This study examined the impact of laser‐assisted liposuction on the quality and differentiation potential of adipose‐derived stromal cells (ASCs). It was found that laser‐assisted liposuction ...negatively impacts the biology of ASCs, and therefore cell harvest using suction‐assisted liposuction is preferable for tissue‐engineering purposes.
Harvesting adipose‐derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third‐generation ultrasound‐assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser‐assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser‐assisted lipoaspirate and suction‐assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical‐sized cranial defect in athymic nude mice. Although ASCs isolated from suction‐assisted lipoaspirate and laser‐assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34+CD31−CD45−) in the stromal vascular fraction were all significantly less with laser‐assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro‐computed tomography revealed significantly more healing with ASCs isolated from suction‐assisted lipoaspirate relative to laser‐assisted lipoaspirate at the 4‐, 6‐, and 8‐week time points (p < .05). Therefore, as laser‐assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction‐assisted liposuction is preferable for tissue‐engineering purposes.
The regenerative abilities of adipose‐derived mesenchymal stem cells (ASCs) harvested via a third‐generation ultrasound‐assisted liposuction (UAL) device versus ASCs obtained via standard ...suction‐assisted lipoaspiration were evaluated. ASC yield and viability, and expression of most osteogenic, adipogenic, and key regenerative genes were equivalent between the two methods. Cells harvested via UAL showed comparable abilities to enhance cutaneous regeneration and appear suitable for cell therapy and tissue engineering applications.
Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose‐derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third‐generation ultrasound‐assisted liposuction (UAL) device versus ASCs obtained via standard suction‐assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface‐marker profile (CD34+/CD31−/CD45−), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL‐ and SAL‐derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real‐time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell‐therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications.
Significance
Adipose‐derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction‐assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound‐assisted (UAL) versus standard suction‐assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.
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.
Cell-based therapy is an emerging paradigm in skeletal regenerative medicine. However, the primary means by which transplanted cells contribute to bone repair and regeneration remain controversial. ...To gain an insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we used a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen, type I, alpha 1 promoter/enhancer sequence (Col1a1(GFP)) and membrane-bound tomato red fluorescent protein constitutively in all cell types (R26(mTmG)). A comparison of healing in parietal versus frontal calvarial defects in these mice revealed that frontal osteoblasts express Col1a1 to a greater degree than parietal osteoblasts. Furthermore, the scaffold-based application of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts derived from these mice to critical-sized calvarial defects allowed for investigation of cell survival and function following transplantation. We found that ASCs led to significantly faster rates of bone healing in comparison to BM-MSCs and osteoblasts. ASCs displayed both increased survival and increased Col1a1 expression compared to BM-MSCs and osteoblasts following calvarial defect transplantation, which may explain their superior regenerative capacity in the context of bone healing. Using this novel reporter system, we were able to elucidate how cell-based therapies impact bone healing and identify ASCs as an attractive candidate for cell-based skeletal regenerative therapy. These insights potentially influence stem cell selection in translational clinical trials evaluating cell-based therapeutics for osseous repair and regeneration.
Current methods for the isolation of fibroblasts require extended ex vivo manipulation in cell culture. As a consequence, prior studies investigating fibroblast biology may fail to adequately ...represent cellular phenotypes in vivo. To overcome this problem, we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of adult mice that bypasses the need for cell culture, thereby preserving the physiological, transcriptional, and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional programs and the surface expression of 176 CD markers in cultured versus uncultured fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest for investigations of fibroblast biology.
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.
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