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

Summary

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Abstract

Retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2′-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Introduction

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells required for maintaining the proper function of the retina¹. RPE dysfunction and death are closely associated with many retinal degenerative diseases, including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease^2,3. RPE replacement therapy is one of the most promising treatment regimens for these diseases^4,5,6,7. A stable supply of donor RPE cells is vital for cel....

Protocol

1. Cell dissociation

1. Maintain RPE cells as previously described^17,22.
   NOTE: All cells are grown at 37 °C in a 5% CO₂ atmosphere throughout the duration of the protocols.
2. Prepare the required amount of PBS and culture medium in a 37 ° C water bath and place the cell dissociation reagent at room temperature.
3. Discard the culture solution and wash the plates twice with 1 mL of preheated PBS .......

Discussion

In the present study, a successful freeze-thaw protocol for hESC-derived RPE cells for research and clinical needs is described. Unlike the immortalized RPE cell line, ARPE-19, RPE cells with proper characteristic epithelial phenotype and function, like stem cell derived-RPE cells, are more sensitive to cryopreservation. Less than 32% of the cells remained at 24 h post thaw if not properly preserved¹⁷. Cryopreservation timing is a critical parameter. An established view for immortalized cell cryop.......

Materials

Name	Company	Catalog Number	Comments
40 μm Cell strainer	Corning	431750
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 Dye	Thermo Fisher Scientific	C10337
Cryo freezing container	Nalgene	5100-0001
CryoStor CS10	Biolife Solutions	07930	cryopreservation medium #1
DPBS, no calcium, no magnesium	Thermo Fisher Scientific	14190144
Genxin	Selcell	YB050050	cryopreservation medium #2
Human embryonic stem cells	provided by Wicell, USA	H9 cell line
Matrigel, hESC-Qualified Matrix	Corning	354277	basement membrane matrix
ThawSTAR CFT2 Automated Cell Thawing System	BioLife Solutions	AST-601
Trypan Blue solution 0.4%	Sigma	T8154
TryPLE Select	Thermo Fisher Scientific	12563029	cell dissociation reagent
XVIVO-10 medium	Lonza	BEBP04-743Q	RPE culture medium
Y-27632	Selleck	S1049