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 78

laser deposition, can be further classified as direct-contact (DC) or direct-write (DW) mechanisms. DC
methods, such as microcontact patterning, load cells suspended in media onto a “stamp” surface and
then bring that “inked” surface in contact with the desired substrate surface. This prints an entire pattern
at once, as opposed to being built over time. Stamps are a priori patterns that require new molds for
new patterns. After the solution is applied to the stamp, there is no way to select certain cells or groups
of cells for printing. Thus, the nature of cell distribution in suspension determines the probabilistic cell
number per print area. DW methods, such as ink-jet printing and laser-assisted transfer, use “bottom-up
techniques” that generate whole constructs on a subunit-by-subunit basis. DW methods can be com-
bined with CAD/CAM techniques to facilitate the controlled transfer of biological “voxels” (volume
pixels) containing desired cells. By designing blueprints and depositing biological voxels accordingly,
researchers can generate 2D arrays or layered 2D patterns to create 3D constructs. However, laser-
assisted cell transfer is distinct from other DW methods for single-cell printing applications.
Unambiguous scientific conclusions rely on reproducibility of experiments to test theories and prove
statistical significance. Single-cell printing reduces sample-to-sample variability and permits clear
quantification in biological studies by repeating addressable units (voxels) to create cell or tissue con-
structs. Inkjet methods that rely on nozzle-ejected droplets and blind laser-assisted methods that depend
on laser-material interactions for droplet formation are capable of printing one cell at a time on average.
This average and reproducibility depend on the probabilistic localization of only a single cell within
the ejection volume ( Liberski et al ., 2011 ; Barron et al ., 2005 ). Caution should be taken, however, as
voxel-to-voxel reproducibility necessarily varies due to inherent cell-to-cell differences. In addition, the
ability to select cells during DW procedures produces less variance compared to blind DW methods
simply by enabling the end-user to visibly target cells. Moreover, the impact of scientific conclusions
increases inversely with the number of cells written with single-cell deposition being paramount.
Rather than rely on distribution statistics, MAPLE-DW and other select DW techniques provide in
situ, real-time optical monitoring and recording to enable users to select cells for printing and confirm
deposition onto the substrate. This distinguishing feature provides feedback to determine precision in
cell placement, relative to target location. MAPLE-DW spatial resolution is ±5 m m ( Schiele, 2010 ).
By using a camera-equipped, laser-assisted DW system, one is able to reduce voxel-to-voxel varia-
tion, monitor deposition in real time, and achieve tight control in spatial printing. This platform for
cell printing enables researchers to explore numerous biological systems, including stem niches,
cancer invasion, and neuron manipulation/functionalization using functional testing platforms, such
as isolated-node single-cell arrays, network-level single- cell arrays, integrated single cells, and 3D
cellular invasion models.
4.2 BASICS OF LASER-ASSISTED PRINTING: OVERVIEW OF SYSTEMS AND
CRITICAL ANCILLARY MATERIALS
4.2.1 LASER-ASSISTED CELL TRANSFER SYSTEM COMPONENTS
Variations and generational iterations of laser-based cell transfer bioprinting systems are based
on similar principles to those of laser-induced forward transfer (LIFT) for inorganic electronic
materials ( Piqué et al., 1999 ). The four most common laser bioprinting systems are LIFT, absorbing
film-assisted LIFT (AFA-LIFT), biological laser processing (BioLP), and MAPLE-DW. They all utilize