ENGINEERING 2D AND 3D CELLULAR MICROENVIRONMENTS USING LASER DIRECT WRITE - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 104

patterning involved controlled spatial adsorption of an adhesive protein, with subsequent seed-
ing of cells that preferentially grow on the patterned protein. While this sort of patterning was first
demonstrated using lithography-based techniques such as microcontact printing ( Chen et al., 1998;
Tien et al., 2002 ), other deposition methods like ink-jet printing (reviewed in ( Calvert, 2001 )) and
LDW ( Ringeisen et al., 2002a ; Wu et al., 2003 ; Colina et al., 2005 ) have also shown successful protein
or nucleic acid deposition. Moreover, ink-jet printing ( Xu et al., 2005 ; Roth et al., 2004 ; Saunders
et al., 2008 ) and LDW ( Odde et al., 2000 ; Pirlo et al., 2006 ; Barron et al., 2004a ; Schiele et al., 2010 ;
Schiele et al., 2011 ) have been used to deposit viable mammalian cells directly to a homogeneous sub-
strate, without requiring the prior patterning of a protein. There are other patterning techniques avail-
able, such as dip-pen nanolithography ( Piner et al., 1999 ) or AFM-based patterning ( Xie et al., 2006 ),
that focus on patterning at submicron scales. Cells certainly sense nanoscale features in their environ-
ments, but in order to study and produce cellular microenvironments where cell- and population-level
interactions are controlled, patterning at the micro- and mesoscale may be most relevant for directing
cellular signaling. However, patterning at this scale can be quite challenging; it is too large to be ac-
complished directly by chemistry, and too small to use many traditional fabrication methods.
Some methods that have proven suitable for patterning at this scale include microcontact printing, ink-
jet printing, and LDW. Each of these has its own specific advantages and disadvantages, and they are quite
complementary. Microcontact printing employs a stamp with relief features attained via photolithography.
The resolution of the pattern is limited only by the wavelength of light, making submicron resolution attain-
able with this technique. By contrast, LDW is a noncontact technique that propels material to a substrate by
laser energy absorption and partial volatilization of a sacrificial layer. The spatial location of the transferred
material is determined by the programmed position of a computer-aided design/computer-aided manu-
facturing (CAD/CAM) stage. The resolution of this technique can be under 10 m m ( Zhang et al., 2003 ),
because of the dynamics of the material transfer event and controlled stage movement. Ink-jet printing, by
comparison, has a printing resolution on the order of 50 m m ( Calvert, 2001 ), which is appreciably lower
but still very good. Material deposition is achieved by one of several methods of propulsion through a
nozzle ( Saunders et al., 2008; Boland et al., 2006; Lee et al., 2009a; Gonzalez-Macia et al., 2010 ), and both
the size of the nozzle and method of material ejection can influence printing resolution.
Because of their different mechanisms and printing resolutions, each of these methods is particular-
ly well suited for a specific subset of applications, summarized in Table 5.1 . Micropatterning excels at
creating patterns of proteins on 2D flat, or even curved ( Jackman et al., 1995 ) surfaces. Cells can be seeded
on adhesive proteins, allowing their behavior in response to the protein or protein patterns to be studied.
However, once the pattern of proteins is set, it is immobilized, and cells generally do not proliferate

Table 5.1 Patterning techniques and applications

Technique Resolution Application

< 1 m m Micropatterning Patterns of adhesive proteins in 2D and cell culture on patterned adhesion

islands. Study of cell behavior in controlled population or cell size and/or
geometry. New mask must be fabricated for each new pattern.

∼ 50 m m Ink-jet printing High-throughput or large constructs in 2D or 3D on any suitable substrate.

Direct patterning of cells or less viscous materials.

∼ 10 m m LDW Patterning of cells or material in 2D or 3D on any suitable flat substrate.

Direct patterning of cells with high spatial resolution. More viscous materials
can be patterned, but with lower throughput than ink-jet printing.