Digital microfabrication of user-defined 3D microstructures in cell-laden hydrogels

Abstract

Complex 3D interfacial arrangements of cells are found in several in vivo biosystems such as blood vasculature, renal glomeruli, and intestinal villi. Current tissue engineering techniques fail to develop suitable 3D microenvironments to evaluate the concurrent effects of complex topography and cell encapsulation. There is a need to develop new fabrication approaches that control cell density and distribution within complex 3D features. In this work, we present a dynamic projection printing process that allows rapid construction of complex 3D structures using custom-defined computer-aided-design (CAD) files. Gelatin-methacrylate (GelMA) constructs featuring user-defined spiral, pyramid, flower, and dome micro-geometries were fabricated with and without encapsulated cells. Encapsulated cells demonstrate good cell viability across all geometries both on the scaffold surface and internal to the structures. Cells respond to geometric cues individually as well as collectively throughout the larger-scale patterns. Time-lapse observations also reveal the dynamic nature of mechanical interactions between cells and micro-geometry. When compared to conventional cell-seeding, cell encapsulation within complex 3D patterned scaffolds provides long-term control over proliferation, cell morphology, and geometric guidance. Overall, this biofabrication technique offers a flexible platform to evaluate cell interactions with complex 3D micro-features, with the ability to scale-up towards high-throughput screening platforms.

Keywords: 3D micro-architecture; cell encapsulation; digital microfabrication; gelatin-based hydrogel.

Figure 1

Fabrication method and examples of hydrogels formed via dynamic projection printing. (a) Cells in a macromer solution are placed in a chamber covered by a methacrylated glass coverslip. Polymerization of the 3D scaffold begins at the coverslip surface, where the reflected UV image from the DMD array is focused [1]. Starting from the bottom portion of the scaffold, which is cross-linked to the coverslip, the complete structure is fabricated in one continuous process. The projection mask on the DMD changes as the servo-controlled platform translates up [2] until the top of the scaffold is reached [3], thereby encapsulating the cells in the user-defined 3D structure. (b) Heatmap representing the cumulative UV exposure at each part of the pattern. Precise specification of the duration and spatial distribution of UV light allows for the creation of cell-laden structures with complex 3D features at high resolution. (c) and (d) DIC micrographs of 10% gelatin methacrylate (GelMA) scaffolds fabricated using the dynamic projection printing system. The patterns generated consist of three-dimensional spiral, pyramid, dome, and flower structures [clockwise from top left]. All scale bars are 100 μm.

Figure 2

Cells respond to the complex 3D geometric cues and interact dynamically with the GelMA scaffold to remodel the position and shape of the structures. (a) GelMA scaffolds with encapsulated NIH/3T3 cells at 12 hrs post-fabrication. (b) Deformation of the structures as observed 4 days post-encapsulation. (c) 3D reconstruction of confocal fluorescence micrographs indicates height-dependent deformation of the scaffold as mediated by cell-cell interactions across two flower structures. Cells were stained for F-actin (red) and nuclei (blue). (d) Individual Z sections of the same flower structures as shown in (c) demonstrate height-dependent deformation when progressing up from the floor to the top of the structures. All scale bars are 100 μm.

Figure 3

Cells that remain encapsulated within the GelMA scaffolds exhibit 3D cell spreading and maintain active cell-material interactions. Confocal fluorescence micrographs (a) reconstructed in 3D and (b) viewed at various Z planes throughout the scaffold reveal NIH/3T3 fibroblasts on the scaffold surface displaying morphology different from cells internal to the structures. (b) Cells that remain embedded within the GelMA scaffold at 4 days post-encapsulation exhibit extension of pseudopodia preferentially towards the surface of the encapsulating structure. Cells were stained for F-actin (red) and nuclei (blue). (c) 10T1/2 cells encapsulated within GelMA scaffolds express a smooth muscle cell phenotype, as shown via staining for α-SM actin (green), and maintain cell-material interactions at 8 days post-encapsulation as indicated by 3D projections of pseudopodia. All scale bars are 50 μm.

Figure 4

Characterization of cell viability and proliferation. (a) Viability of 10T1/2 cells encapsulated in GelMA scaffolds was assessed via calcein-AM (green)/ethidium homodimer (red) LIVE/DEAD assay at 8 hrs after encapsulation. Cell viability within the 3D patterned scaffold was compared to an unpatterned control slab and to a slab that received a second round of UV exposure. These 3 conditions were compared to determine if differences in cumulative UV exposure alone were contributing to the cellular responses to geometric cues. High cell viability was maintained in all scaffolds, and quantification revealed that similar levels of viability were seem across the 3 conditions (c). The Click-iT® EdU assay was performed at day 5 to assess the proliferative ability of 10T1/2 cells throughout the scaffold (b). Incorporated EdU was labeled with Alexa 488 (green) and counter-stained for α-SM actin (red) and for all nuclei (blue) using Hoechst. Images shown were taken at a Z cross-section mid-depth within the scaffold. (d) Quantification of proliferation at different heights throughout the scaffold demonstrates height-dependent rates of proliferation, with cells exhibit higher proliferation within the 3D patterned scaffold. Labels for the height indicate quarter-increment distances from the base of the scaffold to the scaffold surface (e.g. for a scaffold with total height = 200 μm, “1/4 height” refers to the slice located 50 μm from the base of the scaffold). Scale bars are (a) 250 μm and (b) 100 μm. Error bars represent the SD of 3 independent samples.

Figure 5

Differences in cell morphology and α-SM actin expression (green) between seeded vs. encapsulated 10T1/2 cells in GelMA. Comparison between (a) seeded cells on 3D patterned scaffolds at day 3 and (b) encapsulated cells after long-term (1 month) culture reveal a more defined geometric response for encapsulated cells along with differences in α-SM actin expression and cell morphology. All scale bars are 100 μm.