Introduction

The development of 3D in vitro models of skeletal muscle has become a subject of increasing interest, with publications steadily growing in the past 10 years [1, 2]. These culture models are useful for studying muscle function in health and disease, or as screening platforms for clinical research. In order to mimic in vivo muscle physiology, they must recapitulate relevant features of skeletal muscle architecture and function, such as the formation of aligned and compacted muscle fibers, and contractile ability in response to electrical or chemical stimulation [3].

In order to obtain differentiated skeletal muscle cells, some models use primary myoblasts [4, 5] due to their close reproduction of in vivo phenotype; but this requires having a source for extraction of primary myoblasts, which increases animal experimentation. To solve this issue and adhere to the 3R strategy of animal experimentation, numerous immortalized myoblast cell lines have been generated, which can be used in in vitro platforms. One of the most used cell lines is the C2C12 mouse myoblast line [6], which has been extensively characterized and serves as a robust model of mouse skeletal muscle function [7, 8]. Nevertheless, human myoblasts are also needed for more accurate modeling of human skeletal muscle. Several immortalized human myoblast cell lines have been generated in recent years [9–11], from both healthy samples and neuromuscular disease patients.

Concerning culture type, as it occurs with many other tissues and organs, emerging 3D tissue engineering techniques have proven to be a more accurate way of modeling native tissue architecture and function in skeletal muscle. Using 2D culture is more straightforward than developing 3D culture systems, which has allowed scientists to obtain various 2D neuromuscular co-culture models [12–14]; but these models have relevant drawbacks. For instance, when using 2D-cultured myoblasts, differentiation, and subsequent spontaneous contractions of formed myotubes are known to cause detachment of cells from the substrate. To address this issue, it has been proposed to use a hydrogel overlay to ensure cell survival and support appropriate myotube contraction [11]. This 2D method has been largely used in our lab (S1 Fig) but lacked alignment of cells and myotubes adopted a random organization. Alternatively, surface modifications can improve attachment and alignment of individual myotubes [13], but do not recapitulate the formation of compacted bundles of myotubes similar to muscle myofibers.

When using 3D models, another advantageous feature is the ability to mimic the way that muscles are anchored to tendons, which connect the muscle to the bone. For this purpose, free-floating cell-laden hydrogel constructs have been developed, which can be anchored at both ends using different methods. One of the most popular methods is the use of flexible polydimethylsiloxane (PDMS) pillars around which the cells can attach [15–18]. This method has the advantage of allowing quantification of muscle contractile force, through observation of the flexible pillars’ deflection [19, 20] in response to muscle contraction. However, the microscale fabrication of these PDMS-based platforms is complex and time-consuming, often requiring use of cleanroom facilities for fabrication of molds by soft lithography. In addition, other types of anchoring methods without pillars, such as adding hydrogel anchors at the ends of a tubular mold [21, 22] suffer from a higher rate breakage or anchor detachment, which results in the loss of putatively valuable samples after long culture times. Finally, due to the large number of myoblasts needed to ensure proper myotube formation and the small chambers used for these purposes, cultures often display poor oxygen and nutrient diffusion in generated muscle fascicles, affecting cell survival.

As a simplified yet effective alternative, several publications have relied on the use of pieces of VELCRO^TM fabric as anchors for bioengineered muscle constructs [4, 5, 23, 24]. In this method, the loops on the VELCRO^TM successfully entrap the hydrogel-cell mixture, forming a robust anchoring site. Attaching the VELCRO^TM pieces can be achieved by simply gluing them to the culture support of choice. In particular, the simplest manufacturing method is the one published in [4], which directly glues VELCRO^TM pieces onto 6-well culture plates using PDMS glue. Nevertheless, in this method, the selected hydrogel composition (a mixture of Matrigel^TM and Collagen I) differs from the most commonly used hydrogel (Matrigel^TM and Fibrinogen) in other studies using myoblasts [5, 17, 24, 25], which was reported to show the best properties to support muscle differentiation and function in a comparative study on ECM composition [23]. In fact, in our experience, the Matrigel^TM/Collagen I based gels showed an excessive degree of compaction over time, which resulted in excessive rigidity, low oxygen and nutrient diffusion, increased cell death, and breakage of muscle bundles.

Therefore, we were interested in optimizing a simple culture system combining VELCRO™ anchors and the Matrigel^TM/Fibrinogen hydrogel composition. We noticed that, in anchor-based systems, this hydrogel is always used in combination with some type of mold coated with a hydrophobic Pluronic^® solution [5, 23] or bioprinted [17, 25] to achieve the desired shape. This is likely due to the lesser degree of compaction of this gel, which results in an inability to detach from the culture dish on its own, as compared to the Matrigel^TM/Collagen-based gel, which readily detaches with no coating [4]. Thus, we modified the protocol to ensure proper fascicle formation and contraction with easy manipulation and contraction analysis. Indeed, here we use three different kinds of stimulation and two different ways to quantify muscle response in our modified platforms. The protocol has been adapted to allow for individual imaging of muscle bundles, by using cell culture 35 mm ∅ Petri dishes.

The most relevant indicator of functional maturation in muscle cells is their contractile ability. In vivo, muscle contraction is initiated by motor neurons, which innervate muscles at the end-plate. In the vicinity of this area, neuron terminals branch into synaptic boutons releasing the neurotransmitter acetylcholine (ACh), which binds to nicotinic acetylcholine receptors (AChRs) on the muscle membrane [26]. This event causes rapid depolarization of the membrane, resulting in a large post-synaptic excitatory potential [26]. This mechanism of contraction implies that in vitro stimulation of muscle constructs can be achieved either by chemical stimulation with acetylcholine or other depolarizing agents (e.g., potassium chloride), or simply by applying electrical pulses to depolarize the cell membrane.

Electrical stimulation is widely used in in vitro models to study contractile response [20, 23, 25], as well as for maturation and hypertrophy of muscle constructs [27–29]. In this protocol, we use a simple graphite electrode setup for applying electrical pulses to individual plates. The muscle constructs generated with this protocol reproducibly respond to electrical stimulation, showing synchronic contractions. However, despite the effectiveness of electrical stimulation, this stimulus differs from physiological conditions, and may cause excessive depolarization of muscle cells. For this reason, we have also performed chemical stimulation with acetylcholine [5, 15]. In the absence of motor neuron co-culture, we have induced clustering of AChRs by the addition of agrin to the differentiation medium [5, 30].

As an alternative stimulation method, we have included the option of using optogenetic stimulation in our muscle constructs by modifying cultured myotubes to express the genetically encoded light-activated ion channel Channelrhodopsin-2 (ChR2) [22]. As this method is based on the selective activation of a single ion channel, optical stimulation is a gentler method to depolarize cells, as opposed to a general electrical stimulus. In addition, using light is also non-invasive, which reduces the possibility of contamination derived from the introduction of external electrodes or chemical solutions [20].

After the application of these three types of stimulation, we illustrate two different methods for evaluating muscle response, which are contraction analysis in bright-field time-lapse videos, or calcium imaging with the fluorescent calcium indicator Fluo-4 AM. The first method is based on the acquisition of sequential images of contraction using bright-field, from which displacement and contraction speed data is extracted in relation to pixel movement with the MUSCLEMOTION plugin for ImageJ [31, 32]. Thus, this method is compatible with all three proposed stimulation methods. Alternatively, we have also included the option to perform calcium imaging. Calcium changes can be easily analyzed by using a fluorescent calcium indicator such as Fluo-4 AM [33], or a genetically encoded calcium indicator (GECI) such as GCaMP6 [34]. In this protocol, muscle bundles are loaded with Fluo-4 AM and imaged during stimulation with a fluorescence microscope and blue light filter. Then, videos are processed using ImageJ to extract normalized fluorescence data from a set of cell ROIs of each muscle bundle, and quantify fluorescence increases that occur due to stimulation. In all, with the combination of these methods, this step-by-step protocol can serve as a practical guide for studying muscular activity in a versatile manner, while using a straightforward and reliable setup.

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io, updated February 11 2022, dx.doi.org/10.17504/protocols.io.b4qiqvue and is included for printing as S1 File with this article.

Expected results

In the following paragraphs, we describe some of the results that can be obtained using our protocol, as illustrated in Fig 1.

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Fig 1. Expected results.

A) Example of the generation of the muscle constructs. (i) Scheme illustrating the establishment of the muscle strip between the two VELCRO^TM pieces. Photograph of the construct shown in (ii). SEM analysis of the anchor microstructure can be seen in (iii). (iv) Low phase contrast photomicrograph illustrating the dense band of oriented myotubes. This orientation can be easily analyzed by using the ImageJ Directionality plugin (v and vi). B) Imaging of differentiation markers by immunostaining and confocal fluorescence microscopy: sarcomeric α-actinin (SAA) and Hoechst 33342 (i), MHC and Hoechst 33342 (ii) or α-BTX and Hoechst 33342 (iii). Note the generation of sarcomeric banding patterns in (i) and (ii), as well as end-plate formation (arrows in iii). C) Electrical stimulation. A scheme of the basic stimulation setup can be seen in (i). (iii) Example of the analysis of the myotubes contraction by using MUSCLEMOTION. In this analysis, the electrical stimulation was performed as indicated in (ii). D) Chemical stimulation and calcium imaging. Example of ROI selection with ImageJ in mytotubes incubated with Fluo4-AM (i). In this example, 10 ROIS are displayed, and the graph illustrating the calcium changes (indicated as ΔF/F₀) after ACh and KCl treatment is shown in (ii). (iii) Example of contraction analysis of a classical ACh treatment in our device, analyzed with MUSCLEMOTION. E) Optical stimulation. (i) Scheme of the basic setup for optogenetic stimulation of our devices using an inverted microscope, Pulser device, and LED light source. (iii) Example of the contraction analysis using the light exposure protocol illustrated in (ii) after their analysis using ImageJ. Scale bars: A) (ii) 1 mm, (iii) 1 mm, (iv) 200 μm. B) (ii) 15 μm (ii) 15 μm, (iii) 15 μm. D) (i) 100 μm.

https://doi.org/10.1371/journal.pone.0272610.g001

Supporting information

Acknowledgments

We thank Dr. Eduard Gallardo, from Neuromuscular BCN (Hospital de Sant Pau, Barcelona, Spain) for providing the AB1079 immortalized human myoblast cell line [10] and advice on the differentiation protocol. We also thank Dr. Ana López-Mengual for her advice on the calcium imaging protocol, and Dr. Arnau Hervera for advice on the generation of the ChR2⁺ myoblast cell line.