Development of a High-Throughput 3D Cardiac Tissue Assay

מושגי ליבה

A novel high-throughput assay enables the generation of 3D cardiac tissues for drug testing and disease modeling.

תקציר

Abstract:

• Developed a 96-well plate assay for generating 3D cardiac rings.
• Human iPSC-derived cardiomyocytes self-organized into ring-shaped constructs.
• Showed expected inotropic responses to calcium and drugs.

Introduction:

• Cardiac tissue engineering aims to mimic native myocardium properties.
• Biomaterials and microfabrication techniques enable miniature heart tissues.
• Various geometries of engineered heart tissues have been generated.

Design and Characterization:

• Steel molds designed for directing cells into ring-shaped cavities.
• PEG gel structures obtained with known stiffness suitable for cardiac tissue.
• Young's modulus measurements confirmed physiological range compatibility.

Generation of Cardiac Tissues:

• Efficient differentiation of iPSCs into cardiomyocytes observed.
• Fibroblasts essential for tissue structure, optimal ratio determined as 3:1.
• Ring-shaped tissues compacted around central pillar, started beating within 24 hours.

Structure and Organization:

• Immunofluorescence imaging showed fibroblasts at the base, cardiomyocytes forming compact ring above.
• Striated elongated fibers typical of cardiac tissue observed in immunostaining.
• Tissues exhibited toric shape with organized structure at day 14.

Contractility Analysis:

• Tissues started contracting less than 24 hours after seeding, contraction stress increased until day 7 then stabilized.
• Long-term culture possible but not yet optimized, contractile parameters improved at day 28 compared to day 14.

Arrhythmia Analysis:

-Tissues beat regularly without spontaneous extra beats, no reentrant waves detected in live imaging studies.

Physiological Testing:

-Increased extracellular calcium concentration led to positive inotropic response as expected.
-Dose-response to verapamil, isoproterenol, and dofetilide demonstrated expected effects on contractility parameters.

Pharmacological Testing:

-Negative inotropic effect observed with verapamil, positive response with isoproterenol, arrhythmic events induced by dofetilide at higher concentrations.

Discussion:

-Novel platform allows fast generation of multiple cardiac tissues suitable for drug testing and disease modeling applications.
-Limitations include small size compared to native tissue and challenges regarding external pacing methods.

סטטיסטיקה

The average stress during contractions was calculated as 1.4±0.4 mN/mm2.
The efficiency of differentiation into cardiomyocytes was approximately 96.9 ± 0.8%.

ציטוטים

"Our EHTs are easy to use, image, manipulate and obtain readouts from."
"The circular geometry induces homogeneous distribution of cardiomyocytes around the pillar."

תובנות מפתח מזוקקות מ:

A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell derived cardiomyocytes

by Seguret,M., ... ב- www.biorxiv.org 03-29-2023

https://www.biorxiv.org/content/10.1101/2023.03.26.534303v2

שאלות מעמיקות

How can this high-throughput assay be adapted for personalized medicine applications?

This high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes can be adapted for personalized medicine applications by incorporating patient-specific cells. For personalized medicine, patient-derived induced pluripotent stem cells (iPSCs) can be used to generate cardiomyocytes that closely resemble the individual's own cardiac tissue. By utilizing iPSCs derived from patients with specific genetic mutations or conditions, researchers and clinicians can create customized cardiac tissues that mimic the unique characteristics of the patient's heart.
Furthermore, this platform allows for the screening of various drugs and compounds on these personalized cardiac tissues to assess individual responses to different treatments. By exposing the patient-specific cardiac constructs to different pharmaceutical agents, researchers can evaluate drug efficacy and potential side effects in a more tailored manner. This approach holds promise for predicting how an individual may respond to certain medications based on their genetic makeup and disease profile.
In addition, integrating advanced imaging techniques such as live fluorescence imaging with voltage-sensitive dyes into this platform could enable real-time monitoring of electrical activity in these personalized cardiac tissues. This would provide valuable insights into arrhythmogenic tendencies or response to antiarrhythmic drugs specific to each patient.

What are the potential implications of the small size limitation on drug testing outcomes?

The small size limitation of the ring-shaped cardiac tissues in this high-throughput assay may have several implications on drug testing outcomes:

Limited Tissue Complexity: The miniature size of the cardiac constructs may not fully capture all aspects of native myocardial tissue complexity, including cellular heterogeneity and structural organization found in larger heart models. This reduced complexity could impact how drugs interact with different cell types within a complete heart structure.

Mechanical Constraints: The smaller size may limit mechanical properties such as force generation capacity compared to larger tissue models like strips or sheets. This could affect contractile behavior under pharmacological stimulation and potentially alter drug responses observed in larger-scale models.

Diffusion Limitations: In smaller tissues, diffusion gradients across the construct might differ significantly from those seen in actual human hearts or larger engineered heart tissues. Variations in nutrient supply, waste removal efficiency, and drug distribution throughout tiny structures could influence drug metabolism rates and overall pharmacokinetics within these miniaturized systems.

Sensitivity Issues: Due to their reduced dimensions, small-sized constructs might exhibit higher sensitivity towards external factors like temperature fluctuations or changes in media composition during prolonged experiments which could introduce variability into drug testing outcomes.

Scaling Challenges: Translating findings from microscale assays using tiny tissue rings into clinical settings where dosages are calculated based on standard organ sizes poses challenges due to differences in metabolic rates between scaled-down models and actual human organs.

How might the platform be modified to allow external pacing methods for more controlled experiments?

To enable external pacing methods for more controlled experiments using this platform:
1-Microelectrode Integration: Integrate microelectrodes directly onto or around each ring-shaped tissue within wells so that precise electrical stimuli can be delivered externally while recording electrophysiological parameters simultaneously.
2-Optogenetic Stimulation: Incorporate optogenetic tools by genetically modifying cardiomyocytes with light-sensitive ion channels allowing non-invasive optical control over pacing rhythms through light stimulation directed at specific regions within each construct.
3-Electrical Field Stimulation Setup: Develop an automated system capable of delivering programmable electrical field stimulations across multiple wells simultaneously enabling synchronized contractions among all constructed rings according to predefined patterns.
4-Real-Time Monitoring System: Implement a real-time monitoring system equipped with sensors capable of detecting minute changes in contraction patterns triggered by external pacing ensuring accurate data collection during experimental procedures.
5-Customized Software Interface: Design user-friendly software interface enabling researchers easy access controls over external pacing parameters such as frequency rate modulation intensity adjustments facilitating customization according experiment requirements.
By implementing these modifications effectively it will enhance experimental flexibility precision reproducibility when conducting studies involving controlled pacing mechanisms essential understanding electrophysiological behaviors response dynamics engineered cardiovascular model systems