Isolation and Maintenance-Free Culture of Contractile Myotubes from Manduca sexta Embryos

Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS) and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment, combined with the interesting metabolic properties, suggests that this cell source is a promising candidate for further investigation toward bioactuator applications

Index of movement analysis for <i>M. sexta</i> vs. C2C12 muscle.

<p>Plot of average index of movement over time for both cell types for 3 regions per condition. Values are significant for <i>M. sexta</i> t = 16d compared to previous time points, as indicated, and t = 23d compared to previous time points, as indicated. * p<0.001.</p

Medium concentrations of metabolites tracked over time in insect and mouse C2C12 cell cultures, on a per cell basis.

<p>Glucose consumption and lactate production were analyzed for <i>M. sexta</i> control (CON, black circles) and low glucose (LG, black diamonds) conditions, along with C2C12 CON (gray squares) and LG (gray triangles) samples for comparison (A–B). Statistically different values are compared with corresponding time 0 values for glucose (A), and between <i>M. sexta</i> CON and LG for lactate (B). For amino acid analysis, C2C12 low glucose condition was omitted (C–D). * p<0.05.</p

Cell type identification within heterogeneous cultures.

<p>Histological sectioning and H&E staining of a developing embryo 19 h post-ovipositioning (A). Dark regions are cross-sections of the embryo. The rest of the egg contains yolk granules and yolk cells. Yolk cells (vitellophages) are 20–50 µm in diameter and contain a single nucleus, stained dark purple (inset, day 0). These cells are also observed in our cultures, and tend to contain a single, large lipid droplet, as shown in red by Oil Red O staining (B). Myogenic cells are the dominant population present in long-term cultures (C–D). Positive staining of myotubes for insect muscle myosin heavy chain (green) on day 48 confirms mature muscle cell identity. Additionally, sarcomeric striations are visible. Nuclear staining with DAPI (blue) reveals multinucleation Scale bars are as noted.</p

Effects of juvenile hormone (JH) mimic, methoprene, on 20HE action and cell proliferation.

<p>Phase contrast images of cells cultured in 20 ng/mL 20HE and varying levels of methoprene (A–F). Total number of nuclei and BrDU-positive nuclei on day 2 and 6 for varying methoprene concentrations (G–H). * p<0.05, ** p<0.01. Scale bars are 50 µm.</p

Schematic of embryonic <i>M. sexta</i> cell isolation process.

<p>Schematic of embryonic <i>M. sexta</i> cell isolation process.</p

<i>M. sexta</i> muscle cell viability over time.

<p>Phase contrast (A, C, E) and LIVE/DEAD staining (B, D, E) of typical cultures grown in the absence of medium changes. For LIVE/DEAD images, green staining indicates live cells and red staining indicates dead cells. Representative images taken from cultures on day 28 (A–B), day 44 (C–D), and day 75 (E–F). Scale bars are 100 µm.</p

Cryopreservation and resuscitation of <i>M. sexta</i> embryonic cells.

<p>Phase contrast images of freshly isolated (A) and resuscitated cells, after two weeks of cryopreservation, using DMSO and glycerol as cryoprotectants (B–C)). All panels show day 18 results. Scale bars are 100 µm.</p