A simple method to align cells on 3D hydrogels using 3D printed molds

Abstract

Vascular smooth muscle cells align circumferentially around the vessel lumen, which allows these cells to control vascular tone by contracting and relaxing. It is essential that this circumferential alignment is recapitulated in tissue engineered blood vessels. While many methods have been reported to align cells on 2D polymeric substrates, few techniques enable cell alignment on a 3D physiologically relevant hydrogel substrate. We hypothesized that the ridges inherent to the sides of fused deposition modeling 3D printed molds could be used to topographically pattern both stiff and soft substrates and thereby align cells on flat and curved surfaces. Flat and curved molds with 150, 250, and 350 μm ridges were 3D printed and used to topographically pattern polydimethylsiloxane and gelatin-methacryloyl. The ridges transferred to both substrates with less than 10% change in ridge size. Vascular smooth muscle cells were then seeded on each substrate, and nuclear and actin alignment were quantified. Cells were highly aligned with the molded ridges to a similar extent on both the stiffer polydimethylsiloxane and the softer gelatin-methacryloyl substrates. These data confirm that fused deposition modeling 3D printed molds are a rapid, cost-effective way to topographically pattern stiff and soft substrates in varied 3D shapes. This method will enable investigators to align cells on 3D polymeric and hydrogel structures for tissue engineering and other applications.

Keywords: 3D printing; Cell alignment; Hydrogel patterning; Vascular smooth muscle cells.

Fig. 1.

Ridge patterns on 3D printed molds can be successfully transferred to PDMS. (A) Images of 3D printed PLA molds and corresponding patterned PDMS samples acquired with Nikon SMZ745T. (B) Measured ridge thickness versus 3D printer programmed ridge thickness for 3D printed molds (left) and PDMS samples (right).

Fig. 2.

SMC show significant alignment when seeded on flat PDMS patterned with different ridge thicknesses compared to unpatterned samples. (A) Representative confocal microscopy images of SMC on flat PDMS labeled for actin fibers (phalloidin, red) and nuclei (Hoechst, blue) with corresponding normalized alignment angle histograms. (B) Average absolute angles (0°–90°) for actin fibers and nuclei (left) and kurtosis of actin fibers and nuclei angle distributions (right). # p < 0.05, * p < 0.01, ** p < 0.001 compared to cells on flat unpatterned PDMS.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

SMC show alignment when seeded on curved PDMS patterned with ridges compared to unpatterned samples. (A) Representative confocal microscopy images of SMC on curved PDMS with 150 μm ridges labeled for actin fibers (phalloidin, red) and nuclei (Hoechst, blue) with corresponding normalized alignment angle histograms. (B) Average absolute angles (0°–90°) for actin fibers and nuclei (left) and kurtosis of actin fibers and nuclei angle distributions (right). # p < 0.05, * p < 0.01, ** p < 0.001 compared to cells on curved unpatterned PDMS.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Ridge patterns on 3D printed molds can be successfully transferred to GelMA. (A) Images of flat and curved patterned GelMA acquired with Nikon SMZ745T. (B) Measured GelMA ridge thickness versus 3D printer programmed ridge thickness.

Fig. 5.

SMC show alignment when seeded on flat GelMA patterned with different ridge thicknesses compared to unpatterned samples. (A) Representative confocal microscopy images of SMC on flat GelMA labeled for actin fibers (phalloidin, red) and nuclei (Hoechst, blue) with corresponding normalized alignment angle histograms. (B) Average absolute angles (0°–90°) for actin fibers and nuclei (left) and kurtosis of actin fibers and nuclei angle distributions (right). # p < 0.05, * p < 0.01, ** p < 0.001 compared to cells on flat unpatterned GelMA.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

SMC show alignment when seeded on curved GelMA patterned with ridges compared to unpatterned samples. (A) Representative confocal microscopy images of SMC on curved GelMA labeled for actin fibers (phalloidin, red) and nuclei (Hoechst, blue) with corresponding normalized alignment angle histograms. (B) Average absolute angles (0°–90°) for actin fibers and nuclei (left) and kurtosis of actin fibers and nuclei angle distributions (right). #p < 0.05, * p < 0.01, ** p < 0.001 compared to cells on curved unpatterned GelMA.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)