Comparative Analysis of the Physiological and Transport Functions of Various Sources of Renal Proximal Tubule Cells Under Static and Fluidic Conditions in PhysioMimix{trade mark, serif} T12 Platform.

In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.

Reproducibility and Robustness of a Liver Microphysiological System PhysioMimix LC12 under Varying Culture Conditions and Cell Type Combinations.

The liver is one of the key organs for exogenous and endogenous metabolism and is often a target for drug- and chemical-driven toxicity. A wide range of experimental approaches has been established to model and characterize the mechanisms of drug- and chemical-induced hepatotoxicity. A number of microfluidics-enabled in vitro models of the liver have been developed, but the unclear translatability of these platforms has hindered their adoption by the pharmaceutical industry; to achieve wide use for drug and chemical safety evaluation, demonstration of reproducibility and robustness under various contexts of use is required. One of these commercially available platforms is the PhysioMimix LC12, a microfluidic device where cells are seeded into a 3D scaffold that is continuously perfused with recirculating cell culture media to mimic liver sinusoids. Previous studies demonstrated this model's functionality and potential applicability to preclinical drug development. However, to gain confidence in PhysioMimix LC12's robustness and reproducibility, supplementary characterization steps are needed, including the assessment of various human hepatocyte sources, contribution of non-parenchymal cells (NPCs), and comparison to other models. In this study, we performed replicate studies averaging 14 days with either primary human hepatocytes (PHHs) or induced pluripotent stem cell (iPSC)-derived hepatocytes, with and without NPCs. Albumin and urea secretion, lactate dehydrogenase, CYP3A4 activity, and metabolism were evaluated to assess basal function and metabolic capacity. Model performance was characterized by different cell combinations under intra- and inter-experimental replication and compared to multi-well plates and other liver platforms. PhysioMimix LC12 demonstrated the highest metabolic function with PHHs, with or without THP-1 or Kupffer cells, for up to 10-14 days. iPSC-derived hepatocytes and PHHs co-cultured with additional NPCs demonstrated sub-optimal performance. Power analyses based on replicate experiments and different contexts of use will inform future study designs due to the limited throughput and high cell demand. Overall, this study describes a workflow for independent testing of a complex microphysiological system for specific contexts of use, which may increase end-user adoption in drug development.

Analysis of reproducibility and robustness of a renal proximal tubule microphysiological system OrganoPlate 3-lane 40 for in vitro studies of drug transport and toxicity.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.

Analysis of reproducibility and robustness of OrganoPlate® 2-lane 96, a liver microphysiological system for studies of pharmacokinetics and toxicological assessment of drugs.

Establishing the functionality, reproducibility, robustness, and reliability of microphysiological systems is a critical need for adoption of these technologies. A high throughput microphysiological system for liver studies was recently proposed in which induced pluripotent stem cell-derived hepatocytes (iHeps) and non-parenchymal cells (endothelial cells and THP-1 cells differentiated with phorbol 12-myristate 13-acetate into macrophage-like cells) were co-cultured in OrganoPlate® 2-lane 96 devices. The goal of this study was to evaluate this platform using additional cell types and conditions and characterize its utility and reproducibility. Primary human hepatocytes or iHeps, with and without non-parenchymal cells, were cultured for up to 17 days. Image-based cell viability, albumin and urea secretion into culture media, CYP3A4 activity and drug metabolism were assessed. The iHeps co-cultured with non-parenchymal cells demonstrated stable cell viability and function up to 17 days; however, variability was appreciable both within and among studies. The iHeps in monoculture did not form clusters and lost viability and function over time. The primary human hepatocytes in monoculture also exhibited low cell viability and hepatic function. Metabolism of various drugs was most efficient when iHeps were co-cultured with non-parenchymal cells. Overall, we found that the OrganoPlate® 2-lane 96 device, when used with iHeps and non-parenchymal cells, is a functional liver microphysiological model; however, the high-throughput nature of this model is somewhat dampened by the need for replicates to compensate for high variability.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

An in vitro alveolar epithelial cell model recapitulates LRRK2 inhibitor-induced increases in lamellar body size observed in preclinical models.

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.

Freshly isolated primary human proximal tubule cells as an in vitro model for the detection of renal tubular toxicity.

Drug induced kidney injury (DIKI) is a common reason for compound attrition in drug development pipelines with proximal tubule epithelial cells (PTECs) most commonly associated with DIKI. Here, we investigated freshly isolated human (hPTECs) as an in vitro model for assessing renal tubular toxicity. The freshly isolated hPTECs were first characterized to confirm gene expression of important renal transporters involved in drug handling which was further corroborated by confirming the functional activity of organic cation transporter 2 and organic anion transporter 1 by using transporter specific inhibitors. Additionally, functionality of megalin/cubilin endocytic receptors was also confirmed. A training set of 36 compounds was used to test the ability of the model to classify them using six different endpoints which included three biomarkers (Kidney Injury Molecule-1, Neutrophil gelatinase-associated lipocalin, and Clusterin) and three non-specific injury endpoints (ATP depletion, LDH leakage, and barrier permeability via transepithelial electrical resistance) in a dose-dependent manner across two independent kidney donors. In general, biomarkers showed higher predictivity than non-specific endpoints, with Clusterin showing the highest predictivity (Sensitivity/Specificity - 65.0/93.8 %). By using the thresholds generated from the training set, nine candidate internal Takeda compounds were screened where PTEC toxicity was identified as one of the findings in preclinical animal studies. The model correctly classified four of six true positives and two of three true negatives, showing validation of the in vitro model for detection of tubular toxicants. This work thus shows the potential application of freshly isolated primary hPTECs using translational biomarkers in assessment of tubular toxicity within the drug discovery pipeline.

High-Throughput Screening to Evaluate Inhibition of Bile Acid Transporters Using Human Hepatocytes Isolated From Chimeric Mice.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.

Human Pluripotent Stem Cell-Derived Kidney Model for Nephrotoxicity Studies.

Current in vitro models for identifying nephrotoxins are poorly predictive. We differentiated human pluripotent stem cells (hPSCs) into three-dimensional, multicellular structures containing proximal tubule cells (PTCs) and podocytes and evaluated them as a platform for predicting nephrotoxicity. The PTCs showed megalin-dependent, cubilin-mediated endocytosis of fluorescently labeled dextran and active gamma-glutamyl transpeptidase enzymes. Transporters from both the ATP-binding cassette (ABC) and the solute carrier (SLC) families were present at physiological levels in the differentiated cells, but important renal transporters such as organic anion transporter 1 (OAT1), OAT3, and organic cation transporter 2 (OCT2) were present only at lower levels. Radioactive uptake studies confirmed the functional activity of organic cation transporter, novel, type 2 (OCTN2), organic anion transporter polypeptide 4C1 (OATP4C1), and OCTs/multidrug and toxin extrusion proteins (MATEs). When treated with 10 pharmacologic agents as a test of the platform, the known nephrotoxic compounds were distinguished from the more benign compounds by an increase in tubular (PTC, kidney injury molecule 1 (KIM-1), and heme oxygenase 1 (HO-1)) and glomerular (nephrin [NPHS1]/Wilms tumor protein [WT1]) markers associated with nephrotoxicity, and we were able to distinguish the type of nephrotoxin by examining the relative levels of these markers. Given the functions demonstrated and with improved expression of key renal transporters, this hPSC-derived in vitro kidney model shows promise as a platform for detection of mechanistically different nephrotoxins.

Emerging Kidney Models to Investigate Metabolism, Transport, and Toxicity of Drugs and Xenobiotics.

The kidney is a major clearance organ of the body and is responsible for the elimination of many xenobiotics and prescription drugs. With its multitude of uptake and efflux transporters and metabolizing enzymes, the proximal tubule cell (PTC) in the nephron plays a key role in the disposition of xenobiotics and is also a primary site for toxicity. In this minireview, we first provide an overview of the major transporters and metabolizing enzymes in the PTCs responsible for biotransformation and disposition of drugs. Next, we discuss different cell sources that have been used to model PTCs in vitro, their pros and cons, and their characterization. As current technology is inadequate to evaluate reliably drug disposition and toxicity in the kidney, we then discuss recent advancements in kidney microphysiological systems (MPS) and the need to develop robust in vitro platforms that could be routinely used by pharmaceutical companies to screen compounds. Finally, we discuss the new and exciting field of stem cell-derived kidney models as potential cell sources for future kidney MPS. Given the push from both regulatory agencies and pharmaceutical companies to use more predictive "human-like" in vitro systems in the early stages of drug development to reduce attrition, these emerging models have the potential to be a game changer and may revolutionize how renal disposition and kidney toxicity in drug discovery are evaluated in the future.

Identification of direct negative cross-talk between the SLIT2 and bone morphogenetic protein-Gremlin signaling pathways.

Slit guidance ligand 2 (SLIT2) is a large, secreted protein that binds roundabout (ROBO) receptors on multiple cell types, including neurons and kidney podocytes. SLIT2-ROBO-mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP-Gremlin signaling also regulates UB formation. However, direct cross-talk between the ROBO2-SLIT2 and BMP-Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP-Gremlin signaling pathways. We found that the SLIT2-Gremlin interaction inhibited both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 down-regulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMP-mediated repression of SLIT2. BMP2 treatment of nephron progenitor cells derived from human embryonic stem cells decreased SLIT2 expression, further suggesting an interaction between the BMP2-Gremlin and SLIT2 pathways in human kidney cells. In conclusion, our study has revealed direct negative cross-talk between two pathways, previously thought to be unassociated, that may regulate both kidney development and adult tissue maintenance.

Advances and Challenges in Recapitulating Human Pulmonary Systems: At the Cusp of Biology and Materials.

The aim of this review is to provide an overview of physiologically relevant microengineered lung-on-a-chip (LoC) platforms for a variety of different biomedical applications with emphasis on drug screening. First, a brief outline of lung anatomy and physiology is presented followed by discussion of the lung parenchyma and its extracellular matrix. Next, we point out the technical challenges in recapitulating the complexity of lung in conventional static two-dimensional microenvironments and the need for alternate lung platforms. The importance of scaling laws is also emphasized in designing these in vitro microengineered lung platforms. The review then discusses current LoC platforms that have been used for testing the efficacy of drugs or as model systems for investigating disorders of the lung parenchyma. Finally, the design parameters in developing an ideal physiologically relevant LoC platform are presented. As this emerging field of organ-on-a-chip can serve an alternative platform for animal testing of drugs or modeling human diseases in vitro, it has significant potential to impact the future of pharmaceutical research.

Three-dimensionally printed biological machines powered by skeletal muscle.

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 µm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.

3D biofabrication strategies for tissue engineering and regenerative medicine.

Over the past several decades, there has been an ever-increasing demand for organ transplants. However, there is a severe shortage of donor organs, and as a result of the increasing demand, the gap between supply and demand continues to widen. A potential solution to this problem is to grow or fabricate organs using biomaterial scaffolds and a person's own cells. Although the realization of this solution has been limited, the development of new biofabrication approaches has made it more realistic. This review provides an overview of natural and synthetic biomaterials that have been used for organ/tissue development. It then discusses past and current biofabrication techniques, with a brief explanation of the state of the art. Finally, the review highlights the need for combining vascularization strategies with current biofabrication techniques. Given the multitude of applications of biofabrication technologies, from organ/tissue development to drug discovery/screening to development of complex in vitro models of human diseases, these manufacturing technologies can have a significant impact on the future of medicine and health care.

Graphene-based patterning and differentiation of C2C12 myoblasts.

This study aims at generating highly aligned functional myotubes using graphene as the underlying scaffold. Graphene not only supports the growth of C2C12 muscle cells but also enhances its differentiation and leads to spontaneous patterning of myotubes.

Patterned three-dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography.

Controlling the assembly of cells in three dimensions is very important for engineering functional tissues, drug screening, probing cell-cell/cell-matrix interactions, and studying the emergent behavior of cellular systems. Although the current methods of cell encapsulation in hydrogels can distribute them in three dimensions, these methods typically lack spatial control of multi-cellular organization and do not allow for the possibility of cell-cell contacts as seen for the native tissue. Here, we report the integration of dielectrophoresis (DEP) with stereolithography (SL) apparatus for the spatial patterning of cells on custom made gold micro-electrodes. Afterwards, they are encapsulated in poly (ethylene glycol) diacrylate (PEGDA) hydrogels of different stiffnesses. This technique can mimic the in vivo microscale tissue architecture, where the cells have a high degree of three dimensional (3D) spatial control. As a proof of concept, we show the patterning and encapsulation of mouse embryonic stem cells (mESCs) and C2C12 skeletal muscle myoblasts. mESCs show high viability in both the DEP (91.79% ± 1.4%) and the no DEP (94.27% ± 0.5%) hydrogel samples. Furthermore, we also show the patterning of mouse embryoid bodies (mEBs) and C2C12 spheroids in the hydrogels, and verify their viability. This robust and flexible in vitro platform can enable various applications in stem cell differentiation and tissue engineering by mimicking elements of the native 3D in vivo cellular micro-environment.

3-D biofabrication using stereolithography for biology and medicine.

In this paper, we review our recent work on the potential of stereolithography (SL) for different biomedical applications including tissue engineering, neovessel formation, investigating cell-cell and cell matrix interactions, and development of cellular systems. Also, we show that SL technology can be combined with dielectrophoresis (DEP) to create scaffolds with micro-scale organization, a hallmark of in vivo tissues.

Multi-material bio-fabrication of hydrogel cantilevers and actuators with stereolithography.

Cell-based biohybrid actuators are integrated systems that use biological components including proteins and cells to power material components by converting chemical energy to mechanical energy. The latest progress in cell-based biohybrid actuators has been limited to rigid materials, such as silicon and PDMS, ranging in elastic moduli on the order of mega (10(6)) to giga (10(9)) Pascals. Recent reports in the literature have established a correlation between substrate rigidity and its influence on the contractile behavior of cardiomyocytes (A. J. Engler, C. Carag-Krieger, C. P. Johnson, M. Raab, H. Y. Tang and D. W. Speicher, et al., J. Cell Sci., 2008, 121(Pt 22), 3794-3802, P. Bajaj, X. Tang, T. A. Saif and R. Bashir, J. Biomed. Mater. Res., Part A, 2010, 95(4), 1261-1269). This study explores the fabrication of a more compliant cantilever, similar to that of the native myocardium, with elasticity on the order of kilo (10(3)) Pascals. 3D stereolithographic technology, a layer-by-layer UV polymerizable rapid prototyping system, was used to rapidly fabricate multi-material cantilevers composed of poly(ethylene glycol) diacrylate (PEGDA) and acrylic-PEG-collagen (PC) mixtures. The incorporation of acrylic-PEG-collagen into PEGDA-based materials enhanced cell adhesion, spreading, and organization without altering the ability to vary the elastic modulus through the molecular weight of PEGDA. Cardiomyocytes derived from neonatal rats were seeded on the cantilevers, and the resulting stresses and contractile forces were calculated using finite element simulations validated with classical beam equations. These cantilevers can be used as a mechanical sensor to measure the contractile forces of cardiomyocyte cell sheets, and as an early prototype for the design of optimal cell-based biohybrid actuators.

Patterning the differentiation of C2C12 skeletal myoblasts.

Mammalian cells are sensitive to the physical properties of their micro-environment such as the stiffness and geometry of the substrate. It is known that the stiffness of the substrate plays a key role in the process of mammalian myogenesis. However, the effect of geometrical constraints on the process of myogenic differentiation needs to be explored further. Here, we show that the geometrical cues of substrates can significantly influence the differentiation process of C2C12 skeletal myoblasts. Three different geometries including lines of different widths, tori of different inner diameters, and hybrid structures (linear and circular features with different arc degrees) were created by micro-contact printing of fibronectin on the surface of Petri dishes. The differentiation of C2C12 cells was studied over a period of seven days and was quantified; we report the differentiation parameters of (1) fusion index, (2) degree of maturation, (3) alignment, and (4) response to electrical pulse stimulation (EPS). Hybrid structures with the smallest arc degree (hybrid 30°) showed the best results for all four differentiation parameters. The hybrid 30° pattern exhibits an ~2-fold increase in the fusion index when compared to the line patterns and an ~3-fold increase when compared to the toroid patterns. The hybrid 30° also showed a higher maturation index compared to the line or the toroid patterns. In response to electrical stimulation (20 V, 50 ms pulse, 1 Hz), mature myotubes on hybrid 30° patterns showed an ~2-fold increase in cellular displacement when compared to myotubes on the line and torus patterns. We tested the influence of C2C12 cell density on fusion and maturation indices, and the results suggest that density does not exert significant influence on cellular differentiation under these conditions. Our results can have important implications in engineering skeletal muscle tissues and designing muscle cell bio-actuators.