Application of Fluorescent In Situ Hybridization in the Mouse Xenograft Model of Human Fat Grafting

Abstract

Determining the optimal technique for autologous fat grafting requires elucidation of the engraftment process at the cellular level. The nude mouse xenograft model for autologous human fat grafting is an excellent tool to evaluate the incorporation of grafted fat into the recipient. The authors present a murine xenograft model that uses fluorescent in situ hybridization (FISH) to differentiate between murine (host) cells and human (grafted) cells. Fat grafts were harvested from human abdominoplasty specimens, fixed, embedded in paraffin blocks, and cut into 5 µm sections. The sections were stained and used for in situ hybridization with fluorescently labeled murine and human nucleic acid reagents. A fluorescence microscope was used for photographic analysis of the grafts, allowing identification of murine and human cell populations based on the wavelength at which they stained. DAPI (4,'6-diamidino-2-phenylindole) counterstaining was also applied to confirm that the images obtained represented cell nuclei. At 20× magnification, strong species-specific staining of cells was seen within the tissue sections, which allowed the authors to easily discriminate between graft-derived and host-derived cells. Using this approach, human fat xenografts were revealed to comprise a complex matrix of closely interacting graft- and host-derived structures. FISH has the potential to be a powerful technique for distinguishing between murine and human cells in the nude mouse xenograft model of human fat grafting. By applying this technique, it may be possible to evaluate the engraftment process at a cellular level, which may ultimately allow clinicians to obtain more predictable results with grafted fat.