Developing a Functional Human Cardiovascular Micro-Organ System for Modeling Heart Development, Mechanobiology, and Disease

Introduction: : Reflecting limitations of traditional cell culture and animal models for disease modeling, drug development and screening, and the associated high costs and failure rates of clinical trials, there is an urgent need for advanced human in vitro models, including organoids, organ-on-chip (OoC), and microphysiologic systems (MPS), as recently endorsed by the FDA. To help address this need, we are developing a new generation of in situ differentiated MPS that combines the microphysiology of organoids with the tube-shaped functionality and modularity of OoCs, called the “μ-organ”, cultured in a microfluidic cardiovascular chip (μCVchip). The system is supported by custom robotics for automatically seeding cells, culturing micro-organs, and monitoring their structure and functional performance within the chip. With this infrastructure, we have achieved a first-of-its-kind tube-shaped micro-heart model that can autonomously generate oscillatory flow detectable in connected microfluidic channels. The objective of this study is to characterize the function of these tube-shaped cardiovascular micro-organs, aiming to test our hypothesis that such cardiovascular “tuboids” will provide more biomimetic functionality and improved predictive ability, in comparison to “spheroid” controls, for next-generation in vitro modeling of human heart development, physiology, and disease.

Materials and

Methods: : The μCVchip is cast in PDMS from a negative master, and plasma bonded to a PDMS-coated 60-mm dish (Fig. 1). Each chip contains 8 independent circulatory systems, with fluid compartment volumes of 150 μL for the reservoirs, 10 μL for the return shunt, and 1 μL for the μ-organ culture well (μw) and microfluidic channels (μc). The channels are made hydrophilic with poly-dopamine, and coated with laminin/entactin (200 μg/mL) and fibronectin (10μg/mL) to promote cell adhesion for μ-organ culture. Human iPSCs suspended in StemFlex (with 2μM Thiazovivin and 1μM CHIR99021) are seeded into the chip using a custom machine for computer-controlled pipet positioning and precise cell deposition into the 8 μw’s [Sachs+, bioRxiv, 2023]. Additional laminin/entactin is sedimented onto the cells, and after initial cell adhesion (~20 min), the seeded chip is transferred to a custom Automated Monitoring and Imaging Robot (AMIR), that handles up to 12 μCVchips at a time, each with 8 μ-organ systems. To create functional cardiac μ-organs, we use an ontomimetic strategy to mimic early heart development in vitro by seeding the chip with hiPSCs suspended in liquid hydrogel; once a 3D scaffold forms, multi-lineage cardiac-directed differentiation is performed in the μCVchip using an optimized protocol. Within 5-6 days, cells form a primitive μ-organ with a globular body skewed to one side of the μw, and aligned tissue processes extending into the μc’s. After differentiation is completed on day 7, custom defined serum-free media is used for μ-organ maintenance.

Results, Conclusions, and Discussions:: By day 10-12 we have observed tube-like cardiac μ-organs where a hollow channel with distinct borders extends through an optically dense mass of beating cardiac tissue (Fig. 2A), driving pulsatile motion of suspended particles along the tuboid axis, like a biological micro-pump. Troponin-positive cardiomyoytes are constrained to the μw and excluded from the fluidic channels, whereas endoglin-positive endothelial cells migrate and extend into the channels where they form tube-like microvascular structures. Optical flow analysis of video (Fig. 2A) yields vectors indicating contraction of the cardiac tuboid with axial motion of fluid within the hollow channel. Diastolic and systolic tube diameters (63 and 50 μm) yield an area ejection fraction of 38%. Resulting velocity vs. time data show an oscillatory wave form (Fig. 2B), with peak mean velocity of about 500 μm/sec. Assuming Poiseuille flow with a fluid viscosity of 2cP, the systolic shear stress is about 1.6 dyne/cm2 on the inner wall of the tuboid. Figure 2C shows example responses of another cardiac μ-organ to electrical stimulation in the μCVchip; decreased motion with faster pacing reflects a negative frequency-dependence, similar to fetal or newborn heart contraction. The beta-adrenergic agonist, Isoproterenol, increased beat rate from 72±19 bpm at baseline, to 136±29 bpm at steady state (Fig. 2D), which decreased to 40±16 bpm with addition of calcium channel blocker, Verapamil; responses are consistent with known effects of these drugs. To assess μ-organ tube stiffness, the chip was sealed and pressurized with a pneumatic syringe pump with pressure sensor feedback; a pressure change of 5 mmHg caused a 10-μm change in diameter (Fig. 2E), yielding a tissue stiffness of 2.25 kPa. Despite subphysiologic μ-organ function, comparing metrics across different conditions in vitro should allow us to predict responses in vivo, as we and others have done with various human engineered cardiac tissue models [Keung+, Clin Pharmacol Ther, 2019]. Once rigorously validated, we anticipate that our μCV-MPS technology will be valuable for advanced in vitro studies of human-specific cardiac development, mechanobiology, disease modeling, drug screening, and therapeutic discovery.

Acknowledgements and/or References (Optional):: Funding from NIH/NHLBI R21-EB023573; NSF-CASIS 1929028; NIH/NHLBI R01-HL173456; Additional Ventures SVRF-1297839. Technical support from Tamorae Binns, Nicole Ratnapala, Serena Chang, Kennedy Salamat, Audrey Lee, and expert guidance from Nicole Dubois and Irene C. Turnbull.