Cryopreserved 3T3 fibroblasts retain their capacity to enhance the growth of human keratinocyte cultures

Abstract

Cultured epithelial grafting of full-thickness skin defects is a new promising possibility for the successful treatment of patients with large burns. The major obstacle to this method, however, is a 3-4-week interval between burn and grafting, which is necessary for the growth of sufficient quantities of cultured epithelium. Generally, growth-arrested murine 3T3 fibroblasts have been used with success as feeder layers to shorten the cultivation time of keratinocytes. In the present work we have initiated studies to determine whether or not cryopreserved growth-arrested 3T3 fibroblasts retain their capacity to enhance the growth of human keratinocytes in vitro. The results of this study show that the [3H]thymidine incorporation in cultures containing mitomycin C treated 3T3 feeder cells was significantly greater than in cultures without feeder cells. Furthermore, in keratinocyte cultures containing freshly separated or cryopreserved 3T3 fibroblasts, a similar rate of [3H]thymidine incorporation was observed and the activity of incorporation has never differed significantly during the 11 days of culturing, meaning that cryopreserved, growth-arrested 3T3 fibroblasts retain their ability to enhance the growth of human keratinocytes. This observation renders the continuous maintenance of 3T3 cells unnecessary in laboratories which want to culture keratinocytes without delay.