ENGINEERING 2D AND 3D CELLULAR MICROENVIRONMENTS USING LASER DIRECT WRITE - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 115

5.4 LDW APPLICATIONS IN 3D
5.4.1 MICROENVIRONMENTS IN 3D
Planar, 2D cell culture has long been a paradigm for studying mammalian biological phenomena in
vitro , ranging from stem cell differentiation and tissue development to drug testing. However, funda-
mental differences exist in the way cells behave between 2D and 3D microenvironments. Cells with 3D
microenvironment interactions show differences in their cytoskeletal structure, morphology, membrane
protein distribution, and interaction with soluble factors ( Pampaloni et al., 2007 ). Additionally, cells
cultured in a 3D ECM experience limitations for cell migration, while those grown on a 2D substrate
can migrate and proliferate without the same restrictions.
In a 2D environment, cells are only able to interact and attach to the ECM on the substrate. This
produces a difference in the receptor density and orientation along the surface of the cell compared
with receptor orientation in 3D ( Meshel et al., 2005 ). Further, the composition and strength of the
complexes forming the adhesions differ between 2D and 3D microenvironments. As an example, on
a 2D substrate, fibroblasts form adhesions along only the ventral surface ( Berrier et al., 2007 ). The
localization of cell traction forces results in a morphological polarity that does not exist in 3D. In a 3D
environment, adhesion is formed via focal complexes (as opposed to 2D focal adhesions), all along the
cell membrane, and has a different composition. The difference in cell distribution and type of adhesion
site in 3D affects the organization and generation of tension in the cytoskeleton ( Pedersen et al., 2005 ).
The compounding discrepancy between 2D and 3D cell-matrix interactions can yield very different
behaviors and response to stimuli, when explored experimentally.
In a 3D ECM, cell migration occurs either by moving through the material's pores, or by break-
ing down the surrounding ECM with proteases. Highly porous materials, such as sponges and
foams, typically have pore sizes greater than the cell diameter, allowing for nonproteolytic migra-
tion. In hydrogels, the pores are on the nano scale (much smaller than the actual cell). For infiltra-
tion to occur, the cell must produce proteases to break up the restrictive matrix, assuming the matrix
is made from a peptide-based gel. The ease of infiltration will determine the migratory rate of the
cells within the material. Additionally, the pore size will be a factor in determining the rate that
nutrients can diffuse into and waste products can flow out of the bulk material. If the bulk material
is too thick and/or pore size too small, toxins can build up in the environment, or cells can die due
to ischemic effects.
Fundamental biological questions have been, and will continue to be solved using 2D culture
models. However, 3D alternatives are necessary to overcome 2D matrix interactions that fundamen-
tally change cell behavior (e.g. cytoskeletal structure, morphology, membrane protein distribution
and interaction with soluble factors ( Pampaloni et al., 2007 ), and focal adhesions). Additionally, the
creation of large tissue-engineered constructs, for fundamental research or in vivo transplantation, will
require a means of 3D fabrication with precise spatial arrangement of the contents. To overcome the
limitations set by 2D environments, LDW has been adapted to build more physiologically relevant 3D
culture models, in vitro diagnostic tools, and tissue-engineered constructs.
5.4.2 LAYER-BY-LAYER APPROACHES
Layer-by-layer (LbL) printing is a 3D biofabrication approach adapted from industrial rapid pro-
totyping technologies. Traditionally, LbL printing utilizes a 2D method, sequentially, to produce