15502-m-pleumeekers

MATERIALS AND METHODS Chemicals were obtained from Sigma-Aldrich, USA unless stated otherwise. Cell sources To obtain primary bovine chondrocytes from ear ( b ECs) and the cartilaginous part of the nasoseptal ( b NCs) origin, macroscopically intact cartilage was harvested from calves ≤ 6 months old, and washed with saline after careful resection of the perichondrium ( n =3 pools of 3 donors). To isolate cells, cartilage pieces were incubated for 1 hour with 2 mg/mL protease, followed by overnight incubation with 1.5 mg/mL collagenase B (Roch Diagnostics, Germany) in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, USA). Non-expanded primary chondrocytes were harvested and directly cultured in 3D-alginate hydrogel. Human bone-marrow-derived mesenchymal stem cells ( h BMSCs) were isolated from bone- marrow heparinized aspirates, after informed consent had been acquired and with approval of the local Medical Ethics Committee (MEC-2004-142 and Albert Schweitzer Hospital 2011/7) ( n =3: M 67Y; F 75Y; M 22Y) and seeded and cultured overnight in medium consisting of Minimum Essential Medium Alpha (MEM-α; Gibco), supplemented with fetal calf serum, L- ascorbic acid 2-phosphate, and 1 ng/mL basic Fibroblast Growth Factor 2 (bFGF2; AbD Serotec, UK). Second-passaged cells were harvested and cultured in 3D-alginate hydrogel. Chondrogenesis For 3D-alginate culture, 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 (Batch MG-004, CellMed, Germany) as single-cell-type populations or as combination of 80% h BMSCs and 20% b ECs or b NCs. (Table 1) Human stem cells Bovine chondrocytes Source Cell density (x10 6 ) Source Cell density (x10 6 ) h BMSC h BMSCs 4 nc/mL x x b EC x x b ECs 4 nc/mL b NC x x b NCs 4 nc/mL h BMSC/ b EC h BMSCs 3.2 nc/mL b ECs 0.8 nc/mL h BMSC/ b NC h BMSCs 3.2 nc/mL b NCs 0.8 nc/mL Control b EC x x b ECs 0.8 nc/mL Control b NC x x b NCs 0.8 nc/mL Table 1. Construct conditions. Cell density is displayed as the number of cells (nc) in 1 milliliter of alginate. h BMSCs = human Bone-marrow- derived Mesenchymal Stem Cells ; b ECs = bovine Ear Chondrocytes ; b NCs = bovine Nasal Chondrocytes. 111 CO-CULTURE: A PROMISING CELL-BASED THERAPY FOR FACIAL CARTILAGES 6