Supplementary Information. Induction of functional tissue-engineered skeletal muscle. constructs by defined electrical stimulation

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1 Supplementary Information Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation Akira Ito 1,*, Yasunori Yamamoto 2,*, Masanori Sato 1, Kazushi Ikeda 2, Masahiro Yamamoto 1, Hideaki Fujita 3,, Eiji Nagamori 3,, Yoshinori Kawabe 1 & Masamichi Kamihira 1,2 1 Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 7 Motooka, Nishi-ku, Fukuoka , Japan 2 Graduate School of Systems Life Sciences, Kyushu University, 7 Motooka, Nishi-ku, Fukuoka , Japan 3 Toyota Central R&D Laboratories Inc., 1-1 Yokomichi, Nagakute, Aichi , Japan Present address: Laboratory for Comprehensive Bioimaging, Riken Qbic, Furuedai, Suita, Osaka , Japan Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka , Japan * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M. K. ( kamihira@chem-eng.kyushu-u.ac.jp) 1

2 Figure legends Supplementary Figure S1: Schematic of the magnetic forced-based tissue engineering (Mag-TE) technique. To magnetically label C2C12 cells, the cells were seeded in tissue culture dishes containing ml of culture medium in the presence of magnetite cationic liposomes (MCLs) and incubated for 8 h to allow for MCL uptake. A polycarbonate cylinder was placed at the center of the well of a 2-well ultralow-attachment culture plate. MCL-labeled cells were seeded into the ring-shaped gap, and a magnet was placed underneath the plate. Next, the cells were cultured in growth medium for 12 h to form a ring-shaped cellular construct. After culture for 12 h, the extracellular matrix solution consisting of type I collagen and Matrigel was poured into the well and then quickly replaced with culture medium to coat the tissue construct with extracellular matrix. After h, the ring-shaped tissue construct was removed from the polycarbonate cylinder and hooked around two stainless-steel minutien pins on a silicone rubber sheet in a tissue culture dish. To induce myogenic differentiation, the tissue constructs were then cultured in differentiation medium. Supplementary Figure S2: In vitro skeletal muscle tissue fabricated by Mag-TE (day ). (a) Macroscopic appearance (left) and bright-field micrographs (right) of tissue constructs fabricated by a gel-based technique (Gel, upper) or the Mag-TE technique (Mag, lower). To visualize the Gel tissue construct, trypan blue dye was added to the extracellular matrix solution (upper left). The Mag-tissue construct is a black-brown color because of the magnetite (lower left). Representative hematoxylin and eosin (H&E) staining of Gel tissue constructs showing the outermost cell layer and an acellular central core (upper right). Representative H&E staining of Mag tissue constructs showing a thin, densely packed cell layer (lower right). (b) A representative peak of the twitch force generated by the tissue construct using a single electric pulse (voltage:.83 V/mm, width: ms). Blue line: Gel 2

3 tissue construct. Red line: Mag tissue construct. (c) Specific force of tissue constructs on day (voltage:.83 V/mm, width: ms). The data are expressed as mean ± SD of three constructs. *P <.5. Supplementary Figure S3: The percentage of peak twitch force of the in vitro skeletal muscle tissues on day. To determine %Pt, peak twitch force was measured using a single electric pulse at.83 V/mm and ms. The %Pt for each set of electric stimulation parameters was determined by measuring the peak twitch force. Data are shown as mean ± SD of three constructs. Supplementary Figure S: The peak twitch force generation of EPS-treated tissue constructs on day 7. The tissue constructs were cultured with continuous EPS at various frequencies (.5, 1, or 2 Hz), voltages (.1,.3, or.5 V/mm), and pulse widths (2,, or ms) for 3 days starting on day, and peak twitch force was measured on day 7 using a single electric pulse (voltage:.83 V/mm, width: ms). The data are expressed as mean ± SD of three constructs. Supplementary Figure S5: The percentage of peak twitch force of the in vitro skeletal muscle tissue constructs on day 7. Comparisons at ~2, ~5, and ~8%Pt are shown. The data are expressed as mean ± SD of three constructs. Columns that were statistically indistinguishable (P >.5) are marked with the same letter. Supplementary Video 1: Contraction of the in vitro skeletal muscle tissue constructs cultured without EPS. On day 1, the tissue construct was stimulated with the electric pulses at.83 V/mm, ms, and 1 Hz. 3

4 Supplementary Video 2: Contraction of the in vitro skeletal muscle tissue constructs cultured with continuous 1 Hz EPS at.3 V/mm and ms for days starting on day (day 1). On day 1, the tissue construct was stimulated with electric pulses at.83 V/mm, ms, and 1 Hz.

5 Cationic liposome Magnetite nanoparticle Magnetite cationic liposome (MCL) C2C12 cell Magnetically labeled C2C12 cell MCL C2C12 cell Magnetically labeled cells Magnetically labeled C2C12 cell Polycarbonate cylinder Magnet Extracellular matrix solution Silicone rubber sheet Pin Supplementary Fig. S1 Ito et al.

6 (a) Gel Gel mm 2 μm Mag Mag mm μm (b) Force (μn) (c) Specific force (μn/mm 2 ) * Time (s) Gel Mag Supplementary Fig. S2 Ito et al.

7 Percentage of peak twitch force (%) (ms) (V/mm) Supplementary Fig. S3 Ito et al.

8 5 Force (μn) 3 2 (ms) (V/mm) (Hz) Supplementary Fig. S Ito et al.

9 Percentage of peak twitch force (%) a a b b c c (V/mm) (ms) ~2%Pt ~5%Pt ~8%Pt Supplementary Fig. S5 Ito et al.