Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaells differentiation, resulting in decreased signaling by the Th2cytokine Il-13 and increased signaling by the Th1 cytokine IFN-γ.Expression of the goblet cell transcription factor KLK4 increased.Commercial Relationships: Rosa M. Corrales, None; Stephen C.Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb(C), Baylor College of Medicine (P)Support: NIH Grant EY11915 (SCP), Research to Prevent Blindess,Oshman Foundation, William Stamps Farish Fund, HamillFoundationProgram Number: 914 Poster Board Number: B0219Presentation Time: 1:00 PM - 2:45 PMDecellularization of porcine lacrimal gland tissue fordevelopment of a lacrimal gland scaffoldKristina Spaniol 1 , Alexander Kunze 1 , Marco Metzger 2 , GerdGeerling 1 , Stefan Schrader 1 . 1 ophthalmology, university hospitalDüsseldorf, Düsseldorf, Germany; 2 tissue engineering andregenerative medicine, university hospital Würzburg, Würzburg,Germany.Purpose: In cases of severe dry eye due to lacrimal glandinsufficiency engineering of a lacrimal gland tissue construct in vitromight be a promising future treatment approach. Aim of this studywas to evaluate structure and main basement membrane componentsof native porcine lacrimal gland tissue before and after adecellularization process in order to develop an acellular scaffold forlacrimal gland regeneration.Methods: Lacrimal glands were extracted from six domestic pigsafter euthanasia. Six biological replicates were used for this study.Each gland was cut into four pieces, two of them were left native andthe other two were decellularized using sodium desoxychelate inultra-pure water over night. Tissue pieces were embedded in paraffinas well as OCT and the morphology of native and decellularizedtissue was examined histologically by hematoxylin and eosin (H&E)staining. Additionally, expression of basement membrane markers,such as laminin, collagen IV and fibronectin was evaluated byimmunostaining.Results: Histology showed an intact connective tissue matrix afterthe decellularization process. Immunohistochemistry revealed theexpression of major basement membrane components such ascollagen IV and laminin in native lacrimal gland tissue and thesecomponents were still detectable after the decellularization of thelacrimal gland tissue. Efficacy of the decellularization process wasdemonstrated by complete absence of nuclei in the lacrimal glandtissue, as assessed by DAPI-staining.Conclusions: Decellularization of lacrimal gland tissue generates anintact acellular scaffold with preserved acinar structures, containingmajor basement membrane components such as collagen IV andlaminin. An intact basement membrane is a prerequisite for essentialcellular processes like adhesion, migration as well as proliferationand therefore decellularized lacrimal gland tissue might be apromising scaffold for lacrimal gland regeneration in vitro.Commercial Relationships: Kristina Spaniol, None; AlexanderKunze, None; Marco Metzger, None; Gerd Geerling, Alcon (C),Allergan (C), Thea Pharma (C), Novagali (C), Bausch & Lomb (C),Tearlab Inc. (C); Stefan Schrader, NoneProgram Number: 915 Poster Board Number: B0220Presentation Time: 1:00 PM - 2:45 PMFunctional rabbit acinar cells cultured on decellularized lacrimalgland scaffoldHui Lin 1 , Guoying Sun 1 , Hong he 1 , Jennifer Elisseeff 1 , Samuel C.Yiu 1, 2 . 1 Wilmer Eye Institute, Johns Hopkins University, Baltimore,MD; 2 Ophthalmology, 2. King Khaled Eye Specialist Hospital,Riyadh, Saudi Arabia.Purpose: Aqueous tear-deficient dry eye is a multifactorial chronicdisorder in which the lacrimal glands fail to produce enough tears tomaintain a healthy eye surface. The treatment primarily involvespalliation using ocular surface lubricant. Construction of abioengineered lacrimal gland having functional secretory epithelialcells is a potentially promising option for providing long-term reliefto severe dye eye patients. Previously, we have successfullydecellularized the rabbit lacrimal gland to create a novel scaffold for3 dimensional culture of acinar cell. Current study investigates thesecretory function of cells seeded and cultured in decellularizedlacrimal gland tissue.Methods: NZW rabbit lacrimal glands purchased from Pel Freezewere treated with 1% sodium dodecyl sulfate or 1% Triton X-100 for36 to 40 hours for decellularization. The rabbit lacrimal gland acinarcells were isolated by enzyme mixtures of collagenase, hyaluronidaseand DNase. Subsequently, the isolated cells were layered over 5%Ficoll and centrifuged at 50g for purification. Prior to seeding, thedecellularized lacrimal gland scaffold was treated with Matrigel,fibronectin or collagen I in different groups, and with Peter’scomplete medium (PCM) in control group. The lacrimal constructswere cultured in PCM for up to 2 weeks. The live/dead assay for cellviability was performed on day 2, 7 and 14. The beta-hexosaminidasesecretion assay for assessing acinar cell function was performed onday 2 and 7. RNA was also extracted to evaluate the gene expressionof beta-hexosaminidase.Results: Majority of the lacrimal acinar cells in the decellularizedscaffold survived for at least 7 days in all groups, however, cellviability decreased at day 14. Use of extracellular matrix to treat thescaffold during the seeding process helped improve cell viability.Beta-hexosaminidase activity increased in the supernatant mediumfollowing stimulation with 100uM carbachol on day 2 and 7.Carbachol stimulation was also found to up-regulate mRNA level ofbeta-hexosaminidase in the reseeded cells.Conclusions: Isolated acinar cells successfully cultured indecellularized lacrimal gland tissue and maintained the cell viabilityand secretory function for at least 1 week. Further investigation isrequired to optimize the decellularization and seeding process toimprove the duration of cell viability and function.Commercial Relationships: Hui Lin, None; Guoying Sun, None;Hong he, None; Jennifer Elisseeff, Collagen Vitrigel (P); Samuel C.Yiu, NoneSupport: Partially supported by an unrestricted grant from Researchto Prevent Blindness, New York, NY to the Wilmer Eye InstituteProgram Number: 916 Poster Board Number: B0221Presentation Time: 1:00 PM - 2:45 PMENaC in the rabbit lacrimal gland and its changes duringSjögren’s syndrome and pregnancyChuanqing Ding, Michael Lu, Jianyan Huang. Cell &Neurobiology/Doheny Eye Institute, University of SouthernCalifornia, Los Angeles, CA.Purpose: The epithelial sodium channel (ENaC), which is comprisedof α, β, and γ subunits, plays a critical role in the control of Na+balance and has been implicated in the development and progressionof exocrine gland pathology. The aim of the present study was toinvestigate the expression of ENaC in the rabbit lacrimal gland (LG)and its potential changes in rabbits with induced autoimmunedacryoadenitis (IAD), a rabbit model of Sjögren’s syndrome, and interm-pregnant rabbits, a physiological condition that has been shownto exhibit altered LG secretion and ocular surface symptoms.Methods: Total mRNA of α, β, and γ subunits was extracted from©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.