15502-m-pleumeekers

MATERIALS AND METHODS Chemicals were obtained from Sigma-Aldrich, USA unless stated otherwise. Cell sources Ear (EC: n =5, median age 69, range 17-75 years) and nasal cartilage (NC: n =8, median age 24, range 18-46 years) were obtained from patients undergoing reconstructive subtotal septorhinoplasty. For articular cartilage (AC), both healthy ( n =2 , traumatic amputation) and diseased knee cartilage ( n =7, osteoarthritis) were harvested. Since no clear differences in chondrogenic potential were visible between both healthy and diseased AC (data not shown), we combined them for further experiments (total n =9, median age 68, range 43-88 years). To obtain adipose-tissue-derived mesenchymal stem cells (AMSC), subcutaneous abdominal adipose tissue was used from patients undergoing reconstructive breast surgery ( n =7, median age 51, range 34-71 years). All these tissue samples were obtained as waste material after surgery with approval of the local Medical Ethics Committee (MEC-2011-371). Finally, bone- marrow-derived mesenchymal stem cells (BMSC) were harvested from femoral-shaft biopsies during total hip-replacement surgery, after informed consent had been acquired and with approval of the local Medical Ethics Committee (MEC-2004-142) ( n =11, median age 63, range 39-72 years). Cell isolation and culture Expansion To isolate chondrocytes, macroscopically intact cartilage pieces were washed after careful resection of the perichondrium (in the case of nasal and ear cartilage). Cartilage pieces were diced into small fragments and incubated for one hour with protease (2 mg/mL), followed by overnight incubation with collagenase B (Roche Diagnostics, Mannheim, Germany) in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium (HG-DMEM; Gibco, Carlsbad, USA) with 10% fetal calf serum (FCS; Gibco), 50 µg/mL gentamycin (Gibco), and 0.5 µg/mL amphotericin B (Fungizone; Life Technologies, Breda, the Netherlands). To remove small parts of undigested cartilage, the cell suspension was filtered through a nylon 100-µm mesh. Prior to cell seeding, cell viability was tested using the trypan blue exclusion test, and cell number was calculated with a hemocytometer. Chondrocytes were finally seeded at an initial density of 7500 cells/cm 2 in ‘standard chondrocyte expansion medium’ containing HG-DMEM supplemented with 10% FCS, 50 µg/mL gentamycin, and 0.5 µg/mL Fungizone. Bone-marrow-derived heparinized aspirates were seeded at a density of 2-5x10 5 nucleated cells/cm 2 and cultured overnight in ‘standard MSC expansion medium’ containing low glucose (1.5 g/L) Dulbecco's modified Eagle's medium (LG-DMEM), supplemented with 10% FCS; 50 µg/mL gentamycin; 0.5 µg/mL Fungizone; 10 -4 M L-ascorbic acid 2-phosphate; and 1 ng/mL basic Fibroblast Growth Factor 2 (bFGF2; AbD Serotec, Kidlington, UK). After 24 hours, non- adherent cells and cell debris were washed out and adherent BMSC were further expanded using ‘standard MSC expansion medium’. To extract AMSCs, excised human adipose tissue was washed with LG-DMEM, minced, and suspended in 0.1% collagenase type I solution (Invitrogen, Carlsbad, CA, USA) in the 62 CHAPTER 4