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Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo.
Sci Rep. 2021 03 18; 11(1):6286.SR

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.

Authors+Show Affiliations

Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, 6131 Biology 2 Bldg 571, NRI, UC Santa Barbara, Santa Barbara, CA, 93106, USA. bop@ucsb.edu. Regenerative Patch Technologies LLC, Portola Valley, CA, USA. bop@ucsb.edu.Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, 6131 Biology 2 Bldg 571, NRI, UC Santa Barbara, Santa Barbara, CA, 93106, USA. Regenerative Patch Technologies LLC, Portola Valley, CA, USA.Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, 6131 Biology 2 Bldg 571, NRI, UC Santa Barbara, Santa Barbara, CA, 93106, USA. Regenerative Patch Technologies LLC, Portola Valley, CA, USA.Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, 6131 Biology 2 Bldg 571, NRI, UC Santa Barbara, Santa Barbara, CA, 93106, USA. Regenerative Patch Technologies LLC, Portola Valley, CA, USA.College of Creative Studies, Biology, University of California, Santa Barbara, CA, USA.Department of Molecular Cellular and Developmental Biology, University of California, Santa Barbara, CA, USA.Regenerative Patch Technologies LLC, Portola Valley, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA. USC Dr. Allen and Charlotte Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA. USC Dr. Allen and Charlotte Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA. USC Dr. Allen and Charlotte Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA, USA.Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA. Department of Biomedical Engineering, Denney Research Center (DRB) of the University of Southern California, Los Angeles, CA, USA. USC Dr. Allen and Charlotte Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA, USA.Regenerative Patch Technologies LLC, Portola Valley, CA, USA.Regenerative Patch Technologies LLC, Portola Valley, CA, USA.Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, 6131 Biology 2 Bldg 571, NRI, UC Santa Barbara, Santa Barbara, CA, 93106, USA. Regenerative Patch Technologies LLC, Portola Valley, CA, USA. Department of Molecular Cellular and Developmental Biology, University of California, Santa Barbara, CA, USA.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

33737600

Citation

Pennington, Britney O., et al. "Xeno-free Cryopreservation of Adherent Retinal Pigmented Epithelium Yields Viable and Functional Cells in Vitro and in Vivo." Scientific Reports, vol. 11, no. 1, 2021, p. 6286.
Pennington BO, Bailey JK, Faynus MA, et al. Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo. Sci Rep. 2021;11(1):6286.
Pennington, B. O., Bailey, J. K., Faynus, M. A., Hinman, C., Hee, M. N., Ritts, R., Nadar, V., Zhu, D., Mitra, D., Martinez-Camarillo, J. C., Lin, T. C., Thomas, B. B., Hinton, D. R., Humayun, M. S., Lebkowski, J., Johnson, L. V., & Clegg, D. O. (2021). Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo. Scientific Reports, 11(1), 6286. https://doi.org/10.1038/s41598-021-85631-6
Pennington BO, et al. Xeno-free Cryopreservation of Adherent Retinal Pigmented Epithelium Yields Viable and Functional Cells in Vitro and in Vivo. Sci Rep. 2021 03 18;11(1):6286. PubMed PMID: 33737600.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo. AU - Pennington,Britney O, AU - Bailey,Jeffrey K, AU - Faynus,Mohamed A, AU - Hinman,Cassidy, AU - Hee,Mitchell N, AU - Ritts,Rory, AU - Nadar,Vignesh, AU - Zhu,Danhong, AU - Mitra,Debbie, AU - Martinez-Camarillo,Juan Carlos, AU - Lin,Tai-Chi, AU - Thomas,Biju B, AU - Hinton,David R, AU - Humayun,Mark S, AU - Lebkowski,Jane, AU - Johnson,Lincoln V, AU - Clegg,Dennis O, Y1 - 2021/03/18/ PY - 2020/11/17/received PY - 2021/03/03/accepted PY - 2021/3/19/entrez PY - 2021/3/20/pubmed PY - 2021/10/28/medline SP - 6286 EP - 6286 JF - Scientific reports JO - Sci Rep VL - 11 IS - 1 N2 - 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. SN - 2045-2322 UR - https://www.unboundmedicine.com/medline/citation/33737600/Xeno_free_cryopreservation_of_adherent_retinal_pigmented_epithelium_yields_viable_and_functional_cells_in_vitro_and_in_vivo_ L2 - https://doi.org/10.1038/s41598-021-85631-6 DB - PRIME DP - Unbound Medicine ER -