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* These authors contributed equally
This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.
Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming 'first in man' trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.
Drug induced cardiotoxicity is a major cause of market withdrawal1. In the last decade of the 20th century, eight non-cardiovascular drugs were withdrawn from the market as they resulted in sudden death due to ventricular arrhythmias2. In addition, several anti-cancer therapies (while in many cases effective) can lead to several cardiotoxic effects including cardiomyopathy and arrhythmias. For example, both traditional (e.g., anthracyclines and radiation) and targeted (e.g., trastuzumab) breast cancer therapies can result in cardiovascular complications in a subset of patients3. A close....
All animal procedures were in accordance with the institutional guidelines of the University of Louisville and approved by the Institutional Animal Care and Use Committee.
1. Preparation for Slicing (One Day Before Slicing)
Using a commercially available cell culture electrical stimulator that can accommodate eight 6 well plates at once, we emulated the adult cardiac milieu by inducing electrical stimulation at the physiological frequency (1.2 Hz), and screened for the fundamental medium components to prolong the duration of functional pig heart slices in culture13. Since pig and human hearts are similar in size and anatomy15, we developed a biomimetic heart sl.......
Here we describe the detailed video protocol for our recently published method for simplified medium throughput (processes up to 48 slices/device) method that enables culture of pig heart slices for a period sufficiently long to test acute cardiotoxicity13. The proposed conditions mimic the environment of the heart, including frequency of electrical stimulation, nutrient availability, and intermittent oxygenation. We attribute the prolonged viability of heart slices in our biomimetic stimulated cu.......
TMAM is supported by NIH grant P30GM127607 and American Heart Association grant 16SDG29950012. RB is supported by P01HL78825 and UM1HL113530.
....Name | Company | Catalog Number | Comments |
1000ml, 0.22µm, Vacuum Filter/Storage Systems | VWR | 28199-812 | |
2,3-Butanedione monoxime (BDM) | Fisher | AC150375000 | |
500ml, 0.22µm, Vacuum Filter/Storage Systems | VWR | 28199-788 | |
6-well C-Dish Cover (electrical-stimulation-plate-cover) | Ion Optix | CLD6WFC | |
6-well plates | Fisher | 08-772-1B | |
Agarose | Bioline USA | BIO-41025 | |
Antibiotic-Antimycotic | Thermo | 15-240-062 | |
C-Pace EM (cell-culture-electrical-stimulator) | Ion Optix | CEP100 | |
Calcium Chloride (CaCl2) | Fisher | C79-500 | |
Ceramic Blades for Vibrating Microtome | Campden Instruments | 7550-1-C | |
Cooley Chest Retractor | Millennium Surgical | 63-G5623 | |
D-Glucose | Fisher | D16-1 | |
Disposable Scalpel #20 | Biologyproducts.com | DS20X | |
Falcon Cell Strainers, Sterile, Corning | VWR | 21008-952 | |
Fetal Bovine Serum | Thermo | A3160502 | |
Graefe Forceps | Fisher | NC9475675 | |
Heparin sodium salt | Sigma-Aldrich | H3149-50KU | |
HEPES | Fisher | BP310-1 | |
Histoacryl BLUE Tissue glue | Amazon | https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/ | |
Iris Spring scissors | Fisher | NC9019530 | |
Iris Straight Scissors | Fisher | 731210 | |
Isoflurane, USP | Piramal | NDC 66794-017-25 | |
ITS Liquid Media Supplement | Sigma-Aldrich | I3146-5ML | |
Ketamine HCl (500 mg/10 mL) | West-Ward | NDC 0143-9508 | |
Magnesium Chloride (MgCl2) | Fisher | M33-500 | |
Mayo SuperCut Surgical Scissors | AROSurgical Instruments Corporation | AROSuperCutâ„¢ 07.164.17 | |
Medium 199, Earle's Salts | Thermo | 11-150-059 | |
Oxygen regulator | Praxair | ||
Oxygen tanks - | Praxair | ||
Plastic Pasteur pipettes | Fisher | 13-711-48 | |
Potassium Chloride (KCl) | Fisher | AC193780010 | |
Printer Timing Belt | Amazon | https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/ | |
Razor rectangle blades | Fisher | 12-640 | |
Recombinant Human FGF basic | R&D Systems | 233-FB-025/CF | |
Recombinant Human VEGF | R&D Systems | 293-VE-010/CF | |
Retractable scalpels | Fisher | 22-079-716 | |
Sodium Bicarbonate (NaHCO3) | Fisher | AC217125000 | |
Sodium Chloride (NaCl) | Fisher | AC327300010 | |
Vibrating Microtome | Campden Instruments | 7000 SMZ-2 | |
Xylazine HCl (100 mg/mL) | Heartland Veterinary Supply | NADA 139-236 |
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