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Supplementary information 1 Optimisation of the Puramatrix concentration for the immobilization of DEP aggregated cells Introduction Immobilization of the cells in the aggregates after the formation of the aggregates by DEP forms an essential step of any attempt to form stem cell microniches; without it, cells would soon disperse. Previous experiments involving the construction of stem cell microniches using DEP had primarily used fibrin gels for cell immobilisation38,43.56,. Fibrin, however is a natural product, and the presence of growth factors or cytokines could not be excluded. For the formation of aggregates of ESCs BD PuramatrixTM (3 DM Inc, Cambridge, USA) was chosen as the immobilization agent. Puramatrix is a fully synthetic peptide composed of 16 amino acids residues and contains no detectable growth factors or cytokines, allowing one to investigate reproducibly the effects of added growth factors or cytokines. It is supplied as a low ion solution; after addition of medium it forms a nano-fibrous scaffold resembling collagen. Materials and Methods Initial optimization of the immobilization procedures was done with SAOS-2 cells. SAOS-2, a sarcoma-osteogenic cell line of adherent human osteoblast-like cells, was grown in T25 flasks in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 10 units ml-1 penicillin and streptomycin solution (SigmaAldrich). The flasks were incubated for 4 days until near 90% confluence was obtained (3-4 days) and medium was exchanged every 2 days. Cells were inoculated at a density of 2500 cells / cm2. To prepare the cells for DEP, after harvesting with trypsin the cells were washed in fresh medium and then washed twice with 300 mM D-sorbitol in order to lower the conductivity to allow patterning with positive DEP. To establish the most suitable concentration of Puramatrix, aggregates were of SAOS-2 osteoblast-like cells were made by collecting the cells using DEP (1 MHz, 10 Vpk-pk) between the castellations of interdigitated oppositely castellated electrode with a characteristic size of 75 or 100 µm. The electric field was maintained for 10-15 minutes after the aggregation process had finished, to force the cells to adhere to each other. Following this, the sorbitol solution in the chamber was replaced with a Puramatrix solution. The Puramatrix solution (made by mixing the Puramatrix precursor solution supplied by the distributor with 300mM sorbitol solution) was introduced into the chamber by carefully adding the Puramatrix solution at one end of the chamber using a pipette, and removing sorbitol solution from the other end. Once the whole chamber had been filled with Puramatrix solution the electric field was switched off and RPMI growth medium was introduced at the edge of the chamber. The ions in the growth medium then initiated gel formation. Gel formation took 5 to 15 minutes from the introduction of growth medium, depending on the Puramatrix concentration. The chamber was then placed in an incubator at 37C, 5% CO2, and images were taken of the aggregates over a period of 24-72 hours. . Results Figures S1.1-S1.4 show the effects different Puramatrix concentrations have on the distribution of the osteoblast-like cells after 24 hour incubation in RPMI growth medium. At 50% and 30% concentration the Puramatrix precursor solution was very viscous. This caused some cells to become dislodged from the aggregates when the Puramatrix solution was introduced. As shown in Figure S1.1 and S1.2, after 24 hours of incubation the cells remained rounded. SAOS-2 osteoblasts normally grow adherently; rounding is usually an indication that the cells are not happy with the growing conditions. There is little evidence of cell movement or of cell adherence to the glass or ITO surface. FIG. S1.1 SAOS-2 cells immobilized with 50% Puramatrix. Bright field images of aggregates of SAOS-2 cells formed with DEP between interdigitated oppositely castellated ITO microelectrodes on glass. The microelectrodes had a characteristic size of 100 μm. The electric field between the microelectrodes was induced by applying a 10 V pk-pk, 1 MHz signal to the electrodes. (a) Aggregates of osteoblast-like cells at 0 hours. (b) Aggregates after 24 hours. FIG. S1.2 SAOS-2 cells immobilized with 30% Puramatrix. Bright field images of aggregates of SAOS-2 cells formed with DEP between interdigitated oppositely castellated ITO microelectrodes on glass. The microelectrodes had a characteristic size of 100 μm. The electric field between the microelectrodes was induced by applying a 10 Vpk-pk, 1 MHz signal to the electrodes. (a) Aggregate at 0 hours. (b) Aggregate after 24 hours. As shown in Figures S1.3 and S1.4, at Puramatrix concentrations of 25 and 20%, after 24 hours the cells close to the microelectrode surface had become adhered to the electrode surface and started to spread over the surface. There was no difference between the ITO and glass. Cells further away from the microelectrode surface could sometimes be seen to adhere to each other forming dense aggregates; this was particularly prominent at the lower gel concentration (20%) and larger electrode size (100 μm). The gel formed at the lower Puramatrix concentration of 20% was mechanically weak and difficult to handle without disturbing the integrity of the gel or disturbing the aggregates. FIG. S1.3 SAOS-2 cells immobilized with 25% Puramatrix. Bright field images of aggregates of SAOS-2 cells formed with DEP between interdigitated oppositely castellated ITO microelectrodes on glass. The microelectrodes had a characteristic size of 75 μm. The electric field between the microelectrodes was induced by applying a 10 V pk-pk, 1 MHz signal to the electrodes. (a) Aggregates at 0 hours. (b) Aggregates after 24 hours. FIG. S1.4 Bright field images of SAOS-2 cells immobilized with 20% Puramatrix. Bright field images of aggregates of SAOS-2 cells formed with DEP between interdigitated oppositely castellated ITO microelectrodes on glass. The microelectrodes had a characteristic size of 100 μm. The electric field between the microelectrodes was induced by applying a 10 Vpk-pk, 1 MHz signal to the electrodes. (a) Aggregates at 0 hours. (b) Aggregates after 24 hours. The results indicate that the most suitable concentration of Puramatrix for the immobilization of osteoblast-like cells was 25%. At higher concentrations there were problems with cells becoming dislodged from the aggregates during the assembly process. Also the osteoblast-like cells remained rounded, indicating the cells were not satisfied with the growing conditions. Concentrations below 25% did not have sufficient mechanical strength. Therefore all further experiments were carried out at a concentration of 25%. Figure S1.5 shows the longer term behavior of SAOS-2 osteoblast-like cells in aggregates in 25% Puramatrix. At 24 hours the cell can be seen to spread from the aggregates, but the initial location of the DEP aggregated cells can still be observed. This becomes increasingly difficult at later times. FIG. S1.5 Bright field images of aggregates of SAOS-2 cells formed with DEP between interdigitated oppositely castellated ITO microelectrodes on glass. The microelectrodes had a characteristic size of 75 μm. The electric field between the microelectrodes was induced by applying a 10 Vpk-pk, 1 MHz signal to the electrodes. The cells were immobilized with 25% Puramatrix. (a) Aggregates at 0 hours. (b) 24 hours. (c) 48 hours. (d) 72 hours. Conclusion Puramatrix is suitable as a gel matrix for aggregates of cells made with DEP if diluted to 25% with an isoosmotic sorbitol buffer, and subsequently gel formation is induced by introduction of growth medium.