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Christopher Chi Wai Tse, Shea Shin Ng, Jonathan Stringer, et al.

D). Figure 5 show cells aligning to the same orienta-
tion as the channel, and through the use of ImageJ
software, the orientation of cells could be clearly ob-
served following the direction of the channel. Cell
alignment could be investigated with this methodolo-
[8]
gy as in the research reported by Duclos et al. , who
described how NIH-3T3 mouse embryo fibroblasts
aligned on confined strips from 30 µm to 1.5 mm.
After fibroblasts or Schwann cells were seeded,
they adhered, had spread and thereafter proliferated
into the desired positions and orientation. The wax
structures were easily removed from the samples with
a sharp scalpel, which was used to physically peel the
wax off, to leave the cells to grow without any space
limitations. Figure 6 shows images of the fabricated
wax structures, patterned cells before and after wax
removal. Cells maintained their position and orienta-
tion for at least 7 days after seeding as shown.

3.3 Cell Proliferation After Removal of Wax
A wax scaffold with channel widths of 40 µm and 30
µm connected together at one end were created and
fibroblasts were seeded into the structure and cultured.
After 2 days of culture and prior to wax removal, fi-
broblasts had aligned within the shape of the open
channels (Figure 7A). Upon wax removal, cells were
not limited to the channel space (Figure 7B) and had
spread to cover the substrate, which was observed 24
to 48 hours after wax removal (Figure 7C and D). The
wax printing technique could therefore be used in-
itially to deposit cells in the desired areas spatially.
The ability of the wax to be removed from the sub-
strate, without the addition of new substances into the
environment, allowed for a new method to analyse
cell migration and proliferation on open substrates
to be studied over time. We observed a high level of
alignment to the direction of channels with diameters
of less than 160 µm, and consequently the authors of
this paper suggest that this work would have imme-
diate applications in cell migration and “scratch as-
say” type studies, commonly used in wound healing

Figure 3. Micrographs of printed wax on glass. (A–C) show and cancer cell migration research.
different patterns that were produced. Magnified light micro- Cells were able to remain attached onto the sub-
scopy images are shown in the small frame. Bar = 200 µm. strate even after wax removal. An exception to this
occurred when cell concentration exceeded confluence.
cells to grow. From this study, when cell density began to reach con-
2
With isolated arrays, cells proliferated in random fluency (typically more than 300 cells/mm ) an in-
shapes with no obvious direction (Figure 4A and B), but creased proportion of cells detached. All cells within
when a linear patterned structure was created, cells the scaffold were removed when cell density reached
2
aligned in the direction of the structure (Figure 4C and above 700 cells/mm . It was postulated that this was
International Journal of Bioprinting (2016)–Volume 2, Issue 1 39