Understanding Mechanical Emulsification (Nanofat) Versus Enzymatic Isolation of Tissue Stromal Vascular Fraction (tSVF) Cells from Adipose Tissue: Potential Uses in Biocellular Regenerative Medicine

Background: With rapid growth of experiences and techniques in the field of Biocellular Regenerative Medicine, clinical scientists and biotechnical advancements constantly seek to understand and optimize uses of the multipotent heterogeneous cellular populations found in adipose tissue complex. The value of including the living, native bioscaffolding within the adipose tissues has likewise gained importance associated with the biologically trophic effects and cellular attachment capabilities believed to positively influence undifferentiated stromal cells in the native sites and biocellular grafts placed. These bonds are felt necessary for cellular activation, proliferation, and contributing to an auto-amplification system within the processes of homeostasis, regeneration, and repair in a “site specific” manner. Appreciation of uses of biologicals (such as platelet-rich and bone marrow concentrates) has grown, and felt to provide a trophic influence on a variety of sites and applications. Study: This study reports on the differences in use of microcannula lipoaspirates undergoing mechanical emulsification in order to provide adult mature adipocyte lysis, preserving tissue stromal vascular fraction (tSVF) in small particle form which can be injected through small bore needles (25-30 gauge). Comparison of the compressed (centrifuged) lipoaspirates with emulsified adipose specimens is made for differences in viability, cell numbers, and total nuclear counts. Simultaneous harvesting of both specimen groups via closed syringe microcannula system was carried out. Following centrifugation at 800 g-force for 5 minutes, comparative 10 cc compressed specimens were submitted for incubation, agitation, and cell separation using a CentriCyte 1000 closed semi-automated system and Vitacyte Clyzme AS at a 1::1 ratio per manufacturer’s instructions. Cellular testing was carried out using flow cytometry for viability, counts and total nucleated counts (following RBC lysis) and compared. Lastly, each emulsified specimen was tested with 1 cc luer syringes through 25, 27, and 30 gauge needles. NOTE: Compressed adipose-derived tissue stromal vascular fraction (AD-tSVF) specimen harvested through 2.11 mm OD microcannulas will not inject through such small bore needles without plugging, and typically require 18-20 gauge needles to easily pass. Conclusions: Comparative testing of 20 specimens each (n=20) revealed no statistically significant differences in mean cell viabilities, numbers or total nucleated cell counts between the non-emulsified AD-tSVF versus the emulsified AD-tSVF specimens. Examination of the emulsified AD-tSVF using fluorescent microscopy and live-dead staining did reveal many small fragments (extracellular matrix or microvascular remnants) with viable stromal cells remaining attached. This suggests that the mechanical emulsification process was effective in reducing the particle size permitting the small needle injection capability while preserving or promoting potentially important cellular attachments, while maintaining a comparable stromal cell viability (without large size mature adipocytes having been mechanically lysed). This emulsified AD-tSVF has been referred to as “microfat” or “nanofat” in current literature. Uses of such microfat or nanofat injections have important potentials in anti-aging, hair regeneration, radiation/sun damage skin, chronic wounds, abnormal scarring, and many ultrasound guided placements in musculoskeletal applications currently using a compressed AD-tSVF + high density Platelet concentrates. Reduction of needle diameter requirement permits significantly less patient discomfort during injections, and permits intradermal placement and small joint placements, currently challenging therapeutic sites. Each mechanically emulsified AD-tSVF specimen successfully achieved injection ability using 1 cc luer syringes and down to 30 gauge needles. This ability changes many applications which involve intradermal patterned injections, scars, radiation damaged skin and chronic wound areas. Patient comfort is significantly improved with use of smaller bore injection needles, whether using sharp or blunt types. NOTE: It is important to clearly understand that the mechanical emulsification alone does not create a true cellular stromal vascular fraction (cSVF), and should not be thought of as a substitute for true cellular isolation and concentration (often reported in laboratory, pre-clinical research, and clinical studies and papers). Due to the regulatory environment in the