! MODELS AND THERAPEUTICS FOR ENGINEERING THE OCULAR SURFACE by Qiaozhi Lu A dissertation submitted to Johns Hopkins University in conformity with the require- ments for the degree of Doctor of Philosophy Baltimore, Maryland January, 2017 © 2017 Qiaozhi Lu All Rights Reserved ! ABSTRACT The ocular surface is the outermost part of the eye that protects the refractive surface and enables sharp vision. The primary components of the ocular surface are corneal and con- junctival epithelia, lacrimal glands, and meibomian glands that are functionally linked as one system. Because of its location, the ocular surface is subject to traumatic injuries and diseases. Dry eye, due to inadequate tear production or excessive evaporation, is one of the most complicated diseases of the ocular surface, whose exact pathogenesis is not yet fully understood, and more effective treatments need to be investigated. This dissertation will address the development of an in vitro ocular surface system and its application in therapeutic discovery. We first investigated a vitrified collagen membrane and its ability to support ocular surface epithelium, which also promoted the repopulation of goblet cells, a key component in mucin production. Afterwards a more complex ocular surface system was established with both conjunctival epithelium and lacrimal glands, the essential parts in tear film secretion. The three-dimensional coculture system highly resem- bles the structure and tear-secretion function of the native environment. This was further evaluated as a model for dry eye disease and a platform for therapeutic evaluation. One potential therapeutic we had studied is decellularized extracellular matrices (ECM). The particulate form was used as an immunomodulator to reduce inflammation and fibrosis on the ocular surface and to accelerate tissue regeneration. Model systems built based on en- gineering principle provide a new approach for drug discovery. This in vitro ocular surface system is proved to recapitulate the physiology of both healthy and diseased ocular surface. Its potential as a therapeutic screening device is promising and could be further investigated. ii! Thesis committee: Prof. Jennifer H. Elisseeff (BME & TTEC, advisor) Prof. Michael P. Grant (Ophthalmology, co-advisor) Prof. Hai-Quan Mao (MSE & TTEC) Prof. Margarita Herrera-Alonso (MSE) Prof. Samuel C. Yiu (Ophthalmology) iii! For my parents, Hong Lu and Liya Sun iv! ACKNOWLEDGEMENTS I would like to thank many people for their support during my pursuit of the PhD degree in the last few years. First, I truly want to thank my parents, who are always there for me. Although they do not work in academia, their life experience and insights are the most valuable for me. Their persistence and optimism has deeply influenced me and set up a perfect example for me ever since I was a child. I also want to thank my husband Daniel for the incredible support he offered during my PhD, without whom I could not have en- joyed life so much. I want to sincerely thank my advisor, Prof. Jennifer H. Elisseeff, for her guidance through my time working in her lab. I have learned so much from her on conducting research and solving problems. She has inspired me countless times and helped me go though many obstacles. As a scientist and professor, she is a great role model to me. I also want to thank Prof. Michael P. Grant and Prof. Hai-Quan Mao for their support and encouragement on my research projects and PhD study. I have worked with so many amazing people during my time in the JHE lab: Dr. Jeannine Coburn, Dr. Iossif Strehin, Dr. Jacob A. Simson, Dr. Qiongyu Guo, Dr. Jeremy J. Chae, Okhee Jeon, Dr. Huifang Zhou, Dr. Hongbo Yin, Dr. Anirudha Singh, David Lee and many more. I really appreciated the opportunities working with them and have benefited a lot from these experiences. I want to thank many former and present members of the lab for helping me with trouble-shooting and protocols. v! Finally, I want to thank all friends that have supported me during all these years. My life- changing journey at Johns Hopkins opens a new era ahead of me, and I am forever grateful for everything and every moment of it. vi! TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii! ACKNOWLEDGEMENTS ............................................................................................. v! LIST OF TABLES ........................................................................................................... xi! LIST OF FIGURES ........................................................................................................ xii! Chapter 1.! Objectives and Significance ..................................................................... 1! Chapter 2.! Introduction and Background ................................................................. 4! 2.1.! THE OCULAR SURFACE ................................................................................ 4! 2.1.1.! The structure and transparency of cornea ................................................... 4! 2.1.2.! The conjunctiva and goblet cells ................................................................. 5! 2.1.3.! The structure of lacrimal gland and its function ......................................... 6! 2.2.! BIOMATERIALS FOR OCULAR SURFACE RECONSTRUCTION ............ 7! 2.2.1.! Injuries and diseases of the ocular surface .................................................. 7! 2.2.2.! Autologous Tissue Grafts ........................................................................... 7! 2.2.3.! Amniotic Membrane ................................................................................... 8! 2.2.4.! Biomacromolecular Materials ................................................................... 11! 2.2.5.! Collagen Vitrigel ....................................................................................... 12! 2.3.! TEAR FILM AND DRY EYE DISEASE ........................................................ 16! 2.3.1.! Tear film structure and components ......................................................... 16! 2.3.2.! Dry eye disease and treatment .................................................................. 16! 2.4.! IN VITRO MODEL SYSTEMS ....................................................................... 17! 2.4.1.! Biomimetic “organ-on-a-chip” models ..................................................... 17! 2.4.2.! Current ocular model systems ................................................................... 18! 2.5.! SUMMARY ...................................................................................................... 20! Chapter 3.! Vitrified Collagen-Based Conjunctival Equivalent for Ocular Surface Reconstruction .................................................................................................. 21! 3.1.! INTRODUCTION ............................................................................................ 21! vii! 3.2.! MATERIALS AND METHODS ...................................................................... 24! 3.2.1.! Collagen vitrigel preparation .................................................................... 24! 3.2.2.! Transmission electron microscopy (TEM) ............................................... 25! 3.2.3.! Isolation and in vitro culture of rabbit conjunctival epithelial cells (rCjECs) 26! 3.2.4.! Cell proliferation, viability and morphology ............................................ 26! 3.2.5.! Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) ................................................................................................................ 27! 3.2.6.! In vivo rabbit conjunctival defect model .................................................. 28! 3.2.7.! Collagen vitrigel transplantation ............................................................... 29! 3.2.8.! Histology and immunostaining ................................................................. 30! 3.2.9.! Statistical Analysis .................................................................................... 31! 3.3.! RESULTS ......................................................................................................... 31! 3.3.1.! Morphology and proliferation of conjunctival epithelial cells ................. 31! 3.3.2.! Collagen vitrigel maintained the phenotypes of rCjECs in vitro .............. 35! 3.3.3.! Reconstruction of conjunctival defect using CV ...................................... 37! 3.3.4.! Assessment of the repaired conjunctival tissue ........................................ 39! 3.3.5.! Survival and integration of transplanted cells in the defect ...................... 43! 3.4.! DISCUSSION ................................................................................................... 44! 3.5.! CONCLUSIONS .............................................................................................. 50! Chapter 4.! An In Vitro Model for the Ocular Surface and Tear Film System ..... 51! 4.1.! INTRODUCTION ............................................................................................ 51! 4.2.! MATERIALS AND METHODS ...................................................................... 54! 4.2.1.! Primary cell isolation ................................................................................ 54! 4.2.2.! Submerged and airlifting culture of CECs ................................................ 56! 4.2.3.! Spheroids formation