(K-433) Exploring Dynamic Changes in Nuclear Alignment during Fibroblast Tissue Formation and after Fixation

Introduction::

Wound healing occurs in response to tissue injury, helping to repair and heal damaged tissues or organs. However, excessive or prolonged cellular response can lead to the formation of scar or fibrotic tissue, impairing organ function and contributing to various chronic diseases, making its regulation crucial for maintaining tissue homeostasis and overall health. 

In previous studies, fibroblast tissue constructs responded to tensional anisotropy (Alisafi et al. BioRxiv 2022). Boundary conditions that trigger tensional anisotropy induce fibroblast cell alignment and activation to a myofibroblast state. However, although boundary conditions designed to induce little to no anisotropy resulted in less cellular alignment, nuclear elongation and myofibroblast markers, we still observed partial cellular alignment within the parts of the tissue that models predicted would have isotropic tension. Within these areas, we still observed that cells had a preferential axis of alignment and some activation toward a myofibroblast state. We hypothesize that a symmetry breaking event occurred during compaction of the engineered fibroblast tissues, even with boundary conditions designed to induce isotropic stress fields. We hypothesize that this symmetry breaking event can be observed during compaction of the tissue. To test these hypotheses, we are developing methods for live imaging of nuclei and cell bodies in a small subset of cells that make up the compaction tissue.

Materials and Methods::

MicroTUG fabrication involved creating posts made of a PDMS (polydimethylsiloxane) mixture that was first 3D printed in its final form with FormLabs Clear Resin. The devices were then created by Hydrogel Assisted Stereolithographic Elastomer Prototyping (HASTE; Simmons et al. BioRxiv, 2022) with PDMS 527 and PDMS 184 in a weight ratio of 4:1. To secure stability and prevent contamination, these PDMS devices were firmly attached to a PDMS well-insert using PDMS 184 as an adhesive. The 4-post design represents an anisotropic model, while the 8-post design represents an isotropic model.

NIH-3T3 fibroblast cells were cultured in DMEM with 10% fetal bovine serum (FBS). At time of seeding, cells were mixed with collagen (1.5mg/ml)  and seeded onto the PDMS devices at a cell density dependent on the number of viable cells. Additionally, a fraction of cells were dyed with NucSpot Live 488 before seeding.

Live imaging of cellular and nuclear behavior was performed using a Keyence BZ-9000 E microscope every 16 hours during tissue formation and compaction. High-resolution images were captured at magnifications of 10x and 20x at predetermined intervals, enabling monitoring of dynamic changes in nuclear deformation as the tissue compacted over time.

Following tissue compaction, the samples were fixed using 4% paraformaldehyde to preserve cellular and nuclear structures. These fixed samples were imaged using a Keyence BZ-9000 E microscope to provide additional data for the analysis of nuclear deformation during fixation. The imaging settings remained consistent with the parameters used during live imaging.

Results, Conclusions, and Discussions::

To enable an additional method for visualizing cell alignment during tissue compaction, 1µM Octadecyl Rhodamine B Chloride (R18) was introduced to the culture media. The resultant alteration in cell staining procedures is predicted to yield a clear image and improved visualization of the live-cell tissue through a different channel. Attempts were made to seed tissues at a lower cell density, specifically at 5-10 million cells/ml. However, the obtained results revealed that optimal tissue compaction consistently occurred at a higher density, ranging from 15-20 million cells/ml. Tissues maintained a consistent shape after full compaction, with < 5% of tissues exhibiting  instances of tearing. 

During the imaging process while the tissue was undergoing compaction and fixation, discernible indications of nuclear shape changes were observed. These findings suggest the presence of preferential alignment in specific axes, highlighting potential patterns of cellular organization during tissue compaction. However, to precisely ascertain the specific time points at which such preferred alignment and symmetry breaking occur, further experimentation will be essential. Conducting experiments at different time points during tissue compaction and development will be necessary to capture the dynamic process and provide a comprehensive understanding of these events.

While these studies are considered preliminary, they are able to show us how stress anisotropy affects fibrosis. These studies can eventually advance our comprehension of the crucial role of cardiac fibrosis in the wound healing process. Specifically, these studies shed light on the implications of excessive fibroblast proliferation and activation, which can contribute to pathological remodeling and ultimately lead to the onset of arrhythmias. This manifests as an early and clinically relevant phenotype in patients afflicted with hypertrophic cardiomyopathy. As we delve further into these investigations, a deeper understanding of cardiac fibrosis and its impact on cardiac health is anticipated, potentially paving the way for improved therapeutic interventions and management strategies in the future.

Acknowledgements (Optional): : Cardiovascular Research Summer Program (CardS), American Heart Association (AHA), Ghiska Ramahdita, and Dr. Nathaniel Huebsch for their incredible support and guidance throughout this program!

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