CULTIVATED CONJUNCTIVAL EPITHELIAL TRANSPLANTATION FOR THE TREATMENT OF OCULAR SURFACE DISEASE LEONARD PEK-KIANG ANG M.B.B.S. (Singapore), FRCS (Edinburgh), MRCOphth (London), M.Med (Singapore) A DISSERTATION SUBMITTED FOR THE DEGREE OF DOCTOR OF MEDICINE DEPARTMENT OF OPHTHALMOLOGY, YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2004 i ACKNOWLEDGEMENTS This dissertation would not have been possible without the help of the many people who unselfishly gave their time and support. Naturally, I relied heavily on the professional judgement of my supervisors, A/P Donald Tan and Prof Roger Beuerman. I would like to express my deepest appreciation and gratitude to my supervisors for their patient guidance and invaluable advice throughout the course of this project. I would also like to express my deepest appreciation to Dr Phan Toan Thang for his advice and supervision in cell and tissue culture techniques and laboratory methods. This project would also not be possible without the tremendous support and help from Prof Robert Lavker, Prof Pamela Jensen, and Dr Barbara Risse, at the University of Pennsylvania, USA, who taught me the various culture methods and laboratory techniques that were required for this project. My sincere gratitude also goes out to A/Prof Vivian Balakrishnan and A/Prof Ang Chong Lye for their unwavering support and advice. I would also like to thank Dr Howard Cajucom-Uy, Dr Jessica Abano, and members of the Cornea team at the Singapore National Eye Centre who assisted in the patient recruitment, management and follow-up of these patients. Sincere thanks to Dr Wang Ziao Jing, Mr David Wong and Ms Cheyenne Seah from the Singapore Eye Research Institute who assisted in the tissue culture and laboratory experiments. Sincere thanks also go out to all the doctors and staff of the Singapore National Eye Centre and the Singapore Eye Research Institute who contributed in any way to this project. I would also like to thank the various grant funding agencies that supported this project: Singapore National Medical Research Council Grant R226/18/2001, Singapore Biomedical Research Council Grant R268/12/2002 and Singapore Eye Research Institute Grant R198/24/2000. ii CONTENTS TITLE PAGE i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF FIGURES vii LIST OF TABLES ix ABBREVIATIONS x SUMMARY xi xiii LIST OF PUBLICATIONS CHAPTER 1: INTRODUCTION CHAPTER 2: OCULAR SURFACE STEM CELL BIOLOGY AND DISEASE TREATMENT 2.1 2.2 2.3 2.4 2.5 2.6 Introduction Ocular surface stem cells Identification of stem cells Treatment of ocular surface stem cell deficiency Ex vivo expansion of limbal stem cells for transplantation Role of conjunctiva in maintaining the integrity of the ocular surface 5 11 12 CHAPTER 3: AIMS 14 CHAPTER 4: SERIAL PROPAGATION OF HUMAN CONJUNCTIVAL EPITHELIAL CELLS WITH SERUM-FREE MEDIA 17 4.1 4.2 Introduction Methods 4.2.1 Isolation and cultivation of human conjunctival iii 18 20 20 epithelial cells Quantitation of growth and proliferative capacity Immunohistochemistry RT-PCR of MUC5AC 4.3 4.4 4.2.2 4.2.3 4.2.4 Results Discussion CHAPTER 5: THE IN VIVO PROLIFERATIVE CAPACITY OF SERUM-FREE CULTIVATED HUMAN CONJUNCTIVAL EPITHELIAL CELLS 5.1 5.2 23 24 25 25 34 37 Introduction Methods 5.2.1 Isolation and cultivation of human conjunctival epithelial cells 5.2.2 Implantation into athymic mice 5.2.3 Histological and electron microscopic analysis of cysts Results Discussion 38 CHAPTER 6: THE DEVELOPMENT OF A TRANSPLANTABLE SERUM-FREE DERIVED HUMAN CONJUNCTIVAL EPITHELIAL SHEET CULTIVATED ON HUMAN AMNIOTIC MEMBRANE 50 5.3 5.4 39 42 43 43 47 Introduction Methods 6.2.1 Preparation and cultivation of conjunctival epithelial cells on HAM 6.2.2 Growth and proliferative capacity of cells Results Discussion 51 52 53 CHAPTER 7: THE IN VIVO CHARACTERISTICS OF TRANSPLANTED CONJUNCTIVAL TISSUE-EQUIVALENTS 65 6.1 6.2 6.3 6.4 7.1 7.2 7.3 Introduction Methods 7.2.1 Ex vivo expansion of conjunctival epithelial cells on HAM 7.2.2 Xenotransplantation of conjunctival equivalent onto SCID mice Results iv 55 58 63 66 67 68 69 7.4 Discussion 74 CHAPTER 8: THE USE OF HUMAN SERUM FOR THE CULTIVATION OF CONJUNCTIVAL EPITHELIAL CELLS 8.1 8.2 8.3 8.4 Introduction Methods Results Discussion 78 78 81 89 CHAPTER 9: RECONSTRUCTION OF THE OCULAR SURFACE BY TRANSPLANTATION OF A SERUM-FREE DERIVED CULTIVATED CONJUNCTIVAL EPITHELIAL EQUIVALENT 9.1 9.2 9.3 9.4 Introduction Methods 9.2.1 Subjects 9.2.2 Development of human conjunctival equivalents 9.2.3 Ocular surface transplantation Results Discussion CHAPTER 10: AUTOLOGOUS CULTIVATED CONJUNCTIVAL TRANSPLANTATION FOR PTERYGIUM SURGERY 10.1 10.2 10.3 10.4 77 Introduction Methods 10.2.1 Subjects 10.2.2 Development of human conjunctival equivalents 10.2.3 Pterygium surgery and transplantation of conjunctival equivalents Results Discussion 92 93 94 95 96 97 105 110 111 113 114 114 117 125 CHAPTER 11: DISCUSSION 130 CHAPTER 12: CONCLUSIONS 135 REFERENCES 138 v 153 APPENDICES vi LIST OF FIGURES Figure 2.1 Hierarchy of stem cell Figure 2.2 Limbal stem cell division and migration Figure 4.1 Conjunctival epithelial cell culture 31 Figure 4.2 Areas of epithelial cell outgrowth from explant cultures 31 Figure 4.3 Colony-forming efficiency of conjunctival epithelial cells 32 Figure 4.4 Immunocytochemistry of cultured conjunctival epithelial cells 33 Figure 4.5 MUC5ACRT-PCR of cultured cells 33 Figure 5.1 Athymic mice conjunctival cysts 45 Figure 5.2 Transmission electron microscopy of conjunctival cyst 45 Figure 6.1 Phase contrast appearance of conjunctival epithelial cells cultivated on human amniotic membrane 61 Figure 6.2 Conjunctival equivalents cultivated in serum-free and serumcontaining media 61 Figure 6.3 BrdU ELISA cell proliferation assay, colony-forming efficiency and number of cell generations of conjunctival epithelial cells cultivated in serum-free and serum-containing media 62 Figure 7.1 Conjunctival equivalents xenotransplanted into SCID mice 72 Figure 7.2 Immunohistochemistry of conjunctival equivalents 72 Figure 7.3 Transmission electron microscopy of conjunctival equivalent 73 Figure 8.1 Phase contrast appearance of conjunctival epithelial cells in serum-free, FBS-supplemented, and human serumsupplemented media 85 Figure 8.2 Areas of cell outgrowth and BrdU ELISA proliferation assay of conjunctival epithelial cells 86 Figure 8.3 Clonal growth and long-term proliferative capacity of cultivated cells 87 vii Figure 8.4 Histological analysis of conjunctival equivalents in vitro and in vivo Figure 9.1 Phase contrast and histology of conjunctival tissueequivalent 103 Figure 9.2 Transplantation of conjunctival equivalent for extensive conjunctival nevus 103 Figure 9.3 Pterygium excision and cultivated conjunctival transplantation 103 Figure 9.4 Patient with persistent leaking glaucoma filtration bleb 104 Figure 9.5 Patient with superior limbic keratoconjunctivitis 104 Figure 10.1 Kaplan-Meier survival analysis of recurrence after pterygium excision and cultivated conjunctival transplantation 122 Figure 10.2 Pterygium patient undergoing transplantation of cultivated conjunctiva 124 Figure 10.3 Pre and post-operative appearance of patient with doubleheaded pterygium 124 viii 88 LIST OF TABLES Table 4.1 Colony-forming efficiency of cultured cells 32 Table 4.2 Serial passage and population doublings of cultured cells 32 Table 5.1 Athymic mice conjunctival cyst cell number 46 Table 9.1 Surgical data of patients with ocular surface disorders 108 Table 10.1 Data of pterygium patients treated with cultivated conjunctival transplantation 120 ix ABBREVIATIONS BPE Bovine pituitary extract BrdU Bromodeoxyuridine CFE Colony-forming efficiency DAB Diaminobenzidinetetrahydrochloride substrate DMEM Dulbecco’s modified Eagles’s medium EDTA Ethylenediaminetetraacetic acid FBS Fetal bovine serum FITC Fluorescein isothiocyanate HBSS Hank’s balanced salt solution hEGF Human epidermal growth factor HAM Human amniotic membrane IgG Immunoglobulin G KGM Keratinocyte Growth Medium PBS Phosphate-buffered saline SFM Serum-free media SCM Serum-containing media x Schermer A, Galvin S, Sun TT. Differentiation-related expression of a mjor 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J. Cell. Biol. 1986;103:49-62. Schnyder CC, Shaarawy T, Ravinet E, Achache F, Uffer S, Mermoud A. Free conjunctival autologous graft for bleb repair and bleb reduction after trabeculectomy and nonpenetrating filtering surgery. J Glaucoma 2002;11:10-6. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea 2000;19:421-426. Scuderi N, Alfano C, Paolini G, Marchese C, Scuderi G. Transplantation of autologous cultivated conjunctival epithelium for the restoration of defects in the ocular surface. Scand J Plast Reconstr Surg Hand Surg. 2002;36(6):340-8. Shimazaki J, Yang HY, Tsubota K. Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmology 1997;104(12):2068-76. Shimazaki J, Aiba M, Goto E, Kato N, Shimmura S, Tsubota K. Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology 2002;109(7):1285-90. Shimazaki J, Kosaka K, Shimmura S, Tsubota K. Amniotic membrane transplantation with conjunctival autograft for recurrent pterygium. Ophthalmology 2003;110(1):119-24. Shimmura S, Shimazaki J, Ohashi Y, Tsubota K. Antiinflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea 2001;20:408-13. Shimmura S, Ishioka M, Hanada K, Shimazaki J, Tsubota K. Telomerase activity and p53 expression in pterygia. Invest Ophthalmol Vis Sci 2000;41(6):1364-1369. 148 Shipley GD, Pittelkow MR. Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium. Arch Dermatol 1987;123:1541a-1544a. Solomon A, Pires RT, Tseng SC. Amniotic membrane transplantation after extensive removal of primary and recurrent pterygia. Ophthalmology 2001;108:449-60. Solomon A, Espana EM, Tseng SC. Amniotic membrane transplantation for reconstruction of the conjunctival fornices. Ophthalmology 2003;110(1):93-100. Solomon A, Ellies P, Anderson DF, Touhami A, Grueterich M, Espana EM, Ti SE, Goto E, Feuer WJ, Tseng SC. Long-term outcome of keratolimbal allograft with or without penetrating keratoplasty for total limbal stem cell deficiency. Ophthalmology 2002;109(6):1159-66. Sun TT, Green H. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature 1977;269:489-493. Tan D. Conjunctival grafting for ocular surface disease. Curr.Opin.Ophthalmol. 1999;10:277-81. Tan DTH, Ang LPK, Beuerman R. Reconstruction of the Ocular Surface by Transplantation of a Serum-free Derived Cultivated Conjunctival Epithelial Equivalent. Transplantation 2004;77(11):1729-1734. Tan DTH, Chee SP, Dear KBG, Lim ASM. Effect of pterygium morphology on pterygium recurrence in a controlled trial comparing conjunctival autografting with bare sclera excision. Arch Ophthalmol 1997;115:1235-40. Tan DT, Ficker LA, Buckley RJ. Limbal 1996;103(1):29-36. 149 transplantation. Ophthalmology Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc.Natl.Acad.Sci.U.S.A 2000;97:10960-5. Tarr KH, Constable IJ. Late complications of pterygium treatment. Br J Ophthalmol 1980;64:496-505. Tei M, Moccia R, Gipson IK. Developmental expression of mucin genes ASGP (rMuc4) and rMuc5ac by the rat ocular surface epithelium. Invest Ophthalmol Vis Sci 1999;40:1944-1951. Tei, M., Moccia, R., and Gipson, I. K Developmental expression of mucin genes ASGP (rMuc4) and rMuc5ac by the rat ocular surface epithelium. Invest Ophthalmol.Vis.Sci. 1999;40:1944-1951. Thoft RA, Wiley LA, Sundarraj N. The multipotential cells of the limbus. Eye 1989;3:109-13. Ti SE, Chee SP, Dear KBG, Tan DTH. Analysis of variation in success rates in conjunctival autografting for primary and recurrent pterygium. Br J Ophthalmol 2000;84:385-9. Ti SE, Tan DTH. Tectonic corneal lamellar grafting for severe scleral melting after pterygium surgery. Ophthalmology 2003;110:1126-36. Tsao MC, Walthall BJ, Ham RG. Clonal growth of normal human epidermal keratinocytes in a defined medium. J Cell Physiol 1982;110:219-229. Tsai RJ, Tseng SC. Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology. Invest Ophthalmol Vis Sci 1988;29:1565-1576. 150 Tsai RJ, Ho YS, Chen JK. The effects of fibroblasts on the growth and differentiation of human bulbar conjunctival epithelial cells in an in vitro conjunctival equivalent. Invest Ophthalmol Vis Sci 1994;35:2865-2875. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86-93. Tseng SC. Concept and application of limbal stem cells. Eye 1989;3 ( Pt 2):141-57. Tseng SC. Regulation and clinical implications of corneal epithelial stem cells. Mol.Biol.Rep. 1996;23:47-58. Tseng SCG, Kruse FE, Merritt J, Li DQ. Comparison between serum-free and fibroblastcocultured single-cell clonal culture systems: evidence showing that epithelial antiapoptotic activity is present in 3T3 fibroblast-conditioned media. Curr. Eye Res. 1996;15: 973-984. Tseng SC, Prabhasawat P, Lee SH. Amniotic membrane transplantation for conjunctival surface reconstruction. Am J Ophthalmol 1997;124:765-74. Tseng SC, Prabhasawat P, Barton K, Gray T, Meller D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch.Ophthalmol. 1998;116:431-41. Tsubota K. Corneal epithelial stem-cell transplantation. Lancet 1997;349:1556. Tsubota K, Satake Y, Kaido M, Shinozaki N, Shimmura S, Bissen-Miyajima H et al. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N.Engl.J.Med. 1999;340:1697-703. Vrabec MP, Weisenthal RW, Elsing SH. Subconjunctival fibrosis after conjunctival autograft. Cornea 1993;12:181-3. 151 Wagoner MD, Kenyon KR. Conjunctival transplantation for pterygium and other ocular surface disorders, in Bruner WR, Stark WJ, and Maumenee AE (eds). A Manual of corneal Surgery. New York, Churchill Livingstone 1987, pp 119-28. Wei ZG, Wu RL, Lavker RM, Sun TT. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci 1993;34:18141828. Wei ZG, Sun TT, Lavker RM. Rabbit conjunctival and corneal epithelial cells belong to two separate lineages. Invest Ophthalmol Vis Sci 1996;37:523-533. Wei ZG, Lin T, Sun TT, Lavker RM. Clonal analysis of the in vivo differentiation potential of keratinocytes. Invest Ophthalmol.Vis.Sci. 1997;38:753-61. Wille JJ, Jr., Pittelkow MR, Shipley GD, Scott RE. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol 1984;121:31-44. Zieske JD, Bukusoglu G, Yankauckas MA, Wasson ME, Keutmann HT. Alpha-enolase is restricted to basal cells of stratified squamous epithelium. Dev.Biol. 1992;151:18-26. 152 APPENDICES 153 Appendix I Serum-free media for cell culture Appendix II Serum-supplemented media for cell cultue Appendix III Supplements for culture media Appendix IV List of materials and suppliers for cell culture Appendix V Immunohistochemistry and antibodies 154 APPENDIX I SERUM-FREE CULTURE MEDIA Keratinocyte Growth Medium (Biowhittaker, Walkersville, MD, USA) supplemented with : • 10ng/ml human epidermal growth factor • 5µg/ml insulin • 0.5µg/ml hydrocortisone • 30µg/ml bovine pituitary extract • 50µg/ml Gentamicin • 50ng/ml Amphotericin 155 APPENDIX II SERUM-CONTAINING CULTURE MEDIA 1:1 DMEM-Ham’s/F12 nutrient mixture, supplemented with: • 10% FBS • 5µg/ml insulin • 0.5µg/ml hydrocortisone • 8.4ng/ml cholera toxin • 24µg/ml Adenine • 100IU/ml penicillin • 100µg/ml streptomycin. 156 APPENDIX III REAGANTS AND SUPPLEMENTS Hydrocortisone (Sigma H0888) MW 362.5 Stock solution is 5x10-3M (=10,000x). Final concentration in medium is 5x10-7M. Weigh 18mg hydrocortisone and add 10ml 95% EtOH. Store at -20oC. Epidermal Growth Factor: (Gibco #13247-051 recombinant human). Stock solution is 10µg/ml (=1000x). Final concentration in medium is 10µg/liter (=10ng/ml). To a vial containing 100µg, add 10ml sterile deionized H2O. Cholera Enterotoxin: Company: Sigma; Cat.#: C8052; Lot #: 24H4052 Dissolve 1mg cholera toxin in 1.2ml sterile water to give 0.833 mg/ml = 833 µg/ml. Take 0.1ml of this and add to 100ml Earles salt + 0.1% BSA + 25mM Hepes to make a 0.833 µg/ml stock (100x). To get final concentration of 8.33 ng/ml in the medium: for 400ml of medium, add 4ml of 0.833 µg/ml stock. 157 Insulin (from bovine pancreas, Sigma I6634). Stock solution is 5mg/ml (=1000x). Final concentration in medium is 5mg/liter. Weigh 50mg insulin and add 10ml of 0.01M HCl (For Baker #9535-3, 11.6N, use 86.2 µl conc. HCl diluted in 100ml H2O; for Fisher #A-144, 12.1N, use 82.6 µl conc. HCl diluted in 100ml H2O). Vortex to dissolve. Sterile filter through 0.22µ Millex GV syringe filter (Millipore). Store in 1ml aliquots at -20oC. Adenine: Company: Sigma; Cat #: A9795; Lot #: 31H06015 Weigh 242mg of adenine and add 100ml sterile 0.05N HCl to make a 2.42 mg/ml stock (100x). Stir for ≈1hr at RT. Filter-sterilize. Store in 10ml aliquots at -200C. To get a final concentration of 24 µg/ml in the medium: for 400ml of medium, add 4ml of 2.42 mg/ml stock. Ethanolamine (Sigma E9508). Stock solution is 0.1M (=1000x). Final concentration in medium is 0.1mM. Take 59.9µl ethanolamine and bring to 10 ml with deionized H2O. Sterile filter through 0.22µ Millex GV syringe filter (Millipore). Store in 1ml aliquots at -20oC. 158 O-Phosphoethanolamine (Sigma P0503) MW 141.1 Stock solution is 0.1M (=1000x). Final concentration in medium is 0.1mM. Weigh 0.141g phosphoethanolamine and add 10ml deionized H2O. Sterile filter through 0.22µ Millex GV syringe filter (Millipore). Store in 1ml aliquots at -20oC. Penn/Strep Stock solution is 10,000IU/ml and 10,000mcg/ml respectively (=100x). Store in 10ml aliquots at -20o 159 APPENDIX IV LIST OF MATERIALS AND SUPPLIERS CULTURE MEDIA AND SOLUTIONS ITEM SUPPLIER Dulbecco’s modified Eagles’s medium Gibco (Grand Island, NY, USA) Hank’s balanced salt solution Gibco Human epidermal growth factor Gibco Penicillin Gibco Streptomycin Gibco Amphotericin B Gibco Insulin Sigma (St. Louis, MO, USA) Hydrocortisone Sigma Bovine pituitary extract Sigma Cholera toxin Sigma Adenine Sigma Keratinocyte growth medium Biowhittaker (Walkersville, MD, USA) Fetal bovine serum (FBS) Hyclone (Logan, Utah, USA) 160 CHEMICALS AND MATERIALS ITEM SUPPLIER Mitomycin C Sigma Dimethyl sulfoxide Sigma Dispase II Gibco Trypsin Gibco Ethylenediaminetetraacetic acid Gibco Nitrocellulose filter paper Millipore ( Bedford, MA, USA) Tissue culture plastic plates Corning (Corning, NY, USA) BrdU labeling solution Amersham Germany) 161 Biosciences (Freiburg, APPENDIX V IMMUNOHISTOCHEMISTRY AND ANTIBODIES MATERIAL SUPPLIER Vectastain Peroxidase Kit Vectastain Elite Kit (Vector Labs, Burlingame, CA, USA) Diaminobenzidinetetrahydrochloride Sigma Substrate Fluorescein Isothiocyanate Labeled Chemicon (Temecula, CA, USA) Goat Anti-Mouse Igg Propidium Iodide Chemicon (Temecula, CA) Tissue-Tek OCT Freezing Compound Sakura Finetek (Torrance, CA) Vectashield Mounting Medium Vector Labs (Burlingame, CA, USA). 162 ANTIBODIES SUPPLIER Normal Mouse Monoclonal IgG Antibody Sigma Cytokeratin Antibody Sigma Cytokertin 12 Antibody Santa Cruz Biotechnology (CA) Cytokeratin 19 Antibody DakoCytomation (Carpinteria, CA) Cytokeratin (AE-5) Antibody A kind gift from T. T. Sun from New York University Pancytokeratin (AE-1 And AE-3) Antibody Sigma MUC5AC Antibody Chemicon (Temecula, CA, USA) Biotinylated Horse Anti-Mouse Vectastain Elite Kit (Vector Labs, Immunoglobulin G Burlingame, CA, USA) 163 [...]... in developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing grafttake and regeneration of the conjunctival surface following clinical transplantation I report the successful use of these cultivated conjunctival epithelial equivalents for the treatment of patients with ocular surface disorders that required conjunctival excision... 15 MAIN AIMS The main aims of my study were:: 1 To develop a serum-free culture system for the ex vivo expansion of conjunctival epithelial cels propagation 2 To develop a transplantable cultivated conjunctival epithelial equivalent in serum-free media 3 To evaluate the use of cultivated autologous conjunctival tissue-equivalents for the treatment of ocular surface disorders This novel treatment modality... CHAPTER 2 OCULAR SURFACE STEM CELL BIOLOGY AND DISEASE TREATMENT 4 INTRODUCTION The ocular surface is a complex biological continuum responsible for the maintenance of corneal clarity, elaboration of a stable tear film for clear vision, as well as protection of the eye against microbial and mechanical insults The ocular surface epithelium comprises corneal, limbal and conjunctival epithelia, of which the. .. 0.125% trypsin/ 0.02% EDTA for a period of 10 minutes Cultures that were carried out in the presence of feeder layers were pretreated with 0.02% EDTA for 5 minutes to remove the feeder cells The single-cell suspensions of the conjunctival epithelial cells were then plated at a density of 3-4 x 104 cells /cm2 For the purpose of characterization of the cultured conjunctival epithelial cells, cells from... replace only the corneal epithelium The lack of the supportive function of the conjunctiva often contributes to the failure To date, few, if any, investigators have focused on the conjunctiva, in the form of conjunctival and fornix reconstruction and restoration of physiological functions There is a perceived need to develop methods to replace normal conjunctiva The use of bioengineered ocular surface replacement... component of the tear film (Wei et al., 1993) The mechanisms that regulate the stratified squamous epithelial cell and goblet cell proliferation and differentiation are largely unknown Examination of these events in vivo is complicated by the multitude of epithelial and mesenchymal factors that contribute to the complex ocular environment Therefore cell culture is one of the best means for examination of the. .. Capacity Of Serum-Free Cultivated Human Conjunctival Epithelial Cells Current Eye Research 2004;28(5):307-317 2 DTH Tan, LPK Ang, R Beuerman Reconstruction of the Ocular Surface by Transplantation of a Serum-free Derived Cultivated Conjunctival Epithelial Equivalent Transplantation 2004;77(11):1729-1734 3 LPK Ang, DTH Tan, R Beuerman, TT Phan, RM Lavker The Development Of A Conjunctival Epithelial Equivalent... quiescent subpopulation of epithelial cells of the ocular surface The limbus is a 1.5 to 2mm wide area that straddles the cornea and bulbar conjunctiva Corneal epithelial stem cells reside in the basal region of the limbus, and are involved in the renewal and regeneration of the corneal epithelium (Thoft et al., 1989; Tseng, 1989, 1996; Dua, 1995; Dua et al., 2000) Following injury, these limbal basal stem... absence of intercellular communication may be an inherent feature of stem cells, reflecting the need for these cells to maintain the uniqueness of its own intracellular environment TREATMENT OF OCULAR SURFACE STEM CELL DEFICIENCY With the widespread acceptance of the limbus as the site of corneal stem cells, limbal stem cell transplantation was introduced as a definitive means of replacing the corneal... MAINTAINING THE INTEGRITY OF THE OCULAR SURFACE The conjunctiva plays an important role in maintaining the optical clarity of the cornea by providing a lubricated, tear film-producing surface Disorders of the ocular surface, such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid and chemical burns, result in injury to the corneal epithelial stem cells at the limbus, as well as the conjunctiva This . crucial for enhancing graft- take and regeneration of the conjunctival surface following clinical transplantation. I report the successful use of these cultivated conjunctival epithelial equivalents. OF THE OCULAR SURFACE BY TRANSPLANTATION OF A SERUM-FREE DERIVED CULTIVATED CONJUNCTIVAL EPITHELIAL EQUIVALENT 9.1 Introduction 9.2 Methods 9.2.1 Subjects 9.2.2 Development of human conjunctival. i CULTIVATED CONJUNCTIVAL EPITHELIAL TRANSPLANTATION FOR THE TREATMENT OF OCULAR SURFACE DISEASE LEONARD PEK-KIANG