Joris van Dongen

312 Chapter 13 were washed with PBS and again lyophilized. Finally, samples were ground to a fine powderwith an UltraTurrax device (PMTamson Instruments) and stored at -80 º C until further use (Fig. 1). Figure 1. Overview of preparation method of an ECM hydrogel from human adipose tissue. SVF = stromal vascular fraction; ECM = extracellular matrix. Gelation of decellularized adipose derived extracellular matrix ECM (20 mg) was mixed with porcine pepsin powder (2 mg, 3,200 I.U. Sigma-Aldrich) in 1 ml 0.01 M hydrochloric acid (HCl). ECM was digested under constant stirring on a magnetic stirring device at 500 rpm at RT for 6h. Afterwards, pH was raised to 7.4 to neutralize the pepsin with 100 μl of 0.1 M sodium hydroxide (NaOH) to reach a final concentration of 0.01 M NaOH. The pre-gel solution was buffered with 110 μl of 10x PBS to reach a final concentration of 1x PBS. Then, the pre-gel solution was mixed well and incubated for one hour at 37˚C to allow for gelation (Fig.1). Immortalization and lentiviral tagging of ASCs Cultured human ASCs (pool of five donors, 1 million at passage 4) were transfected with 1µg pMC1neo-polyA (Stratagene) which is a plasmid encoding the large T antigen of SV40 with neomycin as selectable marker. 27,28 At 48h post-transfection, transfected cells were selected by adding 250 µg/ml geneticin (G418) to the medium. After approximately three weeks, colonies that remained were picked, subcultured and