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 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 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.
NOTE: hiPSC-CMs used in this study were generated according to previously described hiPSC culturing and CM differentiation protocols26,27. Optionally, the hiPSC-CMs can be expanded and cryopreserved as recently published before starting the CS protocol (section 4)10.
1. Preparation of cell culture media, solutions, and aliquots
2. Preparation of buffers
3. Preparation of small molecules
4. Cardiac spheroid generation
NOTE: For larger amounts of CSs, seed up to 1 million CMs in a 6-well ultra-low attachment plate with 2 mL of hiPSC-CM re-plating media. This study used a minimum of 2,500 (2.5k CS) up to 20,000 (20k CS) hiPSC-CMs per well of a 96-well plate.
5. Cryopreservation of CSs
NOTE: CSs can be cryopreserved for long-term storage. Cryopreservation can be performed from day 3 after the generation of CSs. CSs can be cryopreserved directly in the wells of a 96-well plate or as a CS suspension in cryovials.
6. Thawing of cardiac spheroids
NOTE: Do not thaw more than one plate at a time to ensure a quick thawing process.
7. Assessment of intracellular Ca2+ transients
NOTE: CSs are in culture for a total of 3 weeks; 2 weeks before freezing, and 1 week after thawing. The 'fresh' controls are age-matched.
8. Flow cytometry analyses of dissociated cardiac spheroids
NOTE: In this study, flow cytometry was used to determine the viability of the CSs before and after the thawing process.
9. Immunofluorescence staining of whole 3D spheroids
NOTE: This protocol is based on the protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling, which was previously published29 and adjusted for cardiac spheroids. During the procedure, all pipet tips and conical tubes can be coated with 1% wt/v BSA-PBS in order to prevent the spheroids from sticking to plastics. To coat the materials, dip into the 1% BSA-PBS. Be careful not to damage the spheroids by using the 5 mL pipet, avoiding mechanical disruption.
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 α-actinin and troponin T and displayed regular sarcomere organization (Figure 1C). For the quantification of α-actinin positive cells, flow cytometry analysis was performed. In accordance with the immunofluorescence results, the flow cytometry data demonstrated comparable high levels of α-actinin in both day 0 (76.9% ± 16.6%) and 3-week-old CSs (71.1% ± 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 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 ± 36 µm to 351 ± 65 µm (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'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.
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 µm. Wk represents week. (C) Representative immunofluorescence images for cardiac sarcomeric proteins α-actinin and troponin T in 3-week-old CSs. Immunofluorescence: Hoechst (blue), α-actinin (green), and troponin T (red). Scale bar, 200 µm. The zoomed-in merged picture on the right displays the sarcomere organization. Scale bar, 50 µm. (D) Flow cytometry quantification of α-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 ± SD. NS (non-significant) as calculated by an unpaired t-test. Please click here to view a larger version of this figure.
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 µm. (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 ± SD. Biological replicates = three, technical replicates = 38, 50, 66, and 7, respectively. *p < 0.05, ****p < 0.001; one-way ANOVA followed by Tukey's post hoc multiple-comparisons test. Abbreviations; CTD = calcium transient duration, Wk = week, CSs = human cardiac spheroids. Please click here to view a larger version of this figure.
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 µm. (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 ± SD. Biological replicates = three, technical replicates = 28-39. *p < 0.05, ****p < 0.001; one-way ANOVA followed by Tukey's post hoc multiple-comparisons test. Abbreviations: CTD = calcium transient duration, Wk = week, k = x 1,000 cells, CSs = cardiac spheroids. Please click here to view a larger version of this figure.
Figure 4. Effect of cryopreservation on cardiac spheroids' 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 ± SD. ****p < 0.001; one-way ANOVA followed by Tukey'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 < 0.001, by paired t-test; scale bar, 200 µm). (D) Representative bright-field (left) and immunofluorescence staining for α-actinin and troponin T expression in fresh and thawed CSs. Immunofluorescence: Hoechst (blue), α-actinin (green), and troponin T (red). The merged pictures on the right display sarcomere striations in the CSs. Scale bar, 50 µm. Abbreviations: X = thawing day of choice, PI = propidium iodide, Cal-AM = calcein-AM, EthD-I = Ethidium Homodimer I. Please click here to view a larger version of this figure.
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 ± SD. *p < 0.05, ****p < 0.001; one-way ANOVA followed by Tukey's post hoc multiple-comparisons test. Abbreviations; CTD = calcium transient duration, CSs = cardiac spheroids. Please click here to view a larger version of this figure.
Supplementary Figure 1: Representative gating strategies for flow cytometry analysis. (A) Representative gating strategy for α-actinin positive hiPSC-CMs in a pure population versus negative control and isotype control. The number of α-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). Please click here to download this File.
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 "optically clearing", 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' 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 authors have nothing to disclose.
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 "Materials Driven Regeneration" by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013), and the Marie Skłodowska-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)
24 wells suspenion plate | Corning | 3738 | |
96 wells Ultra-Low Attachment Multiple Well Plate | Corning | CLS3474-24EA | |
Albumax | Thermo Fisher Scientific | 11020021 | |
Anti-α-Actinin (Sarcomeric) antibody | Sigma-Aldrich | A7811 | Dilution: 1:200 |
Anti-Cardiac Troponin T antibody (ab45932) | Abcam | ab45932 | Dilution: 1:200 |
Ascorbic acid | Sigma-Aldrich | A8960 | |
B-27 supplement | Thermo Fisher Scientific | 17504-044 | |
Biotin | Sigma-Aldrich | B4639 | |
Bovine serum albumin fraction V (BSA) | Roche | 10735086001 | |
Cal-520, AM | Abcam | ab171868 | |
Confocal microscope | Leica | DMi8 | |
Confocal microscope software | Leica | Las X | |
Conical tubes 15 mL | Greiner Bio-One | 5618-8271 | |
Creatine monohydrate | Sigma-Aldrich | C3630 | |
DAPI | Thermo Fisher Scientific | D3571 | Concentration: 1 µg/mL |
DMEM no glucose | Thermo Fisher Scientific | 11966025 | |
EDTA | Thermo Fisher Scientific | 15575020 | |
Fructose | Sigma-Aldrich | 76050771.05 | |
Glucose | Sigma-Aldrich | G7021 | |
Glycerol | Boom | 76050771.05 | |
Goat anti-mouse Alexa Fluor 488 | Invitrogen | A11029 | Dilution: 1:500 |
Goat anti-rabbit Alexa Fluor 568 | Invitrogen | A11011 | Dilution: 1:500 |
Horizontal shaker | IKA | 4003000 | |
Human induced pluripotent stem cell lines | (Stanford Cardiovascular Institute (S-CVI) Biobank) | CVI-273 (control 1) | |
Human induced pluripotent stem cell lines | Germany | 141 (control 2) 144 (control 3) | |
Hydrochloric acid (HCl) | Ajax Firechem | 265.2.5L-PL | 10 M stock solution, corrosive |
Isotype control, FITC mouse IgM κ isotype | BD | 556652 | |
KnockOut Serum Replacement | Thermo Fisher Scientific | 10828 | Protect from light |
L-carnitine | Sigma-Aldrich | C0283 | |
Myocyte calcium and contractility system | Leica | Thunder, DMi8 | |
Non essential amino acids (NEAA) | Thermo Fisher Scientific | 11140 | |
Paraformaldehyde solution 4% in 1x PBS, pH 7.0–7.6 | Santa Cruz | SC281692 | Hazardous |
PBS, pH 7.4 | Thermo Fisher Scientific | 10010023 | |
Penicillin/streptomycin | Thermo Fisher Scientific | 15140 | |
PES Membrane Vacuum Filter system | Corning | 431097 | |
PI/RNase Staining Solution | Invitrogen | F10797 | Dilution: 1:1000 |
Pluronic F-127 | Sigma-Aldrich | P2443 | |
PSC Cryopreservation Kit | Thermo Fisher Scientific | A2644601 | |
RevitaCell | Thermo Fisher Scientific | A2644501 | |
RPMI 1640 medium | Thermo Fisher Scientific | 11875 | |
Silicone Elastomer Kit | SYLGARD | 184 | |
Sodium dodecyl sulfate solution (10%) | Sigma-Aldrich | 71736 | |
Sodium L-Lactate | Sigma-Aldrich | 71718 | |
Taurine | Sigma-Aldrich | T0625 | |
Tris Fisher | Scientific | 11486631 | |
Triton X-100 | Merck | X100-1L | Hazardous |
Trypan blue solution, 0.4% | Thermo Fisher Scientific | 15250061 | |
TrypLE Select Enzyme (10x) | Thermo Fisher Scientific | A1217701 | |
Tween-20 | Sigma-Aldrich | P1379 | |
Urea | Sigma-Aldrich | 51456 | |
Vitamin B12 | Sigma-Aldrich | V6629 | |
Y-27632 dihydrochloride (Rho-kinase inhibitor) | Tocris | 1254 | Protect from light |