Acrodisc filter

In the laboratory, ATE was produced by adding Ringer lactate (Baxter Healthcare Corporation, Helsinki, Finland) to the adipose tissue sample at an approximate ratio of 1:1 and then processed according to the different study variables. Incubation was performed at 37°C water bath or at room temperature (RT). The incubation times studied were 15, 30, or 45 min. After incubation, the samples were sterile filtered with different 0.2 μm pore size syringe filters (Acrodisc^® filter, polyethersulfone PES membrane [PALL Life Sciences, New York]; Minisart NML filter, cellulose acetate membrane [Sartorius AG, Germany]; Filtropur S Plus filter, cellulose acetate membrane [Sarstedt & Co, Germany]; and Millex GP filter, PES membrane [Merck, Millipore, Germany]). Once filtered, the ATE was stored at −20°C until sample analysis.

Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, San Diego, CA, USA) were prepared for plasmid transfections by seeding 8 × 10⁶ per 100mm dish (one dish for each reprogramming gene). Plat-E cells were maintained in Fibroblast-Platinum (FP) medium (Dulbecco’s modified Eagle medium (DMEM), 10% FBS, and 50 U of penicillin-streptomycin). After 24 h, we introduced each pMXs retroviral plasmid DNA (Oct4, Sox2, Klf4, c-Myc, and Ds-Red), (Addgene plasmids 13366, 13367, 13370, 13375 and 22724, respectively) into Plat-E cells using X-tremeGENE 9 DNA transfection reagent (Roche, High River, AB, Canada), according to the manufacturer’s recommendations. Furthermore, 18 μL of X-tremeGENE 9 transfection reagent was added to 300 μL of OptiMEM to a 1.5-mL tube. A total of 8 μg of each retroviral vector was added into the prepared XtremeGENE9-OptiMEM tube drop-by-drop and incubated for 15 min. Each vector–XtremeGENE 9 complex was added dropwise into the Plat-E cell–containing dishes and incubated overnight at 37 °C, 5% CO₂. The following day, the medium was replaced with 10 mL of fresh FP medium. Forty-eight hours after transduction, we collected virus-containing medium from each transfection by filtering through a 0.45 μm Acrodisc filter (Pall Life Sciences, Mississauga, ON, Canada).

HAEC were seeded on collagen particles purified from porcine skin (Gelfoam® powder, Pfizer, Tadworth, UK) and cultured at 37 °C, 5 % CO₂ for 15 days with media changes every other day, in accordance with published methods [16 (link), 17 (link)]. On day 15 the number of viable cells was determined in duplicated culture to ensure the desirable cell density of >1.8 × 10⁶ cells/tube was reached. For collection of ECPCM, the growing medium was replaced by collection medium and the cells cultured for an additional 24–27 h. ECPCM was collected, passed through a sterile filter with 0.2 μm pore size (Acrodisc® Filter, Pall Corp, Port Washington, NY), aliquoted and stored at −80 °C.