title: The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.
abstract: Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.
authors.affiliation: Cells for Sight Transplantation and Research Programme, United Kingdom. a.shortt@ucl.ac.uk
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Shortt
authors.forename: Genevieve A
authors.initials: GA
authors.lastname: Secker
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lomas
authors.forename: Stacy P
authors.initials: SP
authors.lastname: Wilshaw
authors.forename: John N
authors.initials: JN
authors.lastname: Kearney
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tuft
authors.forename: Julie T
authors.initials: JT
authors.lastname: Daniels
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000650:Amnion / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D015925:Cryopreservation; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects; D005990:Glycerol; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008422:Materials Testing / Q000379:methods*; D009363:Neoplasm Proteins / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D009363:Neoplasm Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D005990:Glycerol