The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.

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.

Experimental control over progenitor cell lineage specification can be achieved by modulating properties of the cell's microenvironment. These include physical properties of the cell adhesion substrate, such as rigidity, topography and deformation owing to dynamic mechanical forces. Multipotent mesenchymal stem cells (MSCs) generate contractile forces to sense and remodel their extracellular microenvironments and thereby obtain information that directs broad aspects of MSC function, including lineage specification. Various physical factors are important regulators of MSC function, but improved understanding of MSC mechanobiology requires novel experimental platforms. Engineers are bridging this gap by developing tools to control mechanical factors with improved precision and throughput, thereby enabling biological investigation of mechanics-driven MSC function. In this review, we introduce MSC mechanobiology and review emerging cell culture platforms that enable new insights into mechanobiological control of MSCs. Our main goals are to provide engineers and microtechnology developers with an up-to-date description of MSC mechanobiology that is relevant to the design of experimental platforms and to introduce biologists to these emerging platforms.

Bioprocessing applications that derive meat products from animal cell cultures require food-safe culture substrates that support volumetric expansion and maturation of adherent muscle cells. Here we demonstrate scalable production of microfibrous gelatin that supports cultured adherent muscle cells derived from cow and rabbit. As gelatin is a natural component of meat, resulting from collagen denaturation during processing and cooking, our extruded gelatin microfibers recapitulated structural and biochemical features of natural muscle tissues. Using immersion rotary jet spinning, a dry-jet wet-spinning process, we produced gelatin fibers at high rates (~ 100 g/h, dry weight) and, depending on process conditions, we tuned fiber diameters between ~ 1.3 ± 0.1 μm (mean ± SEM) and 8.7 ± 1.4 μm (mean ± SEM), which are comparable to natural collagen fibers. To inhibit fiber degradation during cell culture, we crosslinked them either chemically or by co-spinning gelatin with a microbial crosslinking enzyme. To produce meat analogs, we cultured bovine aortic smooth muscle cells and rabbit skeletal muscle myoblasts in gelatin fiber scaffolds, then used immunohistochemical staining to verify that both cell types attached to gelatin fibers and proliferated in scaffold volumes. Short-length gelatin fibers promoted cell aggregation, whereas long fibers promoted aligned muscle tissue formation. Histology, scanning electron microscopy, and mechanical testing demonstrated that cultured muscle lacked the mature contractile architecture observed in natural muscle but recapitulated some of the structural and mechanical features measured in meat products.

Extracellular matrix (ECM) structure and biochemistry provide cell-instructive cues that promote and regulate tissue growth, function, and repair. From a structural perspective, the ECM is a scaffold that guides the self-assembly of cells into distinct functional tissues. The ECM promotes the interaction between individual cells and between different cell types, and increases the strength and resilience of the tissue in mechanically dynamic environments. From a biochemical perspective, factors regulating cell-ECM adhesion have been described and diverse aspects of cell-ECM interactions in health and disease continue to be clarified. Natural ECMs therefore provide excellent design rules for tissue engineering scaffolds. The design of regenerative three-dimensional (3D) engineered scaffolds is informed by the target ECM structure, chemistry, and mechanics, to encourage cell infiltration and tissue genesis. This can be achieved using nanofibrous scaffolds composed of polymers that simultaneously recapitulate 3D ECM architecture, high-fidelity nanoscale topography, and bio-activity. Their high porosity, structural anisotropy, and bio-activity present unique advantages for engineering 3D anisotropic tissues. Here, we use the heart as a case study and examine the potential of ECM-inspired nanofibrous scaffolds for cardiac tissue engineering. We asked: Do we know enough to build a heart? To answer this question, we tabulated structural and functional properties of myocardial and valvular tissues for use as design criteria, reviewed nanofiber manufacturing platforms and assessed their capabilities to produce scaffolds that meet our design criteria. Our knowledge of the anatomy and physiology of the heart, as well as our ability to create synthetic ECM scaffolds have advanced to the point that valve replacement with nanofibrous scaffolds may be achieved in the short term, while myocardial repair requires further study in vitro and in vivo.

Background: Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)–signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. Methods: Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. Results: Torin1 (200 nmol/L) increased the percentage of quiescent cells (G 0 phase) from 24% to 48% compared with vehicle control ( P <0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. Conclusions: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.

We describe a planar, micro-fabricated device for generating fringing non-uniform electric fields. We used it to measure the mechanical properties of individual mammalian cells in suspension by deforming them in time-varying, non-uniform electric fields. Electrical stresses generated by the planar microelectrodes were used to trap and stretch cells, while cell deformation was observed using optical microscopy. Two distinct cell types were compared after fitting strain data with a three-parameter 'standard linear solid' model of visco-elasticity, and with a two-parameter power-law method. Chinese hamster ovary (CHO) cells were approximately twice as stiff as U937 human promonocytes, and CHO cells displayed an elastic behaviour with recovery of initial shape, while U937 strain data bore witness to plastic deformation. Our results demonstrate that electrical stresses generated by micro-fabricated electrodes permit mechanical characterization of distinct mammalian cell types.

Helical alignments within the heart’s musculature have been speculated to be important in achieving physiological pumping efficiencies. Testing this possibility is difficult, however, because it is challenging to reproduce the fine spatial features and complex structures of the heart’s musculature using current techniques. Here we report focused rotary jet spinning (FRJS), an additive manufacturing approach that enables rapid fabrication of micro/nanofiber scaffolds with programmable alignments in three-dimensional geometries. Seeding these scaffolds with cardiomyocytes enabled the biofabrication of tissue-engineered ventricles, with helically aligned models displaying more uniform deformations, greater apical shortening, and increased ejection fractions compared with circumferential alignments. The ability of FRJS to control fiber arrangements in three dimensions offers a streamlined approach to fabricating tissues and organs, with this work demonstrating how helical architectures contribute to cardiac performance.