PURPOSE: The goal of our study is to find an optimal approach to the preparation and preservation of corneal stromal tissue. We want to compare different methods of corneal stromal tissue creation and storage to optimize the efficacy of this process under the conditions of an eye bank. After we find the most suitable method to create a safe high quality product, we want to prove the possibility of using a single donor cornea for more than one patient. We would also like to verify the feasibility of making more corneal lenticules after the removal of a corneal endothelium for DMEK transplantation. METHODS: We provided morphological (histology, scanning electron microscope) and microbiological analysis in order to compare different methods of corneal lenticule and corneal stromal lamellae preparation and preservation. We also tested the surgical handling of the tissue to secure a safe manipulation of the tissue for clinical use. We compared two methods of corneal lenticule preparation: microkeratome dissection and femtosecond laser. As methods of preservation, we tested hypothermia, cryopreservation at -80 degrees Celsius in DMSO (dimethyl sulfoxide) and storage at room temperature with glycerol. Some intrastromal lenticules and lamellae in each group were previously irradiated with gamma radiation of 25 kGy (KiloGray). RESULTS: Corneal stromal lamellae prepared with a microkeratome have a smoother cut - side surface compared to lamellae prepared with a femtosecond laser. Femtosecond laser preparation caused more irregularities on the surface and we detected more conglomerates of the fibrils, while lamellae made with microkeratome had more sparse network. Using femtosecond laser, we were able to make more than five lenticules from a single donor cornea. Gamma irradiation led to damage of collagen fibrils in corneal stroma and a loss of their regular arrangement. Corneal tissue stored in glycerol showed collagen fibril aggregates and empty spaces between fibrils caused by dehydration. Cryopreserved tissue without previous gamma irradiation showed the most regular structure of the fibrils comparable to storage in hypothermia. CONCLUSION: Our results suggest that formation of a corneal lenticule lamellae by microkeratome results in smoother corneal lenticules, while being much cheaper than formation by femtosecond laser. Gamma irradiation of 25 kGy caused damage of the collagen fibres as well as their network arrangement, which correlated with loss of transparency and stiffer structure. These changes impair possible surgical utilisation of gamma irradiated corneas. Storage in glycerol at room temperature and cryopreservation had similar outcomes and we believe that both methods are appropriate and safe for further clinical use .

3rd Faculty of Medicine Charles University Prague Ruska 87 10000 Prague 10 Czech Republic

Department of Ophthalmology University Hospital Kralovske Vinohrady and 3rd Faculty of Medicine Srobarova 1150 50 Prague 10 100 34 Czech Republic

Department of Pathology University Hospital Kralovske Vinohrady and 3rd Faculty of Medicine Srobarova 1150 50 Prague 10 100 34 Czech Republic

Institute of Histology and Embryology 1st Faculty of Medicine Charles University Prague Albertov 4 Prague 10 128 00 Praha 2 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Krč Prague 10 100 34 Czech Republic

International Eye Bank of Prague University Hospital Kralovske Vinohrady and 3rd Faculty of Medicine Srobarova 1150 50 Prague 10 100 34 Czech Republic

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Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G. Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmol. 2016;134(2):167–173. doi: 10.1001/jamaophthalmol.2015.4776. PubMed DOI

Singh R, Gupta N, Vanathi M, Tandon R. Corneal transplantation in the modern era. Indian J Med Res. 2019 Jul PubMed PMC

Garrido C, Cardona G, Güell JL, Pujol J. Visual outcome of penetrating keratoplasty, deep anterior lamellar keratoplasty and Descemet membrane endothelial keratoplasty. J Optom. 2018;11(3):174–181. doi: 10.1016/j.optom.2017.08.003. PubMed DOI PMC

Ehlers N, Hjortdal J, Nielsen K. Corneal grafting and banking. Dev Ophthalmol. 2009;43:1–14. doi: 10.1159/000223833. PubMed DOI

Oganesyan OG, Neroev VV, Grdikanyan AA, Getadaryan VR. Five Keratoplasties From One Donor Cornea. Cornea. 2018;37(5):667–671. doi: 10.1097/ICO.0000000000001551. PubMed DOI

Vajpayee RB, Sharma N, Jhanji V, Titiyal JS, Tandon R. One donor cornea for 3 recipients: a new concept for corneal transplantation surgery. Arch Ophthalmol. 2007;125(4):552–554. doi: 10.1001/archopht.125.4.552. PubMed DOI

Lazaridis A, Messerschmidt-Roth A, Sekundo W, Schulze S. Refractive Lenticule Implantation for Correction of Ametropia: Case Reports and Literature Review. Klin Monbl Augenheilkd. 2017;234(1):77–89. PubMed

Sun L, Yao P, Li M, Shen Y, Zhao J, Zhou X. The Safety and Predictability of Implanting Autologous Lenticule Obtained by SMILE for Hyperopia. Refract Surg. 2015;31(6):374–379. doi: 10.3928/1081597X-20150521-03. PubMed DOI

Pradhan KR, Reinstein DZ, Carp GI, Archer TJ, Gobbe M, Gurung R. Femtosecond laser-assisted keyhole endokeratophakia: correction of hyperopia by implantation of an allogeneic lenticule obtained by SMILE from a myopic donor. J Refract Surg. 2013;29(11):777–782. doi: 10.3928/1081597X-20131021-07. PubMed DOI

Lim CH , Riau AK, Lwin NC, Chaurasia SS, Tan DT, Mehta JS. LASIK re-implantation: feasibility study of a novel method for treatment of presbyopia. PLoS One. 2013 Dec 11;8(12). PubMed PMC

Jin H, He M, Liu H, Zhong X, Wu J, Liu L, Ding H, Zhang C, Zhong X. Small-Incision Femtosecond Laser-Assisted Intracorneal Concave Lenticule Implantation in Patients With Keratoconus. Cornea. 2019;38(4):446–453. doi: 10.1097/ICO.0000000000001877. PubMed DOI PMC

Mastropasqua L, Nubile M, Salgari N, Mastropasqua R. Femtosecond Laser-Assisted Stromal Lenticule Addition Keratoplasty for the Treatment of Advanced Keratoconus: A Preliminary Study. J Refract Surg. 2018;34(1):36. doi: 10.3928/1081597X-20171004-04. PubMed DOI

Wu F, Jin X, Xu Y, Yang Y. Treatment of corneal perforation with lenticules from small incision lenticule extraction surgery: a preliminary study of 6 patients. Cornea. 2015;34(6):658–663. doi: 10.1097/ICO.0000000000000397. PubMed DOI

Li M, Zhao F, Li M, Knorz MC, Zhou X. Treatment of Corneal Ectasia by Implantation of an Allogenic Corneal Lenticule. J Refract Surg. 2018;34(5):347–350. doi: 10.3928/1081597X-20180323-01. PubMed DOI

Ganesh S(1), Brar S, Rao PA. Cryopreservation of extracted corneal lenticules after small incision lenticule extraction for potential use in human subjects. Cornea. 2014 Dec;33(12):1355–62. PubMed PMC

Liu YC, Williams GP, George BL, Soh YQ, Seah XY, Peh GSL, Yam GHF, Mehta JS. Corneal lenticule storage before reimplantation. Mol Vis. 2017;27(23):753–764. PubMed PMC

Chae JJ, Choi JS, Lee JD, Lu Q, Stark WJ, Kuo IC, Elisseeff JH. Physical and Biological Characterization of the Gamma-Irradiated Human Cornea. Cornea. 2015;34(10):1287–1294. doi: 10.1097/ICO.0000000000000555. PubMed DOI

Xia F, Zhao J, Fu D, Xu Y, Yao P, Li M, Aruma A, Zhou X. Optical transmittance and ultrastructure of SMILE-derived lenticules subjected to three different preservative methods. Exp Eye Res. 2020 Dec;201:108357. 10.1016/j.exer.2020.108357. Epub 2020 Nov 13. PMID: 33197451. PubMed

Riau AK, Angunawela RI, Chaurasia SS, Lee WS, Tan DT, Mehta JS. Reversible femtosecond laser-assisted myopia correction: a non-human primate study of lenticule re-implantation after refractive lenticule extraction. PLoS ONE. 2013;8:e67058. doi: 10.1371/journal.pone.0067058. PubMed DOI PMC

Mohamed-Noriega K, Toh K-P, Poh R, Balehosur D, Riau A, Htoon HM, et al. Cornea lenticule viability and structural integrity after refractive lenticule extraction (ReLEx) and cryopreservation. Mol Vis. 2011;17:3437–3449. PubMed PMC

Armitage J. Cryopreservation for corneal storage. Dev Ophthalmol. 2009;43:63–69. doi: 10.1159/000223839. PubMed DOI

Fu L, Hollick EJ. Artificial Cornea Transplantation. 2023 Mar 6. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023 Jan–. PMID: 33760451 PubMed

Belin MW, Güell JL, Grabner G. Suggested Guidelines for Reporting Keratoprosthesis Results: Consensus Opinion of the Cornea Society, Asia Cornea Society, EuCornea, PanCornea, and the KPRO Study Group. Cornea. 2016;35(2):143–144. doi: 10.1097/ICO.0000000000000703. PubMed DOI