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Live Fibroblast Harvest Exposes Surface Marker Shift in vitro
Graham G. Walmsley, B.A., David D. Lo, M.D., Michael S. Hu, M.D., Daniel T. Montoro, B.S., Adrian McArdle, MB, BCh, BAO, MRCSI, Hermann P. Lorenz, M.D., Irving L. Weissman, M.D., Michael T. Longaker, M.D., M.B.A..
Stanford University School of Medicine, Stanford, CA, USA.
Existing techniques for the harvest of dermal fibroblasts require tissue culture plating of the skin fragments to allow for the migration of fibroblasts out of the tissue and onto the dish. For this reason current knowledge of fibroblast biology is primarily derived from studying their growth and behavior in vitro on plastic substrates as monolayer cultures. Here we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of mice that bypasses the need for cell culture, thereby preserving the physiologic transcriptional and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional and surface marker profiles of cultured vs. live harvested fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest.
We performed extensive studies testing different combinations of enzymatic and mechanical digestion methods for the harvest of dermal fibroblasts from the dorsal skin of mice. Ultimately, a protocol was selected that resulted in the highest yield of live cells. Cells isolated by mechanical/enzymatic digestion from the dorsal skin of CD1 mice were subjected to ACK buffer treatment to facilitate the lysis of red blood cells. Flow cytometry allowed for the depletion of hematopoietic, endothelial and epithelial lineages. The resulting FACS-isolated fibroblasts were either processed immediately for RNA isolation and microarray analysis, analyzed by flow cytometry using a surface marker screening panel containing purified monoclonal antibodies specific for 176 mouse cell surface markers, or expanded in culture.
The live harvest protocol we developed includes a 2-hour collagenase IV digestion with multiple mechanical digestion steps followed by FACS-based lineage depletion. The protocol allowed for the isolation of approximately 200,000 viable fibroblasts per adult mouse dorsum. Many surface markers that were expressed on a small subset of the live harvested fibroblast population were ubiquitously expressed (>90% positivity) on the cultured population. Prominent among these were integrins and other surface molecules that mediate cell adhesion. In comparison, relatively few surface markers were down-regulated in vitro. Furthermore, transcriptional microarray analysis revealed significant differences in gene expression between uncultured and cultured fibroblasts with over 1,000 differentially regulated genes and a coefficient of determination of R^2 = 0.83, suggesting that serum exposure and the process of adherence to tissue culture polystyrene results in a substantial shift in fibroblast phenotype.
The ability of flow cytometry to separate cells based on surface marker expression has become an indispensible tool for furthering our understanding of cellular biology. Critical to this effort is the identification of physiologic surface marker profiles. Given the significant differences between the transcriptional and surface marker profiles of cultured vs. freshly isolated fibroblasts, it is strikingly apparent that the process of culturing of fibroblasts selects for a highly homogenous activated state with distinct transcriptional and surface marker profiles. It is therefore essential that a live harvest approach be employed as researchers seek to identify physiologic surface markers that define specific lineages and discrete subpopulations of fibroblasts in the context of development, wound healing, and cancer stroma.
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