The fusion index is a key indicator for quantifying the differentiation of a myoblast population, which is often calculated manually. In addition to being time-consuming, manual quantification is also error-prone and subjective. Several software tools have been proposed for addressing these limitations, but suffer from various drawbacks including unintuitive interfaces and limited performance. Here, we describe MyoFInDer, a Python-based program for the automated computation of the fusion index of skeletal muscle. At the core of MyoFInDer is a powerful artificial intelligence-based image segmentation model. MyoFInDer also determines the total nuclei count and the percentage of stained area, and allows for manual verification and correction. MyoFInDer can reliably determine the fusion index, with a high correlation to manual counting. Compared to other tools, MyoFInDer stands out as it minimizes the inter-operator variability, minimizes process time and displays the best correlation to manual counting. Therefore, it is a suitable choice for calculating fusion index in an automated way, and gives researchers access to the high performance and flexibility of a modern artificial intelligence model. As a free and open-source project, MyoFInDer can be modified or extended to meet specific needs.

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling.

Objective. Human induced pluripotent stem cells (hiPSCs) represent a clinically relevant cell source for skeletal muscle tissue engineering, yet transgene-free maturation remains challenging. Moreover, the potential of spheroids as building blocks for muscle engineering remains poorly defined. This study evaluated whether hiPSC-derived myogenic spheroids enhance biofabrication outcomes in an in vitro bio-artificial muscle (BAM) system compared to a conventional single cell approach. Methods. CD56+ hiPSC-derived myoblasts were generated using a transgene-free protocol and differentiated toward 2D myotubes. In 3D, myoblasts were aggregated into 140-200 µm spheroids. Spheroids or single cells were encapsulated in a fibrin-based BAM, kept under tension using metal pins. Non-encapsulated spheroids and BAMs were cultured up to 15 days and evaluated for myosin heavy chain (MYH)+ organization, transcriptional profile, and contractile function. Results. In 2D, MYH+ myotubes showing multinucleation and, occasionally, sarcomere striation were formed. Spheroids were successfully generated, increased in size and circularity, and showed an enhancing MYH positivity, particularly during the proliferation phase, and displayed a fetal/perinatal myogenic state. In BAMs, spheroids promoted MYH positivity and established larger continuous MYH+ structures. Transcriptional profiles of spheroid and single cell BAMs were broadly similar. Tetanic forces were generally higher in spheroid BAMs; however, this difference did not reach statistical significance. Conclusion. hiPSC-derived myogenic spheroids advance MYH expression, but terminal maturation is constrained, likely due to their intrinsically isotropic nature. Within BAMs, spheroids enhance MYH+ myotube formation, but do not overcome limitations in myoblast fusion. Incomplete spheroid outgrowth and poor control over their spatial distribution are important challenges for their integration. Future work should focus on improving hiPSC differentiation efficiency, implementing spheroids in earlier differentiation stages, and optimizing spheroid outgrowth and positioning, alongside incorporating remaining muscle components as fibroblasts and vascular cells. This will refine whether spheroid-based assembly can add value in specific biofabrication contexts.

Skeletal muscle tissue engineering aims to create bioartificial muscle (BAM) constructs that recapitulate key structural and functional features of native skeletal muscle for applications including regenerative medicine, disease modeling, and drug screening. While most BAM models are generated using myoblasts, multicellular building blocks such as myogenic spheroids may better mimic native cell-cell interactions and microarchitectural organization. In this study, we compared the use of primary human myogenic spheroids with single cells for skeletal muscle tissue engineering. Myogenic spheroids composed of primary human skeletal myoblasts were generated in a high-throughput manner using nonadhesive micropatterned agarose chips. Single cell myoblasts and myogenic spheroids were subsequently embedded in fibrin hydrogels and cast into silicone molds between two metal pins serving as anchor points. Results showed that maturation stage of the spheroids prior to encapsulation significantly influenced BAM morphology after 7 days. Early maturation stage-spheroids promoted enhanced cellular outgrowth, myoblast fusion, and increased myotube diameter compared with more mature spheroids, while the spheroid size did not significantly affect construct formation. Direct comparison between spheroid-based and single-cell BAMs revealed distinct phenotypic differences. Spheroid-based constructs generated fewer but larger multinucleated myotubes with higher fusion indices, whereas single-cell constructs produced a greater number of smaller myotubes. Despite these differences in morphology, no significant changes were observed in the expression of myosin heavy chain isoforms, indicating comparable myogenic differentiation under the tested conditions. Together, these findings demonstrate that primary human myogenic spheroids represent an effective alternative building block for skeletal muscle tissue engineering and provide insight into how cell assembly strategies influence muscle tissue formation.