Accessible Rheo-Optical Compression Assay for High-Throughput Mechanical Characterization of Cell Spheroids

Significance 

Cell spheroids are crucial three-dimensional models resembling tissue structures. They closely mimic the physiological conditions found in tissues and tumors in vivo, making them indispensable in cancer research, tissue engineering, and regenerative medicine. Spheroids exhibit unique mechanical properties due to their heterogeneous structure with an outer layer of proliferating cells, a middle region of quiescent cells, and an inner necrotic core. Understanding these mechanical properties – such as elasticity, viscoelasticity, and responses to mechanical stress – provides valuable insights into cellular interactions, structural integrity, and the influence of the microenvironment on cell behavior.

Conventional methods to study these properties, like atomic force microscopy, micro-indentation, optical tweezers, and cavitation rheology, often require specialized high-cost equipment and technical expertise which are not accessible to many researchers. To this end, a recent study by Dr. Rosalia Ferraro, Professor Sergio Caserta and Professor Stefano Guido from the University of Naples Federico II in Italy, published in Advanced Materials Technologies, introduces an innovative Rheo-optical compression assay. This method leverages standard laboratory equipment commonly found in cell culture facilities. It involves applying a load to cell spheroids using microscope coverslips and capturing the resulting deformation with an optical microscope or a smartphone equipped with magnification lenses. By analyzing images to correlate applied loads with observed deformations, researchers can accurately determine key mechanical properties such as the Young modulus.

Initially, researchers validated their Rheo-optical compression assay using spherical agarose gel particles to demonstrate its feasibility before applying it to cell spheroids; the choice of agarose is related to its status as a biomimetic gel commonly utilized in literature, as it effectively mimics the mechanical properties of tissues. Gel particles were prepared by immersing droplets of hot agarose solution into silicone oil. These particles were then transferred to cell culture plates filled with silicone oil for compression tests. Using microscope coverslips as compressive loads, researchers applied sequential loads to the agarose particles and observed their deformation under a light microscope. The stress-strain outcomes obtained from these experiments at the steady-state regime showed a linear relationship, allowing the calculation of the Young modulus. Comparing these results with values obtained using conventional rheometry, researchers found the Young modulus determined by their method to be consistent, validating the accuracy and reliability of the assay.

Afterward, the researchers applied the Rheo-optical compression assay to cell spheroids derived from PANC-1 (tumor) and NIH/3T3 (non-tumor, referred as control) cell lines. Spheroids were formed using the liquid overlay technique, which involves seeding cells in wells pre-coated with non-adhesive agarose. This setup promoted cell aggregation and spheroid formation over 5-10 days, depending on the cell type. The formed spheroids were then transferred to multiwell plates filled with culture medium. Compression was applied using microscope coverslips positioned within a 3D printed frame in order to avoid asymmetric load, and the morphological response of the spheroids to varying applied stresses was observed using an inverted microscope.

Spheroids deformed progressively with increasing loads, exhibiting distinct mechanical behaviors between the two cell lines. Specifically, PANC-1 tumor spheroids showed greater deformation compared to NIH/3T3 spheroids under the same applied stress. The researchers observed that the stress-strain data followed a linear trend up to a certain strain, beyond which the response became nonlinear. By applying Hooke’s law to the linear portion of the data, the researchers calculated the Young modulus for both cell lines and found that the PANC-1 spheroids had a significantly lower Young modulus compared to NIH/3T3 spheroids, indicating that tumor cells are softer than their non-tumor counterparts. This difference measured using the new assay can be used to distinguish between cell types based on their mechanical properties.

Furthermore, the authors employed a phenomenological model developed for foams to understand the mechanical behavior of cell spheroids beyond the linear elastic regime. The model provided a good fit for data from both NIH/3T3 and PANC-1 spheroids, suggesting that the mechanical behavior of cell spheroids can be described using principles similar to those proposed to describe foams viscoelastic response.

In addition to steady-state measurements, the researchers investigated the transient deformation of cell spheroids following the application of a load. They observed time-dependent deformation behaviors, which provided valuable insights into the viscosity of the spheroids, encompassing not only elastic but also viscous characteristics. In a more recent publication, the transient deformation was also analyzed using a model-free approach. This tool enabled the derivation of viscoelastic properties directly from the time-dependent stress and strain curves of the samples.

In conclusion, Dr. Rosalia Ferraro, Professor Sergio Caserta, and Professor Stefano Guido have successfully developed the Rheo-optical compression assay, providing a straightforward and cost-effective alternative that is readily deployable in standard cell biology laboratories. This democratization of technology expands the horizons of high-throughput mechanobiology research. Specifically, by measuring the mechanical properties of tumor spheroids, this assay enables deeper insights into the mechanical aspects of cancer progression and metastasis. It also facilitates the identification of mechanical biomarkers crucial for cancer diagnosis, prognosis, and the development of new therapeutics, with potential implementation also on clinical studies.

Accessible Rheo-Optical Compression Assay for High-Throughput Mechanical Characterization of Cell Spheroids - Medicine Innovates

References 

R. Ferraro, S. Caserta, S. Guido, A Low-Cost, User-Friendly Rheo-Optical Compression Assay to Measure Mechanical Properties of Cell Spheroids in Standard Cell Culture Plates. Adv. Mater. Technol. 2024, 9, 2301890. https://doi.org/10.1002/admt.202301890

Go To Adv. Mater. Technol.

R. Ferraro, S. Guido, S. Caserta, M. Tassieri, i-Rheo-optical assay: Measuring the viscoelastic properties of multicellular spheroids. Materials Today Bio 2024, 26, 101066. https://doi.org/10.1016/j.mtbio.2024.101066

Go To Materials Today Bio