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Utilising inkjet printed paraffin wax for cell patterning applications

voltage, rise and echo time, which alters the size and A confocal scanning microscope (Carl Zeiss LSM-
velocity of the wax droplet to create optimum condi- 510-META, Germany) with magnification ×10 and ×40
tions to print on varying surfaces and resolutions. When long-range water-dipping lenses were used. FITC cha-
required during the study, the wax was removed by nnel (λ ex = 485 nm; λ em = 520 nm). DAPI (λ ex 400 nm;
physical lifting with a scalpel. λ em = 460 nm). Image acquisition and analysis were
carried out with Carl Zeiss Laser Scanning Systems
2.5 Cell Seeding LSM 510 software.
After the wax template had been printed onto the sub- 3. Results and Discussion
strate (tissue culture plastic or glass substrates), the
sample was placed in a petri dish and cells were 3.1 Inkjet Printing of Wax Guides
4
seeded at 2 × 10 cells per sample in 1 mL, and left in
the incubator for 60 minutes. After this time, the sample A variety of designs were created that were suitable
was supplemented with 10 mL cell medium to cover for isolating and connecting islands of cells on sub-
the entire substrate, and left to proliferate for up to 7 strates (Figure 3A–C). Under high magnification, as
days and wax removal when necessary, during which exemplified in Figure 3A single wax rows of droplets
images were captured to record cell growth along the appeared to have a slightly uneven topography due to
substrate with and without the patterned wax. scalloping behaviour. The final shape and surface tex-
ture of the resultant printed structure was dependent
2.6 Analysis of Samples on conditions that include the wettability of the sub-

Images were obtained using an inverted Olympus strate and its temperature, print head temperature, gap
CK40 phase contrast microscope. Images were cap- distance from the print head to the substrate and drop-
[34–36]
tured of samples prior to cell seeding, after cell seed- let material . The scalloping behaviour was due to
ing and after wax removal through physical lift off the droplets being cooled quicker than optimal during
with a sharp scalpel. jetted flight, after landing on the substrate, merging
with the previous deposited droplet and partially re-
2.7 Image Processing tained their individual rounded contact lines [37] . Print-
ing wax allowed the creation of a range of different
All image processing was performed with ImageJ (U.S.
National Institutes of Health). The orientation field complexities and channel widths that allowed the cre-
was obtained using the ImageJ plugin, OrientationJ. ation of thick impermeable blocks, to channels as
The colour survey was set with the following settings small as 30 µm. The smallest dimensions that can be
— Hue: Orientation, Saturation: Coherency, Bright- created with the wax struts is a single line of inkjet
ness: Original-Image. With this, it was possible to bet- printed paraffin wax. Using a 50 µm diameter print-
ter visualise the orientation of cells along the patterned head nozzle, wax lines with a minimum width of 50 µm
substrate, with and without the wax template over time. could be created to act as a barrier between each
compartment.
2.8 Confocal Fluorescence Microscopy
3.2 Cell Seeding
For confocal fluorescence imaging, RN22 Schwann
cells and dermal fibroblasts were seeded on the scaf- Human dermal fibroblasts and RN22 rat Schwann
4
folds at 2 × 10 cells per sample, stained with phalloi- cells were seeded and imaged to show cell compart-
din-fluorescein isothiocyanate (FITC) for F-actin fila- mentalisation and connection within the wax struc-
ments and 4’,6-diamidino-2-phenylindole dihydroch- tures. Figure 4A and B show images taken after 24
loride (DAPI) for nuclear staining. hours of cell culture with fibroblasts and Schwann
Samples were fixed with 3.7% formaldehyde in cells on a glass substrate, respectively. Figure 4C and
PBS for 30 minutes at room temperature and permea- D show cells that have proliferated after 5 days, where
bilised with 0.1% (v/v) Triton X-100 in PBS for 30 the cells were able to grow in a wax-containing envi-
minutes. Phalloidin:FITC was added at 1:1000 in PBS ronment on tissue culture plastic. No cells were ob-
in combination with DAPI at 1:1000 (300 nM) for 30 served growing across and over the wax structures,
minutes, washed and stored in PBS at 4°C until imag- showing how this technique was effective at impeding
ing. Cells were washed with PBS (×3) for 5 mi- the cell interactions between individual compartments
nutes between each step. and creating separate environments for collections of

38 International Journal of Bioprinting (2016)–Volume 2, Issue 1