Phenotypic Analysis of Stromal Vascular Fraction after Mechanical Shear Reveals Stress-Induced Progenitor Populations

Abstract

Background: Optimization of fat grafting continues to gain increasing attention in the field of regenerative medicine. “Nanofat grafting” implements mechanical emulsification and injection of standard lipoaspirate for the correction of superficial rhytides and skin discoloration; however, little is known about the cellular constituents of the graft. Based on recent evidence that various stressors can induce progenitor activity, the authors hypothesized that the shear forces used in common fat grafting techniques may impact their regenerative capacities. Methods: Lipoaspirates were obtained from 10 patients undergoing elective procedures. Half of each sample was subjected to nanofat processing; the other half was left unchallenged. The viscosity of each sample was measured for computational analysis. The stromal vascular fraction of each sample was isolated, quantified, and analyzed by means of flow cytometry with two multicolor fluorescence antibody panels. Results: Standard lipoaspirate is ideally suited for mechanical stress induction. The mechanical emulsification involved in nanofat processing did not affect cell number; however, viability was greatly reduced when compared with the stromal vascular fraction of standard lipoaspirate. Interestingly, nanofat processing resulted in stress-induced stromal vascular fraction with a higher proportion of endothelial progenitor cells, mesenchymal stem cells, and multilineage differentiating stress-enduring cells. Single-parameter analysis also revealed significant increases in CD34, CD13, CD73, and CD146 of the stress-induced stromal vascular fraction, markers associated with mesenchymal stem cell activity. Conclusions: Mechanical processing used in techniques such as nanofat grafting induces the up-regulation of progenitor phenotypes consistent with multipotency and pluripotency. These data provide a first step in characterizing the potential regenerative benefits realized through stress induction in fat grafting. CLINCAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Reply: Phenotypic Analysis of Stromal Vascular Fraction after Mechanical Shear Reveals Stress-Induced Progenitor Populations

“…We do concede that broadly defining mesenchymal stem cells as CD45 − /CD34 + captures a progenitor population that includes endothelial cells; however, the relationship of mesenchymal stem cell enrichment mirrored that of both adipose-derived stem cells and mesenchymal stem cells in the mechanically processed groups in our study. 1 We are encouraged by your findings that demonstrated a significant enrichment of adipose-derived stem cells (CD45 − /CD31 − /CD34 + ) that corroborates our findings, and we suspect that the key difference in subpopulations observed is related to your use of a 100-μm filter without collagenase.…”

“…1 Indeed, our investigation focused on the analysis of intersyringe-processed unfiltered fat that was subjected to collagenase digestion before single-cell analysis. We have conducted internal experiments on the cellular constituency of the filtrate and filtrand of intersyringe-processed fat and observed a significant number of retained cells in the filtrand, indicating a large portion of regenerative cells that are closely intimated to the adipose extracellular matrix.…”

Mekani̇k Ve Enzi̇mati̇k Yöntem İle İzole Edi̇len Stromal Vasküler Fraksi̇yon’un Yara İyi̇leşmesi̇ Üzeri̇ndeki̇ Etki̇si̇ni̇n in Vi̇tro İncelenmesi̇

“…Geliştirilen yeni mekanik izolasyon yöntemleri ile birlikte bu oran %50-60'a kadar yükselmiştir (16,17). Ayrıca Banyard DA ve arkadaşları tarafından yayınlanan bir çalışmada hücre sayısının etkisinin çok önemli olmadığı, hücre aktivitesinin daha önemli olduğu vurgulanmıştır (8). Bu ışık çerçevesinde bakıldığında mekanik izolasyon yöntemi ile edilen SVF hücre kokteylinin yara iyileşmesinde çok büyük umut vaat ettiği ön görülmektedir.…”

“…Daha sonra yapılan çalışmalarda, hücre sayısı ve canlılığının az olduğu mekanik izolasyon yöntemlerinde hücre aktivitesinin, enzimatik yöntemler kullanılarak ayrıştırılan hücre popülasyonlarından daha fazla olduğu gösterilmiştir (6). Bu çalışmada; literatürde daha önce yara iyileşmesindeki etkinliği kanıtlanmış olan yağ dokusu kullanılarak mekanik ve enzimatik yöntem ile izole edilen SVF'in yara iyileşmesi üzerindeki etkisinin in vitro incelenmesi hedeflenmiştir (7,8,11).…”

Quantitative and Qualitative Analysis of Human Stromal Vascular Fraction from Different Methods of Liposuction Short Title: Stromal Fraction from Liposuction Types

“…Considering this aspect and the literature discussed, the findings obtained suggest that different mechanical stresses over the subcutaneous tissue may impact the cell yielding of the resident SVF and its cellular subpopulations, a component not discussed in literature, focused in volumes and harvested sites. A similar concept has been described with the nano-fatgrafting technique, were the exvivo mechanical emulsification of lipoaspirates resulted in a SVF with higher proportions of progenitor, mesenchymal and multilineage cells, a stress induction that may enhance the regenerative potential of the non-expanded pellet [31].…”