VIABILITY OF HUMAN LIMBAL EPITHELIAL CELLS CULTURED ON DIFFERENT TYPES OFSCAFFOLDSM. Tominac Trcin¹, T. Dolenec¹, M. Sokol¹, E. Zdraveva², B. Mijović², M. Pauk-Gulić³, I. Dekaris³¹University Department of Traumatology, Sestre Milosrdnice Hospital Center; ²Faculty of Textile Technology, Universityof Zagreb; ³Special Eye Hospital Svjetlost, Department of Ophthalmology, School of Medicine in Rijeka, University ofRijeka, CroatiaPurpose: Limbal stem cell deficiency has been treated for the last 14 years more or less successfullyusing in vitro expansion of autologous or allogenic cells isolated from small bioptate of limbalepithelium.There are various techniques of culturing human limbal epithelial cells. Some of them are using mouse3T3 cells as a feeder layer according to Rheinwald &Green method for keratinocyte cultivation. Inaddition, several cell scaffolds have been used for this purpose like: amniotic membrane, fibrin glueor contact lenses. In the field of tissue engineering, new types of scaffolds made from nanofibers offerfavourable 3D environment for cell attachment and growth thanks to interconnected pores and avery large surface-to-volume ratio. It was our intention to test new scaffolds made from nanofibers forlimbal cell proliferation and viability.Methods: Nanoscaffolds were prepared from solutions of polyurethane and polycaprolactonepolymers using electrospinning method. Prior to the cell seeding they were cut into 14 mm diamterecircles, disinfected with UV-light and hydrated by soaking into 70% 50%, 25%(v/v) ethanol, steriledistilled water and Hank/s balanced salt solution. Fibrin glue scaffolds were prepared from commercialfibrin glue kit (Tisseel, Baxter ) diluted with aprotinin and 1.1% NaCl and 1mM CaCl2 solution.Cryopreserved human amniotic membrane was obtained from our tissue bank and defrosted inthe cell culture medium. Cells were seeded on an intact basal side. Surgical excess of human limbalepithelium from 8 donors were treated with 0.25% trypsin. Trypsinized suspensions of limbal cellswere seeded on an irradiated mouse 3T3 feeder cells in 2:1 ratio. Cells were cultured until 80% ofconfluence and frozen. Defrosted cells were seeded with 3T3 feeder cells in 24 well plates: cell cultureplastic gel alone, amniotic membrane, firbin, polyurethane and polycaprolactone nanoscaffolds andcontact lenses (Focus Night&Day, lotrafilcon A, CIBA Vision, Dublin). Viability of cells was determinedfluorimetrically using Fluoroskan II (Labsystems) with CellTiter-Blue (Promega, Madison, WI, USA)reagents. Presence of CK3, CK12 and p63 markers was determined by immunofluorescence using aconfocal microscope (Leica, TCS SP2 AOBS). Cell cultures were photographed by Scenning ElectronMicroscope as well.Results: Cultured limbal epithelial cells showed the best viability when cultured on plastic andfibrin gel compared to other tested scaffolds. A statistically significant difference in viability ofcultured limbal cells was found between cultures on fibrin gel and cultures on all other scaffolds.Immunofluorescence staining showed the presence of p63 marker of limbal stem cells andCK3 and CK 12 markers of differentiated corneal cells in cultures on all tested scaffolds. SEMphotographs of nanoscaffold surfaces showed good cell attachment and colony spreading on both –polycaprolactone and polyurethane scaffolds.Conclusions: All tested scaffolds showed good cell viability and colony spreading of seeded limbalcells and could be used as a method of cell delivery for therapeutical purposes. Nanoscaffolds hadlower cell viability compared to fibrin and plastic gel. Although high porosity and large surfacepromised superior cell attachment and spreading, this was not the case. The reason could be inhydrophobic properties of nanoscaffold surface. Modifications of these surfaces with chemicals suchas NaOH that could enhance their wettability and hydrophilicity should be tested against limbal cellcutivation.72
EPITHELIAL AND PROGENITOR CELL MARKERS OF OCULAR SURFACE IN MESENCHYMALSTEM CELLS FROM HUMAN ADULT ADIPOSE TISSUEE. M. Martínez-Conesa, M. Pérez, N. Otero, N. Nieto-Nicolau, E. Agustí, A. Vilarrodona, E. Trías, R. P. Casaroli-MaranoTransplant Services Foundation (TSF), University of Barcelona, SpainPurpose: Analyze the expression pattern of cytokeratins and progenitor cell markers in mesenchymalstem cells from human adult adipose tissue (ADS).Methods: We carried out a qualitative and quantitative analysis expression of different cytokeratins(CK) in ADS cells by immunocytochemistry (IFI), Western blot (WB) and real-time PCR (qRT-PCR). Limbicsclerocorneal epithelial cells (LSC) and corneal epithelium cells (CO) were used as controlsResults: ADS cells expressed a set of progenitor cell markers, including p63 and ABCG2. Furthermore,CK expression (CK12, CK76 and CK1/5/10/14) was observed in ADS cell cultures by IFI and WB, whichdemonstrate potential and capability to acquire epithelial-like cell characteristics. The presence ofCK12 was also confirmed by PCR amplification. DNA fragment was amplified, purified, sequenced andcompared with the human CK12 mRNA sequence. Finally, we observed comparative differences in theexpression of progenitor cell markers between LSC, CO and ADS by qRT-PCRConclusions: Adult ADS cells could be a potential source for cell therapy in ocular surfaceregeneration. The expression of putative stem cell markers and CK supports the hypothesis that ADScells have self-renewal capacity and intrinsic plasticity that enables them to acquire some epitheliallikecharacteristics.XXV ANNUAL MEETING OF THE EUROPEAN EYE BANK ASSOCIATION Zagreb, Croatia 18/19 January 2013 73
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