title: The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.
abstract: Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).
authors.affiliation: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway; University of Oslo, Oslo, Norway. Electronic address: meeta.pathak@medisin.uio.no.
authors.forename: Meeta
authors.initials: M
authors.lastname: Pathak
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
authors.forename: O K
authors.initials: OK
authors.lastname: Olstad
authors.affiliation: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway; University of Oslo, Oslo, Norway.
authors.forename: Liv
authors.initials: L
authors.lastname: Drolsum
authors.affiliation: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway; University of Oslo, Oslo, Norway.
authors.forename: Morten C
authors.initials: MC
authors.lastname: Moe
authors.affiliation: Institute of Histology and Embryology, Charles University in Prague, First Faculty of Medicine, Czech Republic.
authors.forename: Natalia
authors.initials: N
authors.lastname: Smorodinova
authors.affiliation: Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General Teaching Hospital, Charles University, Prague, Czech Republic.
authors.forename: Sarka
authors.initials: S
authors.lastname: Kalasova
authors.affiliation: Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General Teaching Hospital, Charles University, Prague, Czech Republic.
authors.forename: Katerina
authors.initials: K
authors.lastname: Jirsova
authors.affiliation: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway; University of Oslo, Oslo, Norway.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Nicolaissen
authors.affiliation: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway.
authors.forename: Agate
authors.initials: A
authors.lastname: Noer
mesh_terms: D000368:Aged; D001706:Biopsy; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000235:genetics / Q000473:pathology / Q000601:surgery; D016039:Corneal Transplantation; D003470:Culture Media; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000096:biosynthesis / Q000235:genetics*; D016850:Limbus Corneae / Q000378:metabolism / Q000648:ultrastructure*; D008297:Male; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D003470:Culture Media; D012313:RNA; D007633:Keratins
keywords: Cornea; Explant culture; Human serum; Limbal epithelium; Limbal stem cell deficiency; Stem cell; Transplantation