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Biomechanics

Biomechanics - Poster Session E

Poster Q7 - The Role of Compression Speed in Cell Viscoelasticity: A Study on Relaxation Dynamics

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cell mechanics is a fundamental aspect of cellular biology that provides significant insights into the state and functionality of cells. Understanding how cells respond to mechanical stimuli is crucial for various applications in biomedical research and clinical diagnostics. The mechanical properties of cells influence their behavior and interactions within biological systems. Studying these properties helps understand diseases, develop diagnostic tools, and create targeted therapies. Mechanical responses can indicate changes in cell health, differentiation status, and metastatic potential in cancer cells.
Hypothesis: A proper model can bridle cell behavior from AFM measurements to microfluidic cases, which exhibit higher deformation rates than AFM.
Expected

Results: We anticipate that the cell relaxation curve will steepen significantly at higher deformation rates. Currently unexplained by existing physical models, this phenomenon will be investigated at moderate, high, and ultra-high strain/strain rates. Using a phenomenological approach, we will compare existing viscoelastic and/or poroelastic models and combine them to construct a comprehensive model that captures a broader spectrum of cell responses.
By investigating these responses, we can develop models that help design more efficient microfluidic systems incorporating higher order of:
Throughput: rate of processing large populations of cells.
Cell Distinctiveness: mechanically distinguishing between different cell types.
Intercellular Delivery: force injecting therapeutic and diagnostic substances into cells.

Materials and

Methods:  Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors were cryopreserved and, after thawing, T-cells were isolated using the EasySep Human T-Cell Isolation Kit. The T-cells were activated with Dynabeads Human T-Activator CD3/CD28 at a 1:1 ratio and cultured in X-VIVO 15 medium supplemented with 10% Fetal Bovine Serum, antibiotics, GlutaMAX, and IL-2. After 24 hours of activation, the Dynabeads were removed. The T-cells, maintained at a density of 1.5 × 10^6 cells/mL, were incubated at 37°C with 5% CO2.

Using force spectroscopy, we derived force-indentation curves with an MFP-3D atomic force microscope integrated with a Nikon optical microscope on a vibration isolation table to analyze cell viscoelastic properties at varying strain rates. Cells were affixed to glass FluoroDishes with CellTak adhesive. We used a cantilever with 3.6 μm spherical bead attached to the tip. Calibration involved acquiring a single force curve on a clean FluoroDish in serum-free RPMI-1640, using the Sader method to calculate cantilever spring constants (the order of magnitude is around 10 pN/nm).

Five measurements per cell were taken at 1-20 μm/s until reaching a force of 5 nN, with a 60-second wait between measurements. The cantilever’s z position was held for 10 seconds to allow for viscous relaxation before retraction. Data were processed using our Python-based web app (https://github.com/HoseynAAmiri/pyrtz2), fitting the force-distance curve to a pointwise Hertzian contact model and a bi-exponential decay function to estimate stiffness modulus and viscous time constants (Figure 1). Measurements led to approximately 30% strain and 3 μm indentation on average.

Results, Conclusions, and Discussions: Figure 2 depicts the results of different strain rates experiments. In the first part of the experiment, the load was applied at an ascending rate from cell 1 to cell 11. Compared to the last part of the experiment, in which we used a descending rate on a cell, the morphology change was observed more severely and frequently. There was no consistent pattern for bleb formation. Moreover, we can see heterogeneity and inconsistencies with stiffness readout, which may be due to one or all of the following reasons:
• the small size of the probe compared to cell sizes,
• microscopic movement of cells that were observed to happen naturally (Figure 3),
• and various activations and attachment states.
Therefore, the stiffness readout might be altered due to local changes in contact mechanics. However, relaxation time constants tend to change with the strain rate more consistently due to viscous dissipation, which should be insensitive to local deformations similar to a capsule.

Figures 4 shows that ratio of the fast time constant to the indentation time (t_ind) is correlated with stiffness, indicating an SLS-like (Standard Linear Solid) viscoelastic response. However, SLS cannot justify the change in the relaxation time with the rate. No significant correlation was found between any readouts and the cell size.

The figures show that the overall fast time constant (τ_1) has a strong correlation with the strain rate, revealing the rate-dependent behavior of cells. It can be seen that at higher rates, the relaxation drop becomes steeper. We postulate that the phenomenon can be explained by fractional viscoelastic models. The analytical solution requires a multi-objective optimization for higher accuracy. The reason is that the initial time of the dwell and the fitness of the curve are both essential factors in capturing the relaxation response. Each optimization takes around 5 minutes for each curve and 50 generations of 20 populations. While the complete data fitting is in progress, we capture two data points with all fits in Figure 5.

Acknowledgements (Optional):  Detailed sources of funding will be on the poster.