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

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, chall...

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Autores principales: Britney O. Pennington, Jeffrey K. Bailey, Mohamed A. Faynus, Cassidy Hinman, Mitchell N. Hee, Rory Ritts, Vignesh Nadar, Danhong Zhu, Debbie Mitra, Juan Carlos Martinez-Camarillo, Tai-Chi Lin, Biju B. Thomas, David R. Hinton, Mark S. Humayun, Jane Lebkowski, Lincoln V. Johnson, Dennis O. Clegg
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Publicado: Nature Portfolio 2021
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spelling oai:doaj.org-article:f19a0c25861141a083ed1e359e044d222021-12-02T13:17:55ZXeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo10.1038/s41598-021-85631-62045-2322https://doaj.org/article/f19a0c25861141a083ed1e359e044d222021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-85631-6https://doaj.org/toc/2045-2322Abstract 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.Britney O. PenningtonJeffrey K. BaileyMohamed A. FaynusCassidy HinmanMitchell N. HeeRory RittsVignesh NadarDanhong ZhuDebbie MitraJuan Carlos Martinez-CamarilloTai-Chi LinBiju B. ThomasDavid R. HintonMark S. HumayunJane LebkowskiLincoln V. JohnsonDennis O. CleggNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Britney O. Pennington
Jeffrey K. Bailey
Mohamed A. Faynus
Cassidy Hinman
Mitchell N. Hee
Rory Ritts
Vignesh Nadar
Danhong Zhu
Debbie Mitra
Juan Carlos Martinez-Camarillo
Tai-Chi Lin
Biju B. Thomas
David R. Hinton
Mark S. Humayun
Jane Lebkowski
Lincoln V. Johnson
Dennis O. Clegg
Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
description 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.
format article
author Britney O. Pennington
Jeffrey K. Bailey
Mohamed A. Faynus
Cassidy Hinman
Mitchell N. Hee
Rory Ritts
Vignesh Nadar
Danhong Zhu
Debbie Mitra
Juan Carlos Martinez-Camarillo
Tai-Chi Lin
Biju B. Thomas
David R. Hinton
Mark S. Humayun
Jane Lebkowski
Lincoln V. Johnson
Dennis O. Clegg
author_facet Britney O. Pennington
Jeffrey K. Bailey
Mohamed A. Faynus
Cassidy Hinman
Mitchell N. Hee
Rory Ritts
Vignesh Nadar
Danhong Zhu
Debbie Mitra
Juan Carlos Martinez-Camarillo
Tai-Chi Lin
Biju B. Thomas
David R. Hinton
Mark S. Humayun
Jane Lebkowski
Lincoln V. Johnson
Dennis O. Clegg
author_sort Britney O. Pennington
title Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
title_short Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
title_full Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
title_fullStr Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
title_full_unstemmed Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
title_sort xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo
publisher Nature Portfolio
publishDate 2021
url https://doaj.org/article/f19a0c25861141a083ed1e359e044d22
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