Fattening chips: hypertrophy, feeding, and fasting of human white adipocytes in vitro.

Adipose is a distributed organ that performs vital endocrine and energy homeostatic functions. Hypertrophy of white adipocytes is a primary mode of both adaptive and maladaptive weight gain in animals and predicts metabolic syndrome independent of obesity. Due to the failure of conventional culture to recapitulate adipocyte hypertrophy, technology for production of adult-size adipocytes would enable applications such as in vitro testing of weight loss therapeutics. To model adaptive adipocyte hypertrophy in vitro, we designed and built fat-on-a-chip using fiber networks inspired by extracellular matrix in adipose tissue. Fiber networks extended the lifespan of differentiated adipocytes, enabling growth to adult sizes. By micropatterning preadipocytes in a native cytoarchitecture and by adjusting cell-to-cell spacing, rates of hypertrophy were controlled independent of culture time or differentiation efficiency. In vitro hypertrophy followed a nonlinear, nonexponential growth model similar to human development and elicited transcriptomic changes that increased overall similarity with primary tissue. Cells on the chip responded to simulated meals and starvation, which potentiated some adipocyte endocrine and metabolic functions. To test the utility of the platform for therapeutic development, transcriptional network analysis was performed, and retinoic acid receptors were identified as candidate drug targets. Regulation by retinoid signaling was suggested further by pharmacological modulation, where activation accelerated and inhibition slowed hypertrophy. Altogether, this work presents technology for mature adipocyte engineering, addresses the regulation of cell growth, and informs broader applications for synthetic adipose in pharmaceutical development, regenerative medicine, and cellular agriculture.

Synchronized stimulation and continuous insulin sensing in a microfluidic human Islet on a Chip designed for scalable manufacturing.

Pancreatic ß cell function is compromised in diabetes and is typically assessed by measuring insulin secretion during glucose stimulation. Traditionally, measurement of glucose-stimulated insulin secretion involves manual liquid handling, heterogeneous stimulus delivery, and enzyme-linked immunosorbent assays that require large numbers of islets and processing time. Though microfluidic devices have been developed to address some of these limitations, traditional methods for islet testing remain the most common due to the learning curve for adopting microfluidic devices and the incompatibility of most device materials with large-scale manufacturing. We designed and built a thermoplastic, microfluidic-based Islet on a Chip compatible with commercial fabrication methods, that automates islet loading, stimulation, and insulin sensing. Inspired by the perfusion of native islets by designated arterioles and capillaries, the chip delivers synchronized glucose pulses to islets positioned in parallel channels. By flowing suspensions of human cadaveric islets onto the chip, we confirmed automatic capture of islets. Fluorescent glucose tracking demonstrated that stimulus delivery was synchronized within a two-minute window independent of the presence or size of captured islets. Insulin secretion was continuously sensed by an automated, on-chip immunoassay and quantified by fluorescence anisotropy. By integrating scalable manufacturing materials, on-line, continuous insulin measurement, and precise spatiotemporal stimulation into an easy-to-use design, the Islet on a Chip should accelerate efforts to study and develop effective treatments for diabetes.

Automated fabrication of photopatterned gelatin hydrogels for organ-on-chips applications.

Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 µm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 µm. The standard deviation of feature height was 0.3 µm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.

A tissue-engineered scale model of the heart ventricle.

Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50-250 times smaller and 104-108 times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure-volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.

Toward improved myocardial maturity in an organ-on-chip platform with immature cardiac myocytes.

In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the ß-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.