Myoblast Growth on Fibers

After drawing our fibers, we tested the ability of muscle to grow on them. 

We found that the gelatin fibers quickly dissolved in media so we cross linked them with microbial transglutaminase (mTG). The zein was more stable in media, however three groups were run to test cell development on just the zein fibers, mTG-treated zein fibers, and mTG and Poly-D-Lysine treated zein fibers. 

The mouse myoblast cell line (C2C12) was seeded on the fibers to test cell viability on the fiber scaffolds. For both gelatin and zein fibers, we found that cells were able to proliferate and survive on the fibers.

 

Fluorescent imaging of cell viability on (A) 50% gelatin, 50% glycerol fibers and (B) 70% gelatin, 30% glycerol fibers. Scale bars are 100 micrometers.

The 50% glycerol fibers were prepared slightly differently from the 30% glycerol fibers in the first image as they were in media for a few weeks after cross linking, and cells were added to hydrated fibers. The 30% glycerol fibers were hydrated immediately prior to seeding.

Differentiation media (proliferation media with added IGF1 and insulin -- growth factors to induce differentiation) was introduced to the gelatin fibers after several days to promote differentiation of the myoblasts (muscle precursors) into muscle cells. We fixed and stained the fibers which can be seen in the image below. We were able to see myosin heavy chain (an indicator of muscle differentiation), in red. The cell nuclei are in blue, and the cells’ actin is green. 

 

Immunofluorescent staining of 30% glycerol, 70% gelatin fibers. Scale bars are 121 micrometers.

The zein fibers all seemed to have similar success with cell viability. Since the zein with no treatment requires the least time and substrates to prepare, future experiments would likely follow untreated zein/glycerol fibers. The zein fibers has autofluorescence in the blue and red ranges with the cell viability stains, so only live (green) cells are shown. A 70% gelatin 30% glycerol fiber (mTG treated) was seeded at the same time.

 

Fluorescent imaging of cell viability on (A) 30% glycerol, 70% gelatin fibers with mTG treatment, (B) zein/glycerol fibers with mTG and PDL treatment, (C) zein/glycerol fibers with control/water treatment, (D) zein/glycerol fibers with mTG treatment, and (E) zein/glycerol fibers with mTG and PDL treatment. Scale bars are 100 micrometers.

Instead of adding differentiation media (which presents added costs/time to prepare), we continued the zein fibers in the original, proliferation media to determine success of myogenesis. Due to zein autofluorescence, we were only able to detect the presence of actin on the zein fibers (in green). The thick fibers appear to be muscle fibers, indicating successful differentiation. However, all zein images were taken on the cross section of the fiber, instead of along the length of them. Longer pieces of fiber can be used in future experiments to better determine muscle fiber formation along the length of the fibers.

 

Immunofluorescent staining of zein/glycerol fibers with (A) control/water treatment, (B) mTG treatment, (C) mTG and PDL treatment. Scale bars are 123 micrometers.

We are very excited about the progress that our project has made at developing a scalable method for production of plant protein fibers for cultured meat.

This wraps up our project! Thank you to everyone who donated — we couldn’t have done it without you. If you have more questions/comments please feel free to leave them below.