Building blocks of microphysiological system to model physiology and pathophysiology of human heart.

Microphysiological systems (MPS) are drawing increasing interest from academia and from biomedical industry due to their improved capability to capture human physiology. MPS offer an advanced in vitro platform that can be used to study human organ and tissue level functions in health and in diseased states more accurately than traditional single cell cultures or even animal models. Key features in MPS include microenvironmental control and monitoring as well as high biological complexity of the target tissue. To reach these qualities, cross-disciplinary collaboration from multiple fields of science is required to build MPS. Here, we review different areas of expertise and describe essential building blocks of heart MPS including relevant cardiac cell types, supporting matrix, mechanical stimulation, functional measurements, and computational modelling. The review presents current methods in cardiac MPS and provides insights for future MPS development with improved recapitulation of human physiology.

Electrophysiological Changes of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes during Acute Hypoxia and Reoxygenation.

Ischemic heart disease is the most common cardiovascular disease and a major burden for healthcare worldwide. However, its pathophysiology is still not fully understood, and human-based models for disease mechanisms and treatments are needed. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to model acute ischemia-reperfusion in our novel cell culture assembly. The assembly enables exchange of oxygen partial pressure for the cells within minutes, mimicking acute ischemic event. In this study, hypoxia was induced using 0% O2 gas for three hours and reoxygenation with 19% O2 gas for 24 hours in serum- and glucose-free medium. According to electrophysiological recordings, hypoxia decreased the hiPSC-CM-beating frequency and field potential (FP) amplitude. Furthermore, FP depolarization time and propagation slowed down. Most of the electrophysiological changes reverted during reoxygenation. However, immunocytochemical staining of the hypoxic and reoxygenation samples showed that morphological changes and changes in the sarcomere structure did not revert during reoxygenation but further deteriorated. qPCR results showed no significant differences in apoptosis or stress-related genes or in the expression of glycolytic genes. In conclusion, the hiPSC-CMs reproduced many characteristic changes of adult CMs during ischemia and reperfusion, indicating their usefulness as a human-based model of acute cardiac ischemia-reperfusion.

Compartmentalized organ-on-a-chip structure for spatiotemporal control of oxygen microenvironments.

Hypoxia is a condition where tissue oxygen levels fall below normal levels. In locally induced hypoxia due to blood vessel blockage, oxygen delivery becomes compromised. The site where blood flow is diminished the most forms a zero-oxygen core, and different oxygenation zones form around this core with varying oxygen concentrations. Naturally, these differing oxygen microenvironments drive cells to respond according to their oxygenation status. To study these cellular processes in laboratory settings, the cellular gas microenvironments should be controlled rapidly and precisely. In this study, we propose an organ-on-a-chip device that provides control over the oxygen environments in three separate compartments as well as the possibility of rapidly changing the corresponding oxygen concentrations. The proposed device includes a microfluidic channel structure with three separate arrays of narrow microchannels that guide gas mixtures with desired oxygen concentrations to diffuse through a thin gas-permeable membrane into cell culture areas. The proposed microfluidic channel structure is characterized using a 2D ratiometric oxygen imaging system, and the measurements confirm that the oxygen concentrations at the cell culture surface can be modulated in a few minutes. The structure is capable of creating hypoxic oxygen tension, and distinct oxygen environments can be generated simultaneously in the three compartments. By combining the microfluidic channel structure with an open-well coculture device, multicellular cultures can be established together with compartmentalized oxygen environment modulation. We demonstrate that the proposed compartmentalized organ-on-a-chip structure is suitable for cell culture.

Human induced pluripotent stem cell-based platform for modeling cardiac ischemia.

Ischemic heart disease is a major cause of death worldwide, and the only available therapy to salvage the tissue is reperfusion, which can initially cause further damage. Many therapeutics that have been promising in animal models have failed in human trials. Thus, functional human based cardiac ischemia models are required. In this study, a human induced pluripotent stem cell derived-cardiomyocyte (hiPSC-CM)-based platform for modeling ischemia-reperfusion was developed utilizing a system enabling precise control over oxygen concentration and real-time monitoring of the oxygen dynamics as well as iPS-CM functionality. In addition, morphology and expression of hypoxia-related genes and proteins were evaluated as hiPSC-CM response to 8 or 24 h hypoxia and 24 h reoxygenation. During hypoxia, initial decrease in hiPSC-CM beating frequency was observed, after which the CMs adapted to the conditions and the beating frequency gradually increased already before reoxygenation. During reoxygenation, the beating frequency typically first surpassed the baseline before settling down to the values close the baseline. Furthermore, slowing on the field potential propagation throughout the hiPSC-CM sheet as well as increase in depolarization time and decrease in overall field potential duration were observed during hypoxia. These changes were reversed during reoxygenation. Disorganization of sarcomere structures was observed after hypoxia and reoxygenation, supported by decrease in the expression of sarcomeric proteins. Furthermore, increase in the expression of gene encoding glucose transporter 1 was observed. These findings indicate, that despite their immature phenotype, hiPSC-CMs can be utilized in modeling ischemia-reperfusion injury.

Covalent immobilization of luminescent oxygen indicators reduces cytotoxicity.

Luminescence-based oxygen sensing is a widely used tool in cell culture applications. In a typical configuration, the luminescent oxygen indicators are embedded in a solid, oxygen-permeable matrix in contact with the culture medium. However, in sensitive cell cultures even minimal leaching of the potentially cytotoxic indicators can become an issue. One way to prevent the leaching is to immobilize the indicators covalently into the supporting matrix. In this paper, we report on a method where platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) oxygen indicators are covalently immobilized into a polymer matrix consisting of polystyrene and poly(pentafluorostyrene). We study how the covalent immobilization influences the sensing material's cytotoxicity to human induced pluripotent stem cell-derived (hiPSC-derived) neurons and cardiomyocytes (CMs) through 7-13 days culturing experiments and various viability analyses. Furthermore, we study the effect of the covalent immobilization on the indicator leaching and the oxygen sensing properties of the material. In addition, we demonstrate the use of the covalently linked oxygen sensing material in real time oxygen tension monitoring in functional hypoxia studies of the hiPSC-derived CMs. The results show that the covalently immobilized indicators substantially reduce indicator leaching and the cytotoxicity of the oxygen sensing material, while the influence on the oxygen sensing properties remains small or nonexistent.

Applying total internal reflection excitation and super critical angle fluorescence detection to a morphine assay.

A surface-sensitive fluorescence measurement platform is utilised in the detection of morphine. The platform is based on a polystyrene parabolic lens that enables the simultaneous application of total internal reflection excitation and supercritical angle fluorescence detection in the measurements. The molecular recognition of morphine is based on two antibodies, one against morphine and the other against the immune complex formed between the anti-morphine antibody and a morphine molecule. The antibodies are applied in a sandwich-like format in a one-step test, where the molecular binding onto a liquid-solid-interface is monitored in real time. Morphine concentrations between 0.6 and 18.2 ng/mL were reliably determined in 60 s, while concentrations down to 2.7 ng/mL were detected already in 20 s. With appropriate recognition molecules the technique is applicable also to other drugs and small analytes.