Speaker
Description
Introduction:
Biofabrication of highly aligned 3D tissues like nerves, tendons, or muscles remains challenging due to insufficient scaffold cues to guide cell alignment, proliferation, extracellular matrix secretion, and maturation into functional tissues. Previously, we showed the ability to create such tissue constructs using filamented light (FLight) biofabrication approach1. We expanded the method by feeding photoresin continuously into a cuvette while exposing the resin to a speckled laser light projection (Fig. 1a), allowing us to create layerless filamented constructs on a larger scale and overcoming light scattering. This study further demonstrates the ability to encapsulate cells directly into the printed resin with high viability and culture them into aligned tissues.
Materials and Methods:
Gelatin-methacrylate (Gel-MA) with a 99% degree of functionalization was first dissolved in PBS to prepare a 5% w/v solution. Photoinitator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate) and photoabsorber (Quinoline Yellow) were added to the Gel-MA solution to reach a final concentration of 0.05 w/v% and 0.0043 w/v%, respectively. C2C12 cells were mixed into the resin at a concentration of 106 cells/ml. The constructs were printed using a 405 nm laser integrated into the custom FLight printer. The resin was extruded constantly at a rate of 11.6 mm3/s at a laser intensity of 34 mW/cm2 into a cuvette with a cross-section of 100 mm2. The light was patterned using a 0.65-inch DLP® chip with a resolution of 1920x1080 pixels. The constructs were cultured in growth media for 7 days and then switched to differentiation media for 5 more days, after which the samples were stained with Hoechst (Nuclei), MF20 (Myosin Heavy Chain), and phalloidin (F-actin) to visualize alignment. Cell viability and cell count were assessed using Propidium Iodide, Calcein AM, and Hoechst staining on days 1,3, and 7.
Results and discussion
4 cm long constructs were printed with C2C12 cells. After 12 days of culture, fusion of cells into contractile myotubes was observed, which were aligned along the filament direction (Fig 1b). Furthermore, this behaviour was found in three different regions of the graft, the top (I), the middle (II), and the bottom (III). The cell viability (Fig 1c) dropped to 70% on the first day after printing, however, it recovered to 90% on day 7. Lastly, we found that the compressive modulus of the printed gel can be modified by varying the energy density delivered by the laser light (Fig 1d) to match material stiffness or even create gradients.
Conclusion
We demonstrate that our biofabrication technique can print cellular constructs with high cell viability. Furthermore, the hydrogel scaffold aids the encapsulated cells in aligning and proliferating along the printed cues, supporting the fusion into myotubes. Lastly, we demonstrate that we can control the stiffness of the printed hydrogels, allowing us to modify cell confinement for the best cell differentiation.
References
[1] Liu H. et al., Adv. Mat. 34, 2204301, 2022.
Acknowledgments
MZW acknowledges Bridge Discovery funding from the Swiss National Science Foundation: 40B2-0_211764
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