THE POWER OF CAD/CAM LASER BIOPRINTING AT THE SINGLE-CELL LEVEL: EVOLUTION OF PRINTING - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 89

AFA-LIFT printing. Cells were printed onto gelatin using an LLG two-pass amplification KrF excimer
laser. Each target was irradiated with a single 30 ns pulse from the laser. Cell viability was inspected
using trypan blue dye, before and after printing, showed that 98-99% of each cell type was alive before
printing and 80-85% of each type of the transferred cells remained alive after printing. In addition, the
cells retained the ability to proliferate and differentiate 2 weeks after initial printing.
Greater survival rates have been demonstrated with MAPLE-DW. Ringeisen et al. (2004) dem-
onstrated 95% cell survival rate when printing with pluripotent embryonic carcinoma cells. This
MAPLE-DW array used an ArF excimer laser from Lambda Physik, model LPX 305, wavelength of
193 nm, and 30 ns full width at half maximum. The target spot on the print ribbon was 100 × 125 m m 2
and incident laser fluence was in a controlled range from 100 to 500 mJ/cm 2 . P19 (pluripotent embryonal
carcinoma) cells were cultured and differentiated to neural and muscle cell phenotypes. Cells were
prepared on the gelatin-coated ribbon. Printing was done in a humidity-controlled environment, as
evaporation of droplet volume reduces cell survivability.
Cell viability was tested 6 h following transfer using live/dead visibility kit. Cells printed onto a
40 m m layer of hydrogel resulted in greater than 95% post-transfer viability. Furthermore, the P19 cells
retained their ability to differentiate in induction media.
This study also investigated DNA damage due to ultraviolet light exposure. Comet assays were per-
formed to detect single- and double-strand breaks. MAPLE-DW was used to perform noncontact cell
transfer from the quartz ribbon directly into a -MEM. After comparison with control groups, there was no
statistically significant damage to the cell DNA as a result of the ultraviolet laser interaction with the cell.
This mitigation is due to the energy absorption by the sacrificial biopolymer on the quartz print ribbon.
With laser-assisted cell transfer methods, researchers benefit from soft cell deposition into
programmable pattern positions to study cell behavior.
4.5 CASE STUDIES AND APPLICATIONS ILLUSTRATING THE IMPORTANCE
OF SINGLE-CELL DEPOSITION
High-throughput, parallel analysis of single cell behavior within the context of biological screening
tools or structured cell-cell interaction interfaces necessitates the ability to isolate and position individ-
ual cells into engineered arrays. The rationale for intentional cell isolation is to identify significant sin-
gularities that may be lost when averaging response across an entire population of heterogeneous cells
( Birtwistle et al., 2012 ). Resolving single-cell characteristics allows researchers to identify cells with
the greatest differentiation potential—for example, pluripotent stem cells or cancer stem cells ( Shack-
leton et al., 2006 ). Similarly, as tissues are composed of various cell types, the dynamics of cells from
an explant or whole organism can drown out the finer nuances of the cell-cell interaction of interest.
Reintegrating individual cells into a spatially controlled, construct-containing discrete voxel of single
cells simplifies the cellular cross-talk. In this section, we explore case studies of single-cell patterns,
organized by general application.
4.5.1 ISOLATED-NODE, SINGLE-CELL ARRAYS
The simplest single-cell arrays are ones wherein each cell is a standalone node for independent analy-
sis. Neighboring cells, isolated by physical or spatial means, cause negligible effects on an individual
cell's response to presented external cues. These types of arrays are particularly useful for studying