Fetal Bovine Serum (FBS) is one of the most commonly used media supplement for the maintenance of mammalian cell types, yet the expensive costs, ethical concerns, and lot-to-lot variation have provoked a clear need for a serum that is standardized and derived from non-animal sources. Several serum-free formulations have been developed in the past, however they are often cell type specific, contain animal-derived components, and lack long-term culture validation. In this study, we developed a novel animal component-free (ACF) media and investigated its effectiveness on four commonly used mammalian cell lines via long-term (up to 90 days) morphological, transcriptomic, and proliferative analyses. Cells cultured in our ACF medium exhibited comparable cellular morphologies and equal or greater growth rates compared with cells cultured with FBS. Additionally, differentially expressed genes between the FBS-grown and ACF-grown groups were predominantly associated with functions linked to proliferation and cell attachment. While the tested cells were initially derived using conventional methods and include non-human lines, this study demonstrates that our medium supports long-term culture without animal-derived supplements. The findings from this study indicate that this medium is a suitable replacement to FBS-containing medium for several common cell lines.Insight Box Traditional cell culture methods often rely on animal-derived components, which can pose ethical and economic challenges. The use of animal serum in vitro is needed to supply nutrients to cells but raises concerns about animal welfare and introduces variability and contaminants that can negatively affect downstream applications. This study presents a novel animal component-free medium designed to support the growth of adherent cell types, providing a sustainable alternative to serum. Here, we demonstrate long-term cell viability, normal morphology, and differential gene expression patterns indicative of enhanced proliferation and attachment in cells cultured in 2D environments. By addressing the demand for ethical and reproducible cell culture methods, this research aims to contributes to the broader adoption of sustainable practices in biotechnology.

Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as its ability to effectively support osteogenic differentiation. The present study further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and energy-dispersive spectroscopy (EDS) confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young's modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as its ability to effectively support osteogenic differentiation. The present study further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and energy-dispersive spectroscopy (EDS) confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young's modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Neural stem cell (NSC)-based therapies are at the forefront of regenerative medicine strategies for various neural defects and injuries such as stroke, traumatic brain injury, and spinal cord injury. For several clinical applications, NSC therapies require biocompatible scaffolds to support cell survival and to direct differentiation. Here, we investigate decellularized plant tissue as a novel scaffold for three-dimensional (3D), in vitro culture of NSCs. Plant cellulose scaffolds were shown to support the attachment and proliferation of adult rat hippocampal neural stem cells (NSCs). Further, NSCs differentiated on the cellulose scaffold had significant increases in their expression of neuron-specific beta-III tubulin and glial fibrillary acidic protein compared to 2D culture on a polystyrene plate, indicating that the scaffold may enhance the differentiation of NSCs towards astrocytic and neuronal lineages. Our findings suggest that plant-derived cellulose scaffolds have the potential to be used in neural tissue engineering and can be harnessed to direct the differentiation of NSCs.

In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for studying the complex microenvironment of this interface remain lacking. Herein, these complex living interfaces by manufacturing modular “blocks” of naturally porous decellularized plant‐derived scaffolds with a computer numerical controlled mill are studied. How each scaffold can be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI) is demonstrated. Cells migrate across the interface formed between an empty scaffold and a scaffold preseeded with cells. However, when both scaffolds contain cells, only a shallow cross‐over zone of cell infiltration forms at the interface. As a proof‐of‐concept study, ETIs to investigate the interaction between lab grown bone and connective tissues are used. Consistent with the above, a cross‐over zone of the two distinct cell types forms at the interface between scaffolds, otherwise the populations remain distinct. Finally, how ETIs are biocompatible in vivo are demonstrated, becoming vascularized and integrated into surrounding tissue after implantation. Herein, new tissue design avenues for understanding biological processes or the development of synthetic artificial tissues are created.

Seasonal modifications in the structure of cellular membranes occur as an adaptive measure to withstand exposure to prolonged environmental change. Little is known about whether such changes occur independently of external cues, such as photoperiod or temperature, or how they may impact the central nervous system. We compared membrane properties of neurons isolated from the retina of goldfish (Carassius auratus), an organism well adapted to extreme environmental change, during the summer and winter months. Goldfish were maintained in a facility under constant environmental conditions throughout the year. Analysis of whole-retina phospholipid composition using mass spectrometry-based lipidomics revealed a twofold increase in phosphatidylethanolamine species during the winter, suggesting an increase in cell membrane fluidity. Atomic force microscopy was used to produce localized, nanoscale-force deformation of neuronal membranes. Measurement of Young's modulus indicated increased membrane-cortical stiffness (or decreased elasticity) in neurons isolated during the winter. Voltage-clamp electrophysiology was used to assess physiological changes in neurons between seasons. Winter neurons displayed a hyperpolarized reversal potential (V-rev) and a significantly lower input resistance (R-in) compared with summer neurons. This was indicative of a decrease in membrane excitability during the winter. Subsequent measurement of intracellular Ca2+ activity using Fura-2 microspectrofluorometry confirmed a reduction in action potential activity, including duration and action potential profile, in neurons isolated during the winter. These studies demonstrate chemical and biophysical changes that occur in retinal neurons of goldfish throughout the year without exposure to seasonal cues, and suggest a novel mechanism of seasonal regulation of retinal activity.

In recent years, plant-derived cellulosic biomaterials have become a popular way to create scaffolds for a variety of tissue engineering applications. Moreover, such scaffolds possess similar physical properties (porosity, stiffness) that resemble bone tissues and have been explored as potential biomaterials for tissue engineering applications. Here, plant-derived cellulose scaffolds were seeded with MC3T3-E1 pre-osteoblast cells. Moreover, to assess the potential of these biomaterials, we also applied cyclic hydrostatic pressure (HP) to the cells and scaffolds over time to mimic a bone-like environment more closely. After one week of proliferation, cell-seeded scaffolds were exposed to HP up to 270 KPa at a frequency of 1Hz, once per day, for up to two weeks. Scaffolds were incubated in osteogenic inducing media (OM) or regular culture media (CM). The effect of cyclic HP combined with OM on cell-seeded scaffolds resulted in an increase of differentiated cells. This corresponded to an upregulation of alkaline phosphatase activity and scaffold mineralization. Importantly, the results reveal that well known mechanosensitive pathways cells which regulate osteogenesis appear to remain functional even on novel plant-derived cellulosic biomaterials.

Numerous biomaterial scaffolds have been developed which provide architectures to support the proliferation of mammalian cells. Scaffolds derived from plant components have been utilized in several tissue engineering applications, including the production of cultured meats. Bread crumb is a common ingredient employed as a texturizer and filler in existing manufacturing processes for the production of animal meat products. Though an unconventional choice as a scaffolding material, we developed a yeast-free "soda bread" with controllable porosity and mechanical properties which is stable over several weeks in culture with fibroblasts, myoblasts and pre-osteoblasts. All cells were able to proliferate throughout the three-dimensional scaffolds, depositing extra-cellular matrix while exhibiting low stress and high viability. Importantly, myoblasts were also able to differentiate into myotubes, a key step required for the culture of skeletal muscle tissue. The results suggest opportunities for the dual-use possibility of utilizing existing texturizer and filler components in future lab grown meat products, however this will of course require further validation. Regardless, the bread-derived scaffolds presented here are simply produced, inherently edible and support muscle tissue engineering, qualities which highlight their utility in the production of future meat products.

Our organs and tissues are in constant motion, exposing epithelial cells to mechanical stretch. How these external forces impact cellular morphology, organization and dynamics in healthy and diseased tissues is still being elucidated. Carcinoma, the most common type of cancer, develops in the sheets of cells forming the epithelium and lining our organs and cavities. It usually begins with the transformation of a single cell via the activation of oncogenes such as Ras. Here, we show in a model system how mechanical stretch in epithelial sheets results in a more invasive phenotype in transformed cells. Cyclic strain impedes the apical extrusion of Ras(V12) cells from the healthy monolayer and prevents the formation of strong circumferential belts of actin in Ras(V12) cells. Concurrently, strain also changes the metastatic phenotype of newly transformed cells by greatly promoting the formation of Ras(V12) protrusions, potentially making them harder to be eliminated from healthy tissues. We also show that Ras(V12) and wild type MDCK cells possess distinct sensitivity to strain. External forces remodel their actin cytoskeletons and adhesion complexes differently, resulting in a more invasive system dynamic. Our work demonstrates that the Rho-ROCK mechanotransduction pathway is involved in regulating a mechanically-induced switch to a more invasive phenotype. The insights gained in this study reveal that the complex dynamics at play in healthy and transformed epithelial cells is drastically different in a mechanically active microenvironment when compared to static conditions.

ABSTRACTPlant-derived cellulose biomaterials have recently been utilized in several tissue engineering applications. These naturally-derived cellulose scaffolds have been shown to be highly biocompatible in vivo, possess structural features of relevance to several tissues, and support mammalian cell invasion and proliferation. Recent work utilizing decellularized apple hypanthium tissue has shown that it possesses a pore size similar to trabecular bone and can successfully host osteogenic differentiation. In the present study, we further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) and analyzed their mechanical properties in vitro and in vivo. MC3T3-E1 pre-osteoblasts were seeded in cellulose scaffolds. Following chemically-induced osteogenic differentiation, scaffolds were evaluated for mineralization and for their mechanical properties. Alkaline phosphatase and Alizarin Red staining confirmed the osteogenic potential of the scaffolds. Histological analysis of the constructs revealed cell invasion and mineralization throughout the constructs. Furthermore, scanning electron microscopy demonstrated the presence of mineral aggregates on the scaffolds after culture in differentiation medium, and energy-dispersive spectroscopy confirmed the presence of phosphate and calcium. However, although the Young’s modulus significantly increased after cell differentiation, it remained lower than that of healthy bone tissue. Interestingly, mechanical assessment of acellular scaffolds implanted in rat calvaria defects for 8 weeks revealed that the force required to push out the scaffolds from the surrounding bone was similar to that of native calvarial bone. In addition, cell infiltration and extracellular matrix deposition were visible within the implanted scaffolds. Overall, our results confirm that plant-derived cellulose is a promising candidate for BTE applications. However, the discrepancy in mechanical properties between the mineralized scaffolds and healthy bone tissue may limit their use to low load-bearing applications. Further structural re-engineering and optimization to improve the mechanical properties may be required for load-bearing applications.

ABSTRACT Numerous synthetic and naturally-occurring biomaterials have been developed to provide such architectures to support the proliferation of mammalian cells in vitro and in vivo. Our group, and others, have shown that scaffolds derived from plants can be utilized for tissue engineering applications in biomedicine and in the burgeoning cultured meat industry. Such scaffolds are ideally straightforward and inexpensive to prepare, allowing researchers to take advantage of their intrinsic 3D microarchitectures. These efforts inspired us to continue to pursue the development of novel and unconventional biomaterials that are easily produced and high performing in vitro . With this in mind, few plant-derived materials are more ubiquitous than bread. Having observed the porosity of the crumb (i.e. the internal bulk) we sought to investigate whether it might support the proliferation of mammalian cells in vitro . Here, we develop and validate a yeast-free “soda bread” that maintains its mechanical stability over several weeks in culture conditions. Importantly, we also demonstrate that control over the mechanical stability of the scaffold can also be achieved with both chemical and enzymatic means. The scaffolding is a heterogeneous and complex structure of isolated and interconnected pores which allow for the proliferation of multiple cell types. We demonstrate here that mouse fibroblasts, myoblasts and pre-osteoblasts are able to proliferate up to four weeks in culture. Immunohistochemistry demonstrates that the fibroblasts are able to deposit their own fibronectin extracellular matrix and that mouse myoblasts are able to differentiate and fuse into myotubes. Although the pre-osteoblasts proliferated over the course of four weeks their ability to differentiate was inconclusive. Metabolic analyses of proliferation, cytotoxicity and oxidative stress reveal that cells remain highly viable and functional on these novel bread scaffolds. While the results presented in this proof-of-concept study create many new questions and opportunities, the results open up novel possibilities in the development of edible scaffolds that may be utilized in future food applications. Bread derived scaffolds represent a surprising alternative to synthetic or animal-derived scaffolds for addressing a diverse variety of tissue engineering challenges in food science. Future studies will delve deeper into investigating these how possibilities might take advantage of the immense breadth of knowledge about bread making and examine their applicability in the development of lab grown foods and broader applications in cellular agriculture.

ABSTRACT As of yet, no standard of care incorporates biomaterials to treat traumatic spinal cord injury (SCI). However, intense development of biomaterials for treating SCI has focused on fabricating microscale channels to support the regrowth of axons while minimizing scar formation. We previously demonstrated that plant tissues could be decellularized and processed to form sterile, biocompatible and implantable biomaterials that support cell infiltration and vascularization in vivo . Vascularized plant tissues contain continuous microscale channels with geometries relevant for supporting neural regeneration. We hypothesized that decellularized vascular bundles would support neural regeneration and motor recovery in SCI. Sprague Dawley rats received a complete T8-T9 spinal cord transection and were implanted with acellular plant-derived scaffolds and allowed to recover over 28 weeks. Animals that received the scaffolds alone, with no other therapeutic compounds, demonstrated a significant and stable partial improvement in motor function compared to control animals as early as week 4 post-injury. Hind-limb motor function did not deteriorate over the remaining 28 weeks. Histological analysis revealed minimal astrocyte scarring at the spinal cord - scaffold interface, aligned axonal projection through the scaffolds, populations of serotonergic neurons and Schwann cells, laminin and collagen deposition and the presence of blood vessels. Axonal reconnection via the scaffold was also confirmed by Fluro-gold retrograde tracing. Taken together, our work defines a novel route for building upon naturally occurring plant microarchitectures to support the repair of the spinal cord post-injury. Notably, these results were achieved without the use of growth factors, stem/progenitor cells, or any other interventions.

Fibrosis is a leading cause of death in developed countries that is characterized by a progressive deterioration of tissue mechanical behavior. Developing methods to study tissue mechanics and myofibroblast activation may lead to new targets for therapeutic treatments that are urgently needed. Microtissue arrays are a promising approach to conduct relatively high throughput research into fibrosis as they recapitulate key biomechanical aspects of the disease through a relevant 3D extracellular environment. In early work, our group developed a device called the MVAS-force to stretch microtissues while enabling simultaneous assessment of their dynamic mechanical behavior. Here we investigated TGF-β1 induced fibroblast to myofibroblast differentiation in microtissue cultures using our MVAS-force device through assessing α-SMA expression, contractility and stiffness. By doing so, we linked cell-level phenotypic changes to functional changes that characterize the clinical manifestation of fibrotic disease. As expected, TGF-β1 treatment promoted a myofibroblastic phenotype and microtissues became stiffer and possessed increased contractility. Furthermore, these changes were partially reversible upon TGF-β1 withdrawal. In contrast, however, long-term cyclic stretching maintained myofibroblast activation. Furthermore stretching had no effect compared static cultures when TGF-β1 receptors were inhibited and stretching promoted myofibroblast differentiation when given latent TGF-β1. Together these results suggest that external mechanical stretch may activate latent TGF-β1 and might be a powerful stimulus for continued myofibroblast activation to progress fibrosis. Further exploration of this pathway with our approach may yield new insights into myofibroblast activation and more effective therapeutic treatments for fibrosis. Insight box Using a novel high-throughput approach, we quantified the effects of dynamic mechanical stretching on the phenotype and function of cells in 3D microtissue cultures during myofibroblast activation with TGF-β1 treatment and subsequent withdrawal. Our findings show that mechanical stretch may activate endogenously produced latent TGF-β1 to maintain the presence and activity of myofibroblasts after tissue injury. Importantly, through this feed forward mechanism, mechanical stretch might be a powerful stimulus that directs tissues away from recovery and towards the development of fibrosis.

Plant-derived cellulose biomaterials have recently been utilized in several tissue engineering applications. Naturally-derived cellulose scaffolds have been shown to be highly biocompatible in vivo , possess structural features of relevance to several tissues, as well as support mammalian cell invasion and proliferation. Recent work utilizing decellularized apple hypanthium tissue has shown that it possesses a pore size and properties similar to trabecular bone. In the present study, we examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE). Confocal microscopy revealed that the scaffolds had a suitable pore size for BTE applications. To analyze their in vitro mineralization potential, MC3T3-E1 pre-osteoblasts were seeded in either bare cellulose scaffolds or in composite scaffolds composed of cellulose and collagen I. Following chemically-induced differentiation, scaffolds were mechanically tested and evaluated for mineralization. The Young’s modulus of both types of scaffolds significantly increased after cell differentiation. Alkaline phosphatase and Alizarin Red staining further highlighted the osteogenic potential of the scaffolds. Histological sectioning of the constructs revealed complete invasion by the cells and mineralization throughout the entire constructs. Finally, scanning electron microscopy demonstrated the presence of mineral aggregates deposited on the scaffolds after differentiation, and energy-dispersive spectroscopy confirmed the presence of phosphate and calcium. In summary, our results indicate that plant-derived cellulose is a promising scaffold candidate for bone tissue engineering applications.

When stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of the cytoskeleton is profoundly influenced by the dimensionality of a cell’s environment. Therefore, in this study we aimed to determine whether cells cultured in a 3D matrix share this softening behavior and to link it to cytoskeletal remodeling. To achieve this, we developed a high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging within physiologically relevant 3D microtissues. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they regained their initial mechanical properties. Furthermore, microtissue prestress decreased with the strain amplitude to maintain a constant mean tension. This adaptation under an auxotonic condition resulted in lengthening. A filamentous actin cytoskeleton was required, and responses were mirrored by changes to actin remodeling rates and visual evidence of stretch-induced actin depolymerization. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a 3D matrix, and connected remodeling of the cytoskeleton to homeostatic mechanical regulation of tissues.

In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for engineering and studying the complex microenvironment of this interface remain lacking1–3. Here, we create and study these complex living interfaces by manufacturing modular “blocks” of decellularized plant-derived scaffolds4–6 with varying shapes and sizes with a computer numerical controlled mill. Each block can then be seeded with different cell types and easily assembled in a manner akin to LEGO™ bricks to create an engineered tissue interface (ETI). As a proof-of-concept study we utilize ETIs to investigate the interaction between lab grown bone and connective tissues. We also demonstrate how ETIs are biocompatible in vivo, stimulating the formation of blood vessels, cell infiltration, and tissue integration after implantation. This work creates possibilities for new tissue design avenues for understanding fundamental biological processes or the development of synthetic artificial tissues.

In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for engineering and studying the complex microenvironment of this interface remain lacking1-3. Here, we create and study these complex living interfaces by manufacturing modular "blocks" of decellularized plant-derived scaffolds4-6 with varying shapes and sizes with a computer numerical controlled mill. Each block can then be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI). As a proof-of-concept study we utilize ETIs to investigate the interaction between lab grown bone and connective tissues. We also demonstrate how ETIs are biocompatible in vivo, stimulating the formation of blood vessels, cell infiltration, and tissue integration after implantation. This work creates possibilities for new tissue design avenues for understanding fundamental biological processes or the development of synthetic artificial tissues.

Characterizing the elastic and dissipative properties of cells is not only necessary to determine how they deform, but also to fully understand how external mechanical forces trigger biochemical-signaling cascades to govern their behavior. Presently mechanical properties are largely assessed by applying local shear or compressive forces on single cells in isolation grown on non-physiological 2D surfaces. In comparison, our microfabricated vacuum actuated stretcher measures tensile loading of 3D multicellular microtissue cultures. With our approach, we assessed here the time-dependency of microtissue mechanics and quantified the spatial remodeling that follows step length changes. Unlike previous results from other micro-rheological techniques, stress relaxation and recovery in microtissues followed stretched exponential behaviors that shared similar amplitudes but differed in their dynamics. In that regard, relaxation time constants changed with an inverse power law with step size, while recovery rates were invariant. Pharmacological responses, however, indicated that the contributions of individual cytoskeletal elements did not qualitatively differ from our existing understanding of their roles. The elasticity and dissipation of microtissues were mainly determined by the actin cytoskeleton but also augmented by myosin motor activity and reduced by the presence of microtubules. These results were reflected in changes to remodeling dynamics and spatial distributions of the integrated strain field. This assessment of microtissues offers insights into how the collective behavior of cells and their cytoskeletal proteins generate the dynamic mechanical properties of tissues, which is necessary for a full understanding of how cell behaviors are regulated in both health and disease.

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.

Characterizing the time-dependent mechanical properties of cells is not only necessary to determine how they deform but also to understand how external forces trigger biochemical-signaling cascades to govern their behavior. At present, mechanical properties are largely assessed by applying local shear or compressive forces on single cells grown in isolation on non-physiological 2D surfaces. In comparison, we developed the microfabricated vacuum actuated stretcher to measure tensile loading of 3D multicellular “microtissue” cultures. Using this approach, we here assessed the time-dependent stress relaxation and recovery responses of microtissues and quantified the spatial viscoelastic deformation following step length changes. Unlike previous results, stress relaxation and recovery in microtissues measured over a range of step amplitudes and pharmacological treatments followed an augmented stretched exponential behavior describing a broad distribution of inter-related timescales. Furthermore, despite the variety of experimental conditions, all responses led to a single linear relationship between the residual elastic stress and the degree of stress relaxation, suggesting that these mechanical properties are coupled through interactions between structural elements and the association of cells with their matrix. Finally, although stress relaxation could be quantitatively and spatially linked to recovery, they differed greatly in their dynamics; while stress recovery acted as a linear process, relaxation time constants changed with an inverse power law with the step size. This assessment of microtissues offers insights into how the collective behavior of cells in a 3D collagen matrix generates the dynamic mechanical properties of tissues, which is necessary to understand how cells deform and sense mechanical forces in vivo.