Low-temperature biopreservation and 3D tissue engineering present two differing routes towards eventual on-demand access to transplantable biologics, but recent advances in both fields present critical new opportunities for crossover between them. In this work, we demonstrate sub-zero centigrade preservation and revival of autonomously beating three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study complex tissue biopreservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves.

Mesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the direct isolation of MSCs from adipose tissue into a 3D environment, avoiding contact to a 2D plastic surface. Furthermore, the use of a cryoprotective medium for the cryopreservation of whole adipose tissue was evaluated. Cryopreservation of fresh adipose tissue with and without a cryoprotective medium was compared with regard to the viability and metabolic activity of cells. After thawing, the tissue was embedded in a novel human platelet lysate-based hydrogel for the isolation of MSCs. The migration, yield, viability, and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment or 2D explant culture. The cryopreservation of whole adipose tissue was found to be feasible, and therefore, adipose tissue can be stored and is available for MSC isolation on demand. Also, we demonstrate the isolation of MSCs from adipose tissue into the 3D matrix. The cells derived from this isolation procedure display a similar phenotype and differentiation capacity like MSCs derived by traditional procedures. The presented approach allows to cryopreserve adipose tissue. Furthermore, for the first time, MSCs were directly isolated from the tissue into a soft 3D hydrogel environment, avoiding any contact to a 2D plastic culture surface.

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug’s efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug’s inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug’s overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

New approach methodologies (NAMs), including microphysiological systems (MPSs), can recapitulate structural and functional complexities of organs. Vanoxerine was reported to induce cardiac adverse events, including torsade de points (TdP), in a Phase III clinical trial. Despite earlier nonclinical animal models and Phase I–II clinical trials, events of QT prolongation or proarrhythmia were not observed. Here, we utilized cardiac NAMs to evaluate the functional consequences of vanoxerine treatment on human cardiac excitation–contraction coupling. The cardiac MPS used in this study was a microfabricated fluidic culture platform with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) capable of evaluating voltage, intracellular calcium handling, and contractility. Likewise, the hiPSC-CM comprehensive in vitro proarrhythmia assay (CiPA) was employed based on multielectrode array (MEA). Vanoxerine treatment delayed repolarization in a concentration-dependent manner and induced proarrhythmic events in both NAM platforms. The complex cardiac MPS displayed a frequency-dependent vanoxerine response such that EADs were eliminated at a faster pacing rate (1.5 Hz). Moreover, exposure analysis revealed a 99% vanoxerine loss in the cardiac MPS. TdP risk analysis demonstrated high to intermediate TdP risk at clinically relevant concentrations of vanoxerine and frequency-independent EAD events in the hiPSC-CM CiPA model. These findings demonstrate that nonclinical cardiac NAMs can recapitulate clinical outcomes, including detection of vanoxerine-induced delayed repolarization and proarrhythmic effects. Moreover, this work provides a foundation to evaluate the safety and efficacy of novel compounds to reduce the dependence on animal studies.