3D PRINTING AND NANOMANUFACTURING - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 48

was no significant difference in results. Rosenbalm et al. concluded that LGDW is a safe method for
cell patterning.
The LIFT technique was applied to print human skin cells, NIH3T3 fibroblasts, and HaCaT kera-
tinocytes, and human mesenchymal stem cells (hMSC) using Nd:YAG laser ( Koch et al. , 2009 ). The
donor slide was sputter-coated with a 55-60 nm thick gold and was covered with about 50 m m thick
cell-containing layer. Laser having 1064 nm wavelength and 3-6 J/cm 2 fluence transferred a cell-
suspended mixture of alginate and blood plasma on the Matrigel ® -coated collector slide. Biological
material droplets were printed in droplet diameter of 80-140 m m with a speed of 1200 droplets per
minute. The cell viabilities were 98% ± 1% for skin cells and 90% ± 10% for human stem cells after
laser printing. Apoptosis and genotoxicity were tested to evaluate the damage to cells and DNA, re-
spectively, and the results proved that LIFT has no significant influence on cells and DNA. The result
of fluorescence activated cell sorting (FACS) analysis described that the phenotype of hMSC was not
affected by LIFT.
Porcine mesenchymal stem cells (MSC) were printed on the hydrogel-covered collector slide using
LIFT ( Gruene et al. , 2010 ). Gold was sputter-coated on the donor slide in 55-60 m m thickness, and
then the cell suspension was covered in ∼ 65 m m thickness on gold layer. Parameters of Nd:YAG laser
were fixed with 1046 wavelength and 2.15 J/cm 2 laser fluence. There was no significant difference in
viability between laser-printed cells and control (cells without laser exposure). No damage to DNA was
observed. ALP activity was measured to evaluate and compare the osteogenic differentiation between
laser-printed cells and control. Immunofluorescence staining and Alcian blue staining were conducted
to detect the presence of type II collagen and aggrecan, and to quantify sulfated glycosaminoglycan
(sGAG). The results supported that MSC differentiated to bone. Koch et al. also three-dimensionally
patterned NIH3T3 fibroblasts and HaCaT keratinocytes ( Koch et al. , 2012 ). Twenty layers of each cell
line were stacked to mimic 3D skin structure.
MAPLEDW was utilized on biomaterials containing pluripotent embryonal carcinoma cells (P19)
onto Matrigel ® -coated quartz substrate ( Ringeisen et al. , 2004 ). Matrigel ® was spin-coated on quartz
10-30 m m thickness, and the substrate had a Matrigel ® layer on its cell receiving face. An ArF excimer
laser was set with 193 nm wavelength and 400 mJ/cm 2 laser fluence. Cell viability was over 95% for
24 h post-transfer. The comet assay was employed to evaluate DNA damage; the results showed no
noticeable damage. Cell differentiation was induced via adding retinoic acid or dimethyl sulfoxide
(DMSO; 1%). The immunofluorescence staining test proved that P19 cells were differentiated into
neuronal and muscle cells.
B35 neuronal cells were transferred onto a Matrigel ® -coated quartz substrate using an ArF laser
(193 nm wavelength) ( Patz et al. , 2006 ). The ribbon consisted of transparent quartz and cell-seeded
Matrigel ® matrix. The matrix was printed onto the substrate via a laser beam having a laser fluence
range of 0.02 to 0.08 J/cm 2 . Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TU-
NEL) immunostaining was used to detect cell apoptosis. The  -tubulin immunofluorescence staining
was employed to examine axon morphology. TUNEL staining image showed that 3% of B35 neuronal
cells were in apoptosis after 96 h. Analysis of axonal projection showed no significant impairment after
the MAPLEDW process. The penetration of cells within the Matrigel ® substrate was observed using a
confocal microscope and the maximum depth was 75 m m.
Human osteosarcomas (MG-63) were codeposited with bioceramic, hydroxyapatite, and zir-
conia, using MAPLEDW ( Doraiswamy et al. , 2007 ). Hydroxyapatite or zirconia powder solvated
in glycerol:water matrices were spin-coated onto a quartz ribbon. ECM was covered onto the