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

Xinyue Bai; Ting Zhang; Xinyue Zhu; Xianyu Huang; Haiyun Liu; Xiaoyan Ding; Mei Jiang; Xiaodong Sun

doi:10.3791/65888

Method Article

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

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DOI:

10.3791/65888

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November 3rd, 2023

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

^* These authors contributed equally

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 disease²^,³. RPE replacement therapy is one of the most promising treatment regimens for these diseases⁴^,⁵^,⁶^,⁷. A stable supply of donor RPE cells is vital for cel....