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

axes (x,y,z) and was therefore not limited to 2D design atmosphere with 5% CO 2 at 37°C. Prior to seeding,
structures. 3D and topologically irregular surfaces cells were grown to near confluence, and detached
could be printed. At any point during the experiment, with 0.05% trypsin/EDTA (GIBCO, Invitrogen, Karl-
the wax could be removed to allow cells to freely mi- sruhe, Germany). A Neubauer chamber was used to
grate on the substrate, permitting the further study of count the cells. Passages 20 to 22 were used for RN22
cell behaviour (Figure 1C). Schwann cells and dermal fibroblasts respectively.
2. Materials and Methods 2.3 Paraffin Wax

2.1 Inkjet Printing System Paraffin wax was placed inside the cartridge reservoir
and the printing system was heated up to 75°C for
A single nozzle piezoelectric inkjet device (MicroFab, printing, while the printing platform was heated to
Texas, USA) was used to print paraffin wax. Specifi- 30°C to improve the topography of the printed struc-
cally, a Jetlab4 xl-A table top printing platform with tures. Wax viscosity was measured with a rheometer
position accuracy and repeatability of 25 µm and 5 µm (AR 2000, TA Instruments) at different temperatures
respectively, with a 50 µm orifice diameter (PH-04a (Figure 2). When the temperature reached above 60°C,
Polymer Jet™) high-temperature, drop-on-demand the viscosity was less than 10 mPa⋅s (10 centipoise).
printhead, was used. This was a drop-on-demand prin- Specification guidelines from Microfab stated that
thead that was connected together to the cartridge re- inks should have a viscosity below 20 mPa⋅s (20 cen-
servoir through an integrated filter. It allows print- tipoise) for successful droplet formation during inkjet
on-the-fly and point-to-point printing, through vector printing. At the temperature of printing (75°C) the
and raster printing modes. viscosity was 6.02 mPa⋅s (6 centipoise), which was
Such a system had a 30 mL stainless steel reservoir within optimal printing limits.
and the system could heat up to 240°C. A CT-PT4
four-channel pressure controller was used, made by 2.4 Wax Patterning
Microfab to maintain a slight negative pressure within Using a combination of Microsoft Paint and MS
the system to control the creation of the correct nozzle Windows-based computer aided design software en-
meniscus level for optimal jetting. The print head was vironment (Jetlab4, Microfab), varying shapes and
made of a glass capillary tapered to the stated orifice designs of wax structures were created with varying
size and encased in a metal body surrounded by a channel widths and complexity onto tissue culture
piezoelectric actuator. JetDrive III software was used plastic and glass substrates. Inkjet printing parameters
to drive the electronics to control the generation of a were optimised to create a single wax droplet per ejec-
waveform to provide complex drive waveforms to tion, with droplet spacing of 40 µm between each
tailor the jetting parameters of the print heads. Prior to droplet to create an impermeable scaffold block of
jetting, all the tubings, reservoirs and the print head wax. The volume of wax that was ejected from the
were flushed with 1% Micro-90 cleaning solution and piezoelectric print head could be manipulated through
distilled de-ionised water. the fine tuning of its printing parameters, such as the
2.2 RN22 Schwann Cells and Dermal Fibroblasts

Rat RN22 Schwann cells were purchased from the
European Collection of Cell Cultures (ECACC) (Pub-
lic Health England, Porton Down, Salisbury, UK).
Human dermal fibroblasts were obtained from abdo-
minoplasty or breast reduction operations according to
local ethically approved guidelines (under an HTA
Research Tissue Bank license number 12179). All cell
types were cultured independently and grown in Dul-
becco’s Modified Eagle Medium (DMEM) containing

10% (v/v) fetal calf serum (FCS), 1% (v/v) glutamine, Figure 2. Viscosity values of paraffin wax over a range of
1% (v/v) penicillin/streptomycin, and 0.5% amphote- temperatures, which was Newtonian when molten. Printing
ricin B (under serum-free conditions) in a humidified parameters below 20 mPa⋅s are optimal for printing.

International Journal of Bioprinting (2016)–Volume 2, Issue 1 37