title: Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels.
abstract: In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (μmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (μprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-μprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS μprinted with FN, myogenic index, myotube width, and myotube length on μmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on μmolded gelatin hydrogels compared to FN-μprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Archana
authors.initials: A
authors.lastname: Bettadapur
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Gio C
authors.initials: GC
authors.lastname: Suh
authors.affiliation: Oxford Instruments Asylum Research, Santa Barbara, CA, 93117, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Geisse
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.|Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, 90033, USA.
authors.forename: Evelyn R
authors.initials: ER
authors.lastname: Wang
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Clara
authors.initials: C
authors.lastname: Hua
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Holly A
authors.initials: HA
authors.lastname: Huber
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Alyssa A
authors.initials: AA
authors.lastname: Viscio
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Joon Young
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Julie B
authors.initials: JB
authors.lastname: Strickland
authors.affiliation: Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, 90089, USA.|Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, 90033, USA.
authors.forename: Megan L
authors.initials: ML
authors.lastname: McCain
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D004129:Dimethylpolysiloxanes / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D005780:Gelatin / Q000737:chemistry / Q000378:metabolism*; D020100:Hydrogels / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018625:Microscopy, Atomic Force; D024510:Muscle Development; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods
chemical_list: D004129:Dimethylpolysiloxanes; D005353:Fibronectins; D020100:Hydrogels; C013830:baysilon; D005780:Gelatin