Biomaterials and Bioprinting of Skeletal Muscle Spheroids for use as Building Blocks for Engineered Muscle Tissue

Skeletal muscle tissue engineering has the potential to address key clinical and societal challenges such as traumatic muscle injury, our fundamental understanding of skeletal muscle development and disease, and the future of meat production <i>via</i> cell cultured meat. Preclinical animal models and monolayer culture have driven our understanding of skeletal muscle biology and differentiation. However, two-dimensional (2D) cell cultures lack complex cell-cell and cell-extracellular matrix (ECM) interactions that provide essential biochemical and biomechanical signals directing cell function <i>in vivo</i>. Engineered tissue must sufficiently model the complexity of native muscle, which has led to the innovation of advanced fabrication methods such as 3D bioprinting.<br/><br/>Bioprinting is a promising biofabrication technique for generating structured tissue due to the potential to precisely pattern multicellular constructs of relevant cell types. The most common bioprinting applications use monodisperse cells, which requires disruption of essential cell-cell and cell-matrix interactions. A growing body of research confirms the benefits of retaining the endogenous ECM to mimic the native extracellular environment and support cell function, hence exposing a key current limitation of bioprinting.<br/><br/>Spheroids are dense cellular aggregates that exhibit promise as building blocks for tissue engineering due to their retention of endogenous ECM, upregulated cytokine production, and increased cell-cell interactions. Spheroids are advantageous for use in tissue engineering due to their enhanced differentiation, angiogenic potential, and cell survival <i>in vitro </i>and <i>in vivo </i>compared with monodisperse cells. Spheroids have been fabricated from a wide variety of cell types yet literature on skeletal muscle spheroids, sometimes referred to as myospheres, is sparse. The study of myospheres has primarily been focused on understanding cell behavior of primary muscle cells, but their application in tissue engineering lags behind other tissues types such as bone, heart, and adipose, among others. While work utilizing muscle spheroids from immortalized cell lines has been limited, initial studies have shown promising results in regard to cell survival and differentiation. For example, C2C12 spheroids, once dissociated, exhibited higher proliferation, upregulated MyoD expression, and enhanced myogenic potential in both 2D and 3D culture. Furthermore, C2C12 spheroids have upregulated myogenic factors compared to monodisperse cells and can differentiate into aligned myotubes on electrospun substrates. However, little is known about how muscle cell spheroids function in contiguous matrices such as bioinks, representing a key information gap in the field.<br/><br/>We hypothesized that skeletal muscle cell spheroids will function as potent building blocks of muscle tissue when embedded in 3D microenvironments. We bioprinted skeletal muscle spheroids to assess cell function and tissue forming potential compared to monodisperse cells. We utilized 3D bioprinting as a proof-of-concept to determine whether bioprinting may adversely affect cell viability. Experiments were first performed using C2C12 spheroids before translating to more clinically and culinarily relevant primary bovine satellite cells. Both C2C12 and primary bovine SCs formed spheroids of similar sizes, remained viable after bioprinting, and exhibited similar spreading characteristics within an alginate matrix. When bioprinted with alginate as a bioink, spheroids of both cell types fused into larger tissue constructs over time and exhibited tissue formation potential similar to monodisperse cells. These data demonstrate that skeletal muscle spheroids are promising building blocks for muscle tissue and validate 3D bioprinting as a compatible fabrication technique. I will also highlight other studies illustrating the synergy of engineered biomaterials and muscle cell response.