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Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

Lipotransfer is a vital tool in the surgeon's armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.(1) However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. (1-3) To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 µl of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes. Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.