Hormonal Control of Manduca sexta Cell Culture for Bioactuator Applications.
Jo, Jonathan Erin.
- Bioactuators are robotic contractile elements that are powered by muscle cells. The motivation behind bioactuator research centers on the cells' natural contractile properties and the fact that they are a biodegradable organic system that runs primarily on glucose. Current bioactuator research is mainly focused on mammalian cells, but these cells are limited by their sensitivity to temperature, pH... read more, and oxygen levels. Muscle cells from insects such as the tobacco hornworm Manduca sexta, on the other hand, are better suited for bioactuators as they can tolerate fluctuations in environmental conditions and can spontaneously contract for over three weeks. However, efficient, controllable cell culture methods are unknown. Therefore, the present research focused on developing a cell harvesting protocol to isolate embryonic M. sexta muscle cells and to enhance their development. 20-hydroxyecdysone (20-HE), an ecdysteroid hormone found during M. sexta development, was added to the media to improve differentiation. A myosin/DAPI immunostaining identified the developing cells as differentiated muscle cells, and the concentration of 20 ng/ml 20-HE was determined to yield the most enhanced differentiation based on its significantly larger myotube width measurements (p<0.001). Methoprene, a juvenile hormone mimic, was added to the media for proliferation assays. A LIVE/DEAD assay indicated that 500 and 1000 ng/ml concentrations were nontoxic to the cells after a 5-day culture. A PicoGreen assay indicated a statistically significant increase in DNA concentrations due to methoprene addition (p<0.05). 500 ng/ml methoprene with 20 ng/ml 20-HE induced the most rapid initial increase in DNA levels during the first two days and was chosen as proliferation media. The long-term developmental effects of this media were determined from a prolonged 10-day exposure, which resulted in significantly less-developed myotubes compared to a control grown on differentiation media (p<0.001). However, switching from methoprene media to differentiation media at day 3 resulted in no significant difference from the positive control (p=0.15). Based on these results, a culture protocol was developed using proliferation media for three days followed by a switch to differentiation media to receive the benefits of both media formulations. These methods were applied to a preliminary bioactuator construct, in which the cells were seeded onto silk or PDMS. The cells differentiated on the PDMS but no on the silk, and they were weakly attached to both materials. Cells were also embedded in a collagen gel prior to seeding, but these cells did not differentiate. The collagen and silk may have interfered with the cells' ability to migrate out of cell masses, which may be an essential part of the differentiation process. Despite the limited success with this construct, it provided useful information regarding future studies and was an acceptable first attempt at a Manduca-based bioactuator system.read less