Evaluation of cellular wound healing using flow cytometry and expanded polytetrafluroethylene implants.

ABSTRACT

In wound healing studies that investigate therapeutic interventions, it is important to characterize cellular responses. In a randomized trial enrolling patients at risk for surgical infection, one goal is to phenotype cells within a polytetrafluoroethylene implant using flow cytometry and immunohistochemistry, together with standard hematoxylin- and eosin-based histology. Subcutaneous implants are removed 8-9 days postoperatively. To obtain single cells associated with the mechanism of wound healing, we initially used a mouse skin digestion protocol. We optimized this to increase the cell yield and isolate sufficient cells for flow cytometry. The modifications increased the total cells recovered per subject from an average of 5.3 x 10(4)-41 x 10(4) with an average viability of 80%. The immunoflourescent staining assay was verified for our samples, which have smaller cell sample numbers than tissue biopsies. Thirty-two samples were stained. Cells from the polytetrafluoroethylene tubes were isolated and stained positively with fluorescent-labeled antibodies to CD3, CD20, CD31, CD34, CD68, CD133, and VEGF receptor type 2. Flow cytometry data correlated with immunohistochemistry data especially with respect to CD68. This antigen was the most prevalent in both the cell analysis methods. Our findings demonstrate that flow cytometry can be used with polytetrafluoroethylene samples as an additional evaluation method to document and describe cellular wound healing responses.