Contractile force assessment methods for in vitro skeletal muscle tissues

  1. Camila Vesga-Castro
  2. Javier Aldazabal
  3. Ainara Vallejo-Illarramendi  Is a corresponding author
  4. Jacobo Paredes  Is a corresponding author
  1. University of Navarra, Tecnun School of Engineering, Manuel de Lardizábal, Spain
  2. University of Navarra, Biomedical Engineering Center, Campus Universitario, Spain
  3. Group of Neurosciences, Department of Pediatrics, UPV/EHU, Hospital Donostia - IIS Biodonostia, Spain
  4. CIBERNED, Instituto de Salud Carlos III, Ministry of Science, Innovation, and Universities, Spain
7 figures and 2 tables


Skeletal muscle structure and requirements for contractile force production.

(Top) Summary of the main requirements to enable contractility in skeletal muscle in vitro models. (Middle) Representation of the muscular hierarchy and (bottom) summary of contractile stimuli and contraction profiles.

Cross-sectional area (CSA) in 2D and 3D muscle models.

(A) Myotube CSA estimated as an elliptical shape from the thickness and the width of the cell. (B) CSA of 3D muscle constructs can be estimated by approximation to different shapes (circle, in left panel), or calculated from immunohistochemical sections. Effective-CSA is known as the area occupied by myotubes (red area in the right panel).

Cantilever deflection setup.

(A) The beam deflects due to myotube contraction (Left). In this case, cantilever deflection is interrogated by a laser beam and detected using a photodetector (Right). Commonly, cantilever arrays are made of Silicon (Si) or PDMS. Different coatings (FN, laminin, collagen I) have been tested to improve cell attachment and longer culture times. (B) Human myotubes on silicon cantilevers in bright field, top view (top) and immunostained for Myosin Heavy chain, side view (above). Scale bar: 50 µm. (C) Representative images from healthy and DMD myotubes at baseline (i and iii) and peak stress (ii and iv). Blue rectangles represent film length. Red lines represent the tracking of the film edge. Yellow arrows represent the distance between the projected film length and the unstressed film length. The yellow horizontal lines represent the change in projected film length from baseline stress (top bar) to peak stress (bottom bar).

© 2014, Elsevier. Figure 3B is reprinted with permission from Figure 1 from Smith et al., 2014. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

© 2016, Nesmith et al. Figure 3C is reprinted with permission from Figure 5B from Nesmith et al., 2016 (published under a CC BY-NC-SA 3.0 license). It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

Post Deflection features.

(A) In vitro skeletal muscle is grown between two micropost which serve as anchors (tendons). As muscle contracts in response to a stimulus, posts bend proportionally. By tracking these displacements and knowing the mechanical characteristics of the platform, the force exerted by the muscle can be quantified. (B) Micropost displacement due to miniature bioartificial muscle (mBAM) contraction in response to a maximum tetanic electrical stimulus. Scale bar: 100 µm. (C) Formation of human skeletal muscle micro-tissue (hMMTs). Phase-contrast images depicting the remodeling of the ECM by human myoblast over time. Muscle construct immunostained (2 weeks) for sarcomeric α-actinin (SAA, red) and counterstained with DRAQ5 (1, 5-bis{[2-(di-methylamino)ethyl]amino}–4, 8-dihydroxyanthracene-9, 10-dione) nuclear stain in blue. Scale bar: 500 µm. Reprinted from Figure 2A and C from Afshar et al., 2020.

© 2008, John Wiley and Sons. Figure 4B is reprinted with permission from Figure 4A from Vandenburgh et al., 2008 with permission from John Wiley and Sons. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

Force transducers.

(A) In vitro 3D tissue is grown between two anchors. To assess contraction force, one of its sides is connected to a force transducer which will evaluate the force exerted by the muscle due to stimuli. (B) Representative contractile properties of hPSC-derived iSKM bundles. TRiPS-derived bundle (4 weeks) shows increases in contractile force with an increase of stimulation frequency up to the formation of tetanic contraction. Specific force and tetanic-to-twitch ratio of H9 and TRiPS-derived bundle (2 weeks) and (C) (Left) two-week differentiated iSKM bundles pair anchored within a nylon frame. (Right) Representative immunostaining of dense, uniformly distributed myotubes in bundle-CSA. Panel B reprinted from Figures 3A, B and 4A from Rao et al., 2018.

Overview of the different techniques used to measure contractile force in vitro.

* Represents de % of studies that have performed this measurement.

Functional characteristics of in vitro 2D and 3D skeletal muscle tissues from C2C12 and human sources (immortalized, iPSC and primary myoblast).

(A) Whole cross-sectional area (CSA) of muscle tissues. (B) Tetanic-to-Twitch ratio was calculated from data within the same study, except for bar with a diagonal pattern in post deflection, which was calculated from two different studies. (C) Twitch and Tetanic specific force measure in the three platforms for C2C12 constructs and (D) Human source. Data is presented as mean ± SEM. *p < 0.05, unpaired t-Test.


Table 1
Maximum contractile force data from in vitro muscle models measured by the three main platforms.
Cell sourceEvaluation timeSizeCSA(mm2)Twitch contractionTetanic contractionTetanic-Twitch Ratio*References
CF (µN)sF (kN/m²)CF (µN)sF (kN/m²)
C2C12 myoblasts (mouse)Day 750 μm (Width)i
33 µm (Thickness)*
0.001308*0.54 ± 0.020.41*1.01 ± 0.140.77*1.87Fujita et al., 2010
Rat myoblasts (embryonic)Day 10–1322.5 µm (Width)i
10 µm (Thickness)
0.000176*0.23*1.3______Wilson et al., 2010
C2C12 myoblasts (mouse)Day 612.75 µm (Width)*
8–9 µm (Thickness)
0.0000851*0.80*9.4 ± 4.6______Sun et al., 2013
Rat myoblasts (embryonic)Day 12–1411.7–23.4 μm (Width)
7.9–13 μm (Thickness)
Stoney’s Eq.
Stoney’s Eq.
______Pirozzi et al., 2013
Primary human myoblastDay 2310 µm (Width)i
6.67 µm (Thickness)*
0.000052*0.14g2.69*______Smith et al., 2014
Rat myoblasts (adult)Day 4–716.74 µm (Width)*
11.16 µm (Thickness)g
0.000146*0.17g1.15*______McAleer et al., 2014
Human myoblastsDay 3–612.11 μm (Width)g
8.07 µm (Thickness)*
0.0000767*0.78*9.98g______Nesmith et al., 2016#
Human induced pluripotent stem cellDay 1411.82 μm (Width)g
10.35 μm (Thickness)g
0.0000961*0.38*3.98g______Badu-Mensah et al., 2020
Human induced pluripotent stem cellDay 10–119.30 μm (Width)g
6.2 μm (Thickness)*
0.0000452*0.12 ± 0.022.65*______Guo et al., 2020
Chick myoblasts3 weeks11.24 µm (Width)g
7.49 μm (Thickness)*
0.0000661*1.44*21.89g3.31*50g2.28Santoso et al., 2021
C2C12 myoblasts (mouse)16.30 µm (Width)g
10.86 μm (Thickness)*
Human myoblasts14.02 µm (Width)g
9.34 μm (Thickness)*
Human induced pluripotent stem cellDay 7–1022.5 μm (Width)*
15 μm (Thickness)
0.000265*0.26*0.986g0.521.986g2.01Al Tanoury et al., 2021
Primary Mouse myoblastsDay 1–122 mmi3.14*____42.5g13.53*__Vandenburgh et al., 2008#
C2C12 myoblasts (mouse)Day 140.14 ± 0.01 mm0.0125 (active)
0.0012 (effective)
1.4*0.11 (active)*
1.12 (effective)*
__Sakar et al., 2012
C2C12 myoblasts (mouse)Day 60.32 mmi0.079*____57.5 ± 12.80.72*Shimizu et al., 2017#
Primary human myoblastsDay 110.85 mm*0.566i79.44i0.14*428.57i0.76*5.42Mills et al., 2019
Derived-Myoblasts from Human Dermal FibroblastDay 4–100.30 mmi0.120*____12.2 ± 5.30.10*__Shimizu et al., 2020
Primary human myoblastsDay 7–140.71 mm*0.395*____192*0.49*__Afshar et al., 2020
Immortalized human myoblastDay 80.4 mm0.125*____28.5 ± 10.50.23__Nagashima et al., 2020
Immortalized human myoblastDay 7–140.47 mm*0.17 ± 0.03200 ± 401.171100 ± 3006.475.52Hofemeier et al., 2021
Immortalized human myoblastDay 100.49 mmi0.189*118.01g0.62*201.89g1.07*1.72Ebrahimi et al., 2021
Rat myoblasts (adult)Day 31 ± 40.49 ± 0.04 mm0.188*215 ± 261.14*440 ± 452.9 ± 0.52.54Dennis and Kosnik, Ii, 2000
Rat myoblast
(Extensor digitorum longus)
Day 32 ± 40.17 mm*0.024 ± 0.009162 ± 1256.75*281 ± 21811.70*1.73Dennis et al., 2001
Rat myoblasts3 weeks0.18 ± 0.01 mm0.0246*329 ± 26.313.37*805.8 ± 5532.752.45Huang et al., 2005
Rat myoblastsDay 16–180.25 mmg0.048*102g2.12*212g4.41*2.08Larkin et al., 2006
C2C12 myoblasts (mouse)Day 5–80.21 mm*0.0978i71.39*0.73 ± 2.1386.06*0.88 ± 0.481.20Fujita et al., 2009#
C2C12 myoblasts (mouse)Day 2–170.2 mm*0.031*33.21.06______Yamamoto et al., 2011#
Rat myoblasts (neonatal)Day 142.7 ± 0.18 mm (Bundle)5.72 (Bundle)*1680 ± 3200.29*
5.5 ± 0.6 (effective)
2840 ± 5000.50*
9.4 ± 0.7 (effective)
1.72Hinds et al., 2011
C2C12 myoblasts (mouse)Day 70.40 mm0.13*18.3 ± 2.40.15*34.5 ± 2.80.276*1.84Sato et al., 2013#
Rat myoblasts2 weeks1.38 mm (Bundle)*
0.9 mm (F-actin+) *
1.50 (Bundle)g
0.63 ± 0.05
17830 ± 100011.89 (Bundle)*
28.30 (F-actin+)*
28800 ± 93019.2 (Bundle)*
43.39 ± 3.82
1.61Juhas and Bursac, 2014#
Primary Human myoblast4 weeks2.5 mmi4.91*701g0.14*1460g0.30*2.14Madden et al., 2015
C2C12 myoblasts (mouse)Day 140.5 ± 0.08 mm0.19*81.26g0.42*151.37g0.79*1.88Ikeda et al., 2016#
C2C12 myoblasts (mouse)3 weeks0.6 mmi0.28*166.3 ± 59.40.59*______Nakamura et al., 2017
hPSC derived human myoblasts2 weeks0.42 mmi0.14*140g1.04402g3.00*2.88Rao et al., 2018
C2C12 myoblasts (mouse)Day 140.98 mmi0.756*48.39 ± 3.490.0647.74 ± 0.310.061Capel et al., 2019
Primary Human Myoblast2 weeks0.62 mmi0.30*1700 ± 1305.70*3400 ± 18011.40*2Khodabukus et al., 2019
hPSC derived human myoblasts4 weeks0.28 mmi0.06*1393 ± 34223.21*2924 ± 51748.73*2.09Xu et al., 2019
C2C12 myoblasts (mouse)10 Days0.99 mm0.77*1360 ± 2101.77*1930 ± 1202.50*1.41Akiyama et al., 2021
Primary Human MyoblastDay 17–191.39 mmi1.51*____175*0.13*__Alave Reyes-Furrer et al., 2021
  1. *Recalculated data; gData extract from a graph; iData extract from an image; #Studies with where maximal instantaneous CF data was used.

Table 1—source data 1

Additional information specific to the experimental setup and stimulation parameters.
Table 2
Summary of several skeletal muscle disease models in post deflection and force transducer platforms.

Key parameters like drugs, change in CF (%∆CF) and other observed effects are detailed.

Disease modelDrugPlatformCell source%∆CF(Dose)Observed effectReference
AtrophyDexamethasonePost deflectionC2C12 myoblast (mouse)–53%
(100 µM)
Increase in the expression of Atrogin-1 (2.6) and MuRF-1 (2.2)
Decrease in number of fibers with striation patterns (20% vs 8%)
Shimizu et al., 2017
Immortalized human myogenic cells–57%
(100 µM)
Increase in the expression of Atrogin-1 and MuRF-1.
Expression of FOXO3 and KLF15
Nagashima et al., 2020
Primary Human myoblast–85%
(10 nM)
Dose-dependent decrease in myotube widthAfshar et al., 2020
C2C12 myoblast (mouse)–48%
(1 mM)
Yoshioka et al., 2020
Force transducerPrimary Human myoblast–67%
(25 µM)
Decrease in myotube diameter (25 µM, 12%)
Decrease in effective-CSA (60%)
Decrease in injury biomarkers CK and LDH
Khodabukus et al., 2020
C2C12 myoblast (mouse)–70%
(100 µM)
Decrease in myotube-CSA (37%)Aguilar-Agon et al., 2021
HypertrophyIGF-1Force transducerRat myoblast+ 31%
(75 ng)
Increase in CF (75 ng, 31%)
Slow Time to peak twitch force (25 ng, 26%)
Huang et al., 2005
Primary Human myoblast+ 28%
(0.5 mg/ml)
Increase in myotube diameter (0.5 mg/ml, 21%)
Increase in injury biomarkers CK and LDH
Khodabukus et al., 2020
Post deflectionPrimary Mouse myoblast+ 66%
(100 ng/ml)
Increase in fiber-CSA (41%)Vandenburgh et al., 2008
Immortalized control C57 and mdx myoblast+ 93%
(0.01 µM)
Vandenburgh et al., 2009
C2C12 myoblast (mouse)+ 25%Increase in CF in Dex-induced atrophic tissues (45%*)Shimizu et al., 2017
Derived myoblast from human dermal fibroblast+ 72%
(100 ng/ml)
Decrease in CF in non-cryopreserved cells (100 ng/ml, 79%*)Shimizu et al., 2020
Statin-induced myopathy
LovastatinForce transducerPrimary Human myoblast–75%
(2 µM)
Dose-dependent lipid accumulationMadden et al., 2015
Primary Human myoblast–53%
(5 µM)
Dose-dependent lipid accumulationAnanthakumar et al., 2020
Post deflectionImmortalized human myogenic cells–75%
(2 µM)
Increase in the expression of Atrogin-1 and MuRF-1.Nagashima et al., 2020
CerivastatinForce transducerPrimary Human myoblast–50%
(50 nM)
Decrease in CF (50 nM, 50%*)
Dose-dependent lipid accumulation
Madden et al., 2015
Primary Human myoblast–85%Reduction in myotube diameter
Dose-dependent decrease in injury biomarkers CK and LDH
Vandenburgh et al., 1996
Human Skeletal myoblast–40%
(100 nM)
Decrease in CF
Decrease in passive force
Myofibers Sarcomere degradation
Zhang et al., 2018
Post deflectionPrimary Human myoblast–62%
(10 nM)
Decrease in CF (10 nM, 62%*)
Dose-dependent decrease in myotube width
Alave Reyes-Furrer et al., 2021
  1. *Recalculated data.

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  1. Camila Vesga-Castro
  2. Javier Aldazabal
  3. Ainara Vallejo-Illarramendi
  4. Jacobo Paredes
Contractile force assessment methods for in vitro skeletal muscle tissues
eLife 11:e77204.