Joris van Dongen

68 Chapter 3 adipose tissue is a fast time sparing inexpensive method. In this study we describe a new method to mechanically dissociate adipose tissue intra-operatively to get hold of its therapeutic components for injection: this procedure is named the FAT procedure (Fractionation of Adipose Tissue). MATERIAL & METHODS Liposuction and FAT procedure Adipose tissue harvesting was performed with a Sorenson lipo-harvesting cannula (Tulip, Medical Products®, San Diego, Calif. QR link video) during normal liposuction procedures in eleven patients (Fig. 1, Supplemental Content). Adipose tissue was harvested after infiltration with 500ml-modified Klein’s solution (per 500ml of saline, 20ml of lidocaine, 2% Epinefrine 1:200.000 and 2ml of bicarbonate was added). For the FAT procedure harvested adipose tissue was centrifuged at 3,000 rpm with a 9.5 cm radius fixed angle rotor for 2.5 min (Medilite™, Thermo Fisher Scientific, NY) at room temperature (RT) after decantation. After one round of centrifugation, the oil and infiltration fluid fractions were discarded, yielding condensed lipoaspirate. One sample of 10ml of condensed lipoaspirate that was centrifuged only once was used as a control, from heron referred as ‘control’. One sample of 10ml of condensed lipoaspirate was used for mechanical dissociation. For mechanical dissociation, two 10ml syringes with a total volume of 10ml of condensed lipoaspirate were connected to the Fractionator, a Luer to Luer transfer with three 1.4 mm holes (Tulip) (Fig. 2, Supplemental Content). Mechanical dissociation was performed by pushing the lipoaspirate through the Fractionator forward and backwards thirty times. After mechanical dissociation, the adipose tissue was centrifuged again at 3,000 rpmwith a 9.5 cm radius fixed angle rotor for 2.5 min at room temperature (Medilite). This resulted in four different fractions: an oily fraction, a stromal vascular fraction (from heron referred as ‘FAT-SVF’) and an aqueous fraction containing a small pellet fraction (Fig. 1). Stromal Vascular Fraction Viability A live dead staining with Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA-SE) and Propidiumiodide (PI) was performed to measure the FAT-SVF viability compared to the control (n=3). For CFDA-SE and PI staining, pre-warmed 0.001% of CFDA-SE and 0.001% of PI in serum free Dulbecco’s Modified Eagle’s Medium (DMEM) was used directly on the FAT-SVF and control. After 30 min of incubation under culture conditions cells were washed with Phosphate Buffered Saline (PBS) three times and fixed with 2% paraformaldehyde (PFA). 0.0002% DAPI in PBS was used to stain the