Non-enzymatic Stromal Vascular Fraction: An Automated Microfluidic Approach To Improve The Syringe-injectable Adipose Therapeutic
Derek A. Banyard, M.D., M.B.A., M.S., Jeremy A. Lombardo, M.S., Alexandria M. Sorensen, Gregory R.D. Evans, M.D., Jered B. Haun, Ph.D., Alan D. Widgerow, M.B.B.Ch, M.Med.
University of California, Irvine, Orange, CA, USA.
Background: Research has intensified towards efficient processing of adipose tissue and isolation of the stromal vascular fraction due to its immense potential as an autologous therapeutic for regenerative therapies. The Nanofat technique, popularized by Tonnard and others, involves manual processing of lipoaspirate via intersyringe transfer and was recently described by our group to contain high proportions of stem and progenitor cells. One major drawback to the Nanofat technique, however, is the lack of standardization and automation potential that are inherent to this methodology.
Methods: A series of microfluidic devices were designed to emulsify, filter and enrich the stem cell components of lipoaspirate, and were fabricated using laser-machining and 3D-printing techniques. Liposuction samples were obtained from both healthy and diabetic patients that served as controls (macrofat), manually intersyringe processed sample (Nanofat), or sequentially processed sample using our automated microfluidic pump-based (NESVF) system under various processing conditions. Each resulting sample was subsequently digested with collagenase to release the remaining cells, and analyzed for cell count, viability, and cell types using multiparameter flow cytometry. RNA was isolated for future downstream analysis.
Results: Both Nanofat and emulsification device processing resulted in decreased total cell recoveries compared to macrofat in both healthy (n=5) and diabetic (n=4) samples, however, they also yielded populations enriched in mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), multilineage stress enduring cells (Muse) and DPP4+/CD55+ cells, an MSC subpopulation implicated in diabetic wound healing. There was a statistically significant increase in MSCs and EPCs after Nanofat (p < 0.05) and emulsification device (p < 0.01) processing in healthy tissue, but only in EPCs in diabetic tissue (p < 0.05). When filtration was added in series, overall cell recovery reduced significantly for both Nanofat and emulsification device samples (p < 0.01), without any effect on viability. Filtered Nanofat and emulsification device healthy tissue suspensions were higher in MSC and EPC concentrations (p < 0.05) compared to macrofat. Interestingly, emulsification device plus filtration also significantly enriched Muse (p < 0.01) and DPP4+/CD55+ (p < 0.05) cells, which was not observed in the Nanofat group or macrofat control. Next, healthy filtered samples were processed via the enrichment device at various flow rates. A direct flow rate-to-cell enrichment relationship was observed for NESVF-processed EPCs, whereas an inverse of this flow rate relationship was observed for Muse cells. Overall, the enrichment device resulted in a higher normalized cell population percentage when compared to emulsification device combined with filtration. Finally, RNA was extracted from samples that were processed at times zero and 24 hours in standard culture conditions that revealed purity acceptable for downstream applications like real-time PCR.
Conclusions: Here we demonstrate a novel microfluidic device platform that emulsifies, filters and enriches stem and progenitor populations implicated in tissue regeneration and diabetic wound healing. This system is superior to manual techniques such as Nanofat processing in that user-variability is eliminated and greater enrichment of various stem populations are observed. Future transcriptional and ex vivo assays will help to determine the clinical significance of these findings.
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