title: Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens.
abstract: The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue-derived fibroblasts and keratinocytes were co-cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate.
authors.affiliation: Section of Cell Biology and Molecular Genetics, Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Bononi
authors.forename: Silvia
authors.initials: S
authors.lastname: Bosi
authors.forename: Gloria
authors.initials: G
authors.lastname: Bonaccorsi
authors.forename: Roberto
authors.initials: R
authors.lastname: Marci
authors.forename: Alfredo
authors.initials: A
authors.lastname: Patella
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferretti
authors.forename: Mauro
authors.initials: M
authors.lastname: Tognon
authors.forename: Paola
authors.initials: P
authors.lastname: Garutti
authors.forename: Fernanda
authors.initials: F
authors.lastname: Martini
mesh_terms: D052685:Alphapapillomavirus / Q000145:classification / Q000235:genetics; D015415:Biomarkers; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005260:Female; D005455:Fluorescent Antibody Technique; D005838:Genotype; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000502:physiology*; D009375:Neoplasms, Glandular and Epithelial / Q000473:pathology*; D012235:Rhodamines; D013194:Staining and Labeling; D002583:Uterine Cervical Neoplasms / Q000473:pathology*; D014777:Virus Diseases / Q000235:genetics
chemical_list: D015415:Biomarkers; D012235:Rhodamines; C029773:rhodamine B