Establishment of a novel method for primary culture of normal human cervical keratinocytes.
Chin Med J (Engl)
; 126(17): 3344-7, 2013.
Article
em En
| MEDLINE
| ID: mdl-24033962
ABSTRACT
BACKGROUND:
Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.METHODS:
Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity.RESULTS:
Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%.CONCLUSION:
A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.
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Base de dados:
MEDLINE
Assunto principal:
Queratinócitos
/
Colo do Útero
/
Técnicas de Cultura de Células
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article