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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 37C, 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.