ENGINEERING 2D AND 3D CELLULAR MICROENVIRONMENTS USING LASER DIRECT WRITE - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 105

beyond the adhesion islands. Moreover, controlled coculture is difficult, because different cell types,
if seeded simultaneously, will all adhere to the protein islands. Coculture patterning (reviewed in ( Kaji
et al., 2011 )) requires either sequential seeding on micropatterns, masking the substrate, switching regions
of the substrate to be favorable to cell binding ( Yamato et al., 2001; Yousaf et al., 2001 ), or combining
multiple substrates ( Hui and Bhatia S, 2007 ).
On the other hand, ink-jet printing and LDW do not require patterning of adhesive proteins to control
cellular placement. Therefore, cells can be directly deposited to a homogeneous substrate, allowing evo-
lution of a printed structure from a prescribed initial condition. Furthermore, both ink-jet printing and
LDW have also been extended to print in 3D, allowing 3D spatial control over the microenvironment.
In ink-jet printing, the biologic payload (e.g. cells, proteins, or other biomolecules) is printed through
the controlled deposition of solution containing the desired payload, much like the multiple colors of
ink in color printing. In this way, the amount of payload-containing solution, and its placement, can be
precisely controlled to rapidly generate patterns with multiple cell types or materials. However, the location
of the cells, or other biopayload, within the areas of dispensed solution is not controlled. For discrete
components like cells, the payload is randomly distributed within a liquid volume, although parameters
such as concentration are controllable. Therefore, ink-jet printing is particularly well suited for rapidly
fabricating larger patterns, including constructs of intricate geometries and multiple cell types, in which
the geometric precision is important, but spatial control of the cells or groups of cells is not.
In a traditional LDW setup, a camera is coincidently focused with the laser, allowing direct visu-
alization of the biologic payload on the print ribbon. As a result, the specific cell or group of cells (or
other biopayload) to be printed can be visualized, targeted, and transferred to the substrate with high
spatial precision. In contrast to ink-jet printing, where a controlled volume of liquid is deposited,
LDW allows targeting of specific cells, so the dispersal of cells within the volume transferred is not
random. LDW is also capable of printing more viscous materials that may not be dispensed by an
ink-jet nozzle. This unique ability to combine the high-resolution placement of selected biological
payloads with various substrate materials, makes LDW particularly attractive to engineer in vitro
cellular microenvironments.
5.1.3 LDW OVERVIEW
LDW is a noncontacting material deposition technique. While some differences in configuration exist,
a typical LDW setup consists of two coplanar plates: a laser-transparent print ribbon, which holds the
material/cells to be deposited, and a receiving substrate onto which material is printed ( Figure 5.2 ).
Both the receiving substrate and print ribbon can be independently moved with CAD-CAM-controlled
stages. A charge-coupled device (CCD) camera also allows real-time visualization of the ribbon and
receiving substrate. The underlying side of the mounted ribbon is coated with two thin layers, the first
is a sacrificial layer, and the second a transfer layer. The sacrificial layer directly interacts with the laser,
while the transfer layer consists of the actual printed material. A pulse from the laser passes through the
transparent ribbon and volatilizes the sacrificial layer.
The consensus mechanism for deposition is that laser energy is absorbed by the sacrificial layer,
forming a vapor pocket at the ribbon-material interface ( Barron et al., 2004a ). Expansion of the
vapor pocket allows the printed material - the donor material - to form a droplet that is ejected
from the surface of the print ribbon, on a trajectory perpendicular to the plane of the ribbon. It is
also generally accepted that for a high-power laser and an appropriate sacrificial layer, mass transfer