15-milliliter tube with medium.

Centrifuge the cells at 250G for three minutes at room temperature. Then discard the supernatant. Resuspend the pellet in two milliliters of warm medium.

Load the cells in a hemocytometer and count them to determine the recovery and survival rates. Culture the thawed cells on basement membrane-coated plates in a culture medium with Y27632. The retinal pigment epithelial cells that were frozen on day five in their exponential phase displayed a higher attachment rate after thawing.

Relative to their characteristic hexagonal morphology, the cells frozen at other time points had a fibroblastic phenotype. P2 day-five cells displayed higher expression and more properly localized cell markers 28 days after thawing. It was noted that different cryopreservation media performed equally well in achieving high cell viability and attachment post-thawing.