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William Whitford and James B. Hoying

3. Accommodating Newest Approaches tecting from shear-stress and/or establishing cellular
orientation) in cell-support, the biomolecular charac-
Newer methods being promoted today include the use teristics are also important.
of hybrid multicomponent bioinks, deposited in a
multi-step and even multi-mode 3D printing process. 3.2 Maintaining Robust Viability
This will obviously demand a higher level of process All printing processes involve an ink being formulated,
monitoring, equipment integration, and process control. stored for significantly variable periods of time before
Co-deposition of two or more bioink streams can in- or during the printing process, pumped through a noz-
tegrate desirable physical properties from each con- zle or applied to a dispensing or culture plate, and fol-
stituent component and exhibit complex phase beha- lowed by the actual integration into the progressing
vior [15] . It is notable that both the effect of the matrix construct. There, the cells remain in the media, buffer
upon a cell type or implantation environment as well or bioink until at least the remainder of the construct
as the inclusion of high complements of cells upon the is completed. From that point onwards, the ambient
properties of the gel or matrix itself must be consi- environment of cells in the nascent construct may or
dered. Surprisingly, the exact nature of the reticulation may not be altered during a post-printing “maturation”.
of many matrix monomers (actually homo- or hetero- Throughout these stages the cell needs must be sup-
oligomeric complexes), as well the matrices’ effects ported, and any of these activities can affect the cells
upon the biological nature of the printed construct, are viability, differentiation, adhesion, state, functionality
only becoming understood. For example, the “func- and up-or down regulation such as specifically induc-
tionalization” of substrate components, including the ing apoptosis.
introduction of soluble cell-binding inducers to matrix
components has demonstrated enhanced cell adhesion 4. Major Considerations
and spreading [17] . Peptide gels with simple composi-
tion and tunable physical properties have been devel- When cells are included in a printing operation, main-
oped to facilitate targeted differentiation [21] . Post tenance of their viability and state of health must be
printing perfusion with growth or differentiation fac- considered. Suspensions of robust eukaryotic cells can
tors can drive multipotent cells to a desired lineage [22] . survive for short periods of time in poorly controlled
Immediate nutrient mass-transport has been facili- environments in simple buffered salt solutions. How-
tated by both anisotropic matrix environments guiding ever, for optimized performance, especially over ex-
cellular alignment and microchannel architectures as tended print periods including pre- or post- printing
well as the engineering of inherently high isotropic staging operations, many factors must be considered
matrix porosity [23] . Finally, issues have arisen in the (Table 2).
application of even elegantly designed approaches — 4.1 Biomolecular Characteristics
such as the observation of an inverse relationship be-
tween printed cell density and robust functionality in First, while there are mechanical properties to be con-
immediate scaffold development. Generally, therefore, sidered in cell-support (such as protecting from shear-
each application must be studied on its own. stress and/or establishing cellular orientation), the bio-
molecular characteristics are also important. Many of
3.1 Cultured Cell-support the common ingredients of modern cell culture media
When cells are included in a printing operation, main- must be supplied either within the bioink formulation,
tenance of their viability and state must be considered. or made available immediately post printing. Further-
Suspensions of robust eukaryotic cells can survive for more, most nascent constructs may be altered during a
short periods of time in poorly controlled environ- post-printing “maturation” operation. Characteristics
ments in simple buffered salt solutions. However, for of the cells ambient fluid media throughout this are
optimized performance and in extended pre- or important.
post-printing incubations, many of the common ingre- 4.2 Environmental Parameters
dients of modern cell culture media must be supplied
either within the bioink formulation, or made available Second, depending upon the cells, bioink formulation
immediately post printing. Thus, while there are me- and printing style, it can be critical to control envi-
chanical properties to be considered (such as in pro- ronmental parameters during these steps. These can

International Journal of Bioprinting (2017)–Volume 3, Issue 1 21