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Medicine

Published: November 3, 2023 doi: 10.3791/65888
Automatic Translation
*1,2,3,4,5,6,7, *1,2,3,4,5,6,7, 1,2,3,4,5,6,7, 1,2,3,4,5,6,7, 1,2,3,4,5,6,7, 8, 1,2,3,4,5,6,7, 1,2,3,4,5,6,7
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

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

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References

  1. Lakkaraju, A., et al. The cell biology of the retinal pigment epithelium. Progress in Retinal and Eye Research. 78, 100846 (2020).
  2. Mcbain, V. A., Townend, J., Lois, N. Progression of retinal pigment epithelial atrophy in stargardt disease. American Journal of Ophthamology. 154 (1), 146-154 (2012).
  3. George, S. M., Lu, F., Rao, M., Leach, L. L., Gross, J. M. The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems. Progress in Retinal and Eye Research. 85, 100969 (2021).
  4. Rizzolo, L. J., Nasonkin, I. O., Adelman, R. A. Retinal cell transplantation, biomaterials, and in vitro models for developing next-generation therapies of age-related macular degeneration. Stem Cells Translational Medicine. 11 (3), 269-281 (2022).
  5. Bertolotti, E., Neri, A., Camparini, M., Macaluso, C., Marigo, V. Stem cells as source for retinal pigment epithelium transplantation. Progress in Retinal and Eye Research. 42, 130-144 (2014).
  6. Da Cruz,, L,, et al. Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration. Nature Biotechnology. 36 (4), 328-337 (2018).
  7. Mehat, M. S., et al. Transplantation of human embryonic stem cell-derived retinal pigment epithelial cells in macular degeneration. Ophthalmology. 125 (11), 1765-1775 (2018).
  8. Buchholz, D. E., et al. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Translational Medicine. 2 (5), 384-393 (2013).
  9. Li, Q. -Y., et al. Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases. Experimental Eye Research. 202, 108305 (2021).
  10. Francois, M., et al. Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-gamma licensing. Cytotherapy. 14 (2), 147-152 (2012).
  11. Moll, G., et al. Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties. Stem Cells. 32 (9), 2430-2442 (2014).
  12. Ekpo, M. D., et al. Incorporating cryopreservation evaluations into the design of cell-based drug delivery systems: an opinion paper. Frontiers in Immunology. 13, 967731 (2022).
  13. Bailey, T. L., et al. Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells. Cryobiology. 71 (3), 472-480 (2015).
  14. Okumura, N., et al. Feasibility of a cryopreservation of cultured human corneal endothelial cells. PLoS One. 14 (6), e0218431 (2019).
  15. Pennington, B. O., et al. Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo. Scientific Reports. 11 (1), 6286 (2021).
  16. Woods, E. J., Thirumala, S., Badhe-Buchanan, S. S., Clarke, D., Mathew, A. J. Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use. Cytotherapy. 18 (6), 697-711 (2016).
  17. Zhang, T., et al. Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells. Stem Cell Research & Therapy. 13 (1), 454 (2022).
  18. Leach, L. L., et al. Induced pluripotent stem cell-derived retinal pigmented epithelium: a comparative study between cell lines and differentiation Methods. Journal of Ocular Pharmacology and Therapeutics. 32 (5), 317-330 (2016).
  19. Reichman, S., et al. Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in Xeno-free and feeder-free conditions. Stem Cells. 35 (5), 1176-1188 (2017).
  20. Brandl, C., et al. In-depth characterisation of retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC). Neuromolecular Medicine. 16 (3), 551-564 (2014).
  21. Hongisto, H., Ilmarinen, T., Vattulainen, M., Mikhailova, A., Skottman, H. Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method. Stem Cell Research & Therapy. 8 (1), 291 (2017).
  22. Foltz, L. P., Clegg, D. O. Rapid, directed differentiation of retinal pigment epithelial cells from human embryonic or induced pluripotent stem cells. Journal of Visualized Experiments: JoVE. (128), 10.3791/56274 (2017).
  23. Marcantonini, G., et al. Natural cryoprotective and cytoprotective agents in cryopreservation: a focus on melatonin. Molecules. 27 (10), 3254 (2022).
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Cite this Article

Bai, X., Zhang, T., Zhu, X., Huang,… More

Bai, X., Zhang, T., Zhu, X., Huang, X., Liu, H., Ding, X., Jiang, M., Sun, X. Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage. J. Vis. Exp. (201), e65888, doi:10.3791/65888 (2023).

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