Name: cardiac spheroids as in vitro bioengineered heart tissues to study human heart pathophysiology
Uploaded: 2021-01-23T14:00:00.000+00:00
Duration: 10 min 41 s
Description: Watch this Scientific Journal Video about Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology at JoVE.com

A special thanks to Nat Johnston for the recording and editing the video.
Poonam Sharma was supported by University of Newcastle with UNIPRS and UNRS Central &#38; Faculty School (UNRSC5050) scholarships. Carmine Gentile was supported by a UTS Seed Funding, Catholic Archdiocese of Sydney Grant for Adult Stem Cell Research and a Sydney Medical School Foundation Cardiothoracic Surgery Research Grant.

NOTE: hiPSC-CMs used for this protocol are commercially available. Please seek institutional human ethics committee approval before commencing this work if required.
1. Human cardiac fibroblast&#160;and endothelial cell culture plating and growth
<ol>
	<li>Thaw cryovials containing HCFs and HCAECs in a water bath at 37 &#176;C for one minute.</li>
	<li>Move cryovials under a sterile laminar flow biosafety cabinet class 2.</li>
	<li>Collect 1 mL of cell suspension from the cryovials using a 1000 &#181;L pipette tip and add into a 15 mL tube containing 7 mL of Human Cardiac Fibroblast Medium for HCFs and 7 mL of Human Meso Endo Growth Medium for HCAECs. 
	NOTE: In order to collect the majority of the cells from each cryovial, rinse them twice with 1 mL of culture medium from the same 15 mL tube.</li>
	<li>Gently mix cell suspensions.</li>
	<li>Transfer cell suspensions to separate T75 culture flasks using a 10 mL serological pipette.</li>
	<li>Incubate cells at 37 &#176;C with 5% CO2.</li>
	<li>After 18 h, aspirate the medium from both culture flasks and rinse them once with sterile phosphate buffered saline (PBS) to remove freezing medium and dead cells.</li>
	<li>Replace PBS with 7 mL of appropriate culture medium to each culture flask and incubate at 37 &#176;C.</li>
	<li>Examine cellular expansion and viability regularly and replace media every other day until cells reach 80-90% confluency.</li>
</ol>
2. iCM&#160;culture plating and growth
<ol>
	<li>Pre-coat two T25 culture flasks with 2 mL of PBS containing 40 &#181;g/mL of fibronectin (FN) and incubate at 37 &#176;C, 5% CO2 for at least 4 hours.</li>
	<li>After 4 hours, collect one cryovial containing iCMs and place it in a water bath at 37 &#176;C for 4 min.</li>
	<li>Move the cryovial under a sterile laminar flow biosafety cabinet class 2.</li>
	<li>Gently transfer the iCMs from the cryovial to a sterile 50 mL centrifuge tube using a 1 mL pipette tip.</li>
	<li>Rinse the empty iCMs cryovial with 1 mL of room temperature plating medium to recover any residual cells. Transfer the 1 mL of plating medium rinse from the cryovial drop-wise over 90 sec to the 50 mL centrifuge tube containing the iCM cell suspension. 
	NOTE: Gently swirl the tube while adding the medium to mix the solution completely and to decrease the osmotic shock on the thawed cells.</li>
	<li>Slowly add 8 mL of room temperature Plating Medium to the 50 mL centrifuge tube. Add the first 1 mL dropwise over 30 - 60 s. Then, add the remaining volume over the next 30 s. Gently swirl the centrifuge tube while adding the Plating medium. Gently mix the contents of the 50 mL centrifuge tube by inverting 2 - 3 times (avoiding vigorous shaking or vortexing).</li>
	<li>Immediately perform the cell counting using a hemocytometer and determine the viable cell density (in cells/mL).</li>
	<li>Take the FN-pre-coated T25 flasks and aspirate the FN-PBS solution without letting the flasks dry. To this add seeding volume of iCMs (1.6 x 106 viable iCMs&#160;in 8 mL room temperature plating medium).</li>
	<li>Culture iCMs in the incubator for 48 h at 37 &#176;C, 5% CO2.</li>
	<li>Thaw the Maintenance Medium overnight at 4 &#176;C a day before use.</li>
	<li>Equilibrate the Maintenance Medium in a 37 &#176;C water bath and use immediately.</li>
	<li>After 2 days, move the iCMs T25 flasks under the biosafety cabinet.</li>
	<li>Gently wash off dead cells and debris by gently pipetting the Plating Medium up and down 5 times.</li>
	<li>Aspirate the Plating Medium and replace with the 8 mL of pre-warmed Maintenance Medium. Place the T25 flasks back in the incubator. Replace the Maintenance Medium every other day and examine the confluency regularly.</li>
</ol>
3. Cell isolation and counting
<ol>
	<li>Start by collecting first HCAECs and HCFs, and then iCMs by following steps 3.2-3.12.</li>
	<li>Prepare CS culture medium by mixing 10 mL of iCMs Maintenance Medium, 5 mL of Human Cardiac Fibroblast Medium and 5 mL of Meso Endo Growth Medium.</li>
	<li>Remove culture medium from each tissue flask containing HCFs and HCAECs and rinse once with 5 mL PBS for T75 flasks. Remove PBS.</li>
	<li>Add 5 mL of 0.25% trypsin EDTA solution to each T75 flask and incubate for 5 min at 37 &#176;C, 5% CO2.</li>
	<li>Once cells detach, immediately neutralize the trypsin EDTA solution with 5 mL of culture medium.</li>
	<li>Transfer cell suspensions to a 15 mL tube and centrifuge cells at 300 x g for 4 min.</li>
	<li>Remove the supernatant carefully from each tube. Add 1 mL of CS medium to each cell pellet and resuspend them. Keep the tube on ice and count cells using Trypan Blue and a hemocytometer.</li>
	<li>Remove Maintenance Medium from tissue flasks containing iCMs and rinse once with 3 mL of PBS.</li>
	<li>Add 1 mL of 0.25% trypsin EDTA solution to each T75 flask and incubate at 37 &#176;C, 5% CO2. Check cells every minute until detached.</li>
	<li>Once cells detach, immediately neutralize the trypsin EDTA solution with 4 mL of culture medium.</li>
	<li>Transfer cell suspensions to a 15 mL tube and centrifuge them at 300 x g for 5 min.</li>
	<li>Remove the supernatant carefully from each tube. Add 1 mL of culture medium to the cell pellet and resuspend it. Keep the tube on ice and count cells using Trypan Blue and a hemocytometer.</li>
</ol>
4. CS&#160;generation and growth
<ol>
	<li>Mix iCMs, HCFs and HCAECs in 2:1:1 ratio by plating 10,000 iCMs, 5,000 HCFs and 5,000 HCAECs per hanging drop culture containing 20 &#181;L of CS medium. Adjust to the final volume for the total number of CSs.</li>
	<li>Pipette 20 &#181;L of cell suspension into each well of the 384 well HDC plate either manually or by using a robotic multichannel pipette for automated liquid handling.</li>
	<li>Pipette 1.5 mL of sterile PBS in each side of the channel around the Hanging Drop Plate to prevent drying out CSs. Incubate HDC plate at 37 &#176;C.</li>
	<li>Examine formation of CSs on a daily basis until a fully formed spheroid is observed in the majority of wells. Add 7.5 &#181;L of CS medium to each well every other day until a CS is formed.</li>
</ol>
5. CS&#160;embedding in collagen gels
<ol>
	<li>Collect CSs with a 1 mL pipette tip. 
	NOTE: It is necessary to cut the tip of pipette around 0.2 cm from the edge before its use with a sterile sharp surface (either a scalpel or a scissor) to prevent any damage gel embedded spheroids during their collection.</li>
	<li>Collect the CS suspension into a 50 mL tube on ice.</li>
	<li>Centrifuge the tube at 300 x g for 5 min. 
	NOTE: the pellet obtained must be kept on ice until use.</li>
	<li>Prepare a collagen gel solution (100 &#181;L/well for 30 wells of 96 well plate) on ice using rat tail collagen and CS medium in a 3:7 ratio.</li>
	<li>Remove the supernatant from the tube containing CSs.</li>
	<li>Mix the pelleted CSs within the collagen gel solution.</li>
	<li>Add 1 &#181;L/mL of 5 mM sodium hydroxide to the CS-collagen gel suspension and mix gently.</li>
	<li>Transfer 100 &#181;L of CS-collagen gel to a clear flat bottom 96 well black polystyrene microplate and incubate at 37 &#176;C for 30 min.</li>
</ol>
6. Viability and toxicity measurements of DOX-treated CSs
<ol>
	<li>After 30 min collect the 96 well plate from incubator</li>
	<li>Prepare a 10 &#181;M DOX (based on previously established protocol for cell death in CSs14). 
	&#8203;NOTE: To potentially test other agents that may protect against HF in CSs, generate solutions containing DOX + Agent A, B, etc.</li>
	<li>Add 100 &#181;L of solutions containing DOX and/or other agents to each well. Control cultures contain media without any DOX.</li>
	<li>Incubate plate for 18 h at 37 &#176;C, 5% CO2.</li>
	<li>On the following day, collect the Live/Dead staining reagent stock solutions and allow them to thaw on ice in the dark in a biosafety cabinet.</li>
	<li>Prepare a solution containing Hoechst stain, 4 &#181;M of ethidium homodimer and 2 &#181;M calcein-AM.</li>
	<li>Add 100 &#181;L of Hoechst stain, calcein-AM/ethidium homodimer solution into each well.</li>
	<li>Measure the fluorescence into each well at 645 nm for ethidium homodimer and at 530 nm for calcein-AM, respectively, using multimode microplate reader.</li>
	<li>Transfer fluorescence measurements into Graphpad PRISM (or an equivalent software for statistical analysis).</li>
	<li>Use GraphPad Prism software for data analysis and statistics.</li>
	<li>For quality control, check under an epifluorescence microscope for the nuclei staining, together with calcein-AM and ethidium homodimer.</li>
</ol>
7. CS contractile function evaluation
<ol>
	<li>Collect the microplate as prepared in step 5.8.</li>
	<li>Turn the on the computer containing the IonOptix software for a video-based edge-detection, the Fluorescence Assistance Interface, and the MyoPacer Field Stimulator.</li>
	<li>Place a new cover slip on the tissue holder platform and assemble water bath with electrodes.</li>
	<li>Gently collect CSs from collagen gels using a 1 mL pipette tip cut 0.5 mm from the edge and transfer them to a falcon tube. Add media onto CS to prevent drying of CSs. Transfer CS (one at a time) with a few &#181;L of media on the stage of the IonOptix system.</li>
	<li>Select the CS to be analysed via setting peaks on left and right side of CS using the IonOptix software.</li>
	<li>Use the computer-based motion analyzer to track the movement of CS edges. 
	NOTE: Normally, contractility is measured in either % cell shortening or % fractional shortening. In this case, we measured % spheroid shortening.</li>
	<li>Stabilize both the peaks adjusting threshold and edge options from the computer.</li>
	<li>Expose CSs to different frequencies (0.3, 0.6, 1, 2 and 3 Hz) and voltages (1, 2, 3 and 5 V) using the Myopacer Field Stimulator.</li>
	<li>Record spheroid shortening as CS length changes of DOX-treated and untreated CSs. Analyse data using the Soft-Edge software and averaged for each CS.</li>
</ol>
8. Microscopy of CSs: fixation and immunolabeling
<ol>
	<li>Collect the 96 well plate after 30 min (as prepared in step 5.8) and fix CSs in 4% paraformaldehyde (PFA) for 1 h at room temperature.</li>
	<li>Remove PFA and rinse three times with PBS containing 0.01% sodium azide (PBSA).</li>
	<li>Remove PBSA.</li>
	<li>Add 200 &#181;L of PBSA containing 0.02% Triton-X-100 to each well for 30 min on a shaker. 
	NOTE: This step permeabilizes CSs for better antibody infiltration.</li>
	<li>Add 200 &#181;L of 3% bovine serum albumin in PBSA solution for 60 min at room temperature. 
	NOTE: This step blocks unspecific antibody binding in CSs.</li>
	<li>Prepare a solution containing 10 &#181;g/mL of primary mouse anti-human antibodies against CD31 diluted in blocking solution.</li>
	<li>Add 100 &#181;L of primary antibody solution to each well and incubate overnight at 4 &#176;C on a shaker.</li>
	<li>Rinse the plate three times with PBSA for 20 min at room temperature on a rocking plate.</li>
	<li>Prepare a solution containing Hoechst DNA stain and 10 &#181;g/mL of Cy3-conjugated secondary donkey anti-mouse antibody diluted in blocking solution.</li>
	<li>Add 100 &#181;L of secondary antibody solution containing Hoechst stain to each well and incubate overnight at 4 &#176;C on a shaker. 
	NOTE: Cover the plate with aluminium foil from this point onwards.</li>
	<li>Rinse the plate three times for 20 min with PBSA at room temperature on a rocking plate.</li>
	<li>Add 100 &#181;L of Vectashield mounting medium to each well.</li>
	<li>Image CSs under a laser scanning confocal microscope. Perform optical sectioning along the Z axis and collapse images into a single focal plane using ImageJ software.</li>
</ol>

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Developmentally, proper vascular network formation is critical for the generation of functional tissues, including the human heart10,12,23,24,25,26. Consideration for the proper vascularization of 3D tissues allow the exchange of oxygen, growth factors, signalling molecules and nutrients, preventing the development of cell necrosis within any tissue thicker than 200 &#956;m6,10,12,17,24,25,26,27,28. Currently available in vitro 3D heart models that present a vascular network are primarily presenting capillary-sized, disorganized vascular networks and lack the hierarchical complex branched vascularization observed in vivo6,8,29. The alternative approach to develop complex cardiac endothelial cell network described in this manuscript presents improved cell viability and function compared to existing models (Figure 1)14,22. 3D in vitro CSs model the human heart by better recapitulating its in vivo microenvironment, including its molecular, cellular and extracellular components14,22. CS generation from stem cell-derived cells in the hanging drops allow their cultures in defined conditions (e.g., cell types and ratio, proper tissue formation). Co-cultures of iCMs together with HCFs and HCAECs within CSs define the molecular and cellular crosstalk that regulates the heart pathophysiology, including its contractile function and response to drugs at concentrations found in the patient&#39;s bloodstream14. Due to these unique features, CSs have been utilized to model cardiac fibrosis, a severe consequence of myocardial infarction and heart failure21. Our previous studies showed how the presence of both endothelial cells and fibroblasts is critical for the recapitulation of the vascular microenvironment in the human heart, allowing the optimal deposition of fibroblast-derived ECM proteins, such as laminin, fibronectin and collagen type IV, localized in proximity of a developing endothelial cell network14,21.
DOX is a well-known cardiotoxic drug that may develop heart failure in cancer patients even 17 years after their treatment30. Nevertheless, it remains a drug of choice for the treatment of leukemia and lymphoma in paediatric patients and breast cancer in women30. DOX treatment in CSs has then been used to model heart failure (HF) in vitro to study both the mechanisms regulating toxicity in cardiac myocytes, endothelial cells and fibroblasts14 and to model HF-induced cardiac fibrosis21. Cell viability was statistically reduced in DOX treated CSs within 24 h when exposed to the drug at the concentration found in the bloodstream of cancer patients (between 5 and 10 &#956;M)14 (Figure 2). Previous studies in our laboratory also demonstrated the toxic effects of DOX on both cardiac endothelial cells and fibroblasts via endothelial nitric oxide synthase (eNOS) using both genetic and chemical inhibitors of this signaling pathway14. The use of genetic (NOS3 shRNA) and chemical (N5-(1-iminoethyl)-L-ornithine, dihydrochloride, or L-NIO) antagonists of the eNOS signaling pathway as a downstream target of DOX prevented its toxic effects in both cardiac endothelial cells and fibroblasts14.
Contractile activity within CSs has also been measured thanks to the electrical coupling of cardiac cells when exposed to field potential stimulation. We found that CSs cultured with control media (DOX 0 &#956;M) contract spontaneously and homogenously at a beating rate that can be paced by field stimulation within 1 and 3 Hz, comparable with a healthy human heart. On the other hand, DOX-treated CSs do not follow the electrical stimulation as they cannot contract. Together with the measurements of cell viability and toxicity using calcein-AM and ethidium homodimer, this functional assay for CS contractile function allow the evaluation of the complex scenario typical of the human heart in vitro, currently not achievable with other models. Compared to contractile activity measurements of single cardiac cells using the same system, we are not able to visualize and measure the sarcomere in CSs. Therefore, we are limited to measurements of % spheroid shortening over time, an assay we had to develop within our laboratory. As we control the number of cells, we co-culture in each CS and therefore the size of each CS, we utilize CSs with similar size that indeed present homogenous contractile function. However, even in case we generated CSs of different sizes, their contractile activity did not change.
It is also important to report that the multicellular nature of CSs makes them heavy enough to localize at the bottom of the coverslip in the Ion-Optix system, even in case they are superfused. Based on the fact that CSs sit by themselves in a specific position, we do not need to make them adhere to the coverlip, on the contrary of what is commonly done with single cardiac cells in most laboratories.
The microscopic analysis of CSs stained with antibodies against cardiac troponin T, CD31/PECAM, and PECAM (as markers for iCMs, HCAECs, and HCFs, respectively) showed the formation of a endothelial cell network (Figure 1, blue). To fully exclude necrosis in the inner part of CSs, spatial evaluation of cell viability was performed in our laboratory by confocal analysis of calcein-AM/ethidium homodimer stained CSs (data not shown). However, it is important to acknowledge that future developments in the biofabrication field to better recapitulate other complex features typical of the human heart in vivo, currently not available in the existing model. These include: i) contractile function typical of adult cardiomyocytes; ii) blood flow and pressure forces; iii) paracrine signaling; iv) immune response, which will be critical to improve this and other in vitro cardiac models6. As any other model aims at recapitulating major features of either a healthy tissue or a disease state, the protocol for the generation and use of CS described in this manuscript aims at helping the researcher at addressing specific questions, that may not be exhaustive using this approach. For instance, the potential use of patient-derived cells for the generation of CSs would provide tools for personalized medicine, currently not available using commonly available high-throughput assays for cardiovascular research.
In conclusion, we demonstrated a simple way to better recapitulate the human heart microenvironment using cardiac cells. Cardiac spheroids present an endothelial cell network that better recapitulates the one present in the human heart compared to monolayer cultures of cardiac cells.Given their unique features, they represent advanced tools for in vitro testing for cardiovascular research. Future studies using patient-derived cells could provide options for personalized medicine and novel therapies to both prevent and better treat cardiovascular disease.

The human heart has a limited regenerative capacity while cardiovascular disease (CVD) remains the main cause of death worldwide despite the recent advances in tissue engineering and stem cell technologies1. The need for novel therapeutics including molecular and cellular approaches to either repair a damaged heart or to prevent a heart from failing is one of the major current clinical needs for patients suffering from heart disease2,3,4. The main goal of cardiac tissue engineering is to fabricate a three-dimensional (3D) heart tissue that presents molecular, cellular, and extracellular features typical of a human heart, including its vascular network and physiological contractile function4,5,6.
In order to bioengineer and fabricate a functional human cardiac tissue that mimics the human heart for in vitro and in vivo applications, several approaches have been investigated including engineered heart tissues (EHTs), cell sheets and spheroid cultures7,8. However, these tissues fail at recapitulating the optimal 3D microenvironment typical of the human heart and their potential use for CVD patients cannot directly translate from the bench to the bedside7. This is because they do not recapitulate the complex biology, morphology, and physiology of in vivo heart tissues9. One of the major challenges in cardiac tissue engineering includes the development of a hierarchical vascular network within the bioengineered cardiac tissue, as any tissue that is bigger than 200 &#956;m in diameter develops cell death in the middle2,10. A properly formed vascular network in a human heart tissue plays an major role for the supply of blood, oxygen and nutrients to cardiac cells11. During embryonic development, coronary capillaries and arteries form via vasculogenesis (de novo blood vessel formation) and angiogenesis (generation of blood vessels from pre-existing ones) from endothelial progenitor cells8,12. Cardiac fibroblasts also play a major role in proper vascular network formation by providing the optimal extracellular matrix (ECM) and growth composition13,14.
The 3D vascular network of bioengineered heart tissues controls cell survival and function by creating oxygen and nutrient gradients and paracrine signaling, such as homotypic cell interaction, heterotypic cell interaction, interaction of cells through secreted soluble proteins and cell to ECM interactions3,10,15,16,17,18. This prevents cell death in the middle of the tissue and promotes cell viability and physiological function in bioengineered heart tissues16,18,19.
Spheroid cultures from stem cells have been recently explored as in vitro models of the human heart20. To further improve the cardiac microenvironment in vitro, they have included the use of all the main cell types found in the human heart, such as cardiac myocytes, endothelial cells, and fibroblasts. Spheroid cultures present the required 3D structural support for cells to grow and function and can be used to bioengineer a vascular network14,20,21,22. In this context, our laboratory has developed human cardiac spheroids (CSs) by co-culturing cardiac myocytes, endothelial cells and fibroblasts at ratios found in the human heart14. This model is an expansion of the rat ventricular cardiac cells spheroid model, generated by co-culturing cardiac cells in hanging drop cultures, used to model cardiac fibrosis21. Human CSs can be used as toxicity assays by treating them doxorubicin (DOX, an anti-cancer agent used to treat leukemia, lymphoma and breast cancer), which is well-known to induce cardiac fibrosis and heart failure (HF) even 17 years following its somministration14.
In this manuscript, we describe how to generate human CSs by co-culturing human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs or iCMs), human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop cultures. In order to use and image CSs for in vitro testing, they are embedded in a collagen gel. The confocal analysis of CSs stained with antibodies against CD31, a marker for endothelial cells, showed that these cells form a network similar to the one observed in vivo. To induce HF and potentially test novel agents that may treat or prevent it, CSs were treated with 10 &#181;M DOX (a concentration found in the bloodstream of cancer patients receiving the drug). When stained with calcein-AM and ethidium homodimer (staining live and dead cells, respectively), DOX-treated CSs present a significant decrease in viability in comparison to CSs that did not receive the drug. CSs also present a homogeneous contractile activity when paced using field potential stimulation between 1 and 3 Hz.

<table><tbody><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A1933</td><td></td></tr><tr><td>Donkey anti-mouse Secondary Antibodies</td><td>Jackson Immunological Research Labs, Inc.</td><td>715-165-150</td><td>Cyanine Cy3-conjugated secondary antibody</td></tr><tr><td>Doxorubicin hydrochloride</td><td>Sigma-Aldrich</td><td>D1515</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141-1MG</td><td>From Bovine Plasma</td></tr><tr><td>Human cardiac fibroblasts (HCFs)</td><td>Cell Applications, Inc., San Diego, CA, USA</td><td>306AK-05a</td><td>5x10^5 Cells (Adult), Medium &amp;amp; Subculture Reagents</td></tr><tr><td>Human coronary artery endothelial cells (HCAECs)</td><td>Cell Applications, Inc., San Diego, CA, USA</td><td>300K-05a</td><td>5x10^5 Cells (Adult), Medium &amp;amp; Subculture Reagents</td></tr><tr><td>Human iPSC-derived cardiomyocytes (iCMs)</td><td>Fujifilm Cellular Dynamics, Inc.</td><td>R1057</td><td>iCell Cardiomyocytes Kit, 01434</td></tr><tr><td>HCF Growth medium</td><td>Cell Applications, Inc., San Diego, CA, USA</td><td>316-500</td><td></td></tr><tr><td>Human MesoEndo Cell Growth Medium</td><td>Cell Applications, Inc., San Diego, CA, USA</td><td>212-500</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit</td><td>Invitrogen, Carlsbad, CA, USA</td><td>L3224</td><td></td></tr><tr><td>Maintenance Medium (iCells)</td><td>Fujifilm Cellular Dynamics, Inc.</td><td>R1057</td><td>iCell Cardiomyocytes Kit, 01434</td></tr><tr><td>Mouse Monoclonal anti-human CD31/PECAM</td><td>BD Pharmingen, San Diego, CA, USA</td><td>566177</td><td></td></tr><tr><td>NucBlue Live ReadyProbe Reagent (Hoechst 33342)</td><td>Invitrogen, Carlsbad, CA, USA</td><td>R37605</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Phosphate-Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Plating Medium (iCells)</td><td>Fujifilm Cellular Dynamics, Inc.</td><td>R1057</td><td>iCell Cardiomyocytes Kit, 01434</td></tr><tr><td>Rat Tail Collagen</td><td>Sigma-Aldrich</td><td>C3867</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Trypsin&amp;ndash;EDTA, 0.25%</td><td>Gibco, Thermofisher Scientific</td><td>25200072</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco, Thermofisher Scientific</td><td>15250061</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Tissue culture flasks (T25)</td><td>Thermofisher Scientific</td><td>156367</td><td></td></tr><tr><td>96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates</td><td>Corning, New York, USA</td><td>3603</td><td></td></tr><tr><td>384-Well Hanging Drop Plate</td><td>3D Biomatrix, Ann Arbor, MI, USA</td><td>HDP1385</td><td></td></tr></tbody></table>

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.

The protocol described in this manuscript represents an alternative approach to develop complex cardiac endothelial cell network within a bioengineered cardiac tissue with improved cell viability and function compared to existing models (Figure 1).&#160;The recapitulation of the 3D in vivo heart microenvironment within CSs promoted their response to DOX at the concentration found in the bloodstream of cancer patients (between 5 and 10 &#956;M, Figure 2). DOX treated CSs presented a statistically significant reduction in cell viability compared with control (no DOX) CSs within 24 h (Figure 2), a toxic effect that is observed in human cancer patients even 17 years after their treatment with the drug.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61962/61962fig01.jpg" /> 
Figure 1. CS Formation and Vascularization Analysis. (A) Protocol showing the steps for the formation of a CS from the co-culture of iCMs, HCAECs and HCFs in hanging drops. Brightfield images on the right side show the progressive spheroid formation from single cells in hanging drops. (B) Collapsed Z-stacks of confocal images of a CS stained with antibodies against cardiac Troponin T (cTNT), PECAM and vimentin, staining cardiac myocytes, endothelial cells and fibroblasts, respectively. This figure has been modified from14. <a href="https://www.jove.com/files/ftp_upload/61962/61962fig01large.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/61962/61962fig02.jpg" /> 
Figure 2. Viability and Toxicity in CSs. Statistical analyses of calcein-AM (A) and ethidium homodimer (B) fluorescence of CSs treated in presence of either media only (DOX 0 &#956;M) or doxorubicin (10 &#956;M). <a href="https://www.jove.com/files/ftp_upload/61962/61962fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology at JoVE.com

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

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 ...

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Bioengineering

7.5K Views.  University of Newcastle. This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops. 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.

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

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Biochemistry

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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 ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

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.

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 ...