(C-113) Biomaterials Design for Bioengineered Meats

Bioengineered meat is the recreation of animal livestock meat via the process of tissue engineering and cell culturing, aiming to address the issue of rapid live animal product consumption and attempts to find a more efficient usage of resources and lower the risk of climate change damage, animal welfare issues, and overall food sustainability for the human population. Skeletal muscle is mainly composed of aligned myotubes, which contribute most to muscle structure and as such, the C2C12 mouse myoblast cell line was used to for the cultivated meat strategy in this project. A custom 3D printed microscale mold was designed to serve as a template for a hydrogel to provide both stiffness and topographic cues to C2C12s. Once optimizing both the mold and the hydrogel, the myoblasts were seeded for 2 days, and their orientation observed via fluorescence microscopy to quantify their alignment with respect to hydrogel ridges. Un-patterned and patterned hydrogels of 10 kPa and 23 kPa were created to measure the effects of stiffness. Immunofluorescence data indicates that there was an increased alignment and linear growth along the ridged hydrogel as opposed to the smooth hydrogel. This model can be furthered by using edible materials, e.g., gelatin-based, or other natural hydrogels, as well as by improving the topographic features using soft lithography to create more accurate molds and hydrogels for seeding as opposed to a 3D printed mold.

A mold was designed using the Prusa Slicer SL1S 3D printer to serve as a template for hydrogel synthesis. After designing the geometry on Onshape, it was exported and printed using the PrimaCreator Tough Clear resin with a 25-micron layer height for maximum resolution. The hydrogel was then synthesized atop this mold via the addition of the hydrogel solution composed of variable amounts of acrylamide and bis-acrylamide to create two physiologically relevant stiffnesses (10kPa and 23 kPa). After coating with collagen, the C2C12 cells were seeded atop the hydrogels in growth media and spun down into the ridges. Cells were fixed and immunostained after two days for actin, tubulin, nucleus, and collagen. Both widefield and confocal fluorescence microscopy were carried out and images were analyzed via ImageJ for alignment and expression.

When observing the confocal IF images, a notable nuclear and cell alignment difference was seen between the nuclei on the 23 kPa patterned hydrogel and the smooth hydrogels, as shown in Figure 1. The nuclei on the patterned hydrogel appear elongated and elliptical, while those on the smooth hydrogel maintain a circular morphology. Although the 10 kPa patterned hydrogel was unable to be created due to mold detachment issues, it is still clear that stiffness plays a role in determining nuclear orientation and differentiation when comparing 10 kPa vs. 23 kPa smooth hydrogels. Moreover, an increase in stiffness seems to promote a higher tendency for the cells to align themselves vertically at a 90-degree angle and follow the direction of the ridges, as can be seen in Figure 2. This is also visible via the elongated stretch of tubulin and actin stains indicating that the myoblasts matured and fused to show their oriented morphology and alignment. 

We have shown that using ridged hydrogels containing patterned lines where cells could be seeded and grown cause myoblasts to fuse and form “myotube-like” morphologies, like that of meat. More specifically, the nuclei of the cells on the patterned hydrogel tend to display a more elongated, elliptical and directional orientation as opposed to their control smooth hydrogel counterpart. The cytoskeleton fragments along the patterned ridges take on a more structured and connected pathway in the 90-degree direction compared to a random orientation on the smooth hydrogel. These results indicate that there could be a preference towards the vertical 90-degree orientation that lines along the ridges showing that the myoblasts fuse and alter their direction due to the geometry.

I would like to thank Dr. Jennifer Young for, Faris for mentoring and guiding us throughout the, Jennifer for aiding in 3D printer training and lab introduction, Hepi for the cell passaging, culturing and hydrogel training, Marteen for the hydrogel synthesis training, Martin for the GelMa introduction and synthesis training, Jashan for the protocols for hydrogel coating and synthesis and image analysis, and the entirety of the Mechanobiology Institute for providing us with support and all of the resources and equipment needed to perform my research.

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