Isolation of Corneal and Limbal Cells From Human Donor Cornea

Year 2025, Volume: 11 Issue: 3, 120 - 131, 30.09.2025

Sibel Cetinel , Sevilay Burcu Sahin , Ece Turan-vural

https://doi.org/10.30934/kusbed.1678793

Abstract

Objective: Isolation of corneal cells while preserving their specific phenotypes is crucial for regenerative medicine applications. Here, we aimed to evaluate corneal cell isolation techniques from human corneal tissue.
Methods: We isolated corneal cells and limbal stem cells from human donor corneas obtained from the eye bank by employing both enzymatic and explant cell culture approaches and monitored the morphological characteristics of the isolated cells.
Results: Explant culture demonstrated suitability for the isolation of corneal endothelial and epithelial cells, while both explant and enzymatic culture processes yielded successful results for limbal and corneal stromal tissues. The optimal conditions for enzymatic culture were determined to be 2 mg/mL enzyme solution for 30 minutes at 37 °C. On the other hand, the feasibility of explant cultures was increased by re-transferring and culturing tissue specimens to isolate an enlarged quantity of cells in an extended culture periods. However, this approach resulted in morphological changes in the isolated cells. The earliest passage at which isolated primary cells (keratocyte and limbal stem cells) could be immortalized was Passage 1, enabling subculturing and cell stock creation. On the other hand, isolated primary limbal stem cells demonstrated the capacity to differentiate into corneal epithelial and keratocyte cells.
Conclusion: This study provides a comprehensive and detailed presentation that will serve as an informative resource in the literature for corneal cell culture, corneal tissue engineering, and drug delivery studies.

Keywords

cornea , primary human corneal cells , limbus , limbal stem cells , differentiation , immortalization

İnsan Donör Korneasından Korneal ve Limbal Hücrelerin İzole Edilmesi

Abstract

Amaç: Gözün en dış tabakasında yer alan kornea, ışığın merceğe etkin bir şekilde iletilmesini sağlayan, saydam ve damarsız bir dokudur; bu yönüyle görme keskinliğine önemli ölçüde katkı sağlar. Dıştan içe doğru sırasıyla epitel, Bowman membranı, stroma, Descemet membranı ve endotel olmak üzere beş farklı tabakadan oluşan korneada, her bir tabakada bulunan özelleşmiş hücre tipleri, dokunun bütünlüğü için hayati öneme sahip spesifik işlevler üstlenir. Ayrıca, korneaya komşu bir doku olan limbus bölgesinde, kornea dokusunun yenilenmesini sağlayan kök hücreler bulunmaktadır. Korneal hücrelerin kendi özgün fenotiplerini koruyacak şekilde izole edilmesi, rejeneratif tıp uygulamaları açısından büyük önem taşımaktadır. Bu çalışmada, insan kornea dokusundan korneal hücre izolasyon tekniklerinin değerlendirilmesi amaçlanmıştır.
Yöntem: Göz bankasından temin edilen insan donör kornealarından korneal hücreler ve limbal kök hücreler, enzimatik ve eksplant hücre kültürü yaklaşımları kullanılarak izole edildi ve elde edilen hücrelerin morfolojik özellikleri gözlemlendi.
Bulgular: Eksplant kültürü yöntemi, korneal endotel ve epitel hücrelerinin izolasyonu için uygunluk göstermiştir. Bununla birlikte, limbal ve korneal stromal dokuların izolasyonunda hem eksplant hem de enzimatik kültür yöntemleri başarılı sonuçlar vermiştir. Enzimatik kültür için en uygun koşul; 2 mg/mL enzim çözeltisinin 37°C’de 30 dakika inkübasyonu olarak belirlenmiştir. Diğer yandan, eksplant kültürlerin verimliliği, doku örneklerinin yeniden transfer edilerek kültüre alınmasıyla artırılmış; bu da daha uzun süreli kültürlerde hücre sayısının artırılmasını sağlamıştır. Ancak bu yaklaşım, izole edilen hücrelerin morfolojisinde değişikliklere yol açmıştır. Keratosit ve limbal kök hücreler gibi izole edilen primer hücrelerin ölümsüzleştirilebildiği en erken pasaj, Pasaj 1 olarak belirlenmiş ve bu da alt kültürleme ile hücre stoğu oluşturulmasına olanak tanımıştır. Öte yandan, izole edilen primer limbal kök hücrelerin korneal epitel ve keratosit hücrelerine farklılaşma kapasitesi gösterdiği tespit edilmiştir.
Sonuç: Bu çalışma, korneal hücre kültürü, kornea doku mühendisliği ve ilaç salınımı çalışmaları için literatürde yol gösterici nitelikte kapsamlı ve ayrıntılı bir kaynak sunmaktadır.

Keywords

kornea , insan primer korneal hücre izolasyonu , limbus , limbal kök hücreler , farklılaşma , ölümsüzleştirme

References

• Sridhar MS. Anatomy of cornea and ocular surface. Indian J Ophthalmol. 2018;66(2):190-194. doi:10.4103/ijo.IJO_646_17
• Gonzalez-Andrades M, Argüeso P, Gipson I. Corneal Anatomy: Therapy and Surgery. In: In Alió JL, Barrio JLAd, Arnalich-Montiel F, eds. Corneal Regeneration Switzerland: Springer Nature; 2019:3-12.
• Ehlers N, Heegaard S, Hjortdal J, Ivarsen A, Nielsen K, Prause JU. Morphological evaluation of normal human corneal epithelium. Acta Ophthalmol. 2010;88(8):858-861. doi:10.1111/j.1755-3768.2009.01610.x
• Ehlers N, Hjortdal J. The Cornea: Epithelium and Stroma. In: Fischbarg J, ed. The Biology of the Eye. vol 10. Amsterdam: Elsevier; 2005:83-111.
• Lavker RM, Sun TT. Epithelial stem cells: the eye provides a vision. Eye (Lond). 2003;17(8):937-942. doi:10.1038/sj.eye.6700575.
• Cubitt CL, Tang Q, Monteiro CA, Lausch RN, Oakes JE. IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes. Invest Ophthalmol Vis Sci. 1993;34(11):3199-3206.
• Ramaesh T, Ramaesh K, Riley SC, West JD, Dhillon B. Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells. Eye (Lond). 2012;26(8):1138-1144. doi:10.1038/eye.2012.135.
• Chen CC, Chang JH, Lee JB, Javier J, Azar DT. Human corneal epithelial cell viability and morphology after dilute alcohol exposure. Invest Ophthalmol Vis Sci. 2002;43(8):2593-2602.
• Kitazawa K, Hikichi T, Nakamura T, Sotozono C, Kinoshita S, Masui S. PAX6 regulates human corneal epithelium cell identity. Exp Eye Res. 2017;154:30-38. doi:10.1016/j.exer.2016.11.005.
• Araki-Sasaki K, Ohashi Y, Sasabe T, et al. An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci. 1995;36(3):614-621.
• Suzuki T, Richards SM, Liu S, Jensen RV, Sullivan DA. Influence of sex on gene expression in human corneal epithelial cells. Mol Vis. 2009;15:2554-2569.
• Zhang J, Sisley AM, Anderson AJ, Taberner AJ, McGhee CN, Patel DV. Characterization of a Novel Collagen Scaffold for Corneal Tissue Engineering. Tissue Eng Part C Methods. 2016;22(2):165-172. doi:10.1089/ten.TEC.2015.0304.
• Chen S, Mienaltowski MJ, Birk DE. Regulation of corneal stroma extracellular matrix assembly. Exp Eye Res. 2015;133:69-80. doi:10.1016/j.exer.2014.08.001.
• Ho LT, Harris AM, Tanioka H, et al. A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea. Matrix Biol. 2014;38:59-68. doi:10.1016/j.matbio.2014.06.004.
• Espana EM, Birk DE. Composition, structure and function of the corneal stroma. Exp Eye Res. 2020;198:108137. doi:10.1016/j.exer.2020.108137.
• Pinnamaneni N, Funderburgh JL. Concise review: Stem cells in the corneal stroma. Stem Cells. 2012;30(6):1059-1063. doi:10.1002/stem.1100.
• West-Mays JA, Dwivedi DJ. The keratocyte: corneal stromal cell with variable repair phenotypes. Int J Biochem Cell Biol. 2006;38(10):1625-1631. doi:10.1016/j.biocel.2006.03.010.
• Pei Y, Reins RY, McDermott AM. Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes. Exp Eye Res. 2006;83(5):1063-1073. doi:10.1016/j.exer.2006.05.011.
• Surovtseva MA, Poveshchenko OV, Krasner KY, et al. Morphofunctional Properties of Corneal Stromal Cells. Bull Exp Biol Med. 2021;172(1):96-99. doi:10.1007/s10517-021-05339-5.
• Choong PF, Mok PL, Cheong SK, Then KY. Mesenchymal stromal cell-like characteristics of corneal keratocytes. Cytotherapy. 2007;9(3):252-258. doi:10.1080/14653240701218508.
• Sosnová M, Bradl M, Forrester JV. CD34+ corneal stromal cells are bone marrow-derived and express hemopoietic stem cell markers. Stem Cells. 2005;23(4):507-515. doi:10.1634/stemcells.2004-0291.
• Nagymihály R, Veréb Z, Facskó A, Moe MC, Petrovski G. Effect of Isolation Technique and Location on the Phenotype of Human Corneal Stroma-Derived Cells. Stem Cells Int. 2017;2017:9275248. doi:10.1155/2017/9275248.
• Engelmann K, Bednarz J, Valtink M. Prospects for endothelial transplantation. Exp Eye Res. 2004;78(3):573-578. doi:10.1016/s0014-4835(03)00209-4.
• Joyce NC. Cell cycle status in human corneal endothelium. Exp Eye Res. 2005;81(6):629-638. doi:10.1016/j.exer.2005.06.012.
• Peh GS, Toh KP, Wu FY, Tan DT, Mehta JS. Cultivation of human corneal endothelial cells isolated from paired donor corneas. PLoS One. 2011;6(12):e28310. doi:10.1371/journal.pone.0028310.
• Li W, Sabater AL, Chen YT, et al. A novel method of isolation, preservation, and expansion of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2007;48(2):614-620. doi:10.1167/iovs.06-1126.
• Su CC, Chen CW, Ho WT, Hu FR, Lee SH, Wang IJ. Phenotypes of trypsin- and collagenase-prepared bovine corneal endothelial cells in the presence of a selective Rho kinase inhibitor, Y-27632. Mol Vis. 2015;21:633-643. Published 2015 Jun 4.
• Sabater AL, Guarnieri A, Espana EM, Li W, Prósper F, Moreno-Montañés J. Strategies of human corneal endothelial tissue regeneration. Regen Med. 2013;8(2):183-195. doi:10.2217/rme.13.11.
• Choi JS, Kim EY, Kim MJ, et al. Factors affecting successful isolation of human corneal endothelial cells for clinical use. Cell Transplant. 2014;23(7):845-854. doi:10.3727/096368913X664559.
• Engler C, Kelliher C, Speck CL, Jun AS. Assessment of attachment factors for primary cultured human corneal endothelial cells. Cornea. 2009;28(9):1050-1054. doi:10.1097/ICO.0b013e3181a165a3.
• Hsueh YJ, Ma DH, Ma KS, et al. Extracellular Matrix Protein Coating of Processed Fish Scales Improves Human Corneal Endothelial Cell Adhesion and Proliferation. Transl Vis Sci Technol. 2019;8(3):27. doi:10.1167/tvst.8.3.27.
• Branch MJ, Hashmani K, Dhillon P, Jones DR, Dua HS, Hopkinson A. Mesenchymal stem cells in the human corneal limbal stroma. Invest Ophthalmol Vis Sci. 2012;53(9):5109-5116. doi:10.1167/iovs.11-8673.
• Schlötzer-Schrehardt U, Kruse FE. Identification and characterization of limbal stem cells. Exp Eye Res. 2005;81(3):247-264. doi:10.1016/j.exer.2005.02.016.
• Li GG, Zhu YT, Xie HT, Chen SY, Tseng SC. Mesenchymal stem cells derived from human limbal niche cells. Invest Ophthalmol Vis Sci. 2012;53(9):5686-5697. doi:10.1167/iovs.12-10300.
• Gaujoux T, Touzeau O, Laroche L, Borderie VM. Morphometry of corneal epithelial cells on normal eyes and after anterior lamellar keratoplasty. Cornea. 2010;29(10):1118-1124. doi:10.1097/ICO.0b013e3181d5d93b.
• Writing Committee for the Cornea Donor Study Research Group, Sugar A, Gal RL, et al. Factors associated with corneal graft survival in the cornea donor study. JAMA Ophthalmol. 2015;133(3):246-254. doi:10.1001/jamaophthalmol.2014.3923.
• Schroeter J, Rieck P. Endothelial evaluation in the cornea bank. Dev Ophthalmol. 2009;43:47-62. doi:10.1159/000223838.
• Peh GS, Chng Z, Ang HP, et al. Propagation of human corneal endothelial cells: a novel dual media approach. Cell Transplant. 2015;24(2):287-304. doi:10.3727/096368913X675719.
• Choi JS, Kim EY, Kim MJ, et al. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins. Biomed Mater. 2013;8(1):014108. doi:10.1088/1748-6041/8/1/014108.
• Systems AE. FNC Coating Mix. https://athenaes.com/ PromotionalMaterials/Promotional%20Materials/FNCFlier%20wdatasheet%20v2017.pdf. Accessed September 10, 2025.
• Hendijani F. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues. Cell Prolif. 2017;50(2):e12334. doi:10.1111/cpr.12334.
• Volatier TLA, Figueiredo FC, Connon CJ. Effect of isolation method on human corneal stromal cell behaviour. Exp Eye Res. 2021;203:108400. doi:10.1016/j.exer.2020.108400.
• Xie HT, Chen SY, Li GG, Tseng SC. Isolation and expansion of human limbal stromal niche cells. Invest Ophthalmol Vis Sci. 2012;53(1):279-286. doi:10.1167/iovs.11-8441.
• Faragher RG, Kipling D. How might replicative senescence contribute to human ageing?. Bioessays. 1998;20(12):985-991. doi:10.1002/(SICI)1521-1878(199812)20:12<985::AID-BIES4>3.0.CO;2-A.
• de Bardet JC, Cardentey CR, González BL, et al. Cell Immortalization: In Vivo Molecular Bases and In Vitro Techniques for Obtention. BioTech (Basel). 2023;12(1):14. Published 2023 Jan 28. doi:10.3390/biotech12010014.
• Arras W, Vercammen H, Ní Dhubhghaill S, Koppen C, Van den Bogerd B. Proliferation Increasing Genetic Engineering in Human Corneal Endothelial Cells: A Literature Review. Front Med (Lausanne). 2021;8:688223. doi: 10.3389/fmed.2021.688223.
• Innoprot. Immortalized Human Keratocytes. https://innoprot.com/product/immortalized-human-keratocytes/. Accessed August 14, 2024.
• Cell Applications I. Human Corneal Keratocytes: HCK. https://www.cellapplications.com/human-corneal-keratocytes-hck. Accessed August 14, 2024.
• Fehrenbacher N, Gyrd-Hansen M, Poulsen B, et al. Sensitization to the lysosomal cell death pathway upon immortalization and transformation. Cancer Res. 2004;64(15):5301-5310. doi:10.1158/0008-5472.CAN-04-1427.
• Shukla S, Shanbhag SS, Tavakkoli F, Varma S, Singh V, Basu S. Limbal Epithelial and Mesenchymal Stem Cell Therapy for Corneal Regeneration. Curr Eye Res. 2020;45(3):265-277. doi:10.1080/02713683.2019.1639765.
• Robertson DM, Li L, Fisher S, et al. Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line. Invest Ophthalmol Vis Sci. 2005;46(2):470-478. doi:10.1167/iovs.04-0528.
• Kim HS, Jun Song X, de Paiva CS, Chen Z, Pflugfelder SC, Li DQ. Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. Exp Eye Res. 2004;79(1):41-49. doi:10.1016/j.exer.2004.02.015.
• Vedicherla S, Buckley CT. Rapid Chondrocyte Isolation for Tissue Engineering Applications: The Effect of Enzyme Concentration and Temporal Exposure on the Matrix Forming Capacity of Nasal Derived Chondrocytes. Biomed Res Int. 2017;2017:2395138. doi:10.1155/2017/2395138.
• Gonzalez G, Sasamoto Y, Ksander BR, Frank MH, Frank NY. Limbal stem cells: identity, developmental origin, and therapeutic potential. Wiley Interdiscip Rev Dev Biol. 2018;7(2):10.1002/wdev.303. doi:10.1002/wdev.303.
• StemBio. Türkiye'de Limbal Kök Hücre Yetmezliğine Artık Hücresel Tedavi Ürünü ile Çare Üretilebiliyor. https://stembio.com.tr/turkiyede-limbal-kok-hucre-yetmezligine-artik-hucresel-tedavi-urunu-ile-care-uretilebiliyor/. Accessed October 16, 2024.
• Barut Selver Ö, Yağcı A, Eğrilmez S, et al. Limbal Stem Cell Deficiency and Treatment with Stem Cell Transplantation. Turk J Ophthalmol. 2017;47(5):285-291. doi:10.4274/tjo.72593. Trosan P, Svobodova E, Chudickova M, Krulova M, Zajicova A, Holan V. The key role of insulin-like growth factor I in limbal stem cell differentiation and the corneal wound-healing process. Stem Cells Dev. 2012;21(18):3341-3350. doi:10.1089/scd.2012.0180.
• Sidney LE, Branch MJ, Dua HS, Hopkinson A. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells. Cytotherapy. 2015;17(12):1706-1722. doi:10.1016/j.jcyt.2015.08.003.