mTOR Inhibitors Modulate the Physical Properties of 3D Spheroids Derived from H9c2 Cells

Area of Science:

• Cardiovascular Biology
• Biomedical Engineering
• Cellular and Molecular Medicine

Background:

• Establishing accurate in vitro models for cardiomyocyte local environments is crucial for cardiac research.
• Conventional 2D cell cultures do not fully replicate the complex microenvironment of native cardiomyocytes.

Purpose of the Study:

• To develop and characterize a three-dimensional (3D) spheroid model using H9c2 cardiomyoblasts as an in vitro model for the local environment of cardiomyocytes.
• To evaluate the morphological, biophysical, and biochemical characteristics of these 3D spheroids.

Main Methods:

• Preparation of 3D spheroids from H9c2 cardiomyoblasts.
• Evaluation of spheroid morphology, biophysics (phase contrast), and biochemistry.
• Quantitative Polymerase Chain Reaction (qPCR) analysis to assess gene expression.
• Treatment with mTOR modulators (rapamycin and Torin 1) to study cellular responses.

Main Results:

• Successfully obtained 3D H9c2 spheroids that matured without cell multiplication, unlike 2D cultures.
• Observed increased expression of collagen4 (COL4), fibronectin (FN), connexin43 (CX43), β-catenin, N-cadherin, STAT3, and HIF1 in 3D spheroids compared to 2D cultures.
• Found differential responses to mTOR modulators in 3D spheroids, including reduced size and stiffness, and altered gene expression.

Conclusions:

• 3D H9c2 spheroids exhibit unique characteristics, including spontaneous expression of gap junction (GJ)-related molecules and core hypoxia, associated with maturation.
• This 3D spheroid model shows promise as a valuable in vitro tool for replicating the local environment of cardiomyocytes.
• The distinct metabolic responses to mTOR modulators in 3D cultures highlight the model's utility in studying drug effects in a more physiologically relevant context.