A method for removal of fibroblasts from human tissue culture systems.
The phenomenon of fibroblast overgrowth is one of the major problems encountered during long-term culture of more slowly growing specialized cell types. A cell surface glycoprotein, Thy-1, which was originally found to be present on murine T-lymphocytes and brain cells, is also found to be present on only a few human cell types, mainly fibroblasts and neuronal cells. We have taken advantage of this fact, using a solid-phase immunoadsorption technique termed "panning", to rid our culture system (normal human keratinocytes) of contaminating dermal fibroblasts. A mouse monoclonal antibody raised against human brain Thy-1 was used to attach dermal fibroblasts to a goat anti-mouse immunoglobulin-coated plastic surface. By this method we were able to separate a 1:1 mixture of human dermal fibroblasts and keratinocytes with greater than 97.5% efficiency. Furthermore we have successfully removed dermal fibroblasts from naturally arising contaminated keratinocyte cultures, where the proportion of fibroblasts (less than 10%) was considerably less than that of the artificially mixed populations. These results compare favorably with those expected of the fluorescence-activated cell sorter (FACS) method of cell separation. In addition this technique is comparatively simple and inexpensive and is thought to be of use to other primary tissue culture systems (especially human) where contamination and subsequent overgrowth with fibroblasts remains a problem.
Linge
C
C
Department of Anatomy and Human Biology, Kings College London, United Kingdom.
Green
M R
MR
Brooks
R F
RF
D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods*; D046508:Culture Techniques / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D008858:Microscopy, Phase-Contrast; D012867:Skin / Q000166:cytology