A high yield method for growing primary canine keratinocytes.
From a small amount of starting material, a large quantity of canine keratinocytes can be generated for experimental purposes using a refined method of explant culture to initiate the growth of basal cells with a high proliferative potential. The dividing capacity of cultures was promoted by a system selecting clonogenic cells onto an i3T3 feeder layer in combination with carefully monitoring cell morphology and passaging to select out excessive numbers of differentiated keratinocytes. Levels of contaminating dermal fibroblasts, which if left unchecked will overgrow keratinocytes, were kept to a minimum by a combination of careful explant micro-dissection to remove dermis, eliminating explants with signs of fibroblast growth as well as using cholera toxin, EGF and i3T3 feeder layers. The advantage of the method described is that it does not rely on the provision of large quantities of starting material thereby reducing the need for repeated tissue sampling, and passage numbers of five or six can be routinely achieved. This technique can therefore be useful to experimenters who require a regular and reliable source of cells for their studies.
Watson
Adrian
A
Waltham Centre for Pet Nutrition, Freeby Lane, Waltham-on-the-Wolds, Leicestershire LE14 4RT, UK. adrian.watson@eu.effem.com
Baker
Claire
C
Bailey
Julie
J
Fray
Tim
T
Markwell
Peter
P
D000818:Animals; D002478:Cells, Cultured / Q000166:cytology; D046508:Culture Techniques / Q000379:methods / Q000662:veterinary*; D004285:Dogs; D005260:Female; D015603:Keratinocytes / Q000166:cytology*; D008297:Male; D012867:Skin / Q000166:cytology*