The self-assembly of the scaffolds is driven primarily by spatial differences of stresses, elastic moduli and Poisson ratios of rigid panels, flexible hinges and open segments within 2D thin film sheets. These differences are engineered by multiple steps of photolithography, thin film deposition and etching. Guidelines and geometric design rules required in the patterning of 2D sheets to enable assembly of specific 3D curved structures are described elsewhere [23
]. However, in previous work the criterion for material selection was based on ease of patterning and accessibility of different materials. In order to utilize these structures as in vitro
scaffolds it was necessary to construct them with bio-inert materials, so that cells could be cultured for extended periods of time (e.g. up to a month). Hence, metals like Cu and Ni were replaced by Au and SU-8 [26
]; thicknesses were retargeted with these materials to achieve self-assembly. The process flow required two major modifications. First, lift-off metallization and electrodeposition were used to pattern the Cr/Au bilayer due to concerns regarding poor wet and dry etching selectivity of Au. Second, the use of a Cu film as compared to a previously used polymeric sacrificial layer provided several desirable features. It served as a conductive seed layer for electrodepositions and reduced cracking of the 2D sheets. Cu could also be rapidly and selectively etched which enabled the lift-off metallization step and the release of the 2D templates leading to self-assembly.
To demonstrate the feasibility of growing cells on the 3D micro-patterned scaffolds, we investigated the biocompatibility of individual materials for fibroblast culture. In both the 24 and 48 hour cell viability assays, all components with the exception of Cu and the negative, latex exposure control were found to be non-toxic when compared to the media-only positive control (p < .05). The viability assay confirmed the suitability of the materials for cell culture. We then evaluated the viability of fibroblasts grown on the self-assembled scaffolds.
Fibroblasts were cultured on a patterned cylindrical volute and the viability of the population was monitored over 9 days (). It should be noted that by day 9, the 3D scaffold was completely covered with a film of cells. We have independently verified that cells are viable on these structures for at least 30 days. Calcein AM was used to visualize the live fibroblasts since it could be administered on multiple days without eliciting a cytotoxic effect. Initially, cells were interspersed along the scaffold () and exhibited lamellipodia and an elongated morphology characteristic of fibroblasts grown on planar tissue culture substrates [28
]. By day 5 (), the rigid panels and flexible hinges were covered by a confluent layer of cells. The open regions of the scaffold were clearly distinguishable by the absence of fluorescent cells and are an important feature of the design of the scaffolds since they can improve the diffusion of oxygen and growth factors to the cells, which becomes severely limited in cell layers 100–200 μm thick [30
]. By day 7 () the cells began to grow into multiple layers and the open regions began to fill-in until, by day 9 (), these regions were completely covered by a film of cells.
We fabricated scaffolds with varying geometries, materials, feature sizes, and curvatures. Shown in are zoomed-in optical images of cylinders formed from 2D arrays of 15×15, 160 μm square panels of Au or SU-8 polymer, respectively. The rigid panels were connected to their neighbours by 80 μm long hinges, resulting in triangular hollow regions. Fibroblasts could be seen on the reflective surfaces of both scaffolds. The SU-8 panels were transparent, which may be useful for cell-migration experiments. We believe that these scaffolds can be constructed with most photopatternable materials; however, retargeting of the width, length and thickness of individual layers may be required based on the thin film stresses, moduli and Poisson ratios of the materials.
Figure 4 Versatility of geometry in 3D cell culture. (a–f) Cylindrical cultures grown on scaffolds with varying materials, feature sizes, and diameters. (a,c,d) Cylinder consisting of a 15×15 array of 160 μm square rigid panels connected (more ...)
Another highlight of the construction methodology is that many 2D patterns can be included while retaining a similar overall 3D curvature and geometry. The cylinder shown in was processed simultaneously with that of and although they have different sized patterns, they have approximately the same radius of curvature. This similar curvature was accomplished by halving the dimensions of all the rigid, flexible and open areas while doubling their number. As a consequence of the change in feature size, the cylinders also had similar porosities (21±1%), but different pore densities (approximately 1 pore per 254 μm2
for the cylinder in versus 1 pore per 120 μm2
for ). Additionally, since the rigid panels and hinges can be coated with specific cell adhesion proteins, one could in principle manipulate the initial cell-cell spacing on the scaffolds. The self-assembly strategy thus enables the precise control over the porosity and patterning on 3D scaffolds which is of importance in studying cell behaviour [7
Recent work by Rumpler et al
suggests that cells are able to sense radii of curvature many times greater than their size [31
]. Our 3D scaffolds can enable studies of the influence of curvature on cell behaviour since the curvature can be readily manipulated. Scaffolds constructed with () and without () the rigid PR layer within the hinges showed a variation in curvature of approximately 30%. The local curvature within the scaffold can also be easily manipulated by changing the thickness of the Cr/Au/PR layers within the hinge. For example, while the folding angle of hinges was approximately 20° in the cylindrical scaffolds () the folding angle was approximately +/− 90° in the bi-directionally curved 3D scaffold (). This bi-directional scaffold was formed from a single continuous 2D array of 450 μm square Au panels connected by 45 μm long hinges. The bi-directional curvature was generated by micropatterning both Cr/Au and Cr/Au/Cr hinges. The undulations of this cell culture geometry are reminiscent of intestinal villi and the gyri/sulci in the brain, where the increased surface area for a given volume is critical.
Finally, there is considerable versatility in the overall geometry of the curved structure. Shown in is a curved, spiral-like geometry. Unlike the highly interconnected cylinders and bi-directional sheet, the spiral-like geometry was formed by culturing cells on a scaffold ribbon formed by joining two strips of 40 μm square rigid panels connected to their neighbours by 40 μm long hinges. As the ribbons spiralled outward from the core of the scaffold, the flexible hinges were less constrained in their position and could twist into highly convoluted yet less predictable geometries.
Shown in are SEM images of fibroblasts cultured on a cylindrical double volute scaffold after 5 days. The difference in this volute scaffold design as compared to a normal cylinder was that the square rigid panels of the double volute were only connected to their neighbours at the corners of each panel. Volutes tended to roll up much faster at the edges as compared to the centres of the 2D sheets. These double volutes are interesting since they allow easy visualization of cellular interactions across different layers of the scaffold which come in to close contact along a central plane.
Figure 5 SEM of fibroblasts cultured on a cylindrical double volute scaffold fixed after 5 days of culture. (a) Confluent layer of cells covered the entire scaffold with clearly visible open regions. (b) Zoom-in of the scaffold showed that fibroblasts were adhered (more ...)
The fibroblasts populated the entire length of the double volute () and were observed on both the inner (Cr) and outer (Au/PR) scaffold surfaces (). Interestingly, the cells that were adhered to the inner surface exhibited fewer filipodia and had elongated cell bodies compared to cells adhered to the outer surface. The filipodia were approximately 200 nm in diameter, which closely matches observed values for adherent fibroblasts [32
]. We hypothesize that the difference in cell morphology can be attributed to the dissimilar material properties, which we are currently exploring. In addition to these morphological differences, the two cylindrical halves of the double volute scaffold were in close proximity to one another along a central plane. In this region, fibroblasts were observed to adhere to adjacent panels (). At higher magnification (), we observed that fibroblasts were not only in contact with cells on adjacent faces via filipodia but many cells bridged the physical gap between the two faces. Although it is not surprising that fibroblasts, a migratory cell line, would be able to populate the entire scaffold, the potential for monitoring cell-cell interactions and migration between two discontinuous regions has important implications for multilayered cultures and tissue engineering [21
From the multi-day cell culture and the SEM images, it is evident that fibroblasts cultured on thin film scaffolds not only adhere to the inner and outer surfaces but over time they become overgrown and form multilayer thick films. We therefore envision multilayered 3D cultures with different cell types populating well-defined regions of the scaffold. To this end, we are exploring methods to culture cells on the 2D templates prior to their self-assembly, which would provide a hands-free alternative to manually stacked and rolled multicellular scaffolds. Our fabrication method allows dissimilar materials to be patterned on the surfaces of the 3D scaffold. Hence, scaffolds can be patterned with regions where the adhesion of proteins and cells is either promoted or eliminated [14
]. For example, the Au and SU-8 surfaces utilized in the scaffolds can be functionalized using specific thiols and silanes respectively [12
] to manipulate cell adhesion in different regions on the surfaces of the 3D scaffold. Since the methodology is compatible with conventional 2D lithography, other micro- and nanoscale factors (such as surface topography) [16
] that are observed to influence cell behaviour can be readily incorporated onto the surfaces as well. Furthermore, adapting the self-assembly process to generate biodegradable scaffolds using polymers such as polycaprolactone will be of value for in vivo
tissue engineering applications [7