Joris van Dongen

132 Chapter 6 3. Place a Sorenson type lipoharvesting cannula into the stab incision. 4. Connect a 50 ml Luer Lock syringe to a Sorenson type harvesting cannula. 5. Pull the plunger backwards to allow for negative pressure and use a surgical clamp or syringe snap lock to maintain the negative pressure. 6. Start harvesting by moving the harvesting cannula forward and backwards. 7. Replace the full 50 ml syringe by an empty 50 ml syringe and start again from step 4. Fractionation of adipose tissue procedure 1. Divide the harvested adipose tissue in 10 ml Luer Lock syringes without plunger. 2. Decant the adipose tissue for 5 min. at room temperature. 3. Remove infiltration fluid by opening the cap of the 10 ml syringe. 4. Refill syringe to 10 ml and centrifuge the syringe without plunger at 3,000 rpm with a 9.5 cm radius fixed angel rotor or at 960 xg for 2.5 min. at room temperature. 5. Remove infiltration fluid by opening the cap of the 10 ml syringe (see Note 1). 6. Remove oil (disrupted adipocytes) by turning the syringe upside down and prevent the adipose tissue from leaking with the use of a gauge. 7. Refill syringe to 10 ml of centrifuged adipose tissue and place the plunger back. 8. Connect the 10 ml syringe with centrifuged adipose tissue to the fractionator and connect an empty 10 ml syringe with plunger to the other side of the fractionator. 9. Push the adipose tissue extensively forward and backwards thirty times (see Note 2). 10. Centrifuge the syringe without plunger at 3,000 rpm with a 9.5 cm radius fixed angle rotor or at 960 xg at room temperature for 2.5 min. 11. Remove infiltration fluid by opening the cap of the 10 ml syringe. 12. Remove oil (disrupted adipocytes) by turning the syringe upside down and prevent the stromal vascular fraction (SVF) from leaking with the use of a gauge. Live/dead assay of SVF 1. SVF is mixed with pre-heated (37°C) 0.001% CFDA-SE and 0.001% (PI) in serum free DMEM and allow for 30 min. of incubation under normal culture conditions (37°C).