A novel miniaturized multimodal bioreactor for continuous in situ assessment of bioartificial cardiac tissue during stimulation and maturation.
ABSTRACT: Stem cell-based cardiac tissue engineering is a promising approach for regenerative therapy of the injured heart. At present, the small number of stem cell-derived cardiomyocytes that can be obtained using current culture and enrichment techniques represents one of the key limitations for the development of functional bioartificial cardiac tissue (BCT). We have addressed this problem by construction of a novel bioreactor with functional features of larger systems that enables the generation and in situ monitoring of miniaturized BCTs. BCTs were generated from rat cardiomyocytes to demonstrate advantages and usefulness of the bioreactor. Tissues showed spontaneous, synchronized contractions with cell orientation along the axis of strain. Cyclic stretch induced cardiomyocyte hypertrophy, demonstrated by a shift of myosin heavy chain expression from the alpha to beta isoform, together with elevated levels of atrial natriuretic factor. Stretch led to a moderate increase in systolic force (1.42?±?0.09?mN vs. 0.96?±?0.09?mN in controls), with significantly higher forces observed after ?-adrenergic stimulation with noradrenalin (2.54?±?0.11?mN). Combined mechanical and ?-adrenergic stimulation had no synergistic effect. This study demonstrates for the first time that mechanical stimulation and direct real-time contraction force measurement can be combined into a single multimodal bioreactor system, including electrical stimulation of excitable tissue, perfusion of the culture chamber, and the possibility of (fluorescence) microscopic assessment during continuous cultivation. Thus, this bioreactor represents a valuable tool for monitoring tissue development and, ultimately, the optimization of stem cell-based tissue replacement strategies in regenerative medicine.
Project description:The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to ?-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.
Project description:Tissue engineering enables the generation of three-dimensional (3D) functional cardiac tissue for pre-clinical testing in vitro, which is critical for new drug development. However, current tissue engineering methods poorly recapitulate the architecture of oriented cardiac bundles with supporting capillaries. In this study, we designed a microfabricated bioreactor to generate 3D micro-tissues, termed biowires, using both primary neonatal rat cardiomyocytes and human embryonic stem cell (hESC) derived cardiomyocytes. Perfusable cardiac biowires were generated with polytetrafluoroethylene (PTFE) tubing template, and were integrated with electrical field stimulation using carbon rod electrodes. To demonstrate the feasibility of this platform for pharmaceutical testing, nitric oxide (NO) was released from perfused sodium nitroprusside (SNP) solution and diffused through the tubing. The NO treatment slowed down the spontaneous beating of cardiac biowires based on hESC derived cardiomyocytes and degraded the myofibrillar cytoskeleton of the cardiomyocytes within the biowires. The biowires were also integrated with electrical stimulation using carbon rod electrodes to further improve phenotype of cardiomyocytes, as indicated by organized contractile apparatus, higher Young's modulus, and improved electrical properties. This microfabricated platform provides a unique opportunity to assess pharmacological effects on cardiac tissue in vitro by perfusion in a cardiac bundle model, which could provide improved physiological relevance.
Project description:We describe here a bioreactor capable of applying electrical field stimulation in conjunction with static strain and on-line force of contraction measurements. It consisted of a polydimethylsiloxane (PDMS) tissue chamber and a pneumatically driven stretch platform. The chamber contained eight tissue microwells (8.05 mm in length and 2.5 mm in width) with a pair of posts (2.78 mm in height and 0.8 mm in diameter) in each well to serve as fixation points and for measurements of contraction force. Carbon rods, stimulating electrodes, were placed into the PDMS chamber such that one pair stimulated four microwells. For feasibility studies, neonatal rat cardiomyocytes were seeded in collagen gels into the microwells. Following 3 days of gel compaction, electrical field stimulation at 3-4 V cm(-1) and 1 Hz, mechanical stimulation of 5% static strain or electromechanical stimulation (field stimulation at 3-4 V cm(-1), 1 Hz and 5% static strain) were applied for 3 days. Cardiac microtissues subjected to electromechanical stimulation exhibited elevated amplitude of contraction and improved sarcomere structure as evidenced by sarcomeric ?-actinin, actin and troponin T staining compared to microtissues subjected to electrical or mechanical stimulation alone or non-stimulated controls. The expression of atrial natriuretic factor and brain natriuretic peptide was also elevated in the electromechanically stimulated group.
Project description:Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.
Project description:In the past two decades, relevant advances have been made in the generation of engineered cardiac constructs to be used as functional in vitro models for cardiac research or drug testing, and with the ultimate but still challenging goal of repairing the damaged myocardium. To support cardiac tissue generation and maturation in vitro, the application of biomimetic physical stimuli within dedicated bioreactors is crucial. In particular, cardiac-like mechanical stimulation has been demonstrated to promote development and maturation of cardiac tissue models. Here, we developed an automated bioreactor platform for tunable cyclic stretch and in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. To demonstrate the bioreactor platform performance and to investigate the effects of cyclic stretch on construct maturation and contractility, we developed 3D annular cardiac tissue models based on neonatal rat cardiac cells embedded in fibrin hydrogel. The constructs were statically pre-cultured for 5 days and then exposed to 4 days of uniaxial cyclic stretch (sinusoidal waveform, 10% strain, 1 Hz) within the bioreactor. Explanatory biological tests showed that cyclic stretch promoted cardiomyocyte alignment, maintenance, and maturation, with enhanced expression of typical mature cardiac markers compared to static controls. Moreover, in situ monitoring showed increasing passive force of the constructs along the dynamic culture. Finally, only the stretched constructs were responsive to external electrical pacing with synchronous and regular contractile activity, further confirming that cyclic stretching was instrumental for their functional maturation. This study shows that the proposed bioreactor platform is a reliable device for cyclic stretch culture and in situ monitoring of the passive mechanical response of the cultured constructs. The innovative feature of acquiring passive force measurements in situ and along the culture allows monitoring the construct maturation trend without interrupting the culture, making the proposed device a powerful tool for in vitro investigation and ultimately production of functional engineered cardiac constructs.
Project description:Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1 × 10(8) cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5 × 10(8). Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.
Project description:The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive Gs-protein coupled receptor JellyOp enables optogenetic stimulation of Gs-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological β-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized Gs-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca2+ currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential Gs-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted Gs stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile Gs activation.
Project description:The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, ?-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.
Project description:Mesenchymal stem cell (MSC) therapy has demonstrated applications in vascular regenerative medicine. Although blood vessels exist in a mechanically dynamic environment, there has been no rigorous, systematic analysis of mechanical stimulation on stem cell differentiation. We hypothesize that mechanical stimuli, relevant to the vasculature, can differentiate MSCs toward smooth muscle (SMCs) and endothelial cells (ECs). This was tested using a unique experimental platform to differentially apply various mechanical stimuli in parallel. Three forces, cyclic stretch, cyclic pressure, and laminar shear stress, were applied independently to mimic several vascular physiologic conditions. Experiments were conducted using subconfluent MSCs for 5 days and demonstrated significant effects on morphology and proliferation depending upon the type, magnitude, frequency, and duration of applied stimulation. We have defined thresholds of cyclic stretch that potentiate SMC protein expression, but did not find EC protein expression under any condition tested. However, a second set of experiments performed at confluence and aimed to elicit the temporal gene expression response of a select magnitude of each stimulus revealed that EC gene expression can be increased with cyclic pressure and shear stress in a cell-contact-dependent manner. Further, these MSCs also appear to express genes from multiple lineages simultaneously which may warrant further investigation into post-transcriptional mechanisms for controlling protein expression. To our knowledge, this is the first systematic examination of the effects of mechanical stimulation on MSCs and has implications for the understanding of stem cell biology, as well as potential bioreactor designs for tissue engineering and cell therapy applications.
Project description:Tissue engineering enables the generation of functional human cardiac tissue with cells derived in vitro in combination with biocompatible materials. Human-induced pluripotent stem cell-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electric pacing on the maturation of human-induced pluripotent stem cell-derived cardiac tissues.Cardiomyocytes derived from human-induced pluripotent stem cells were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electric pacing conditions.The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm2 vs 0.055±0.009 mN/mm2 for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 (Sarco/Endoplasmic Reticulum Calcium ATPase 2) expression, which correlated with a less negative force-frequency relationship. When electric pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm2), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 (Ryanodine Receptor 2) and SERCA2 further increased by combined static stress and electric stimulation.These studies demonstrate that electric pacing and mechanical stimulation promote maturation of the structural, mechanical, and force generation properties of human-induced pluripotent stem cell-derived cardiac tissues.