Microfluidic Constriction Assay Reveals Fibrotic Stiffening in a Spheroid Model of Myocardial Ischemia

This recent study, generation of self-induced myocardial ischemia in large-sized cardiac spheroids without alteration of environmental conditions recreates fibrotic remodeling and tissue stiffening revealed by constriction assays¹, aims to recapitulate the in vivo environment of the ischemic human myocardium by generating large cardiac spheroids (40.000 cells) that self-induce the ischemic core, without modification of culture conditions. Spheroids are composed of hiPSC-CM and human cardiac fibroblast to recapitulate biomechanical features of fibrotic remodeling. Implementation of a microfluidic constriction system allows to investigate the associated stiffening of the cardiac spheroids.

What is Myocardial ischemia?

Myocardial ischemia is a cardiovascular condition characterized by insufficient blood flow to the heart muscle, leading to oxygen deprivation, cell damage, and potential heart failure. Myocardial ischemia remains a major global killer, claiming countless lives each year. This condition is often triggered by the blockage of a coronary vessel, cutting off vital blood flow to the surrounding myocardial tissue. This deprivation leads to the death of cardiomyocytes and biomechanical alterations in the nearby microenvironment. Indeed, under the mechanical constraints experienced by the heart during each pulsation, oxygen deprivation and cell death result in improper and fibrotic tissue remodeling. This often leads to impaired heart function due to the reduced contractility and flexibility of the resulting scar tissue.

The need for accurate Myocardial ischemia models

In order to advance treatments for ischemic heart conditions, a comprehensive in vitro model is essential to accurately replicate the biomechanical features of fibrotic remodeling. To achieve this, two key factors must be addressed:

1. 3D structure and media gradients

Tissue damage during cardiac ischemia tends to be related to an insufficient oxygen supply, but it is also due to a reduced availability of nutrients and an inadequate removal of metabolic end-products. In fact, diseases only caused by hypoxia, like cyanosis, severe anemia or lung diseases, cause less notorious effects than ischemia. Therefore, modelling an ischemic myocardium in vitro requires the recreation of its particular 3D architecture, closely related to the formation of nutrient, oxygen and cellular products concentration gradients; and generating a spatial increase in cellular damage. Spheroids represent the simplest approach to culture cells in 3D and generate gradients of cellular access to the media. However, achieving an ischemic core in cardiac spheroids still relies on the culture under hypoxic atmosphere or deprived nutrient media.

2. Ischemic myocardial remodeling

In the last decades, development of protocols to generate cardiomyocytes (CM) from human induced pluripotent cells (hiPSC) has boosted the generation of cardiac in vitro models. However, cardiomyocytes are not the only cells affected by an ischemic event in vivo (Fig. 1).

Phase I: Inflammation: Along with cardiomyocyte’s death, there is a massive influx of inflammatory cells to the damaged tissue, which will clear the cell debris and help maintain the integrity of the cardiac wall.
Phase II: Proliferation: the inflammatory infiltrate is progressively cleared away, whilst fibroblasts are recruited to the injured site. The presence of inflammatory and profibrotic signals, added to the mechanical stress, trigger the transition of these cells to a myofibroblasts phenotype.
Phase III: Maturation: Myofibroblasts deposit a collagenous scar, similar to the healing of a skin cut. However, the chronification of this collagen deposition leads to the appearance of a hyper-stiff scar, against which the remaining contractile CMs must function, also leading to an alteration of the normal cardiac 3D architecture.

This is the fibrotic remodeling with as a consequence, a dramatic decrease of the efficiency of contraction.

Microfluidic constriction assay for spheroid model characterization setup

Materials

hiPSC-CM
Primary human cardiac fibroblast
U-shape 96 well plates to generate spheroids
THUNDER Leica DMi8 Microscope
Custom-made constriction methacrylate microfluidic device (designed by BEOnChip S.L. and manufactured by Aitiip Centro Tecnológico)
OB1 MK4 Microfluidic Flow Controller (Elveflow)
Elveflow Software Interface (Elveflow)
ImageJ software

Spheroid generation

hiPSC-CMs and human cardiac fibroblast, mixed at a 70:30 ratio, were suspended in the appropriate media and seeded in U-well bottom 96-well plates (40.000 cells/well). After 2 days, cells in each well were aggregated into one large spheroid, and aggregates were maintained for up to 17 days.

Biological characterization

At selected timepoints, cell death at the core of the spheroid was assessed by calcein/propidium iodide viability staining. Immunofluorescence of histological cryo-sections of spheroids allowed determination of hypoxia (HIF-1) and disposition of hiPSC-CM (c-Troponin) and cardiac fibroblast (vimentin) within the spheroid.

Mechanical characterization

Spheroid stiffness was determined using a custom-made constriction methacrylate microfluidic device, consisting of a single 400 μm tubular channel, which is reduced to 200 μm (Figure 2A).

For the constriction assay, the microdevice was connected to the Elveflow OB1 Microfluidic Flow Control System, and placed in an optical inverted microscope for inspection. After measuring spheroid diameter, each spheroid was captured with a micropipette and injected into the system (Figure 2B), previously perfused with culture media. Pressures lower than 1000 Pa were applied to place the spheroid at the entrance of the microchannel constriction, blocking the passage of liquid flow. Then, the pressure was increased at a constant rate of 100 Pa/s, pushing the spheroid through. The spheroid stiffness was determined as a pressure/deformation relationship (ΔP/Δδ, Figure 2A). To obtain this relationship, after each 10-mbar pressure increment, the length occupied by the deformed spheroid in the 200 μm diameter channel was measured (ImageJ software) and normalized by the initial spheroid diameter to calculate deformation. For small deformations, experimental results could be fitted to a linear curve from which the pressure/deformation relationship was obtained (Figure 2C).

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Key Findings

Culture of large size cardiac spheroids results in quick establishment of ischemia without external oxygen deprivation.

40.000 cells spheroids Z-stack projection of confocal images (Figure 3A) showed external distribution of living cells and internal congregation of dead cells since day 2 of spheroid culture. Furthermore, at day 10, the frontier between live and dead cells became sharper, with living cells gathering at the spheroid surface and a clearly defined dead core. Quantification of the dead core radius (Figure 3B) across all the spheroids and conditions showed significant differences between day 2 and 10, indicating an increased progression of dead cells within the spheroid core. The dead core evolution overtime correlated with the expression of hypoxia-inducible factor 1-alpha (HIF-1-α) as observed by immunofluorescence (Figure 3C).

Figure 3. A) Confocal images and z-stack projections of cellular viability inside cardiac spheroids. Living cells stained with calcein (green) and dead cells stained with propidium iodide (red), followed by spheroid clarification. Scale bar = 200 μm. B) Percentage of normalized quantified dead core radius across the multiple spheroids studied for each condition. C) HIF-1-α (green) immunostaining images. Nuclei are depicted in blue.

Fibrotic cellular reorganization can be observed in ischemic cardiac spheroids.

In the first days of culture, vimentin (fibroblast marker) and cTnT (cardiomyocyte marker) were randomly arranged within the spheroid, a thin layer of vimentin+ cells coating the spheroid surface could already be seen at day 2 (figure 4A).

After 10 days of culture, the initial disposition evolved to present a central aggrupation of cTnT+ cells (i.e, hiPSC-CM) flanked by areas of vimentin+ cells (i.e., cardiac fibroblast), similar to previous observations in smaller cardiac spheroids subjected to external hypoxia, suggesting a shift in fibroblast organization towards the edge of the spheroid². Fibroblast redistribution was confirmed by quantifying the fluorescence signal across the spheroids´ radius provided by vimentin immunostaining (Figure 4B). This cellular reorganization matches in vivo observations of activation and local enrichment of vimentin+ cells in the peri-infarction border zone, around myocardial cells that undergoes remodeling, in contrast with necrotic myocardium that remains negative for vimentin³.

Figure 4. Ischemic-related fibrotic remodeling assessed by immunofluorescence. A) Vimentin (CF marker, in red) and cardiac troponin T (cTnT) (CM marker, in green) disposition across cardiac spheroids cryosections. Nuclei stained with Hoechst (blue). Scale bar = 100 μm. B) Quantification of spatial distribution of vimentin. Statistical significance was assessed at 50% and 90% spheroid radius by one-way ANOVA with Dunnet’s multiple comparison test. *: p < 0.05. **: p < 0.01 and ***: p < 0.001, compared to day 2.

Fibrotic reorganization leads to an increase of tissue stiffness.

In order to study the stiffness of hypoxic spheroids, they were subjected to a constriction assay, where spheroids were forced to move through a microchannel constriction (Figure 2A) by increasing applied pressure (Figure 5A). Stiffness was determined as a pressure/deformation relationship (example in Figure 5B). It was found that cardiac spheroids significantly increased their stiffness from day 7 to day 14 of culture (Figure 5C). To discard spheroid compaction as cause of stiffen, stiffness increment was compared to spheroids size (Figure 5D). Spheroids suffered a slight shrinkage during the first 7 days of culture, while stiffness increment was predominant from day 7 to day 14 of culture, discarding compaction as a cause for stiffness increment. Moreover, the timeline of stiffness changes matched the timeline of cellular reorganization. These observations correlate with known consequences of post-ischemic fibrotic remodeling, where recruited fibroblasts increase deposition of collagen, generating a stiffen scar that may dramatically reduce myocardium pumping capacity.

Figure 5. Characterization of cardiac spheroid stiffness and size through time by constriction assay. A) Microscopic images of a spheroid penetrating the narrowest microchannel of the constriction microdevice as pressure increases. B) For lower pressures, the spheroid deformation (i.e., the penetration length inside the microchannel, normalized by spheroid initial diameter), and the applied pressure are linearly correlated. C) Spheroid stiffness calculated as pressure/deformation ratio from constriction assay. D) Initial spheroid radius measured with Fiji.

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Conclusion

In this work, Laura Paz-Artigas and collaborators, assessed the biomimetic recreation of human cardiac ischemia in a 3D in vitro model, based on self-induction of nutrient, pH, and oxygen gradients that lead to ischemia. Cardiac spheroids also recreated hints of ischemic damage pointing to the development of fibrosis, including tissue remodeling and stiffen. Data presented in this work entail the first steps to establish a complex biomimetic spatial recreation of human cardiac ischemia gradients. This microfluidic constriction assay allows stiffness characterization of spherical microparticles and has also been used for the mechanical characterization of alginate-based microcapsules for cell therapy encapsulation^4,5. Spheroids are easy to culture, do not rely on external matrix that may alter tissue intrinsic features, and are directly accessible for the measure of several parameters, from cellular secretions to beating and response to electric stimulus. Generation of ischemic spheroids that accurately recreate fibrotic remodeling and scar formation, along with proper monitoring systems, including stiffness characterization through constriction assay, are fundamental to better understand ischemia consequences in the tissue. Modeling and characterization of biomechanical features of an ischemic heart will help developing therapies focused not only on regenerating damaged cardiomyocytes, but also on recovering mechanical properties of a healthy myocardium.

Take home message:

Myocardial ischemia modeling: A 3D in vitro model was developed using large cardiac spheroids (40,000 cells) to replicate the biomechanical and biochemical processes of myocardial ischemia.
Self-induced ischemia: Spheroids recreate ischemia naturally, without external manipulation of oxygen or nutrient levels, mimicking in vivo conditions.
Fibrotic remodeling: The model replicates key stages of ischemic injury—cell death, fibroblast recruitment, and collagen deposition—leading to increased tissue stiffness.
Innovative mechanical characterization: A custom-made microfluidic constriction assay, using Elveflow’s OB1 MK4 flow controller, measures spheroid stiffness as a pressure/deformation relationship.
Stiffness progression over time: The timeline of stiffness changes correlates with cellular reorganization and fibrotic remodeling, providing insights into ischemic progression.

Authors Information

Laura Paz-Artigas holds a PhD in Biomedical Engineering and her recent research focus on the development of biomimetic models of the human heart, from spheroids to heart-on-chip models. She is currently a Substitute Professor at the Faculty of Medicine of the University of Zaragoza, Spain.

Ignacio Ochoa, PhD is professor at the Faculty of Medicine of the University of Zaragoza and Head of the Tissue Microenvironment Lab (TME LAB) of the Institute for Health Research Aragón (IIS ARAGON) and coordinator of the program of Technologies and innovation applied to the health of the same institute. His current research goals are focused on understanding the role of the microenvironment in the progression of several tumors and cardiovascular diseases as well as on the development of microfluidic devices for cell culture applications (Organ on Chips).

Jesús Ciriza, PhD is professor at the University of Zaragoza and PI in the Tissue MicroEnvironment Lab (TMELab) group at the Instituto de Investigación en Ingeniería de Aragón (I3A), Spain. He is currently working on the development of Heart-on- chip and Skin-on-chip models to understand, simulate, and preserve the environment of cells and tissues in their pathophysiological conditions to find new therapeutic strategies and personalized solutions for patients.

Written and reviewed by Laura Paz-Artigas and Louise Fournier, PhD in Chemistry and Biology Interface. For more content about microfluidics, you can have a look here.

References

Paz-Artigas L, González-Lana S, Polo N, et al. Generation of Self-Induced Myocardial Ischemia in Large-Sized Cardiac Spheroids without Alteration of Environmental Conditions Recreates Fibrotic Remodeling and Tissue Stiffening Revealed by Constriction Assays. ACS Biomater. Sci. Eng. 2024; 10(2): 987-997.
Richards DJ, Li Y, Kerr CM, et al. Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity. Nat. Biomed. Eng. 2020; 4(4): 446-462.
Kondo T, Takahashi M, Yamasaki G, et al. Immunohistochemical analysis of vimentin expression in myocardial tissue from autopsy cases of ischemic heart disease. Legal Medicine. 2022; 54: 102003.
Virumbrales-Muñoz M, Paz-Artigas L, Ciriza J, et al. Force Spectroscopy Imaging and Constriction Assays Reveal the Effects of Graphene Oxide on the Mechanical Properties of Alginate Microcapsules. ACS Biomater. Sci. Eng. 2020; 7(1): 242-253.
Paz-Artigas L, Ziani K, Alcaine C, et al. Benefits of cryopreservation as long-term storage method of encapsulated cardiosphere-derived cells for cardiac therapy: a biomechanical analysis. Int. J. Pharm. 2021; 607: 121014.