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.

Pneumatic equiaxial compression device for mechanical manipulation of epithelial cell packing and physiology.

It is well established that mechanical cues, e.g., tensile- compressive- or shear forces, are important co-regulators of cell and tissue physiology. To understand the mechanistic effects these cues have on cells, technologies allowing precise mechanical manipulation of the studied cells are required. As the significance of cell density i.e., packing on cellular behavior is beginning to unravel, we sought to design an equiaxial cell compression device based on our previously published cell stretching system. We focused on improving the suitability for microscopy and the user-friendliness of the system. By introducing a hinge structure to the substrate stretch generating vacuum chamber, we managed to decrease the z-displacement of the cell culture substrate, thus reducing the focal plane drift. The vacuum battery, the mini-incubator, as well as the custom-made vacuum pressure controller make the experimental setup more flexible and portable. Furthermore, we improved the efficiency and repeatability of manufacture of the device by designing a mold that can be used to cast the body of the device. We also compared several different silicone membranes, and chose SILPURAN® due to its best microscopy imaging properties. Here, we show that the device can produce a maximum 8.5% radial pre-strain which leads to a 15% equiaxial areal compression as the pre-strain is released. When tested with epithelial cells, upon compression, we saw a decrease in cell cross-sectional area and an increase in cell layer height. Additionally, before compression the cells had two distinct cell populations with different cross-sectional areas that merged into a more uniform population due to compression. In addition to these morphological changes, we detected an alteration in the nucleo-cytoplasmic distribution of YAP1, suggesting that the cellular packing is enough to induce mechanical signaling in the epithelium.

Cardiac Ischemia On-a-Chip: Antiarrhythmic Effect of Levosimendan on Ischemic Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Ischemic heart disease (IHD) is one of the leading causes of mortality worldwide. Preserving functionality and preventing arrhythmias of the heart are key principles in the management of patients with IHD. Levosimendan, a unique calcium (Ca2+) enhancer with inotropic activity, has been introduced into clinical usage for heart failure treatment. Human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs) offer an opportunity to better understand the pathophysiological mechanisms of the disease as well as to serve as a platform for drug screening. Here, we developed an in vitro IHD model using hiPSC-CMs in hypoxic conditions and defined the effects of the subsequent hypoxic stress on CMs functionality. Furthermore, the effect of levosimendan on hiPSC-CMs functionality was evaluated during and after hypoxic stress. The morphology, contractile, Ca2+-handling, and gene expression properties of hiPSC-CMs were investigated in response to hypoxia. Hypoxia resulted in significant cardiac arrhythmia and decreased Ca2+ transient amplitude. In addition, disorganization of sarcomere structure was observed after hypoxia induction. Interestingly, levosimendan presented significant antiarrhythmic properties, as the arrhythmia was abolished or markedly reduced with levosimendan treatment either during or after the hypoxic stress. Moreover, levosimendan presented significant protection from the sarcomere alterations induced by hypoxia. In conclusion, this chip model appears to be a suitable preclinical representation of IHD. With this hypoxia platform, detailed knowledge of the disease pathophysiology can be obtained. The antiarrhythmic effect of levosimendan was clearly observed, suggesting a possible new clinical use for the drug.

Vasculogenic Potency of Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells Results in Differing Vascular Network Phenotypes in a Microfluidic Chip.

The vasculature is an essential, physiological element in virtually all human tissues. Formation of perfusable vasculature is therefore crucial for reliable tissue modeling. Three-dimensional vascular networks can be formed through the co-culture of endothelial cells (ECs) with stromal cells embedded in hydrogel. Mesenchymal stem/stromal cells (MSCs) derived from bone marrow (BMSCs) and adipose tissue (ASCs) are an attractive choice as stromal cells due to their natural perivascular localization and ability to support formation of mature and stable microvessels in vitro. So far, BMSCs and ASCs have been compared as vasculature-supporting cells in static cultures. In this study, BMSCs and ASCs were co-cultured with endothelial cells in a fibrin hydrogel in a perfusable microfluidic chip. We demonstrated that using MSCs of different origin resulted in vascular networks with distinct phenotypes. Both types of MSCs supported formation of mature and interconnected microvascular networks-on-a-chip. However, BMSCs induced formation of fully perfusable microvasculature with larger vessel area and length whereas ASCs resulted in partially perfusable microvascular networks. Immunostainings revealed that BMSCs outperformed ASCs in pericytic characteristics. Moreover, co-culture with BMSCs resulted in significantly higher expression levels of endothelial and pericyte-specific genes, as well as genes involved in vasculature maturation. Overall, our study provides valuable knowledge on the properties of MSCs as vasculature-supporting cells and highlights the importance of choosing the application-specific stromal cell source for vascularized organotypic models.

Self-assembled cellulose nanofiber-carbon nanotube nanocomposite films with anisotropic conductivity.

In this study, a nanocellulose-based material showing anisotopic conductivity is introduced. The material has up to 1000 times higher conductivity along the dry-line boundary direction than along the radial direction. In addition to the material itself, the method to produce the material is novel and is based on the alignment of cationic cellulose nanofibers (c-CNFs) along the dry-line boundary of an evaporating droplet composed of c-CNFs in two forms and conductive multi-walled carbon nanotubes (MWCNTs). On the one hand, c-CNFs are used as a dispersant of MWCNTs, and on the other hand they are used as an additional suspension element to create the desired anisotropy. When the suspended c-CNF is left out, and the nanocomposite film is manufactured using the high energy sonicated c-CNF/MWCNT dispersion only, conductive anisotropy is not present but evenly conducting nanocomposite films are obtained. Therefore, we suggest that suspending additional c-CNFs in the c-CNF/MWCNT dispersion results in nanocomposite films with anisotropic conductivity. This is a new way to obtain nanocomposite films with substantial anisotropic conductivity.

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.

A Portable Microscale Cell Culture System with Indirect Temperature Control.

A physiologically relevant environment is essential for successful long-term cell culturing in vitro. Precise control of temperature, one of the most crucial environmental parameters in cell cultures, increases the fidelity and repeatability of the experiments. Unfortunately, direct temperature measurement can interfere with the cultures or prevent imaging of the cells. Furthermore, the assessment of dynamic temperature variations in the cell culture area is challenging with the methods traditionally used for measuring temperature in cell culture systems. To overcome these challenges, we integrated a microscale cell culture environment together with live-cell imaging and a precise local temperature control that is based on an indirect measurement. The control method uses a remote temperature measurement and a mathematical model for estimating temperature at the desired area. The system maintained the temperature at 37±0.3 °C for more than 4 days. We also showed that the system precisely controls the culture temperature during temperature transients and compensates for the disturbance when changing the cell cultivation medium, and presented the portability of the heating system. Finally, we demonstrated a successful long-term culturing of human induced stem cell-derived beating cardiomyocytes, and analyzed their beating rates at different temperatures.

Cellulose Nanofiber Alignment Using Evaporation-Induced Droplet-Casting, and Cell Alignment on Aligned Nanocellulose Surfaces.

This work investigates droplet-evaporated cellulose nanofiber (CNF) alignment and cell responses on CNF surfaces. Surfaces of unmodified (u-), anionic (a-), and cationic (c-) CNFs were fabricated using an evaporation-induced droplet-casting method and characterized in terms of degree of orientation. Circular variance (CV) values obtained using Cytospectre software to analyze the degree of orientation from AFM images showed a significantly higher degree of orientation on c- and u-CNF surfaces (average CV 0.27 and 0.24, respectively) compared to a-CNF surfaces (average CV 0.76). Quantitative analysis of surface roughness plots obtained from AFM images confirmed the difference between the direction of alignment versus the direction perpendicular to alignment. AFM images as well as observations during droplet evaporation indicated c-CNF alignment parallel to a dry-boundary line during droplet evaporation. Fibroblasts were cultured on the u-, a-, and c-CNF surfaces with or without a fibronectin (FN) coating for 48 h, and the cell response was evaluated in terms of cell viability, proliferation, morphology, and degree of orientation. Cell viability and proliferation were comparable to that on a control surface on the a-CNF and c-CNF surfaces. Although an FN coating slightly enhanced cell growth on the studied surfaces, uncoated a-CNF and c-CNF surfaces were able to support cell growth as well. The results showed cell orientation on aligned c-CNF surfaces, a finding that could be further utilized when guiding the growth of cells. We also showed that the alignment direction of c-CNFs and thus the cell orientation direction can be controlled with a contact-dispensing technique.

Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array.

BACKGROUND: Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO2 supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential. NEW METHOD: We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber. RESULTS: We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments. COMPARISON WITH EXISTING METHODS: Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO2 gas. Utilizing dry gas supply makes the proposed chamber simple to use. CONCLUSION: Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture.