15502-m-pleumeekers

Cellular interaction To further understand the complex cellular communication pathways between MSCs and ACs, cell types ( h AMSCs (F 53Y) ; h BMSCs (M 22Y) ; b ACs pool of 3 donors) were co-cultured as follows: (1) h MSCs and b ACs were combined and cultured in alginate as previously described; (2) h MSCs and b ACs were cultured in pellets, allowing direct cell-cell contact. Furthermore, h MSCs and b ACs were encapsulated in alginate separately and co-cultured in (3) a Transwell® system ; as well as (4) in medium conditioned by the other cell type. (Figure 1) The ratio of h MSCs to b ACs in each culture system was kept 80:20 for all conditions. All constructs were cultured under standardized nutritional conditions. Medium was changed 3 times a week. After 3 weeks, alginate beads and pellets were processed for biochemical or gene-expression analysis. (1) Co-culture h MSCs and b ACs were suspended at a density of 4x10 6 cells/mL in clinical grade alginate solution as a mixed-cell-type population at a 80:20 ratio as described above. (Figure 1A) (2) Pellet culture To study the effects of direct cell-cell contact in co-cultures, h MSCs and b ACs were cultured in pellets. Therefore, a mixture of 80% h MSCs and 20% b ACs was suspended in basic medium and a total number of 2,5x10 5 cells in 0.5 mL were transferred into polypropylene tubes and pellet were formed by centrifuging at 200 G for 8 minutes. To induce proper pellet formation, addition of Transforming Growth Factor β1 (TGFβ1 ; R&D Systems, USA) for 24 hours was required. This exposure was not sufficient to induce chondrogenesis in h MSCs (data not shown). After 24 hours, pellet were exposed to the ‘basis medium’ without addition of any growth factors. (Figure 1A) (3) Transwell® system h MSCs and b ACs were suspended at a density of 4x10 6 cells/mL in clinical grade alginate solution as single-cell-type populations and transferred into a 10-mL sterile syringe. Thereafter, the cell-suspension was slowly passed through a 23-gauge needle to produce drops, which fell into a 102 mM CaCl 2 creating alginate beads. Following instantaneous gelation, beads were allowed to further gelate for a period of 10 minutes in the CaCl 2 -solution. After being washed once with 0.9% NaCl and HG-DMEM, the beads were transferred to a Transwell® system (Corning Life Science, USA). The Transwell® inserts separated h MSCs and b ACs by a porous membrane of 8 µm, allowing paracrine signaling between h MSCs and b ACs. (Figure 1B) (4) Conditioned medium Alginate beads containing h MSCs or b ACs were produced as described above and cultured in medium conditioned by the other cell types. To obtain b ACs, h AMSCs and h BMSCs conditioned media, alginate beads were cultured in ‘basic medium’ for 3 days. After 3 days of culture, conditioned media were collected, enriched with 1:1 ‘basic medium’ and immediately added 90 CHAPTER 5