15502-m-pleumeekers

MATERIALS AND METHODS Chemicals were obtained from Sigma-Aldrich, USA unless stated otherwise. Cell sources All human samples were obtained after approval by the Erasmus MC Medical Ethical Committee. Human mesenchymal stem cells ( h MSCs) were isolated from either adipose tissue ( h AMSCs) or bone-marrow aspirates ( h BMSCs). h AMSCs were obtained from subcutaneous abdominal adipose tissue as waste material without the need for informed consent (protocol # MEC-2011-371) ( n=3 independent donors : F 52Y ; F 51Y ; F 53Y). h BMSCs were isolated from bone-marrow heparinized aspirates, after written informed consent had been acquired (protocol # MEC-2004-142 and Albert Schweitzer Hospital 2011/7) ( n=3 independent donors : M 67Y ; F 75Y ; M 22Y). Both h AMSCs and h BMSCs were seeded and cultured overnight in medium consisting of Minimum Essential Medium Alpha (MEM-α ; Gibco, USA), supplemented with 10% fetal calf serum (FCS ; Lonza, the Netherlands), 10 -4 M L-ascorbic acid 2-phosphate, and 1 ng/mL basic Fibroblast Growth Factor 2 (bFGF2 ; AbD Serotec, UK). [248-250] Articular chondrocytes (ACs) were selected, to study the trophic effect of h AMSCs or h BMSCs on chondrocytes. To obtain primary bovine articular chondrocytes ( b ACs), macroscopically intact cartilage was harvested from the metatarsophalangeal joints of calves ≤ 6 months old (T. Boer & Zn., Nieuwerkerk aan den IJssel, the Netherlands), and washed with saline ( n=4 pools of 3 donors each). To isolate cells, cartilage pieces were incubated for 1 hour with 2 mg/mL protease (type XIV derived from Streptomyces griseus), followed by overnight incubation with 1.5 mg/mL collagenase B (Roch Diagnostics, Germany) in High Glucose - Dulbecco's Modified Eagle's Medium (HG-DMEM ; Gibco) with 10% FCS, 50 µg/mL gentamycin (Gibco), and 0.5 µg/mL amphotericin B (Fungizone ; Life Technologies, Breda, the Netherlands). To extract small parts of undigested cartilage, the cell suspension was filtered through a nylon 100-µm mesh. Prior to cell culture, cell viability was tested using the trypan blue exclusion test, and cell number was calculated with a hemocytometer. Chondrogenesis For in-vitro and in-vivo studies, all cells were encapsulated in alginate (Batch MG-004, CellMed, Germany), a hydrogel known of its high biocompatibility [251] and chondrogenic capacity [89]. Moreover, alginate hydrogels enable homogeneous cell distribution and allow paracrine factors to access all cells equally [89], making them suitable scaffolds for following research purposes. Second-passaged h MSCs and non-expanded primary b ACs were harvested and cultured in a 3D-alginate hydrogel. Cells were suspended at a density of 4x10 6 cells/mL in clinical grade 1.1% low viscosity alginate solution dissolved in 0.9% NaCl as single-cell-type populations or as a combination of 80% h MSCs (either h AMSCs or h BMSCs) and 20% b ACs. (Table 1) A 4:1 ratio was selected based on our previous experience [252] and that of others [74, 253]. 87 AMSCs OR BMSCs FULFILL A TROPHIC ROLE IN CO-CULTURE 5