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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 wells suspenion plate&#160;</td>
			<td>Corning&#160;</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-&#945;-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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement&#160;</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&#160;</td>
			<td>Abcam</td>
			<td>ab171868</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica&#160;</td>
			<td>DMi8&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope software&#160;</td>
			<td>Leica&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C3630</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3571</td>
			<td>Concentration: 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>DMEM no glucose&#160;</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&#160;</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&#160;</td>
			<td>141 (control 2) 144 (control 3)</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)&#160;</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 &#954; isotype</td>
			<td>BD</td>
			<td>556652</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement&#160;</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&#160;</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&#8211;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&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>PES Membrane Vacuum Filter system</td>
			<td>Corning&#160;</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&#160;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&#160;</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&#160;</td>
			<td>Scientific&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea&#160;</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&#160;</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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3.3K Views.  University Medical Center Utrecht. 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.

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

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

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 ...