BIOPRINTING FOR SKIN - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 282

layer thickness at unvaried laser pulse energies. A specific viscosity for every layer thickness exists, at
which the printed droplet volume reaches its maximum. This specific viscosity increases with hydrogel
layer thickness. Usually, the quantity of cells per droplet is proportional to the droplet volume and the
initial cell density in the hydrogel layer on the donor slide, and is subject to statistical variations. The
alternative of printing single cell droplets requires a low cell density and is time-consuming, since each
cell needs to be targeted separately. The number of printed droplets per second depends on the laser
pulse repetition rate and the velocity of the mechanical setup, and might be in the kilohertz range. For
printing of tissue as well as high-throughput assembly of cell arrays, studies on multiple cell responses
in parallel high printing speeds are needed.
Koch et al. (2010) used a laser-assisted bioprinting technique for the printing of skin. For the print-
ing process, a Nd:YAG-laser (DIVA II; Thales Laser, Orsay, France) with 1064 nm wavelength, about
10 ns pulse duration, and 20 Hz repetition rate was used. The laser pulses were focused with a 60 mm
achromatic lens into an ablation spot size with 45 m m diameter (FWHM). The laser pulse energy was
set to 40 m J, corresponding to laser fluency of averaged 1.26 J/cm 2 in the focal spot. As an absorption
layer, a 60 nm thin gold layer was deposited on the donor glass slide by sputter coating and a 60 m m
thick layer of collagen with embedded skin cells was coated onto the absorption layer by blade coating.
13.3.4.2 The Printing Process Does Not Affect the Cells
Several groups have demonstrated successful cell printing with various cell types. However, for applica-
tions it is essential that the printing process does not affect the cells in their vitality, behavior, genotype,
and phenotype. The impact of the printing process on skin cells and other cell types was therefore exten-
sively studied. For the skin tissue printing described later, NIH3T3 fibroblasts and HaCaT keratinocytes
were used. Investigations into the effects of the printing process on these cell lines are discussed here.
The cell survival rate was examined directly after printing ( Koch et al., 2010 ). A survival rate of
98.4% ± 0.8% for NIH3T3 and 98.6% ± 0.3% for HaCaT was calculated, which is in accordance
with studies on other cell types ( Hopp et al., 2005 ). Furthermore, genotoxicity was investigated via a
single-cell gel electrophoresis (Comet Assay). It was demonstrated that the printing does not induce
DNA strand breaks ( Koch et al., 2010 ). The genotypes of the cells remained unaffected. These findings
are consistent with similar investigations by Ringeisen et al. (2004) with P19 pluripotent embryonal
carcinoma cells. Apoptosis as a parameter of possible cell death caused by LaBP was assessed by
measurement of the activity of caspases 3/7. Up to 48 h after printing, no increase in apoptosis was
detected, neither compared to nonprinted control cells nor compared at different test intervals ( Koch
et al., 2010 ). Also, the influence of the printing process on cell proliferation was studied by cell counting
up to 6 days after printing. In accordance with experiments of other groups on other cell types ( Barron
et al., 2005; Hopp et al., 2005 ), no difference in the proliferation behavior of NIH3T3 fibroblasts and
HaCaT keratinocytes compared to nonprinted control cells could be found ( Koch et al., 2010 ). Since
high temperature is induced in the absorption layer by the laser pulse energy, potential cell damage by
heat was investigated via immunocytochemical studies (not shown). No increased expression of heat
shock proteins by printed cells was demonstrated ( Gruene et al. 2011b ). This is in line with experiments
of other groups ( Barron et al., 2004, 2005 ). Furthermore, in studies with stem cells, it was shown that
the printing process does not affect the immunophenotype ( Koch et al., 2010 ) or the differentiation
potential ( Gruene, et al. 2011b ; Gruene et al., 2011c ) of the printed cells. In summary, all studies so far
consistently show that the laser printing procedure has no significant effect on the cell; printed cells are
viable and fully functional.