Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro – A Stepwise Linear Regression Algorithm

Abstract

Purpose To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm.

Methods Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO₂ atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm.

Results The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007).

Conclusions The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

Zusammenfassung

Hintergrund Um verschiedene potenzielle Faktoren auf das Wachstum humaner limbaler Epithelzellen (LEC) aus Hornhautspendergewebe nach Langzeitlagerung (Organkultur) in vitro zu untersuchen, wurde ein schrittweiser linearer Regressionsalgorithmus verwendet.

Methoden Für unsere Experimente wurden 304 Hornhautringe von 215 Spendern verwendet. Zunächst wurden die Gewebestücke mit 4,0 mg/mL Kollagenase A bei 37 °C mit 95% relativer Luftfeuchtigkeit und 5% CO₂-Atmosphäre über Nacht inkubiert. Danach wurden die limbalen Epithelzellen mithilfe eines 20 µm CellTricks-Filters von den limbalen Keratozyten getrennt. Die humanen LECs wurden in KSFM-Medium, 1% Penicillin/Streptomycin (P/S), 0,02% epidermalem Wachstumsfaktor (EGF) und 0,3% Rinderhypophysenextrakt (BPE) kultiviert. Der potenzielle Einfluss des Spenderalters (Kovariate), der postmortalen Zeit (Kovariate), des Mediums (Kovariate), der Größe des verwendeten korneoskleralen Rings (360°, 270°180°, 120°, 90°, weniger als 90°; Kovariate), der Endothelzelldichte (ECD; Kovariate), Geschlecht (Faktor), Anzahl der Kulturmediumwechsel während der Organkultur (Faktor) und Herkunft des Spenders (spendende Institution und aufbewahrende Institution, Faktor) auf das Wachstum der LEC wurde mit einem schrittweisen linearen Regressionsalgorithmus analysiert.

Ergebnisse Die Rate der erfolgreichen Kultivierung humaner LEC betrug 37,5%. Die mit dem schrittweisen linearen Regressionsalgorithmus berechneten relevanten Einflussparameter auf das LEC-Wachstum waren: Intercept (p < 0,001), Spenderalter (p = 0,002), Anzahl der Kulturmediumwechsel während der Organkultur (p < 0,001), Gesamtmediumzeit (p = 0,181), Größe des verwendeten korneoskleralen Rings (p = 0,007) sowie Mediumzeit und Größe des korneoskleralen Rings (p = 0,007).

Schlussfolgerungen Der Erfolg der LEC-Kultivierung steigt mit abnehmendem Alter des Spenders, der geringeren Anzahl von Wechseln des Organkulturmediums während der Lagerung, der kürzeren Mediendauer in der Organkultur und der kleineren Größe des korneoskleralen Rings. Dieser von uns berechneter schrittweiser linearer Regressionsalgorithmus kann uns bei der Optimierung der LEC-Kultur in vitro helfen.

Key words

limbal epithelial cell culture - organ culture - stepwise linear regression algorithm

Schlüsselwörter

limbale Epithelzellkultur - Organkultur - schrittweiser linearer Regressionsalgorithmus

• References

• 1 Bobba S, Di Girolamo N. Contact Lenses: A Delivery Device for Stem Cells to Treat Corneal Blindness. Optom Vis Sci 2016; 93: 412-418
• 2 Echevarria TJ, Di Girolamo N. Tissue-regenerating, vision-restoring corneal epithelial stem cells. Stem Cell Rev Rep 2011; 7: 256-268
• 3 Li W, Hayashida Y, Chen YT. et al. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res 2007; 17: 26-36
• 4 Schlötzer-Schrehardt U, Kruse FE. Identification and characterization of limbal stem cells. Exp Eye Res 2005; 81: 247-264
• 5 Cotsarelis G, Cheng SZ, Dong G. et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989; 57: 201-209
• 6 Lavker RM, Tseng SC, Sun TT. Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Exp Eye Res 2004; 78: 433-446
• 7 Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 1986; 103: 49-62
• 8 Pellegrini G, Golisano O, Paterna P. et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 1999; 145: 769-782
• 9 Dua HS, Saini JS, Azuara-Blanco A. et al. Limbal stem cell deficiency: concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol 2000; 48: 83-92
• 10 Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995; 102: 1476-1485
• 11 Pellegrini G, Traverso CE, Franzi AT. et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997; 349: 990-993
• 12 Basu S, Ali H, Sangwan VS. Clinical outcomes of repeat autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol 2012; 153: 643-650 650.e1–2
• 13 Shortt AJ, Secker GA, Notara MD. et al. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 2007; 52: 483-502
• 14 Shahdadfar A, Haug K, Pathak M. et al. Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement. Exp Eye Res 2012; 97: 1-9
• 15 Menzel-Severing J, Kruse FE, Schlötzer-Schrehardt U. Stem cell-based therapy for corneal epithelial reconstruction: present and future. Can J Ophthalmol 2013; 48: 13-21
• 16 Schwab IR. Cultured corneal epithelia for ocular surface disease. Trans Am Ophthalmol Soc 1999; 97: 891-986
• 17 Koizumi N, Inatomi T, Suzuki T. et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001; 108: 1569-1574
• 18 Brzeszczynska J, Ramaesh K, Dhillon B. et al. Molecular profile of organ culture-stored corneal epithelium: LGR5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells. Int J Mol Med 2012; 29: 871-876
• 19 James SE, Rowe A, Ilari L. et al. The potential for eye bank limbal rings to generate cultured corneal epithelial allografts. Cornea 2001; 20: 488-494
• 20 Armitage WJ. Preservation of Human Cornea. Transfus Med Hemother 2011; 38: 143-147
• 21 Kim HS, Jun Song X, de Paiva CS. et al. Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. Exp Eye Res 2004; 79: 41-49
• 22 Vemuganti GK, Kashyap S, Sangwan VS. et al. Ex-vivo potential of cadaveric and fresh limbal tissues to regenerate cultured epithelium. Indian J Ophthalmol 2004; 52: 113-120
• 23 Zito-Abbad E, Borderie VM, Baudrimont M. et al. Corneal epithelial cultures generated from organ-cultured limbal tissue: factors influencing epithelial cell growth. Curr Eye Res 2006; 31: 391-399
• 24 Baylis O, Figueiredo F, Henein C. et al. 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem 2011; 112: 993-1002
• 25 Gavrilov JC, Borderie VM, Laroche L. et al. Influencing factors on the suitability of organ-cultured corneas. Eye (Lond) 2010; 24: 1227-1233
• 26 Armitage WJ, Easty DL. Factors influencing the suitability of organ-cultured corneas for transplantation. Invest Ophthalmol Vis Sci 1997; 38: 16-24
• 27 Armitage WJ, Jones MN, Zambrano I. et al. The suitability of corneas stored by organ culture for penetrating keratoplasty and influence of donor and recipient factors on 5-year graft survival. Invest Ophthalmol Vis Sci 2014; 55: 784-791
• 28 Bourne WM, Nelson LR, Hodge DO. Central corneal endothelial cell changes over a ten-year period. Invest Ophthalmol Vis Sci 1997; 38: 779-782
• 29 Norman RD, Smith H. Applied Regression Analysis. Hoboken, NJ: Wiley-Interscience; 1998: 307-312
• 30 Notara M, Shortt AJ, OʼCallaghan AR. et al. The impact of age on the physical and cellular properties of the human limbal stem cell niche. Age (Dordr) 2013; 35: 289-300
• 31 Constantinou M, Jhanji V, Tao LW. et al. Clinical review of corneal ulcers resulting in evisceration and enucleation in elderly population. Graefes Arch Clin Exp Ophthalmol 2009; 247: 1389-1393
• 32 Parmar P, Salman A, Kalavathy CM. et al. Microbial keratitis at extremes of age. Cornea 2006; 25: 153-158
• 33 Ibrahim YW, Boase DL, Cree IA. Epidemiological characteristics, predisposing factors and microbiological profiles of infectious corneal ulcers: the Portsmouth corneal ulcer study. Br J Ophthalmol 2009; 93: 1319-1324
• 34 Zheng W, Wang S, Ma D. et al. Loss of proliferation and differentiation capacity of aged human periodontal ligament stem cells and rejuvenation by exposure to the young extrinsic environment. Tissue Eng Part A 2009; 15: 2363-2371
• 35 Pels E, Schuchard Y. Organ-culture preservation of human corneas. Doc Ophthalmol 1983; 56: 147-153
• 36 Pels L. Organ culture: the method of choice for preservation of human donor corneas. Br J Ophthalmol 1997; 81: 523-525
• 37 van der Want HJ, Pels E, Schuchard Y. et al. Electron microscopy of cultured human corneas. Osmotic hydration and the use of a dextran fraction (dextran T 500) in organ culture. Arch Ophthalmol 1983; 101: 1920-1926
• 38 Borderie VM, Baudrimont M, Lopez M. et al. Evaluation of the deswelling period in dextran-containing medium after corneal organ culture. Cornea 1997; 16: 215-223
• 39 Hamon L, Quintin A, Mäurer S. et al. Reliability and efficiency of corneal thickness measurements using sterile donor tomography in the eye bank. Cell Tissue Bank 2022; 23: 695-706
• 40 Sharma N, Shaikh F, Nagpal R. et al. Evaluation of various preservation media for storage of donor corneas. Indian J Ophthalmol 2021; 69: 2452-2456
• 41 Shehab A, Gram N, Ivarsen A. et al. The importance of donor characteristics, post-mortem time and preservation time for use and efficacy of donated corneas for posterior lamellar keratoplasty. Acta Ophthalmol 2022; 100: 269-276
• 42 Mistò R, Giurgola L, Pateri F. et al. A new storage medium containing amphotericin B versus Optisol-GS for preservation of human donor corneas. Br J Ophthalmol 2022; 106: 184-189
• 43 Medin W, Davanger M. Swelling tendency of rabbit cornea in organ culture medium. Influence of button size. Acta Ophthalmol (Copenh) 1988; 66: 369-375
• 44 Crewe JM, Armitage WJ. Integrity of epithelium and endothelium in organ-cultured human corneas. Invest Ophthalmol Vis Sci 2001; 42: 1757-1761
• 45 Valtink M, Donath P, Engelmann K. et al. Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro . Graefes Arch Clin Exp Ophthalmol 2016; 254: 285-295
• 46 Redbrake C, Salla S, Nilius R. et al. A histochemical study of the distribution of dextran 500 in human corneas during organ culture. Curr Eye Res 1997; 16: 405-411
• 47 Salla S, Redbrake C, Becker J, Reim M. Remarks on the vitality of the human cornea after organ culture. Cornea 1995; 14: 502-508
• 48 Molvaer RK, Andreasen A, Heegaard S. et al. Interactive 3D computer model of the human corneolimbal region: crypts, projections and stem cells. Acta Ophthalmol 2013; 91: 457-462
• 49 Robert PY, Camezind P, Drouet M. et al. Internal and external contamination of donor corneas before in situ excision: bacterial risk factors in 93 donors. Graefes Arch Clin Exp Ophthalmol 2002; 240: 265-270
• 50 Samy MM, Shaaban YM, Badran TAF. Age- and sex-related differences in corneal epithelial thickness measured with spectral domain anterior segment optical coherence tomography among Egyptians. Medicine (Baltimore) 2017; 96: e8314
• 51 Spelsberg H, Reinhard T, Sundmacher R. [Epithelial damage of corneal grafts after prolonged storage in dextran-containing organ culture medium – a prospective study]. Klin Monbl Augenheilkd 2002; 219: 417-421
• 52 Abdin A, Daas L, Pattmöller M. et al. Negative impact of dextran in organ culture media for pre-stripped tissue preservation on DMEK (Descemet membrane endothelial keratoplasty) outcome. Graefes Arch Clin Exp Ophthalmol 2018; 256: 2135-2142
• 53 Lim P, Fuchsluger TA, Jurkunas UV. Limbal stem cell deficiency and corneal neovascularization. Semin Ophthalmol 2009; 24: 139-148
• 54 Cauchi PA, Ang GS, Azuara-Blanco A. et al. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol 2008; 146: 251-259
• 55 Kim JY, Djalilian AR, Schwartz GS. et al. Ocular surface reconstruction: limbal stem cell transplantation. Ophthalmol Clin North Am 2003; 16: 67-77
• 56 Cheung AY, Eslani M, Kurji KH. et al. Long-term Outcomes of Living-Related Conjunctival Limbal Allograft Compared With Keratolimbal Allograft in Patients With Limbal Stem Cell Deficiency. Cornea 2020; 39: 980-985