TY - JOUR
T1 - High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System
AU - Vonk, Leander A.
AU - Esen, Osman
AU - Yuen, Michaela
AU - Kirby, Tyler J.
N1 - Funding Information: The authors would like to thank Sylvia Bogaards, Sanna Luijcx, Valentijn Jansen, Michiel Helmes, and Emmy Manders for their technical expertise in helping develop this protocol. This work was supported by awards from the Muscular Dystrophy Association (Development Award MDA603238 to T.J.K), the Dutch Cardiovascular Alliance (Talent Grant to T.J.K), and the National Health and Medical Research Council (NHMRC, Australia; Fellowship APP1121651 to M.Y). Publisher Copyright: © 2023, Journal of Visualized Experiments. All rights reserved.
PY - 2023/5/5
Y1 - 2023/5/5
N2 - In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.
AB - In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.
UR - http://www.scopus.com/inward/record.url?scp=85159772248&partnerID=8YFLogxK
U2 - https://doi.org/10.3791/65103
DO - https://doi.org/10.3791/65103
M3 - Article
C2 - 37212577
SN - 1940-087X
VL - 2023
JO - Journal of visualized experiments : JoVE
JF - Journal of visualized experiments : JoVE
IS - 195
M1 - e65103
ER -