For decades, two-dimensional cell culture has been regarded as a major tool in cellular and molecular biology due to its simplicity, reproducibility and reliable nature. However, it is now recognized that 2D cell culture underrepresents the in vivo environment of living cells. The development and use of 3D scaffolds and biomaterials provide researchers an ability to more closely mimic the in vivo environment. However, many biomaterials are of animal origin, leading to variability, environmental and ethical concerns. Here we present three animal-free scaffolds: decellularized plant tissue, chitin/chitosan and recombinant collagen. Decellularized plant tissue provides a wide array of structures with varying biochemical, topographical and mechanical properties; chitin/chitosan-based scaffolds have shown synergistic bactericidal effects and improved cell-matrix interaction; and lastly, recombinant collagen has the potential to closely resemble native tissue, as opposed to the other two. These benefits, alongside potential scalability and tunability, open the door to applications beyond the biomedical realm, such as innovations in cellular agriculture and future food technologies.

Alignment and orientation of cells in vivo plays a crucial role in the functionality of tissue. A challenged faced by traditional cell culture approaches is that the majority of two-dimensional substrates fail to induce a controlled alignment of cells in vitro. To address this challenge, approaches utilizing mechanical stresses, exposure to electrical fields, structurally aligned biomaterials and/or textured microfabricated substrates, have been developed to control the organization of cells through microenvironmental stimuli. In the field of muscle tissue engineering it is often desirable to control the alignment and fusion of muscle precursor cells as it more closely resembles in vivo conditions. In this study, we utilize plant-derived cellulose biomaterials to control the in vitro alignment of C2C12 murine myoblasts. We hereby report that cells display a clear sensitivity to the highly aligned vascular bundle architectures found in decellularized celery (Apium graveolens). Conveniently, the xylem and phloem channels lie within the 10-100μm diameter, which has been shown to be optimal diameter for myoblast alignment through contact guidance. Following 10 days in proliferation media, F-actin filaments were observed to be aligned parallel to the longitudinal axis of the vascular bundle. Subsequently, following 5 days in differentiation media, myoblast maintained an aligned morphology, which led to the formation of aligned myotubes. We therefore conclude that the microtopography of the vascular bundle guides muscle cell alignment. The results presented here highlight the potential of this plant-derived scaffold for in vitro studies of muscle myogenesis, where structural anisotropy is required to more closely resemble in vivo conditions.

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