Growth and differentiation of an established rat keratinocyte line in serial culture

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Abstract
This paper describes the growth and differentiation of an established, feeder layer independent line of rat keratinocytes originally developed from tongue epithelium. The cells grew from any seeding density with a population doubling time of 14 to 16 h and a plating efficiency of 60 to 90%. The cells were kept in continuous culture for more than 3 yr and were cloned several times during this period. After more than 700 population doublings the cultures maintained typical expressions of the keratinocyte phenotype such as desmosomes and tonofilaments. The cells required 10 to 15% fetal bovine serum but no additional supplement of growth factors. Single colonies, as well as confluent multilayers, keratinized and displayed the whole complement of keratinization markers including keratin filaments, cornified envelopes, increased plasma membrane permeability, and destruction of cytoplasmic and nuclear components. However, the ability to stratify in a regular manner was lost although sporadic attempts of stratification were present. In suspension culture the cells terminally differentiated in 1 to 2 wk and developed highly cross-linked cornified envelopes that were resistant to boiling detergent solutions under reducing conditions. Chromosome numbers were in the diploid range (2N=38 to 46), but aberrations were frequent.