An Additive Effect of Promoting Thermogenic Gene Expression in Mice Adipose-Derived Stromal Vascular Cells by Combination of Rosiglitazone and CL316,243.
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ABSTRACT:
SUBMITTER: Li YL
PROVIDER: S-EPMC5454915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAdipose stromal vascular fraction (SVF) isolation with enzymatic digestion is the gold standard, but is expensive, having practical and legal concerns. The alternative mechanical SVF isolation methods provide lower cell yields as they employ either centrifugation, emulsification, or digestion steps alone. We combined mechanical processing with buffer incubation and centrifugation steps into an isolation method called "mechanical digestion" and compared the cell yields with that of enzymatic digestion.MethodsA total of 40-mL lipoaspirate was harvested from 35 women undergoing liposuction and was submitted to conventional enzymatic digestion for SVF isolation or mechanical digestion using a closed unit harnessing 3 ports with blades, followed by buffer incubation and centrifugation. Culture of the SVFs and flow cytometry were performed.ResultsThe SVF cell yield obtained by enzymatic digestion was significantly higher 3.38 × 106/mL (±3.63; n = 35) than that obtained by mechanical digestion 1.34 × 106/mL (±1.69; n = 35), P = 0.015. The average cell viability was 82.86% ± 10.68 after enzymatic digestion versus 85.86% ± 5.74 after mechanical digestion, which was not significant. Mechanical digested SVF expressed 2-fold higher stem cell surface markers compared with enzymatically digested SVF. Mechanical digestion was less time consuming, cost effective, and did not require a specific laboratory environment.ConclusionsMechanically digested SVF was comparable to enzymatically digested SVF in terms of stromal cell composition and viability. With mechanical digestion, we can isolate 30%-50% SVF cells of that isolated with enzymatic digestion. Further studies are warranted to determine the clinical outcomes.
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Project description:Adipose tissue-derived stem cells (ASCs) are a promising tool for the treatment of bone diseases or skeletal lesions, thanks to their ability to potentially repair damaged tissue. One of the major limitations of ASCs is represented by the necessity to be isolated and expanded through in vitro culture; thus, a strong interest was generated by the adipose stromal vascular fraction (SVF), the noncultured fraction of ASCs. SVF is a heterogeneous cell population, directly obtained after collagenase treatment of adipose tissue. In order to investigate and compare the bone-regenerative potential of SVF and ASCs, they were plated on SmartBone®, a xenohybrid bone scaffold, already used in clinical practice with successful results. We showed that SVF plated on SmartBone, in the presence of osteogenic factors, had better osteoinductive capabilities than ASCs, in terms of differentiation into bone cells, mineralization, and secretion of soluble factors stimulating osteoblasts. Indeed, we observed an increasing area of new tissue over time, with and without OM. These data strongly support an innovative idea for the use of adipose SVF and bone scaffolds to promote tissue regeneration and repair, also thanks to an easier cell management preparation that allows a potentially larger use in clinical applications.
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Project description:Endometrial dysfunction affects approximately 1% of infertile women, and there is currently no standard therapy for improving fertility treatment outcomes in these patients. In our study, we utilized a rodent model of thin endometrium to test whether intrauterine application of adipose-derived stromal vascular fraction cells (SVF) could improve morphological and physiological markers of endometrial receptivity. Using anhydrous ethanol, endometrial area and gland density were significantly reduced in our model of thin endometrium. Application of SVF was associated with a 29% reduction in endometrial vascular endothelial growth factor (VEGF) expression and significant increases in uterine artery systolic/diastolic velocity ratios and resistance index values, suggesting reduced diastolic microvascular tone. However, no significant improvements in endometrial area or gland density were observed following SVF treatment. 3D confocal imaging demonstrated poor engraftment of SVF cells into recipient tissue, which likely contributed to the negative results of this study. We suspect modified treatment protocols utilizing adjuvant estrogen and/or tail vein cell delivery may improve SVF retention and therapeutic response in subsequent studies. SVF is an easily-obtainable cell product with regenerative capability that may have a future role in the treatment of infertile women with endometrial dysfunction.
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Project description:Background:The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Alternatively, adipose tissue-derived microvascular fragments (ad-MVF) may serve as vascularization units. In contrast to SVF single cells, they represent a mixture of intact arteriolar, capillary and venular vessel segments. Therefore, we herein hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF single cells-based approach. Results:SVF single cells and ad-MVF were enzymatically isolated from epididymal fat pads of green fluorescent protein (GFP)+ donor mice to assess their viability and cellular composition using fluorescence microscopy and flow cytometry. Moreover, collagen-glycosaminoglycan matrices (Integra®) were seeded with identical amounts of the isolates and implanted into full-thickness skin defects within dorsal skinfold chambers of GFP- recipient mice for the intravital fluorescent microscopic, histological and immunohistochemical analysis of implant vascularization and incorporation throughout an observation period of 2 weeks. Non-seeded matrices served as controls. While both isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes and stem cells, ad-MVF exhibited a significantly higher viability. After in vivo implantation, the vascularization of ad-MVF-seeded scaffolds was improved when compared to SVF-seeded ones, as indicated by a significantly higher functional microvessel density. This was associated with an enhanced cellular infiltration, collagen content and density of CD31+/GFP+ microvessels particularly in the center of the implants, demonstrating a better incorporation into the surrounding host tissue. In contrast, non-seeded matrices exhibited a poor vascularization, incorporation and epithelialization over time. Conclusions:The present study demonstrates that ad-MVF are highly potent vascularization units that markedly accelerate and improve scaffold vascularization when compared to the SVF.
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Project description:BackgroundAdipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation.MethodsSVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses.ResultsCultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater Pparg, Plin1, and Lep expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix.ConclusionNewborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation.
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