Described here is an improved method to create wound cell suspensions for examination by flow cytometry, allowing study of immune cell infiltrate, including rare populations such as the NKT cell. The previous isolation techniques provided minimal serum and/or media for cell nutritional support during isolation, lacked proper cofactors for specific enzymatic activity, limited anti-microbial coverage predominately to gram-negative bacteria and did not utilize a method for removal of adherent cells from tissue culture plastic during the isolation procedure (Sepulveda-Merrill et al., 1994
; Wilson et al., 2002
). Specifically, previous methods cultured cells overnight in HBSS supplemented with 3% FBS (Wilson et al., 2002
). In this isolation procedure, cells were cultured in RPMI with 10% FBS to further promote cell survival. In the subsequent enzymatic digestion, magnesium chloride hexahydrate was added to promote DNase I enzymatic activity. In both digests, penicillin was added to provide broader gram-positive organism coverage, as gram-positive organisms are common in the epidermal microflora. By adjusting these factors we have generated a high cell yield and preserved cell viability for functional assays. Additionally, utilization of Accutase to remove cells from tissue culture plastic allows for a more accurate characterization of the adherent cell populations, such as macrophages, neutrophils, fibroblasts or keratinocytes within the wound that may not have been adequately accounted for in past methods. Further, this isolation procedure would also allow for study of non-immune cell subsets such as keratinocytes or fibroblasts by flow cytometry or additional immunological and molecular techniques.
The alternate method for studying immune cell infiltration following cutaneous injury employs the PVA sponge method (Daley et al., 2010
; Efron and Barbul, 2003
; Gosain et al., 2009
; Swift et al., 2001
). As mentioned, this does not allow for examination of the cellular milieu in other wound healing models such as the commonly used model of excisional cutaneous wound injury. Additionally, this method requires applied pressure to the sponge to release the wound fluid and cellular components. Pressure and tension are known activators of intracellular signaling cascades in both fibroblast and keratinocytes, and thus this isolation method may alter the phenotype of these cells (Eckes et al., 2006
; Tomasek et al., 2002
). Our method only applies minimal mechanical force, primarily gentle agitation and washing, which may limit pressure-induced signaling activation. Further, previous reports using the PVA model yield 1×106
cells/animal at day 1 and 3×106
cells/animal at day 3 (Daley et al., 2005
). Using the technique described in our excisional wound model, we have obtained an increased cell yield in comparison to the PVA sponge models (). Moreover, we demonstrate that these cells maintain functional capabilities following isolation (), allowing further examination of the subpopulations that comprise wound tissue. The ability to isolate cellular components and examine ex vivo
functional aspects such as phagocytosis or chemotaxis will provide more relevant information about the impact of the wound environment on cell function. Additionally, examination of keratinocyte functions, such as antimicrobial peptide generation and expression, could potentially be evaluated using this technique. This may be useful in examining how various disease states impact keratinocyte function in response to wound injury. Considering our findings, this technique is ideal for examining the cutaneous cellular composition and can be extended for use in other models of cutaneous injury, such as incisional wound or burn injuries, as well as cutaneous malignancies. Finally, modifications of this protocol may allow for study of other tissues, such as the cornea or gut.
Despite the benefits of this tissue dispersion protocol, limitations do exist. Due to the fibrous nature of skin tissue, excess debris can be observed during cell counting which can ultimately compromise flow cytometric analysis. To combat this, an additional filtration step with a 70uM filter can be added. Additionally, this protocol is designed to allow examination of the combined cellular composition of the epidermis and dermis. Modification of this protocol could allow for examination of cells isolated from a specific tissue layer. Prior to finely dicing the wound tissue, one may use the “Dispase Solution” to carefully dissect the epidermis away from the dermis, proving better characterization of the cell subset present in respective tissue layers. Furthermore, it is difficult to ascertain the impact of this protocol on cell surface markers and cell phenotype of the isolated wound cells. A potential method of comparison is subjecting the spleen, a leukocyte rich lymphoid organ, to a standard dispersion protocol and the procedure described above. However, this method of comparison is not adequate as the splenocytes are more directly exposed to the enzymes used using this protocol as compared to cell subsets present in the fibrous skin matrix. Alternatively, validating the flow cytomtery results with IHC is difficult for rare cellular subsets and complex phenotypic characterization. Overall, this procedure allows for relative comparison of isolated cells between experimental groups. Though these limitations exist, use and further modifications of this protocol will allow for enhanced understanding of wound and skin biology in a variety of experimental and clinical settings.