We would like to acknowledge VALA sciences for the Cyteseer software package and optimization of the automated 3D calcium analysis. We wish to acknowledge grant support from the PLN foundation (RM). P.A.D. and F.S. are supported by CUREPLaN Leducq. J.P.G.S. is supported by H2020-EVICARE (#725229) of the European Research Council (ERC). J.W.B. is supported by the UMC Utrecht Clinical Fellowship, Netherlands Heart Institute Fellowship, and CVON-Dosis young talent grant; Netherlands Heart Foundation (CVON-Dosis 2014-40). N.C. is supported by the Gravitation Program &#34;Materials Driven Regeneration&#34; by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013), and the Marie Sk&#322;odowska-Curie Actions (Grant agreement RESCUE #801540). V.S.-P. is supported by the Alliance Fund (UMCU, UU, TU/e). A.v.M. is supported by the EU-funded project BRAVE (H2020, ID:874827)

<ol>
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The authors have nothing to disclose.

Cardiac drug discovery is hampered by a reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately perform readouts. hiPSC-CM biology coupled with HT instrumentation and physiological probes has the potential to re-introduce human models into the earliest stages of cardiac disease modeling and drug discovery. We developed a 3D cardiac tissue generation method that produces high-quality and functional CSs for an optimal cardiac disease modeling and drug screening platform. Additionally, combining the spheroid technology in 3D bioreactor systems for industrial EV production allows a necessary step toward the clinical translation of EV-based therapy. The method described here relies on several crucial factors and is a variant of existing protocols9,10,28,29. These methods include: 1) the generation of 3D tissue constructs, 2) the optimal cell number and timing before the screening, 3) improving sensitivity and high-throughput capacity of instruments, and 4) being able to freeze the spheroids before any functional analysis. Unlike previously described protocols, the proposed protocol describes the generation of up to 1,500 spheroids per day and the suitability for HTS. Conventional analysis of a hundred compounds over 6 x 0.5 log doses for 10 replicates using existing 96-well calcium imaging systems or 24-well multiplexed engineered heart tissues require approximately 500 million to 3 billion hiPSC-CMs31,32. The proposed application makes cardiac screenings less costly and time effective compared to the conventional systems since the 96-well plates required only 10% of the seeding density compared to the described method. Moreover, compared to previous protocols, such as the hanging-drop method, the generation of spheroids by self-aggregation in ultra-low attachment plates enables high-quality automated imaging of single microtissues33.
This small 3D model mimics the biological and physiological phenotype of the in vivo cardiovascular environment. As previously demonstrated, calcium transients dramatically increase in 3D cardiac tissue constructs as compared to 2D monolayer cell cultures34.
Next, we found that the seeding density and proper culturing time are also critical factors for a successful CS screening. The densities of 10K-20K hiPSC-CMs per spheroid and screening between weeks 2-3 after generation were optimal, whereas too small or too old spheroids show disturbed calcium handling (Figure 2 and Figure 3). Therefore, it is of importance to maintain seeding densities as consistent as possible, since size influences the functional parameters. Also, although this optical method provides good results for live 3D cultures as a whole tissue, obtaining data within larger spheroids at (sub-)cellular level is challenging without relying on time-consuming histology methods. Recently, several approaches have been published that used &#34;optically clearing&#34;, which enables the acquisition of whole 3D spheroids with the opportunity for single-cell quantification of markers. Here, we adapted a 3-day protocol from CS harvesting to image analysis, which is optimized for 3D imaging using confocal microscopy29 (Figure 1C and Figure 4D).
Lastly, with the increase in 3D cardiac tissue applications and commercial applications, the demand for long-term storage and patient-specific biobanking from various donors is rising. Cryopreservation is an effective strategy to generate HTS-plates from multiple batches over time. The freezing of hiPSC-CMs has been described previously and is not different compared to other cultured cell types10,35,36. Recently, approaches for freezing plates with 2D cells have been described37. Here, we found the PSC cryopreservation kit is the most optimal condition as compared to three others (data not shown) and used this medium for the efficient freezing of spheroids. After cryopreservation, viability remains high (Figure 4B,C), but CSs&#39; electrophysiological properties are affected and a period of incubation after thawing is required. Indeed, 1 week after thawing, CSs displayed spontaneous beating activity and calcium handling. However, it has been described that fresh and recovered hiPSC-CMs do not always show identical molecular and physiological properties38. This limitation needs to be considered when cryopreserved hiPSC-CMs are used for assessing drug-induced cardiac read-outs. Moreover, although we effectively modulate the number of cells per spheroid and the optimal timing of the calcium transient imaging, the cardiac spheroids could be improved by mixing hiPSC-derived cardiomyocyte cells with endothelial cells, fibroblasts, cell-cell junctions, and extracellular matrices, such as chitosan, collagen IV, fibronectin, matrigel, or laminin, mimicking the in vivo cardiac environment39,40. Overall, we propose a step-by-step protocol to efficiently generate CSs which are suitable for downstream applications such as disease modeling and HT drug screening.

The discovery of human-induced pluripotent stem cells (hiPSCs) offered unprecedented opportunities to study human development and disease at the cellular level. Over the past decade, using developmental lessons, various protocols have been established to ensure the efficient differentiation of hiPSCs into cardiomyocytes (CMs)1,2,3,4. hiPSC-derived cardiomyocytes (hiPSC-CMs) can serve as a resource for modeling genetically inheritable cardiovascular diseases (CVDs), testing cardiac safety for new drugs, and cardiac regenerative strategies5,6,7,8. Despite the directed cardiac differentiation of hiPSCs, indefinite CM numbers remain a challenge in the cardiac field, since matured hiPSC-CMs generally are non-proliferative, and primary human cells are not available in high quantities.
Recently, we described that concomitant Wnt signaling activation with low cell-density culture resulted in a massive proliferative response (up to 250-fold) of hiPSC-CMs9,10. This cost-effective strategy for the massive expansion of hiPSC-CMs via serial passaging in culture flask format facilitates the standardization and quality control of large numbers of functional hiPSC-CMs. Additionally, to keep up with the demand for large batches of hiPSC-CMs from various donors, the biobanking of hiPSC-CMs has been described10. However, cardiomyocyte monolayers seeded in these standard culture dishes are not representative of the complex 3D structure present in the heart. Moreover, the immaturity of hiPSC-CMs has remained an obstacle, thus falling short in mimicking the biological and physiological phenotype of the in vivo cardiovascular environment.
Novel 3D in vitro models have been developed where hiPSC-CMs show closer physiological behavior such as self-organization11,12, extracellular matrix (ECM) remodeling13, enhanced maturation14,15,16, and synchronized contraction17,18,19. 3D models have been utilized for drug discovery, drug cardiotoxicity testing, disease modeling, regenerative therapies, and even the first clinical trials20,21,22,23,24. One of the most used models is the fibrin-based engineered heart tissue (EHT), which exhibits a tissue-like arrangement and cardiac contractility13,17,25. Previously, we showed that EHTs generated from expanded hiPSC-CMs displayed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion9. Nevertheless, even though the generation of EHTs from hiPSC-CMs has been well established, further developments are anticipated regarding the establishment of an HT assessment platform. Here, the rapid generation of large numbers of self-aggregating cardiac spheroids (CSs) in 96-well format allows an improvement in 3D conditions for high-throughput screening (HTS) purposes.
Overall, the advantage of CSs as 3D cell culture is their high reproducibility and scalability. In particular, CSs combined with robotic sample handling can standardize and automate CS culture, drug treatment, and high-content analysis20. Here, we describe optimized protocols to generate high-purity and high-quality CSs, which can be efficiently cryopreserved and screened for cardiac function by performing Ca2+ transient measurements using an optical calcium acquisition and analysis system. This model provides a simple yet powerful tool to perform high-throughput screens on hundreds to thousands of spheroids17,18.

<table><tbody><tr><td>24 wells suspenion plate&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>3738</td><td></td></tr><tr><td>96 wells Ultra-Low Attachment Multiple Well Plate</td><td>Corning</td><td>CLS3474-24EA</td><td></td></tr><tr><td>Albumax</td><td>Thermo Fisher Scientific</td><td>11020021</td><td></td></tr><tr><td>Anti-&amp;alpha;-Actinin (Sarcomeric) antibody</td><td>Sigma-Aldrich</td><td>A7811</td><td>Dilution: 1:200</td></tr><tr><td>Anti-Cardiac Troponin T antibody (ab45932)</td><td>Abcam</td><td>ab45932</td><td>Dilution: 1:200</td></tr><tr><td>Ascorbic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>B-27 supplement&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>17504-044</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4639</td><td></td></tr><tr><td>Bovine serum albumin fraction V (BSA)</td><td>Roche</td><td>10735086001</td><td></td></tr><tr><td>Cal-520, AM&amp;nbsp;</td><td>Abcam</td><td>ab171868</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica&amp;nbsp;</td><td>DMi8&amp;nbsp;</td><td></td></tr><tr><td>Confocal microscope software&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>Las X</td><td></td></tr><tr><td>Conical tubes 15 mL</td><td>Greiner Bio-One</td><td>5618-8271</td><td></td></tr><tr><td>Creatine monohydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C3630</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>D3571</td><td>Concentration: 1 &amp;micro;g/mL</td></tr><tr><td>DMEM no glucose&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11966025</td><td></td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td></td></tr><tr><td>Fructose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>76050771.05</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glycerol</td><td>Boom</td><td>76050771.05</td><td></td></tr><tr><td>Goat anti-mouse Alexa Fluor 488</td><td>Invitrogen</td><td>A11029</td><td>Dilution: 1:500</td></tr><tr><td>Goat anti-rabbit Alexa Fluor 568</td><td>Invitrogen</td><td>A11011</td><td>Dilution: 1:500</td></tr><tr><td>Horizontal shaker</td><td>IKA</td><td>4003000</td><td></td></tr><tr><td>Human induced pluripotent stem cell lines</td><td>(Stanford Cardiovascular Institute (S-CVI) Biobank)</td><td>CVI-273 (control 1)</td><td></td></tr><tr><td>Human induced pluripotent stem cell lines</td><td>Germany&amp;nbsp;</td><td>141 (control 2) 144 (control 3)</td><td></td></tr><tr><td>Hydrochloric acid (HCl)&amp;nbsp;</td><td>Ajax Firechem</td><td>265.2.5L-PL</td><td>10 M stock solution, corrosive</td></tr><tr><td>Isotype control, FITC mouse IgM &amp;kappa; isotype</td><td>BD</td><td>556652</td><td></td></tr><tr><td>KnockOut Serum Replacement&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10828</td><td>Protect from light</td></tr><tr><td>L-carnitine</td><td>Sigma-Aldrich</td><td>C0283</td><td></td></tr><tr><td>Myocyte calcium and contractility system</td><td>Leica&amp;nbsp;</td><td>Thunder, DMi8</td><td></td></tr><tr><td>Non essential amino acids (NEAA)</td><td>Thermo Fisher Scientific</td><td>11140</td><td></td></tr><tr><td>Paraformaldehyde solution 4% in 1x PBS, pH 7.0&amp;ndash;7.6</td><td>Santa Cruz</td><td>SC281692</td><td>Hazardous</td></tr><tr><td>PBS, pH 7.4</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15140</td><td></td></tr><tr><td>PES Membrane Vacuum Filter system</td><td>Corning&amp;nbsp;</td><td>431097</td><td></td></tr><tr><td>PI/RNase Staining Solution</td><td>Invitrogen</td><td>F10797</td><td>Dilution: 1:1000</td></tr><tr><td>Pluronic&amp;nbsp;F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>PSC Cryopreservation Kit</td><td>Thermo Fisher Scientific</td><td>A2644601</td><td></td></tr><tr><td>RevitaCell</td><td>Thermo Fisher Scientific</td><td>A2644501</td><td></td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11875</td><td></td></tr><tr><td>Silicone Elastomer Kit</td><td>SYLGARD</td><td>184</td><td></td></tr><tr><td>Sodium dodecyl sulfate solution (10%)</td><td>Sigma-Aldrich</td><td>71736</td><td></td></tr><tr><td>Sodium L-Lactate</td><td>Sigma-Aldrich</td><td>71718</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr><tr><td>Tris Fisher&amp;nbsp;</td><td>Scientific&amp;nbsp;</td><td>11486631</td><td></td></tr><tr><td>Triton X-100</td><td>Merck</td><td>X100-1L</td><td>Hazardous</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>TrypLE Select Enzyme (10x)</td><td>Thermo Fisher Scientific</td><td>A1217701</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Urea&amp;nbsp;</td><td>Sigma-Aldrich</td><td>51456</td><td></td></tr><tr><td>Vitamin B12</td><td>Sigma-Aldrich</td><td>V6629</td><td></td></tr><tr><td>Y-27632 dihydrochloride (Rho-kinase inhibitor)</td><td>Tocris&amp;nbsp;</td><td>1254</td><td>Protect from light</td></tr></tbody></table>

generation, high-throughput screening, and biobanking of human-induced pluripotent stem cell-derived cardiac spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

The protocol shown in Figure 1A describes the generation of CSs from previously expanded hiPSC-CMs. The CSs acquire a 3D structure by day 1 post-seeding in ultra-low attachment round-bottom plates and can be cultured for up to 6 weeks (Figure 1B). As assessed by immunofluorescence staining, the majority of the cells in 3-week-old CSs expressed sarcomeric proteins such as &#945;-actinin and troponin T and displayed regular sarcomere organization (Figure 1C). For the quantification of &#945;-actinin positive cells, flow cytometry analysis was performed. In accordance with the immunofluorescence results, the flow cytometry data demonstrated comparable high levels of &#945;-actinin in both day 0 (76.9% &#177; 16.6%) and 3-week-old CSs (71.1% &#177; 22.7%) (Figure 1D), indicating a constant and highly pure cellular composition during culturing. There was an increased expression of the cardiac genes for junctions (GJA1, JPH2, and PKP2), desmosomes (DES), and mitochondria (ATP5A) in hiPSC-CM derived spheroids (day 42) versus hiPSC-CMs cultured in 2D for 90 days (Figure 1E). The expression of these genes is a hallmark of cell-cell interaction and maturation30.
Subsequently, the functional properties of CSs, namely beating rate and Ca2+ handling, were assessed at different time points (Figure 2). Calcium transient parameters such as rise time, peak time, decay time, and calcium transient duration (CTD90) were evaluated as indicated in Figure 2A,B. The percentage of beating CSs is similar in the first 3 weeks post-generation but significantly dropped in week 6 (Wk6) CSs (Figure 2C). The beating rate was significantly reduced at Wk3 compared to Wk1 and, similar to the percentage of beating CSs, dramatically dropped at Wk6 (Figure 2D). At Wk6, CS deterioration was observed, which can explain the drop in both the beating rate and the number of beating CSs. Measurement of calcium transient parameters indicated a significantly higher peak value at Wk2 (Figure 2E), while the rise time, decay time, and CTD90 were significantly increased at Wk3 compared to Wk1 (Figure 2F-H). Taken together, these results show that hiPSC-CM-derived spheroids are functionally optimal at around weeks 2 and 3 post-generation.
Figure 3&#160;shows the effect of spheroid size on the beating rate and calcium handling. CSs were generated by seeding 2.5 x 104, 5 x 104, 10 x 104, and 20 x 104 hiPSC-CMs in a well of a 96-well plate for a total of 24 CSs/wells per condition (Figure 3A). As expected, the spheroid size increased as the number of cells used increased, ranging from 178 &#177; 36 &#181;m to 351 &#177; 65 &#181;m (Figure 3A, right panel). Ca2+ transients were measured in 3-week-old CSs at the four different seeding densities (Figure 3B). Measurements of beating CSs indicated that only about 50% of the smaller size-CSs (2.5K- and 5K-CSs) were beating, while the percentage of bigger size-beating CSs (10K- and 20K-CSs) was significantly higher (about 85%) (Figure 3C). A similar beating rate (approximately 28 bpm) was shown by 5K-, 10K-, and 20K-CSs, which was significantly higher compared to 2.5K-CSs (Figure 3D). The peak values of calcium images were similar in all tested conditions (Figure 3E), however, rise time (Figure 3F), decay time (Figure 3G), and CTD90 (Figure 3H) were significantly increased in larger size-CSs (10K- and 20K-CSs) compared to the smaller ones (2.5K- and 5K-CSs). Taken together, these results show that hiPSC-CM-derived spheroids are optimal for calcium handling screening when a seeding density between 10K- and 20K hiPSC-CMs/well is used.
Next, we evaluated the impact of cryopreservation on CS&#39;s viability and function. Before analysis, thawed CSs were maintained in culture for 1 week (Figure 4A). As shown by both flow cytometry (Figure 4B) and Calcein-AM (Figure 4C) cell viability tests, cryopreservation did not affect cell viability within the CSs. Additionally, thawed CSs showed similar expression levels of sarcomeric proteins as compared to the fresh age-matched CSs (Figure 4D). These data indicate that CSs can be efficiently cryopreserved for subsequent cardiac function analysis and high-throughput screening.
Finally, the beating activity and Ca2+ handling were measured in both fresh and cryopreserved CSs (Figure 5). The percentage of beating CSs was measured at different time points after thawing, respectively, at 2, 5, and 7 days. While most of the fresh CSs showed beating activity over time, clearly the cryopreserved CSs needed up to 1 week of culturing in order to recover their beating activity (Figure 5B). There was no significant change in the beating rate of thawed CSs versus fresh; however, no spontaneous beating activity was observed in some frozen CSs (Figure 5C). Although peak values were significantly reduced in frozen/thawed CSs compared to fresh (Figure 5D), no significant changes were observed in rise time, decay time and the CTD90 of frozen/thawed CSs compared to fresh (Figure 5E-G). These data indicate that, after thawing, it is important to let the CSs recover in the incubator for at least 1 week before measuring beating activity and Ca2+ transient.
Taken together, these results show that cryopreservation of hiPSC-CM-derived spheroids preserves cardiomyocyte viability, the sarcomeric structure, and their functional characteristics such as spontaneous beating activity and calcium handling. Thus, hiPSC-CM-derived spheroids represent a suitable model to accurately recapitulate cardiac electrophysiology in vitro.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64365/64365fig01.jpg" /> 
Figure 1: Generation of cardiac spheroids. (A) Schematic representation of Wnt-based directed cardiac differentiation, the subsequent expansion of hiPSC-CMs, and the generation of CSs. Created with biorender.com. (B) Bright-field images at different time points of CS culturing. Scale bar, 200 &#181;m. Wk represents week. (C) Representative immunofluorescence images for cardiac sarcomeric proteins &#945;-actinin and troponin T in 3-week-old CSs. Immunofluorescence: Hoechst (blue), &#945;-actinin (green), and troponin T (red). Scale bar, 200 &#181;m. The zoomed-in merged picture on the right displays the sarcomere organization. Scale bar, 50 &#181;m. (D) Flow cytometry quantification of &#945;-actinin positive cells before (day 0) and 3 weeks after the formation of CSs. (n = 14-23 per condition. (E) RT-qPCR performed on hiPSC-CMs cultured for 90 days (2D) and spheroid samples cultured for 42 days to establish expression levels of different cardiac genes related to cell junctions, intermediate filaments, and mitochondria. (n = 1-3 batches). Data are represented as mean &#177; SD. NS (non-significant) as calculated by an unpaired t-test. <a href="https://www.jove.com/files/ftp_upload/64365/64365fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64365/64365fig02.jpg" /> 
Figure 2: Beating rate and Calcium handling in CSs at different weeks post generation. (A) Examples of calcium transient parameters calculated by the Vala sciences analysis algorithm in Cyteseer Software. (B) Representative calcium transient traces and time-lapse images of the CSs at different time points (weeks) post-generation. Scale bar, 200 &#181;m. (C) Time course quantification of spontaneous beating activity is expressed as the percentage of beating CSs. (D) Beating rate of CSs during culturing time. (E-H) Quantification of the calcium transients showing peak value, rise time, decay time, and CTD90. Data shown are mean &#177; SD. Biological replicates = three, technical replicates = 38, 50, 66, and 7, respectively. *p &#60; 0.05, ****p &#60; 0.001; one-way ANOVA followed by Tukey&#39;s post hoc multiple-comparisons test. Abbreviations; CTD = calcium transient duration, Wk = week, CSs = human cardiac spheroids. <a href="https://www.jove.com/files/ftp_upload/64365/64365fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64365/64365fig03.jpg" /> 
Figure 3: Beating rate and calcium handling in CSs generated using different cell seeding densities. (A) Bright-field imaging (left) and size measurements (right) of CSs generated using different numbers of hiPSC-CMs. Scale bar, 200 &#181;m. (B) Representative calcium transient traces and time-lapse images of the 2.5K-20K-CSs. (C,D) Beating percentage and beating rate of 2.5K-20K-CSs. (E-H) Peak value, rise time, decay time, and CTD90 in 2.5K-20K-CSs. Data are mean &#177; SD. Biological replicates = three, technical replicates = 28-39. *p &#60; 0.05, ****p &#60; 0.001; one-way ANOVA followed by Tukey&#39;s post hoc multiple-comparisons test. Abbreviations: CTD = calcium transient duration, Wk = week, k = x 1,000 cells, CSs = cardiac spheroids. <a href="https://www.jove.com/files/ftp_upload/64365/64365fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64365/64365fig04.jpg" /> 
Figure 4. Effect of cryopreservation on cardiac spheroids&#39; viability and structure. (A) Schematic representation of CS generation, subsequent biobanking, and thawing. (B) Flow cytometry cell viability test in both fresh and cryopreserved CSs. As a positive control, a treatment with 10% Triton-X solution for 5 min was used. (n = 4 per condition). Data are represented as mean &#177; SD. ****p &#60; 0.001; one-way ANOVA followed by Tukey&#39;s post hoc multiple-comparisons test. (C) Calcein-AM cell viability test in fresh versus thawed CSs after 7 days of culturing (n = 15-17 per condition, ****p &#60; 0.001, by paired t-test; scale bar, 200 &#181;m). (D) Representative bright-field (left) and immunofluorescence staining for &#945;-actinin and troponin T expression in fresh and thawed CSs. Immunofluorescence: Hoechst (blue), &#945;-actinin (green), and troponin T (red). The merged pictures on the right display sarcomere striations in the CSs. Scale bar, 50 &#181;m. Abbreviations: X = thawing day of choice, PI = propidium iodide, Cal-AM = calcein-AM, EthD-I = Ethidium Homodimer I. <a href="https://www.jove.com/files/ftp_upload/64365/64365fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64365/64365fig05.jpg" /> 
Figure 5: Calcium transients in fresh versus thawed CSs. (A) Representative calcium transient traces and time-lapse images of the CSs before cryopreservation and 1 week after thawing. (B) Beating percentage of fresh and frozen/thawed cardiac spheroids. Bars represent individual experiments. (C) Beating rate of fresh and frozen/thawed cardiac spheroids. (D-G) Quantification of calcium transient parameters: peak value, rise time, decay time, and CTD90. Data are mean &#177; SD. *p &#60; 0.05, ****p &#60; 0.001; one-way ANOVA followed by Tukey&#39;s post hoc multiple-comparisons test. Abbreviations; CTD = calcium transient duration, CSs = cardiac spheroids. <a href="https://www.jove.com/files/ftp_upload/64365/64365fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Representative gating strategies for flow cytometry analysis. (A) Representative gating strategy for &#945;-actinin positive hiPSC-CMs in a pure population versus negative control and isotype control. The number of &#945;-actinin positive analyzed cells is 25 x 105. Abbreviations; SSC = side scatter, PI+ = propidium iodide positive. (B) Representative gating strategy for the viability analysis in both fresh, thawed, the positive control (Triton-X), and the negative control (unstained). <a href="https://www.jove.com/files/ftp_upload/64365/figures version 2_6-end.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids at JoVE.com

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

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Bioengineering

3.6K Views.  University Medical Center Utrecht. Dit protocol beschrijft een workflow voor het genereren, onderhouden en optisch analyseren van cardiale sferoïden. Deze cardiale sferoïden zijn essentieel om de leemte in de huidige in vitro ziektemodellen op te vullen. Deze techniek stelt onderzoekers in staat om next-generation, high-throughput screening en functionele opslag van cardiale sferoïden te creëren.Begin met het toevoegen van één milliliter per vierkante centimeter steriele hartloslatingsoplossing aan elke put van ee...

Video: Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Developmental Biology

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biology

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

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