Stem Cell Reports Article Engineered Macroscale Cardiac Constructs Elicit Human Myocardial Tissue-like Functionality Maria Valls-Margarit,1,10 Olalla Iglesias-Garcı́a,2,3,10 Claudia Di Guglielmo,2,3 Leonardo Sarlabous,3,4,5 Karine Tadevosyan,2,3 Roberto Paoli,6 Jordi Comelles,1 Dolores Blanco-Almazán,3,4,5 Senda Jiménez-Delgado,2,3 Oscar Castillo-Fernández,7 Josep Samitier,3,6,8 Raimon Jané,3,4,5 Elena Martı́nez,1,3,8,* and Ángel Raya2,3,9,* 1Biomimetic Systems for Cell Engineering, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, Spain 2Center of Regenerative Medicine in Barcelona (CMRB), L’Hospitalet de Llobregat, Barcelona, Spain 3Center for Networked Biomedical Research on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain 4Biomedical Signal Processing and Interpretation, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, Spain 5Department of Automatic Control, Universitat Politècnica de Catalunya - BarcelonaTech (UPC), Barcelona, Spain 6Nanobioengineering, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, Spain 7Institute of Microelectronics of Barcelona, IMB-CNM (CSIC), Campus UAB, Bellaterra, Spain 8Department of Electronics and Biomedical Engineering, University of Barcelona (UB), Barcelona, Spain 9Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 10Co-first author *Correspondence: emartinez@ibecbarcelona.eu (E.M.), araya@cmrb.eu (Á.R.) https://doi.org/10.1016/j.stemcr.2019.05.024 SUMMARY In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and imple- mentation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term ‘‘CardioSlice.’’ PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram- like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients. INTRODUCTION tials of 1 mV, easily recorded on the body surface. That the shape of these voltage potential waves (electrocardio- Cardiac tissue engineering aims at producing constructs gram [ECG]) is a reliable indicator of cardiac performance, with structural, physiological, and functional properties used on a routine basis in clinical cardiology for over a cen- resembling human native cardiac tissue. Ultimately, such tury (Fermini and Fossa, 2003), clearly attests to the inti- in vitro models will find applications in disease modeling, mate dependence of structure and function in the heart. drug screening, and toxicology, and replacing or regenerat- Due to the high dependence found between cardiac mus- ing damaged heart tissue. The structural organization of cle structural organization and its function, it has been hy- the cardiac tissue is complex, comprising a vast array of pothesized that growing cardiac constructs in engineering diverse cell types (including fibroblasts, cardiac myocytes, systems mimicking relevant physicochemical stimuli and smooth muscle, pacemaker, and endothelial cells), ar- found in vivo would be advantageous to achieve tissue- ranged in a precise and stereotypical architecture to ensure like properties (Fleischer et al., 2017). In the last years, tis- that the critical function of pumping blood throughout the sue-engineering methods have significantly advanced in body is maintained (Chien et al., 2008). Efficient blood generating functional 3D cardiac constructs (Radisic pumping requires the 5 billion cardiomyocytes that et al., 2004a; Shimizu et al., 2002; Zimmermann et al., make up an average human adult heart contracting and re- 2002). A key issue that has deserved much attention in laxing in a coordinated and timely order. Multiscale struc- this field is the degree of cardiomyocyte maturation tural features characteristic of the heart’s intracellular and achieved within the engineered cardiac constructs (re- intercellular organization enable the necessary coordina- viewed in Parsa et al., 2016). This issue is particularly tion for the entire heart muscle to form a functional syncy- important when using cardiomyocytes derived from tium (Hunter et al., 2003). This results in electrochemical pluripotent stem cells (PSCs), which are typically imma- processes of such magnitude that generate voltage poten- ture, fetal-like under standard two-dimensional (2D) Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 j ª 2019 The Authors. 207 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). (legend on next page) 208 Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 differentiation conditions (Feric and Radisic, 2016). Hu- mented a platform to produce macrotissues of engineered man engineered cardiac constructs developed thus far reca- cardiac muscle from human PSCs, which we termed ‘‘Car- pitulate some of the structural complexity and electrome- dioSlice.’’ PSC-derived cardiomyocytes differentiated un- chanical functionality of the native myocardium, leading der standard 2D conditions were seeded, together with hu- to improved performance compared with standard 2D man fibroblasts, into 3D porous scaffolds of 10 mm in in vitro cultures (Ma et al., 2015; Nunes et al., 2013; Schaaf diameter and 1 mm in thickness. Constructs were cultured et al., 2011; Thavandiran et al., 2013). Exogenous stimuli for up to 14 days in a parallelized perfusion bioreactor such as mechanical and electrical signals have been shown equipped with custom-made culture chambers endowed to further improve the electrophysiological properties, the with electrostimulation capabilities. The constructs ob- cellular and ultrastructural organization, and the expres- tained developed into macroscopically contractile struc- sion of cardiac-specific proteins of cardiac constructs (God- tures in which cardiomyocytes showed signs of increased ier-Furnemont et al., 2015; Radisic et al., 2004a; Tandon cell maturation compared with those cultured under 2D et al., 2009). These strategies have resulted in microengi- conditions, and similar to those of microtissues. More neered models of human cardiac muscle, which emerged importantly, continuous electrical stimulation of the car- as promising platforms for preclinical toxicology and diac macrotissues for 2 weeks promoted cardiomyocyte drug screening assays (Amano et al., 2016; Hansen et al., alignment and synchronization, and the emergence of car- 2010; Mathur et al., 2015). However, microtissues are min- diac tissue-like properties. These translated into sponta- imal units with some cardiac functionality, but are inher- neous electrical activity that could be readily measured ently limited in size and cannot fully capture the on the surface of CardioSlice constructs as ECG-like signals, complexity of the native cardiac tissue structure (Kurokawa and a response to proarrhythmic drugs that was predictive and George, 2016). Unfortunately, the production of hu- of their effect in human patients. man macroscale tissues displaying in vivo-like complexity and, therefore, tissue-like functionality, is still an unmet challenge (Fleischer et al., 2017). RESULTS The design of tissue-engineering constructs on the macroscale (greater than 300 mm in thickness) is met with Development of a Parallelized Bioreactor for the challenge that effective mass transfer cannot rely on Continuous Perfusion and Electrical Stimulation/ passive diffusion alone (Lovett et al., 2009). This is critical Recording of Cardiac Macroscale Constructs for cardiac tissue constructs due to the comparatively To generate CardioSlice constructs, we designed a parallel- high metabolic demand of cardiac muscle cells, which re- ized perfusion bioreactor including electrical stimulation quires a controlled microenvironment with the appro- (Figure 1). Our a priori design requirements included: (1) priate supply of oxygen and nutrients (Carrier et al., suitability for the perfusion of macroscale constructs 2002; Radisic et al., 2004b, 2008). Perfusion bioreactor sys- (10 mm in diameter and 1–2 mm in thickness); (2) capa- tems pioneered in Vunjak-Novakovic’s laboratory have bility for in vivo-like electrical stimulation; (3) ability to pro- proved to be valuable in the generation of thick cardiac tis- duce interstitial flow with proper shear; (4) amenability to sue constructs full of viable cells with aerobic metabolism parallelization for the simultaneous culture of multiple (Radisic et al., 2004b). Here, we have designed and imple- constructs; and (5) on-line recording of the construct Figure 1. Parallelized Perfusion Bioreactor for the Generation of CardioSlice Constructs (A) Scheme of the custom-made perfusion chamber and 3D modeling of the electric field generated when applying a differential voltage of 5 V. The chamber scheme depicts two stimulation electrodes (black), one at each side of the construct (pink), and a central gold electrode (yellow) underneath the construct acting as internal reference. Two gaskets (gray) hold the construct in place and direct the fluid (red) flow through the scaffold. This scheme of the chamber is used to model the electrical field. Electrode configuration (red and blue cylinders in the geometry, positive and negative, respectively) and predicted electric field and current density values are displayed both in 3D and top views. Black arrowheads indicate the direction of the electric field. Plots show electric field and current density values at the positions where cells are seeded in the scaffold (central circle in the top view) (from x = 2.5 to x = 2.5). (B) Horizontal and vertical planes of the perfusion chamber with electrical stimulation. The cardiac construct (scaffold + cells; pink) is held in place by two gaskets (blue). Black rectangles represent graphite electrodes, and the yellow rectangle and dot represent the gold electrode. Interstitial flow of culture medium (red arrow) is routed through the pores of the construct. (C) Voltage measurements in the perfusion chamber with electrical stimulation. Measured electric field values match the values predicted by the model. (D) Overall view of the parallel bioreactor system: medium reservoir (1), luer manifold (2), de-bubblers (3), flow restrictors (4), perfusion chambers (5), gas exchanger (6), and peristaltic pump (7). The bioreactor supports the culture of up to four cardiac constructs simultaneously. Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 209 electrical activity. To address these requirements, we first perfusion bioreactor (Figure 1D). Parallelization of the cul- formulated an electric field model to determine the appro- ture system allowed the production of multiple cardiac priate size and allocation of the stimulating electrodes macrotissues under the same physicochemical conditions within the chamber (Figure 1A). We simulated the electric and from the same pool of cells. Fluidic restrictors were field and current density generated by two graphite elec- installed (Rrestrictor 1013 >> Rmanifold 107) to ensure that trodes (5 mm in diameter) separated 1 cm apart and in con- the flow was homogeneously distributed through all tact with culture medium. Solving the model resulted in an branches. Finally, on-line recording of the electrophysio- electric field of around 400 V/m and a current density of logical activity of cardiac tissue constructs was carried out around 600 A/m2 in the center of the chamber, when a dif- using the two graphite electrodes used for electrical stimu- ferential voltage of 5 V was applied. Similar values were ob- lation (one acting as the source and the other one as the tained for the entire region intended for use to allocate the reference), and a gold electrode as an internal reference cardiac construct containing cells, thus making this config- (Figures 1B and S1B). Recorded signals were amplified and uration suitable for cardiomyocyte stimulation in terms of noise-filtered to obtain the electrical activity of the magnitude and uniformity according to the literature (Bar- constructs. ash et al., 2010; Tandon et al., 2009). We then fabricated custom-made perfusion chambers including electrodes Structural Signs of Cell and Tissue Maturation in with the specifications included in the simulation (Fig- CardioSlice Constructs ure 1B). Upon fabrication, we measured the voltage values To create human cardiac macrotissues, we differentiated in the actual perfusion chamber to verify that the expected cardiomyocytes from human induced PSCs (hiPSCs) stimuli were properly delivered (Figure 1C). Rectangular following a robust and reproducible protocol based on pulses at 3 Hz of frequency, 2 ms of duration, and modulation of Wnt/b-catenin signaling with small-mole- increasing differential voltages from 1 to 5 V were applied cule inhibitors (Lian et al., 2013). hiPSCs differentiated in to the chamber. Electric potentials between stimulating this way formed contracting monolayers comprising graphite electrodes and between each electrode and the high percentages of cardiomyocytes, as defined by coex- center of the chamber (gold electrode) were measured. pression of cardiac troponin I (cTnI) and myosin heavy The intended waveform was reliably applied for all voltages chain (MHC) proteins (71.6% ± 5.3% cTnI+/MHC+ cells) tested. Moreover, when applying 5 V between graphite after 20 days of differentiation (Figure S2). In preliminary electrodes, 2 V were measured in the center of the chamber, studies, we determined that fully differentiated cardio- representing an electric field of 400 V/m, in agreement myocytes (i.e., after 20 days of differentiation from with the values predicted by the model. hiPSCs), but not undifferentiated hiPSCs or early The shear stress to which cells would be exposed in the committed mesoderm progenitors (as defined by the perfusion bioreactor was calculated as previously described expression of T/Brachyury), could be cultured within (Radisic et al., 2008). The minimum flow rate needed for the porous scaffolds under continuous perfusion for up efficiently perfusing cardiac macrotissues depends on the to 21 days while maintaining a differentiated phenotype overall mass balance of oxygen, and was estimated to be (data not shown). We also verified that addition of fibro- 0.1 mL/min for rat cardiac tissue constructs and 0.2 mL/ blasts to iPSC-derived cardiomyocytes improved the min for human cardiac tissues. Shear stress affecting the reproducibility of obtaining viable cardiac constructs, in cells within the scaffold was then calculated by taking agreement with previous studies (Amano et al., 2016; into account the flow rate, culture medium viscosity Liau et al., 2017; Tiburcy et al., 2017). Based on those pre- (0.0078 dyn,s/cm2) (Bacabac et al., 2005), and the volume liminary results, for the present studies we used a (19.6 mm3), void fraction (0.94), and mean pore radius mixture of hiPSC-derived cardiomyocytes and human (8.5 mm, Figure S1A) of the scaffold. Perfusion of culture dermal primary fibroblasts seeded in a 7:1 ratio. We chose medium at 0.1 mL/min evoked a shear stress of around a commercial, clinical-grade porous scaffold made up of 0.7 dyn/cm2 while perfusion at 0.2 mL/min yielded a shear collagen and elastin to culture the cells, on account of stress of around 1.3 dyn/cm2. According to the literature, its biocompatibility and myocardium-like pore size the values selected for the perfusion flow rate are sufficient and stiffness (Figure S1A). A total of 5 million cells to satisfy mass transport requirements associated with car- were seeded into the scaffolds using an on-purpose de- diac muscle tissue (Radisic et al., 2008), while the associated signed perfusion loop (see details in Experimental Pro- shear stress values do not compromise cardiomyocyte func- cedures and Figure S1B). tionality and viability (Cheng et al., 2009; Radisic et al., In a first set of experiments, we tested the effects of 2008; Shachar et al., 2012). electrical stimulation on human cardiac macrotissues. For Next, the custom-made perfusion chambers including this purpose, constructs were introduced into perfusion electrical stimulation were installed in the parallelized chambers and randomly assigned to the control group 210 Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 Figure 2. Morphology and Ultrastructural Organization of Human Cardiac Macrotissues (A) Representative cross-sections of cardiac constructs after 14 days of culture without (Control) or with electrical stimulation (CardioSlice). hiPSC-derived cardiomyocytes positive for cardiac troponin (cTnT; green) and phalloidin (Ph; red) were detected in the scaffold. Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; blue). Cardiac macrotissues were analyzed by Second Harmonic Generation for the detection of collagen (Col; violet). Scale bars, 500 mm (left); higher magnifications (left to right), 100 mm, 50 mm, and 10 mm, respectively. (B) Standard 2D culture of hiPSC-derived cardiomyocytes in monolayer. Cardiomyocytes were positive for cardiac troponin (cTnT; green) and phalloidin (Ph; red). Scale bar, 50 mm. (C) Ultrastructural analysis of hiPSC-derived cardiac macrotissues. Representative transmission electron microscopy images of cardiac macrotissues after 7 and 14 days of culture. Scale bars, 1 mm (bundles) and 0.5 mm (sarcomere width and cell junctions). (D) Morphometric analysis showing sarcomere width. Each individual sarcomere width is represented as a dot in the graph and at least 200 sarcomeres per independent experiment were measured in three independent experiments (***p < 0.001, N = 3, n R 200 per group; Mann- Whitney U test). Data are expressed as mean ± standard deviation (SD). (only perfusion) or the CardioSlice group (perfusion and expressed cardiac contractile proteins, including cardiac electrical stimulation), and maintained in culture for up troponin T (cTnT) and a-sarcomeric actin (Figure 2A), to 14 days. Human cardiac macrotissues exhibited cardio- with signs of increased cardiomyocyte maturation myocytes and fibroblasts distributed throughout the compared with cells cultured for the same time period un- scaffold thickness (Figure S3A). Cardiomyocytes strongly der conventional 2D conditions (Figure 2B). No differences Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 211 Figure 3. Functional Assessment of Human Cardiac Macrotissues (A) Top-view images of human cardiac macrotissues after 7 and 14 days of culture, both without (Control) or with (CardioSlice) electrical stimulation. Scale bars, 2.5 mm. (B) Contraction amplitude analysis of Control and CardioSlice constructs after 7 and 14 days of culture. Area oscillation is represented over time. (legend continued on next page) 212 Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 in cardiomyocyte proliferation or viability, as judged by the of fetal human heart than to those of adult ventricular tis- number of Ki67-positive cardiomyocytes and the number sue, consistent with previous analyses of human iPSC- of TUNEL-positive cardiomyocytes, respectively, were derived cardiomyocytes (reviewed in Yang et al., 2014). found when comparing CardioSlice and control constructs These results indicate that any differences observed in the after 14 days of culture (Figures S3B and S3C). Cardiomyo- functionality of cardiac macrotissues should be attributed cytes in CardioSlice constructs were mostly aligned along to changes in the maturation of the multicellular structure the direction of electric field vector used for stimulation, rather than to the maturation of its individual cellular in contrast with the random distribution found in control constituents. constructs (Figure 2A), a finding also observed in rat- derived cardiac macrotissues (Figure S4A). At the ultrastruc- Biomechanical Signs of Tissue Maturation in tural level, cardiomyocytes in CardioSlice constructs dis- CardioSlice Constructs played a statistically significant increase in sarcomere Upon harvesting, both control and CardioSlice constructs width compared with those in control constructs, after displayed spontaneous beating, which was much more both 7 and 14 days in culture (day 7: 0.70 ± 0.38 mm for apparent macroscopically in the case of CardioSlice con- CardioSlice and 0.52 ± 0.29 mm for control; day 14: structs (Videos S1, S2, S3, and S4). We did not find differ- 0.83 ± 0.38 mm for CardioSlice and 0.64 ± 0.35 mm for con- ences in the spontaneous beating rate between CardioSlice trol; p < 0.001) (Figures 2C and 2D), indicating a higher de- and control constructs, which we monitored after 4, 7, and gree of maturation. Moreover, after 14 days in culture, more 14 days of culture, and ranged from 0.6 to 1.1 beats/s. Elec- developed intercalated discs were detected in CardioSlice tric field stimulation had a direct impact on cell distribu- than in control constructs (Figure 2C). This was again in tion within the CardioSlice constructs, as cells concen- agreement with the improved ultrastructural signs of trated in the region were delimited by the stimulating cardiomyocyte maturation observed in rat-derived cardiac electrodes (Figure 3A). The amplitude of contraction, macrotissues, where wider sarcomeres and better devel- determined by measuring the changes in the fractional oped intercalated discs were present in electrostimulated area during spontaneous beating, was significantly compared with control constructs (Figures S4B and S4C). higher in CardioSlice constructs compared with control The expression level of key cardiac genes was measured ones (day 7: 6.54% ± 0.94% for CardioSlice constructs in control and CardioSlice constructs and compared with compared with 0.54% ± 0.09% for control constructs; day those of cells cultured for equivalent periods of time under 14: 7.85% ± 0.75% for CardioSlice constructs compared conventional 2D conditions (Figure S3D). As expected, with 2.08% ± 0.12% for control constructs; p < 0.001), indi- maturity-related cardiac genes (MYH7, ACTC1, TNNT2, cating improved maturation of the electromechanical MLC2V, GATA4, and GJA1) and genes encoding cardiac coupling machinery (Figures 3B and 3C). Consistently, ion channels (RYR2, SERCA2A, CACNA1C, SCNA5A, CardioSlice constructs also exhibited significantly higher KCNH2, and KCNQ1) were expressed under all conditions values for the maximum capture rate (MCR) parameter analyzed. However, no conspicuous differences were noted when compared with control samples. This was evident in cells cultured under either 2D or 3D conditions when after 14 days of culture: 4.28 ± 1.63 Hz in CardioSlice comparing control and CardioSlice constructs (Figure S3D). constructs versus 2.43 ± 0.75 Hz in control constructs Overall, the expression levels of maturity-related cardiac (p < 0.001) (Figure 3D). The effects of continuous electric genes and cardiac ion channels were more similar to those stimulation were not specific to human cells, since similar (C) Bar chart showing the percentage of fractional area change (FAC) for each cardiac macrotissue (average ± SEM) (***p < 0.001, N R 3, n R 3 per group; Mann-Whitney U test). (D) Maximum capture rate (MCR) of Control and CardioSlice constructs after 7 and 14 days of culture (average ± SD) (***p < 0.001, N R 3, n R 3 per group; Mann-Whitney U test). (E) Representative velocity maps of beating human cardiac macrotissues after 14 days of culture. Red colors and longer arrows represent higher velocities, while blue colors and shorter arrows represent lower velocities. Scale bar, 2.5 mm. (F) Analysis of the alignment between beating direction of human cardiac macrotissues and direction of the electric field. The order parameter was used, with values close to 0 meaning random distribution and values close to 1 meaning parallel alignment (average ± SD) (***p < 0.001, N = 4, n R 4 per group; Mann-Whitney U test). (G) Strain rate of representative macrotissues over time. The curve is obtained by integrating the local strain rate (e.g., local area change per frame) over the whole construct at every time point. Positive and negative values correspond to contractions and relaxations, respectively. (H) Strain rate per beat (average ± SD) (**p < 0.01, N = 4, n R 4 per group; one-way ANOVA). The positive values of the strain rate (contractions) were integrated over time and divided by the number of beats. Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 213 (legend on next page) 214 Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 improvements in contractile behavior were obtained with ECG for human ventricular myocardium (Figure 4A). More- rat-derived cardiac constructs (Figures S4D–S4F). over, ECG-like signals recorded from CardioSlice constructs Velocity maps generated with particle image velocimetry were of statistically significantly higher amplitude, shorter demonstrated that the velocity in each contraction was QRS duration, and shorter QT interval than those of higher in CardioSlice constructs than in controls, corrobo- control constructs (Figures 4B–4D). These results indicate rating their enhanced contractile performance (Figure 3E). that cardiomyocytes within CardioSlice constructs are Directionality of contractions was calculated by deter- electrically better coupled than within control constructs, mining the alignment of velocity vectors with respect to attaining the fast conduction velocity and improved the direction of the stimulating electric field, and expressed action potential propagation characteristic of a functional by the order parameter (Figure 3F). CardioSlice syncytium. constructs contracted parallel to the direction of the elec- We next tested whether cardiomyocytes within macrotis- tric field applied during culture ( close to 1), sue constructs retained the ability to respond to positive whereas control constructs displayed random distribution and negative inotropic factors. Similar to iPSC-derived of velocity vectors ( = 0). Regarding the contractile cardiomyocytes cultured under standard 2D conditions strain generated in each contraction, CardioSlice con- and to control macrotissue constructs (data not shown), structs displayed higher strain rates compared with control CardioSlice constructs increased or decreased beating rate constructs (Figure 3G), with a significantly higher (p < 0.01) upon treatment with isoproterenol or carbachol, respec- average strain rate per beat (Figure 3H). Taken together, tively (Figures 4E and 4F). these results demonstrate that paced electrical stimulation of cardiac macrotissues over the 2 weeks of culture dramat- Prediction of Drug-Induced Cardiotoxicity Using ically enhances contractile performance, and suggest CardioSlice Constructs improved maturation at the tissue level under these To investigate the ability of human cardiac macrotissues to conditions. predict drug-induced cardiotoxicity, we incubated macro- tissues at day 14 of culture with sotalol, a human ether-a- CardioSlice Constructs Generate ECG-like Signals go-go (hERG) potassium channel blocker and adrenergic Human cardiac macrotissues produced a bioelectrical antagonist. Upon 10 min of treatment, control cardiac signal that was a sum of the action potentials generated macrotissues showed no consistent alterations in the fre- by the individual cardiomyocytes constituting the quency or morphology of ECG-like signals (Figures 5A construct. Such signals could be registered on the surface and S5A), making it difficult to determine any drug effect. of constructs using the electrodes embedded within the In contrast, CardioSlice constructs displayed a progressive perfusion chamber (Figure 4A). In control constructs the re- decrease in beating frequency, consistent with sotalol ac- corded electrical signals were of low amplitude and long tion as adrenergic antagonist (Figure S5B). More impor- duration, which is consistent with the incomplete syn- tantly, we could detect features typical of arrhythmia, chronization of contractions visualized across the macro- such as ectopic QRS, prolongation of RR intervals, and reg- tissue (Video S2). In contrast, CardioSlice constructs eli- ular blockades (Figure 5B). These features appeared progres- cited bioelectrical signals composed of narrow and steep sively, were more frequent over time of incubation with so- waveforms that include the QRS complexes and repolariz- talol, and coincided with abnormal prolongation of the QT ing T waves, remarkably similar to those of standard surface interval, which experienced increases greater than 20% Figure 4. Electrocardiogram-like Signals Generated by Human Cardiac Macrotissues during Spontaneous Beating (A) Representative electrocardiogram (ECG)-like signals (voltage recorded as a function of time). Left panel shows non-stimulated (Control) and electrically stimulated (CardioSlice) human cardiac macrotissues during spontaneous beating. Right panel shows the QRS complex and T wave over the averaged ECG-like beats (signals for 10 min were considered). (B–D) Box plots showing the QRS complex duration (B), the QT interval duration (C), and the amplitude of the QRS complex (D) from the signals generated by human cardiac macrotissues. Each experimental pair (Control and CardioSlice) has been normalized by the corre- sponding mean CardioSlice value. Box plot represents median, 25%, and 75%, and whiskers correspond to maximum and minimum values (***p < 0.001, N R 4, n R 4 per group; F test). (E and F) Representative ECG-like signals recorded from CardioSlice constructs before (first minute, black lines) and after (10-min in- cubation, light-gray lines) treatments with isoproterenol (E) and carbachol (F). Time intervals between two consecutive R peaks (RR) of QRS complexes are indicated by arrows. Bar charts show the change of the beat rate (defined as 1/RR) induced by isoproterenol and carbachol treatments. Data plotted show relative changes with respect to the mean beat rate value before treatment (baseline) for each experiment (average ± SEM) (***p < 0.001, N = 3, n = 3 per group; Mann-Whitney U test). All ECG-like bioelectric signals were bandpass filtered (zero-phase fourth-order Butterworth filter with cutoff frequency of 0.2 and 40 Hz, respectively). Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 215 Figure 5. Effects of Sotalol on the ECG- like Signal of CardioSlice Constructs (A, B, and D) Representative ECG-like signals for control (A) and CardioSlice (B and D) constructs cultured for 14 days after treat- ment with sotalol. (A) ECG-like signals recorded from control constructs upon 14 and 18 min of treatment with sotalol. (B) ECG-like signals recorded from Car- dioSlice constructs upon 14 and 18 min of treatment with sotalol. Prolongations in time intervals between two consecutive R peaks (RR) of QRS complex are represented by solid points. (C) QT interval prolongation in CardioSlice constructs, estimated by signal averaging at basal conditions and after 5 and 10 min of incubation with sotalol. QT interval is shown over the averaged ECG-like signals at basal conditions by arrows. The QRS complex and T wave are also shown. The smaller vertical lines indicate the end of T wave. The effects of sotalol on the ECG-like of control and CardioSlice constructs were analyzed in four independent experiments (Control N = 4, n = 5; CardioSlice N = 4, n = 4). (D) ECG-like signals for CardioSlice constructs upon 14, 18, and 23 min of treatment with sotalol. Inversion of ECG-like signals are represented by asterisks. All ECG-like bioelectric signals were bandpass filtered (zero-phase fourth-order Butterworth filter with cutoff frequency of 0.2 and 40 Hz, respectively). (Figure 5C). After prolonged incubation times with of the human myocardium. Electrical stimulation, alone or sotalol, CardioSlice constructs displayed severe alterations in combination with mechanical loading, has been widely in ECG-like signals, with QRS complexes becoming applied in tissue engineering to generate cardiac constructs smaller, inverted, and even disappearing after 30 min of that recapitulate some aspects of the native physiology drug treatment (Figure 5D). The observed electrophysiolog- (Godier-Furnemont et al., 2015; Nunes et al., 2013). Electri- ical alterations translated, at the mechanical level, into cal pacing has also been applied to the generation of hu- dyssynchronous contractile patterns in sotalol-treated man cardiac microtissues (Nunes et al., 2013; Ruan et al., CardioSlice constructs (Figure S5B). Therefore, sotalol 2016; Thavandiran et al., 2013; Xiao et al., 2014). Such mi- impaired the electrical conduction within CardioSlice con- crotissues are usually fabricated by using cell-laden natural- structs and affected their contractile behavior, demon- based hydrogels cast on posts (Fennema et al., 2013; Hin- strating the proarrhythmic potential of the drug. On the son et al., 2015; Huebsch et al., 2016; Kensah et al., 2013; whole, these results indicate that CardioSlice constructs Ruan et al., 2016; Schaaf et al., 2011; Soong et al., 2012; are suitable in vitro models for prediction of drug-induced Thavandiran et al., 2013; Tiburcy et al., 2017; Turnbull cardiotoxicity. et al., 2014; Zhang et al., 2013) or around a wire template (Nunes et al., 2013; Xiao et al., 2014). While some of those constructs could be quite large in surface area, even larger DISCUSSION than ours (Nakane et al., 2017; Shadrin et al., 2017; Tiburcy et al., 2017), their thickness was constrained by Environmental cues are key determinants of cardiomyo- the 300-mm limit of oxygen diffusion in passive culture cyte phenotype and function, and regulate their capacity systems (Lovett et al., 2009). To overcome such size limita- to form functional tissue units. Despite recent advances, tion, the production of thicker cardiac constructs can in vitro generation of human heart tissue is still limited by be then performed by medium perfusion bioreactors, the existing tissue-engineering technologies, which are which are able to maintain cell viability over time (Radisic not able to recapitulate the complex structure and function et al., 2008). Perfusion bioreactors incorporating electrical 216 Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 stimulation have been previously applied for in vitro culture ECG-like signals elicited by CardioSlice constructs showed of murine cardiomyocyte 3D tissue structures (Barash et al., a uniform and reproducible pattern of narrow, steep, and 2010; Kensah et al., 2011; Maidhof et al., 2012). However, well-defined QRS complexes that very much resembled this research had been rarely transferred to human cardiac actual ECG heart recordings. In contrast with CardioSlice tissue-engineering models (Ma et al., 2014; Tiburcy et al., constructs, the bioelectrical signals generated by control 2017) and not previously used to combine iPSC-derived constructs were highly heterogeneous, of comparatively cells and 3D thick scaffolds with simultaneous perfusion lower amplitude and longer duration of waveforms, indic- and electrical stimulation to develop macrotissues of ative of slowly conducting tissues. human cardiomyocytes. Herein, we have developed a par- By incorporating electrodes in our bioreactor chamber, allelized bioreactor system equipped with electrodes to we were able to not only stimulate our constructs but also electrically stimulate human iPSC-derived cardiomyocytes provide the system with the capability of on-line moni- during perfusion in culture. toring of the electrophysiological behavior of thick cardiac Electrical stimulation has been proposed to be a require- constructs in a non-destructive manner. As no standard ment for the electrophysiological maturation of cardiac method exists for the assessment of electrophysiological constructs, favoring cardiomyocyte contractility, align- information from intact macroscale-sized heart tissue ment, and organization within cardiac tissue constructs (Tzatzalos et al., 2016), the technology developed here is (Nunes et al., 2013; Radisic et al., 2004a). In our case, after unique in this context. The bioelectrical signal obtained 14 days in culture, cardiomyocytes in CardioSlice con- in this way is extremely informative of the electrically structs displayed small, but measurable, signs of increased active cells within the construct and, most importantly, cell maturation compared with control constructs, of the intercellular coupling at the macro (whole construct) showing wider sarcomeres and more developed intercel- scale, very similar to the case of surface ECG recordings in lular unions (Figures 2C and 2D). Nevertheless, the expres- humans. sion levels of maturity-related cardiac genes and cardiac ion Typically, safety of cardiac drugs is assessed by measuring channels indicated immature fetal-like cardiomyocyte currents on hERG ion channels ectopically expressed in characteristics, still far from those of adult myocardium non-cardiac cell lines (HEK293, CHO). These assays pro- (Figure S3D). Notably, however, continuous electrical stim- duce false positives and false negatives, as in the cases of ulation did have a profound effect on the properties of verapamil and alfuzosin, respectively (Mathur et al., CardioSlice constructs at the tissue level, resulting in 2015). Our system allowed us to determine the effects of greatly improved electromechanical coupling. We believe proarrhythmic drugs on the ECG-like signal generated by that the implications of these findings are important in CardioSlice constructs. Upon treatment with the proar- two respects: First, our findings illustrate a viable approach rhythmic drug sotalol, CardioSlice constructs showed a to engineering human myocardial surrogates with value for pathological prolongation of the QT interval, and alter- cardiac toxicity testing. In this respect, most previous ations in the morphology and polarity of the QRS complex studies aimed at producing adult-like cardiac constructs in ECG-like signals and arrhythmias, as seen in vivo (Straus focused on increasing the degree of maturation of cardio- et al., 2006). Such responses were not identified in control myocytes (with only relative success to date: see Nunes constructs, even though they contained the same numbers et al., 2013; Ronaldson-Bouchard et al., 2018; Ruan et al., of cardiomyocytes and fibroblasts, cultured in parallel un- 2016; Tiburcy et al., 2017), while our findings highlight der identical conditions (except, of course, for the contin- the usefulness of promoting the maturation at the tissue uous electrical stimulation applied to CardioSlice con- level, in a way that supersedes and does not strictly depend structs). Because the proarrhythmic behavior of drugs is on the maturation at the cellular level. Second, on a more often only found after they have been administered to conceptual note, our findings provide experimental sup- patients, we propose that the predictive capabilities of port for the appearance of emergent behaviors in the CardioSlice constructs reflect their higher degree of close- context of a complex system (Gao et al., 2018) such as to-human tissue-like functionality. myocardial tissue organization and function, where rela- In summary, the novelty of our studies is twofold. On the tively small changes in the properties of the system ele- one hand, we have developed a method and associated ments (cardiomyocytes) can lead to higher-order behaviors technology for the production of thick human cardiac at greater scales (tissue). In the case of myocardial tissue this macrotissues, with capabilities for electric stimulation appears to be particularly relevant, probably because of the and on-line electrophysiological recording. On the other system’s normal operation as a functional syncytium hand, our findings show that continuous electrical stimula- (Hunter et al., 2003). Tissue-like maturation of CardioSlice tion of cardiac macrotissues resulted in minor (albeit constructs resulted in contractions with an amplitude measurable) improvements in cardiomyocyte maturation, 6-fold higher than that of control constructs. Moreover, but greatly enhanced maturation at the tissue level. Stem Cell Reports j Vol. 13 j 207–220 j July 9, 2019 217 CardioSlice constructs showed superior electromechanical as well as to evaluate the beating-rate change after drug treatments coupling and were able to predict drug-induced cardiotox- (Figures 4E and 4F). Regarding the average strain per contraction icity, illustrating the enhanced tissue-like functionality (Figure 3H) and for experiments involving more than two groups achieved by our model. The physiological relevance of (gene expression analysis), data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test. The difference be- CardioSlice constructs, together with their parallel and tween the variance of experimental groups in QRS duration, QT in- scalable nature and the on-line electrophysiological moni- terval, and QRS amplitude (Figures 4B–4D) was analyzed using toring feature, take our technology to the forefront of the two-sample test for variances (F test), and data were normalized production of engineered human cardiac macrotissues. to electrical pacing conditioning. Software used were Origin 8.5 (OriginLab, Northampton, USA) and GraphPad Prism 6, and differ- ences were considered significant when p < 0.05. EXPERIMENTAL PROCEDURES Experimental procedures are provided in detail in Supplemental SUPPLEMENTAL INFORMATION Information. Supplemental Information can be found online at https://doi.org/ 10.1016/j.stemcr.2019.05.024. Use of Human and Animal Subjects The use of animals for experimentation in these studies was done AUTHOR CONTRIBUTIONS following a protocol approved by the Animal Experimentation Ethics Committee of the University of Barcelona (Barcelona, E.M. and A.R. conceived the study and supervised the project. Spain). The use of human iPSCs in this work was approved by M.V.-M., O.I.-G., R.J., E.M., and A.R. planned experiments. the Spanish competent authorities (Commission on Guarantees M.V.-M., O.I.-G., C.D.G., L.S., K.T., R.P., J.C., D.B.-A., S.J.-D., concerning the Donation and Use of Human Tissues and Cells of O.C.-F., and R.J. performed experiments. J.S. provided research the Carlos III Spanish National Institute of Health). advice. M.V.-M., O.I.-G., E.M., and A.R wrote the manuscript with contributions from all the authors. Design, Fabrication, and Characterization of the ACKNOWLEDGMENTS Perfusion Chamber and Bioreactor System Custom-made perfusion chambers with electrical stimulation and The authors would like to thank L. Mulero, C. Pardo, and C. Casal parallel bioreactors for continuous perfusion were designed and for excellent technical assistance with histological analysis and fabricated as described in Supplemental Information. J.M. Vaquero for assistance with flow-cytometry analysis. We thank J. Martı́n from Leventon S.A.U. (WerfenLife Company) for kindly donating parts from the elastomeric infusion system Generation of Engineered Human Cardiac DOSI-FUSER. This work was supported by grants from the Spanish Macrotissues Ministry of Economy and Competitiveness-MINECO (RTI2018- Cardiomyocytes were differentiated from hiPSCs (FiPS Ctrl1- 095377-B-100 and Severo Ochoa Program for Centers of Excel- mR5F-6; cell line registered in the National Stem Cell Bank, Carlos lence in R&D 2016-2019) and Departament de Salut, Generalitat III Spanish National Institute of Health) as described in Supple- de Catalunya (PERIS SLT002/16/00234). Additional support was mental Information, and seeded into porous collagen- and provided by grants from Instituto de Salud Carlos III-ISCIII/FEDER elastin-based scaffolds of 1-mm thickness in dry conditions (Red de Terapia Celular - TerCel RD16/0011/0024), ACCIÓ/FEDER (Matriderm). Detailed methods are provided in Supplemental (AdvanceCat), Generalitat de Catalunya-AGAUR (2017-SGR-1079, Information. 2017-SGR-1770, and 2017-SGR-899), Fundació la Marató de TV3 (201534-30), and CERCA Programme/Generalitat de Catalunya. Morphological, Functional, and Molecular Analyses Immunostaining, flow cytometry, qRT-PCR, confocal and electron Received: May 17, 2018 microscopy, contractility analyses, and electrophysiological Revised: May 23, 2019 recording and processing of cardiac macrotissues were performed Accepted: May 24, 2019 as described in Supplemental Information. Published: June 20, 2019 Statistical Analyses REFERENCES The number of replicated measurements per experiment (n) and of independent experiments (N) is specified in the figure legends. Amano, Y., Nishiguchi, A., Matsusaki, M., Iseoka, H., Miyagawa, S., Normal distribution of data was tested using the Shapiro-Wilk Sawa, Y., Seo, M., Yamaguchi, T., and Akashi, M. (2016). Develop- and Kolmogorov-Smirnov normality tests. 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