(G-254) Engineering a Functional Model of Human Ischemia-Reperfusion Injury on a Chip

Introduction:: After a myocardial infarction, timely reperfusion is essential to restore blood and oxygen flow to cardiac tissue. However, the restoration of flow also induces ischemia-reperfusion injury (IRI), resulting in further cardiomyocyte (CM) death and alterations in calcium homeostasis and contractility. The temporal profile and oxygen level dependency of such changes is not fully understood. Additionally, the lack of adequate human models of IRI has been indicated as a major factor for the failure of preclinical research to translate into clinical applications. To fulfill these unmet needs, we have engineered a human relevant oxygen gradient microdevice model of IRI to measure contractile force and calcium transients in parallel. Advanced controllability enables investigation into how the severity and duration of ischemia impact human CM function.

Materials and Methods:: An oxygen gradient was generated via continuous perfusion of compressed gases through underlying microfluidic channels and diffusion across a thin, gas permeable polydimethylsiloxane (PDMS) membrane on which an array of human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) microtissues was cultured. First, a surface layer of a 1:97 (w/w) mix of Sylgard 184 and 527 was cured on the device. This formulation generated an 8 kPa elastic modulus substrate to allow for traction force microscopy (TFM). Substrates were functionalized with 0.2 µm diameter fluorescent beads using (3-Aminopropyl) triethoxysilane (APTES). Then, an array of 100 µm x 200 µm fibronectin islands was deposited by microcontact printing fibronectin onto polyvinyl alcohol (PVA) films using PDMS stamps, transferring the fibronectin onto the low elastic modulus surface by conformal contact of the film, and dissolving away the PVA with water. WTC-GCaMP iPSCs were differentiated for 35 days into CMs using a chemically-defined differentiation protocol (Stem Cell Technologies) and replated onto the micropatterned fibronectin islands. The genetically encoded calcium indicator GCaMP allowed for monitoring of calcium transients. Image sequences during electrical pacing were collected to determine both the calcium transients and contractile forces of iPSC-CMs as a function of oxygen level exposure (determined by the position of the microtissue in the spatial oxygen gradient). Ischemia-reperfusion conditions were modeled by replacing hiPSC-CM culture media with simulated ischemia buffer and flowing 10% oxygen and nitrogen through the microchannels to establish the range of discrete oxygen levels for the separate cardiac microtissues.

Results, Conclusions, and Discussions:: The PVA film microcontact printing process generated 100 µm x 200 µm fibronectin islands onto which iPSC-CMs were replated (Fig 1A). A linear oxygen gradient was generated from 10% to 0% oxygen. The superimposed microtissue array enabled increased sampling to achieve higher throughput. The spatial oxygen gradient added finer granularity in the data by exposing microtissues to a range of oxygen levels, thereby allowing examination of CM function as it correlated with the oxygen level. Each column of microtissues was exposed to a distinct oxygen level and multiple rows added experimental replicates (Fig 1A). The WTC-GCaMP iPSC-CMs were cultured on the device to determine the calcium transients (Fig. 1B), and TFM was used to map contractile stress of the microtissues (Fig. 1C) during 0.5 Hz electrical pacing. Human in vitro models of IRI are necessary for understanding disease mechanisms, testing therapeutic strategies, and improving translation of preclinical research. Overall, our microdevice aims to broaden our understanding, monitoring, and mitigating of the progression of IRI. Future directions of this iPSC-based tool include development of a diabetic IRI model to test therapies in the setting of a common comorbidity.



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