Screening method to identify hydrogel formulations that facilitate myotube formation from encapsulated primary myoblasts

Abstract Hydrogel‐based three‐dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of tissue specific cells embedded within a support material, such as a hydrogel. As hydrogel properties directly affect cell function, hydrogel composition is often tailored to the cell type(s) of interest and the functional objective of the model. Here, we develop a parametric analysis and screening method to identify suitable encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence‐based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.

encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence-based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.

K E Y W O R D S
3D culture, immunostaining, muscle cells, parametric analysis, primary murine myoblasts, rapid screening INTRODUCTION Three-dimensional (3D) cellular models are increasingly applied to study cell and tissue biology or pathology outside of the body as well as to test potential therapeutic modalities. 1 3D cellular models are often composed of one or more cell types embedded within a synthetic or natural hydrogel that is designed to mimic relevant aspects of the extracellular matrix (ECM). 2,3 Synthetic or semisynthetic hydrogels are particularly attractive as they can be designed with similar physical and biochemical properties as the ECM, while providing a reproducible scaffold to investigate physiology ex vivo. 4 However, the properties of a hydrogel scaffold can influence different cell types in unique ways. For example, soft and macroporous hydrogels have been designed for neuronal cell growth and enzyme-degradable hydrogels have been engineered for intestinal organoid culture. 5,6 Therefore, hydrogel compositions are often tailored to the cell or tissue type and for the specific biological goal of the 3D model. Skeletal muscle is one specific tissue of interest for the design of 3D cellular models, which can be used to study myogenesis and investigate the pathophysiology of common muscle disorders. 7 Skeletal muscle is comprised of contractile myofibers and innervating motor neurons embedded within a surrounding vascularized matrix composed of connective tissue cells. During muscle development and repair, myogenic progenitor cells differentiate to myoblasts, which fuse to form myotubes, further maturing into contractile myofibers. 8 Myoblasts can be sourced using the C2C12 cell line or through the isolation and culture of primary myoblasts from animal or human tissue. 9,10 C2C12 is an immortalized cell line that shows lower expression of muscle specific markers as compared with primary myoblasts. 11 One critical requirement for these models is the functional fusion of myoblasts into contractile myotubes, which comprise the base unit of skeletal muscle and are useful for studying myogenesis and muscle biology ex vivo. Researchers have encapsulated myoblasts in a range of natural, synthetic, or semisynthetic hydrogels, including collagen, fibrin, Matrigel, gelatin, alginate, and poly(ethylene glycol) (PEG)-based gels. In these experiments, it was found that several matrix parameters, such as gel stiffness, cell density, and molecular effectors, such as matrix proteins or growth factors, can influence myoblast proliferation as well as myotube formation and function. 12 C2C12 myoblasts proliferation increased in alginate gels with low degradability; however, myotube fusion was favored in gels with high degradability. 13 Cell density influenced contractile force generation of primary murine myoblasts in collagen gels; increased contractile force was observed with 6 × 10 6 cells ml −1 as compared with 4 or 8 × 10 6 cell ml −1 . 14 In another work, contractile muscle tissue models were bioprinted using myogenic progenitor cells (~5 × 10 6 cells ml −1 ) in methacryloyl gelatin (GelMA). 15 During 2D culture, primary myoblasts are often cultured on Matrigel-coated tissueculture polystyrene, as Matrigel facilitates myoblast adhesion. 16 To improve myoblast function in 3D, researchers have also encapsulated primary human skeletal muscle cells in hydrogels containing a mixture of collagen and Matrigel. 17 Together, these studies illustrate that primary myogenic cells, or myogenic cell lines, can be encapsulated within hydrogel materials to develop 3D models of skeletal muscle; however, standardized conditions do not exist for developing 3D models of skeletal myotubes.
Generally, the selection of hydrogel compositions for 3D cellular models, such as a model of skeletal myotube formation and function, is carried out through a series of iterative encapsulation experiments, based on known hydrogel materials and guidance from the literature.
While this trial-and-error approach is often able to identify suitable material compositions, the process can be time consuming and may overlook suitable mechanical properties and biochemical cues beneficial to the specific cell of interest.
In this work, we applied a parametric analysis to identify appropriate hydrogel compositions for myotube formation from primary murine myoblasts in 3D GelMA hydrogels ( Figure 1). In particular, we investigated the effect of GelMA weight percent (wt%), cell density, and Matrigel concentration on myotube formation. This was identified by myosin heavy chain (MyHC) immunocytochemistry, 7 days after initiating differentiation. To improve the throughput of the parametric analysis, we developed a fluorescence-based assay using a multi-well plate reader to rapidly quantify MyHC expression in the different gel formulations.
The rapid screen refined the material compositions that facilitated myotube formation, which were confirmed by fluorescence microscopy and optical imaging of spontaneously contracting myotubes. We observed that increasing GelMA wt% had the strongest inhibitory effect on myotube formation; that the presence of Matrigel improved myotube formation as compared with pure GelMA samples; and that cell density did not significantly affect myotube formation. In total, the combination of design of experiments using parametric analysis and facile plate reader-based assays offers an attractive approach to screen for hydrogel compositions that are suitable for engineering 3D cellular models.

RESULTS AND DISCUSSION
In preliminary experiments to form myotubes in 3D hydrogel culture, we encapsulated primary murine myoblasts in GelMA and matrix metalloproteinase (MMP) degradable PEG-based hydrogels ( Figure S1).
We observed that myoblasts proliferated more in GelMA compared with PEG-MMP gels. Limited myotube formation occurred in GelMA hydrogels, and myotube formation increased by supplementing GelMA hydrogels with 1.2 mg ml −1 Matrigel ( Figure S1). To determine if hydrogel formulation could be used to increase the extent of myotube formation, a parametric analysis was designed to screen the effect of GelMA hydrogel formulation on myotube formation A core finding of this study is that experimental design and common plate reader-based assays can be used to guide hydrogel formulation selection for 3D cell culture. Here, we found that myotube formation from primary myoblasts was favored in lower wt% GelMA hydrogels supplement with Matrigel. However, we did not identify the specific properties of these gels that facilitate or drive myogenesis in these samples. Lower GelMA wt% affects both the stiffness of the network as well as the bioactive ligand density. The presence of Matrigel can also affect matrix stiffness, porosity, and bioactive ligand density. Therefore, additional controlled studies should be carried out to isolate the specific signals that encourage myogenesis in 3D hydrogels. Understanding the specific extracellular effects that drive myogenesis could further guide the development of biomaterials for skeletal muscle repair and the design of in vitro cellular models.

CONCLUSION
In total, these findings suggest that low wt% GelMA hydrogels with Matrigel as an additive comprise suitable hydrogel formulations to facilitate myotube formation from primary murine myoblasts in 3D.
Further, we demonstrated that commonly available fluorescencebased plate readers can be used to develop rapid screening methods

MATERIALS AND METHODS
All materials were purchased from Sigma-Aldrich unless stated otherwise.

Preparation and characterization of methacryloyl gelatin
Gelatin was functionalized with methacrylate moieties following previously published protocols. [18][19][20] Briefly, methacrylic anhydride (12 g; 276685) was added to gelatin (20 g; 300 bloom, type A; G2500-100G) dissolved in deionized water (50 C) and allowed to react for 120 min. The reaction solution was then cooled to room temperature and unreacted methacrylic anhydride was removed by centrifugation (3,500 RCF for 5 min). The supernatant was dialyzed (SnakeSkin TM dialysis tubing, 3.5 MWCO, Ref 88244, Thermo Scientific) and lyophilized. The degree of functionalization of the obtained powder was characterized via NMR spectroscopy and determined to be~45% ( Figure S7).

Cell culture and encapsulation
Primary murine myoblasts were isolated from C57BL/6J mice following established protocols. 21 The mice were housed at ETH Zürich, tis- Immunostaining and plate reader screening To measure the fluorescence signal in the hydrogel disks, we performed a 30 × 30 raster scan of each well in the 96-well plate (Excitation: 544/20 nm; Emission: 575/20 nm). From each raster scan, two areas (4 × 4) were chosen (area of interest near the center of the gel and the background from the well and surrounding medium). The fluorescence value of each measurement point in these areas were averaged and the difference of these two values (center of gel less the background) was taken as the fluorescence value for that particular hydrogel sample ( Figure S6). Similarly, a value was assigned for the blank gels (same hydrogel composition but without cells). This value was then subtracted from the fluorescence value of the corresponding hydrogel sample will cells to calculate the representative MyHC fluorescence.

Microcopy
Contracting myotubes that formed within the samples were imaged

Statistical analysis
Three-way ANOVA was conducted to analyze the results obtained from the fluorescence-based plate reader assay. GelMA wt%, cell density, and Matrigel concentration were chosen as independent continuous variables. Another ANOVA was conducted considering Matrigel concentration as a categorical variable. A MATLAB code was used to calculate the influence of these factors on the plate reader values.
More details about the statistical analysis can be found in Supporting Information.