Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo

Britney O Pennington,Vignesh Nadar,Tai-Chi Lin,Mitchell N Hee,Biju B Thomas,Cassidy Hinman,Mohamed A Faynus,David R Hinton,Lincoln V Johnson,Juan Carlos Martinez-Camarillo,Jeffrey K Bailey,Dennis O Clegg,Jane Lebkowski,Danhong Zhu,Mark S Humayun,Debbie Mitra,Rory Ritts

doi:10.1038/s41598-021-85631-6

Abstract

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.

Highlights

We report that the DMSO concentration in the final rinse solution is < 20 μM (Supplementary Fig. S2d), which suggests that the cryopreservation medium was adequately removed from REPS and the risk of transferring residual DMSO into the eye is minimal
REPS were generated by seeding custom-designed parylene support m atricies[17,18] with frozen suspension stocks of RPE cells that were generated by spontaneous differentiation45 from WA09 human embryonic stem cells (WiCell Research Institute)
RPE cells were seeded at 1.5–2.9E5 cells/cm2 to achieve optimal post-thaw morphology and viability

Summary

Introduction

Three days prior to the assay, thawed REPS were transferred to a fresh tissue culture plate and incubated in culture medium supplemented with 15% FBS (Atlas Biologics, EF-0500-A) at 37 °C and 5% CO2. At 7 DPT or 28 DPT, approximately three million FITC-labelled POS per REPS were added to the culture medium; treated REPS were incubated at 37 °C and 5% C O2 for 16 h. Thirty minutes prior to POS exposure and throughout the POS incubation, REPS were treated with either 60 μg/mL anti-ɑvβ5-integrin function blocking antibody (abcam, ab177004) or 60 μg/mL mouse IgG1 isotype control (Thermofisher, Cat# 14-471482). REPS were rinsed five times using PBS with Ca2+ and Mg2+ (Gibco) and fixed using 4% paraformaldehyde. Immunofluorescence and image quantification were performed by a blinded analyst as described abo

Methods

Results

Conclusion