Skip to main content Accessibility help

Cultured Human Retinal Pigment Epithelial (hRPE) Sheets: A Search for Suitable Storage Conditions

  • Ayyad Z. Khan (a1) (a2), Tor P. Utheim (a1) (a3) (a4) (a5) (a6) (a7) (a8), Sjur Reppe (a1) (a9), Leiv Sandvik (a10), Torstein Lyberg (a1), Borghild B.-H. Roald (a2) (a11), Ibrahim B. Ibrahim (a2) and Jon R. Eidet (a1) (a3)...

The advancement of human retinal pigment epithelial cell (hRPE) replacement therapy is partly dependent on optimization of cell culture, cell preservation, and storage medium. This study was undertaken to search for a suitable storage temperature and storage medium for hRPE. hRPE monolayer sheets were cultured under standard conditions at 37°C and then randomized for storage at six temperatures (4, 16, 20, 24, 28, and 37°C) for 7 days. After revealing a suitable storage temperature, hRPE sheets were subsequently stored with and without the silk protein sericin added to the storage medium. Live/dead assay, light microscopy, pH, and phenotypic expression of various proteins were used to assess cell cultures stored at different temperatures. After 7 days of storage, hRPE morphology was best preserved at 4°C. Addition of sericin to the storage medium maintained the characteristic morphology of the preserved cells, and improved pigmentation and levels of pigmentation-related proteins in the cultured hRPE sheets following a 7-day storage period at 4°C.

Corresponding author
* Author for correspondence: Ayyad Z. Khan, E-mail:
Hide All
Ahmado, A, Carr, A-J, Vugler, AA, Semo, M, Gias, C, Lawrence, JM, Chen, LL, Chen, FK, Turowski, P, da Cruz, L and Coffey, PJ (2011) Induction of differentiation by pyruvate and DMEM in the human retinal pigment epithelium cell line ARPE-19. Invest Ophthalmol Vis Sci 52(10), 71487159.
Aramwit, P, Damrongsakkul, S, Kanokpanont, S and Srichana, T (2010) Properties and antityrosinase activity of sericin from various extraction methods. Biotechnol Appl Biochem 55(2), 9198.
Berson, JF, Harper, DC, Tenza, D, Raposo, G and Marks, MS (2001) Pmel17 initiates premelanosome morphogenesis within multivesicular bodies. Mol Biol Cell 12(11), 34513464.
Boron, WF and Boulpaep, EL (2012) Medical Physiology, 2e Updated Edition: With STUDENT CONSULT Online Access. Philadelphia, PA: Elsevier Health Sciences.
Cao, TT and Zhang, YQ (2017) The potential of silk sericin protein as a serum substitute or an additive in cell culture and cryopreservation. Amino Acids 49(6), 10291039.
Casten, R and Rovner, B (2008) Depression in age-related macular degeneration. J Vis Impair Blind 102(10), 591599.
Cavallotti, C and Cerulli, L (2008) Age-Related Changes of the Human Eye. Totowa, NJ: Humana Press.
Chlapanidas, T, Farago, S, Lucconi, G, Perteghella, S, Galuzzi, M, Mantelli, M, Avanzini, MA, Tosca, MC, Marazzi, M, Vigo, D, Torre, ML and Faustini, M (2013) Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities. Int J Biol Macromol 58, 4756.
da Cruz, L, Chen, FK, Ahmado, A, Greenwood, J and Coffey, P (2007) RPE transplantation and its role in retinal disease. Prog Retin Eye Res 26(6), 598635.
Double, KL (2006) Functional effects of neuromelanin and synthetic melanin in model systems. J Neural Trans 113(6), 751756.
Duarte, LT, Tiba, JB, Santiago, MF, Garcia, TA and Freitas Bara, MT (2012) Production and characterization of tyrosinase activity in Pycnoporus sanguineus CCT-4518 crude extract. Braz J Microbiol 43(1), 2129.
Eidet, JR, Reppe, S, Pasovic, L, Olstad, OK, Lyberg, T, Khan, AZ, Fostad, IG, Chen, DF and Utheim, TP (2016) The silk-protein sericin induces rapid melanization of cultured primary human retinal pigment epithelial cells by activating the NF-kappaB pathway. Sci Rep 6, 22671.
Friedman, DS, O’Colmain, BJ, Munoz, B, Tomany, SC, McCarty, C, de Jong, PT, Nemesure, B, Mitchell, P and Kempen, J, Eye Diseases Prevalence Research Group (2004) Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 122(4), 564572.
Jager, RD, Mieler, WF and Miller, JW (2008) Age-related macular degeneration. New Eng J Med 358(24), 26062617.
Khadka, A, Iacovelli, J and Saint-Geniez, M (2015) PGC1a rescue retinal pigment epithelial cells from oxidative damage: Implication for age-related macular degeneration. FASEB J 29(1 Suppl), 148.
Khan, AZ, Utheim, TP, Jackson, CJ, Reppe, S, Lyberg, T and Eidet, JR (2016) Nucleus morphometry in cultured epithelial cells correlates with phenotype. Microsc Microanal 22(3), 612.
Kuznetsova, AV, Kurinov, AM and Aleksandrova, MA (2014) Cell models to study regulation of cell transformation in pathologies of retinal pigment epithelium. J Ophthalmol 2014, 801787.
Kwon, BS, Chintamaneni, C, Kozak, CA, Copeland, NG, Gilbert, DJ, Jenkins, N, Barton, D, Francke, U, Kobayashi, Y and Kim, KK (1991) A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12. Proc Natl Acad Sci U S A 88(20), 92289232.
Maminishkis, A, Chen, S, Jalickee, S, Banzon, T, Shi, G, Wang, FE, Ehalt, T, Hammer, JA and Miller, SS (2006) Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci 47(8), 36123624.
Memoli, S, Napolitano, A, d’Ischia, M, Misuraca, G, Palumbo, A and Prota, G (1997) Diffusible melanin-related metabolites are potent inhibitors of lipid peroxidation. Biochim Biophys Acta 1346(1), 6168.
Oduntan, O and Masige, K (2011) A review of the role of oxidative stress in the pathogenesis of eye diseases. Afr Vision Eye Health 70(4), 191199.
Pasovic, L, Utheim, TP, Maria, R, Lyberg, T, Messelt, EB, Aabel, P, Chen, DF, Chen, X and Eidet, JR (2013a) Optimization of storage temperature for cultured ARPE-19 cells. J Ophthalmol 2013, 216359.
Pasovic, L, Utheim, TP, Maria, R, Lyberg, T, Messelt, EB, Aabel, P, Chen, DF, Chen, X and Eidet, JR (2013b) Optimization of storage temperature for cultured ARPE-19 cells. J Ophthalmol 2013, 216359.
Quinn, PJ (1988) Effects of temperature on cell membranes. Symposia Soc Exp Biol 42, 237258.
Reinisalo, M, Putula, J, Mannermaa, E, Urtti, A and Honkakoski, P (2012) Regulation of the human tyrosinase gene in retinal pigment epithelium cells: The significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site. Mol Vision 18, 3854.
Rovner, BW and Casten, RJ (2002) Activity loss and depression in age-related macular degeneration. Am J Geriatr Psychiatry 10(3), 305310.
Salero, E, Blenkinsop, TA, Corneo, B, Harris, A, Rabin, D, Stern, JH and Temple, S (2012) Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives. Cell Stem Cell 10(1), 8895.
Sanyal, S and Zeilmaker, GH (1988) Retinal damage by constant light in chimaeric mice: Implications for the protective role of melanin. Exp Eye Res 46(5), 731743.
Schwartz, SD, Regillo, CD, Lam, BL, Eliott, D, Rosenfeld, PJ, Gregori, NZ, Hubschman, JP, Davis, JL, Heilwell, G, Spirn, M, Maguire, J (2015) Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: Follow-up of two open-label phase 1/2 studies. Lancet 385(9967), 509516.
Shimura, M, Ishizaka, Y, Yuo, A, Hatake, K, Oshima, M, Sasaki, T and Takaku, F (1997) Characterization of room temperature induced apoptosis in HL-60. FEBS Lett 417(3), 379384.
Shimura, M, Osawa, Y, Yuo, A, Hatake, K, Takaku, F and Ishizaka, Y (2000) Oxidative stress as a necessary factor in room temperature-induced apoptosis of HL-60 cells. J Leukocyte Biol 68(1), 8796.
Stover, NP, Bakay, RA, Subramanian, T, Raiser, CD, Cornfeldt, ML, Schweikert, AW, Allen, RC and Watts, RL (2005) Intrastriatal implantation of human retinal pigment epithelial cells attached to microcarriers in advanced Parkinson disease. Arch Neurol 62(12), 18331837.
Strauss, O (2005) The retinal pigment epithelium in visual function. Physiol Rev 85(3), 845881.
Utheim, TP, Raeder, S, Utheim, OA, Cai, Y, Roald, B, Drolsum, L, Lyberg, T and Nicolaissen, B (2007) A novel method for preserving cultured limbal epithelial cells. Br J Ophthalmol 91(6), 797800.
Van Buskirk, RG, Baust, JM, Snyder, KK, Mathew, AJ and Baust, JG (2004) Hypothermic storage and cryopreservation. BioProcess Int 2(10), 4248.
Van Itallie, CM and Anderson, JM (2006) Claudins and epithelial paracellular transport. Annu Rev Physiol 68, 403429.
Wang, N and Hebert, DN (2006) Tyrosinase maturation through the mammalian secretory pathway: Bringing color to life. Pigment Cell Res 19(1), 318.
Watts, RL, Raiser, CD, Stover, NP, Cornfeldt, ML, Schweikert, AW, Allen, RC, Subramanian, T, Doudet, D, Honey, CR and Bakay, RA (2003) Stereotaxic intrastriatal implantation of human retinal pigment epithelial (hRPE) cells attached to gelatin microcarriers: A potential new cell therapy for Parkinson’s disease. J Neural Trans Suppl 65, 215227.
Winder, A, Kobayashi, T, Tsukamoto, K, Urabe, K, Aroca, P, Kameyama, K and Hearing, V (1993) The tyrosinase gene family – Interactions of melanogenic proteins to regulate melanogenesis. Cell Mol Biol Res 40(7–8), 613626.
Wright, B (2013) Corneal Regenerative Medicine: Methods and Protocols. New York: Springer.
Yin, F, Tian, ZM, Liu, S, Zhao, QJ, Wang, RM, Shen, L, Wieman, J and Yan, Y (2012) Transplantation of human retinal pigment epithelium cells in the treatment for Parkinson disease. CNS Neurosci Ther 18(12), 10121020.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed