Abstract
Cultured epidermal cell sheets and bioengineered skin equivalents are widely used for skin reconstruction. Cryostorage of keratinocytes is one of the steps of the bioengineered products manufacture; therefore, it is important to study its effect on the viability of these cells. We investigated the preservation of morphogenetic potential by keratinocytes after cryostorage and found that cryostorage did not alter the dynamics of their proliferation. The population of keratinocytes is known to be highly heterogeneous in terms of proliferative and regenerative abilities. Our analysis of the pattern of keratinocyte death in suspension before and after cryostorage revealed the most vulnerable population with a high level of α6 integrin expression and a low level of transferrin receptor expression. This data must be taken into account in the course of tissue engineering.
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REFERENCES
Carley, E., Stewart, R.M., Zieman, A., Jalilian, I., King, D.E., Zubek, A., Lin, S., Horsley, V., and King, M.C., The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation, Elife, 2021, vol. 10, p. e58541. https://doi.org/10.7554/eLife.58541
Giangreco, A., Qin, M., Pintar, J.E., and Watt, F.M., Epidermal stem cells are retained in vivo throughout skin aging, Aging Cell, 2008, vol. 7, no. 2, pp. 250–259. https://doi.org/10.1111/j.1474-9726.2008.00372.x
Hibino, Y., Hata, K., Horie, K., Torii, S., and Ueda, M., Structural changes and cell viability of cultured epithelium after freezing storage, J. Craniomaxillofac. Surg., 1996, vol. 24, no. 6, pp. 346–351. https://doi.org/10.1016/s1010-5182(96)80035-7
Janes, S.M., Ofstad, T.A., Campbell, D.H., Eddaoudi, A., Warnes, G., Davies, D., and Watt, F.M., PI3-kinase-dependent activation of apoptotic machinery occurs on commitment of epidermal keratinocytes to terminal differentiation, Cell Res., 2009, vol. 19, no. 3, pp. 328–339. https://doi.org/10.1038/cr.2008.281
Metral, E., Bechetoille, N., Demarne, F., Rachidi, W., and Damour, O., α6 Integrin (α6high)/transferrin receptor (CD71)low keratinocyte stem cells are more potent for generating reconstructed skin epidermis than rapid adherent cells, Int. J. Mol. Sci., 2017, vol. 18, p. 282. https://doi.org/10.3390/ijms18020282
Palackic, A., Duggan, R.P., Campbell, M.S., Walters, E., Branski, L.K., Ayadi, A.E., and Wolf, S.E., The role of skin substitutes in acute burn and reconstructive burn surgery: an updated comprehensive review, Semin. Plast. Surg., 2022, vol. 36, no. 1, pp. 33–42. https://doi.org/10.1055/s-0042-1743455
Pasch, J., Schiefer, A., Heschel, I., Dimoudis, N., and Rau, G., Variation of the HES concentration for the cryopreservation of keratinocytes in suspensions and in monolayers, Cryobiology, 2000, vol. 41, no. 2, pp. 89–96. https://doi.org/10.1006/cryo.2000.2270
Reginato, M.J., Mills, K.R., Paulus, J.K., Lynch, D.K., Sgroi, D.C., Debnath, J., Muthuswamy, S.K., and Brugge, J.S., Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis, Nat. Cell Biol., 2003, vol. 5, no. 8, pp. 733–740. https://doi.org/10.1038/ncb1026
Ringstad, H., Reppe, S., Schøyen, T.H., Tønseth, K.A., Utheim, T.P., and Jackson, C.J., Stem cell function is conserved during short-term storage of cultured epidermal cell sheets at 12°C, PLoS One, 2020, vol. 15, no. 5, p. e0232270. https://doi.org/10.1371/journal.pone.0232270
Tiberio, R., Marconi, A., Fila, C., Fumelli, C., Pignatti, M., Krajewski, S., Giannetti, A., Reed, J.C., and Pincelli, C., Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner, FEBS Lett., 2002, vol. 524, nos. 1–3, pp. 139–144. https://doi.org/10.1016/s0014-5793(02)03040-5
Wray, H., Mackenzie, I.C., Storey, A., and Navsaria, H., α6 Integrin and CD44 enrich for a primary keratinocyte population that displays resistance to UV-induced apoptosis, PLoS One, 2012, vol. 7, no. 10, p. e46968. https://doi.org/10.1371/journal.pone.0046968
Yanaga, H., Udoh, Y., Yamamoto, M., Yoshii, S., Mori, S., Yamauchi, T., Kiyokawa, K., Koga, M., and Yanaga, K., Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: Early wound closure and suppression of scarring, Regen. Ther., 2017, vol. 6, pp. 74–82. https://doi.org/10.1016/j.reth.2017.03.002
ACKNOWLEDGEMENT
The authors thank Anna Popova, a former member of the laboratory group, for the idea of this study.
Funding
This work was supported by the Ministry of Science and Higher Education of the Russian Federation under the Agreement no. 075-15-2021-1063 from September 28, 2021 and by the Government program of basic research in Koltzov Institute of Developmental Biology of the Russian Academy of Sciences in 2023 no. 0088-2023-0001.
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The work was carried out in accordance with the Declaration of Helsinki of the World Association of Physicians and the protocol of the Commission on Bioethics of the Koltzov Institute of Developmental Biology, Russian Academy of Sciences (no. 51 of September 9, 2021).
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Rogovaya, O.S., Sutyagina, O.I., Alpeeva, E.V. et al. Studying the Effect of Cryostorage on Cell Viability Revealed the Most Vulnerable Population in the Keratinocyte Suspension. Biol Bull Russ Acad Sci 50 (Suppl 3), S270–S276 (2023). https://doi.org/10.1134/S1062359023604664
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DOI: https://doi.org/10.1134/S1062359023604664