Cell adhesion experiments are fundamental in various scientific disciplines, including tissue engineering and medical device development. The nascent adhesion, representing the initial state of cell attachment, is a critical aspect that is currently under intense research focus.
Schematic illustration of the measurement with single-cell resolution optical sensor. The cells were added to 12 wells in the microplate and measured (A). The sensor can detect refractive index change above the surface in an approximately 150 nm thick layer, which corresponds to the evanescent field [(B), ‘side view’ part, red zone]. The integrin-ligand binding happens in this layer, thus the sensor can monitor cell adhesion and provide kinetic data of the process (C). Image Credit: https://www.nature.com/articles/s41598-024-61485-6
Proper handling of adherent cell types involves the dissociation of cells from their substrates for further analysis and experimentation. In a recent article published in the journal Scientific Reports, researchers from Hungary investigated the intricate relationship between cell dissociation methods and cell adhesivity, emphasizing the early and nascent phases of the adhesion process.
Background
Understanding the mechanisms behind cell adhesion is crucial for decoding fundamental cellular behaviors and developing treatments for diseases linked to adhesion. In cell culture experiments, cell dissociation methods are employed to separate adherent cells from their substrates for subsequent analysis. However, the dissociation technique chosen can affect several aspects of cell behavior, such as adhesion properties, viability, and signaling pathways.
Furthermore, the impact of enzymatic versus non-enzymatic dissociation agents on the dynamics of cell adhesion is not well understood. Therefore, studying the effects of various dissociation methods on cell adhesion is vital for the accurate interpretation of experimental outcomes and for advancing our knowledge of cellular processes.
The Current Study
In this study, HeLa cells were cultured within a controlled environment in a humidified incubator. The researchers then utilized three distinct cell dissociation methods: enzymatic dissociation with a trypsin-EDTA (ethylenediaminetetraacetic acid) mix, non-enzymatic dissociation solely using EDTA, and a proprietary dissociation buffer. The experiments were conducted on three different types of surfaces: an uncoated biocompatible metal-oxide, a fibronectin-coated surface, and a surface coated with the tripeptide Arg-Gly-Asp (RGD).
Preparation of the non-coated surface involved cleaning the metal-oxide substrate with ethanol, followed by sterilization under UV light. The fibronectin-coated surface was prepared by immersing the substrate in a fibronectin solution dissolved in phosphate-buffered saline (PBS). For the RGD-coated surface, RGD peptides were attached using standard coupling chemistry protocols.
Cell adhesion was quantitatively assessed using a label-free optical sensor designed for high-resolution monitoring. This sensor directed a laser beam at the cells and recorded changes in the reflected light spectrum to measure cell adhesion. Optical measurements were performed continuously in real-time, enabling uninterrupted monitoring of adhesion events without disrupting the cells' physiological state.
The analysis of adhesion signal distribution among HeLa cells across different surfaces and dissociation methods was conducted using statistical techniques. Data was then modeled using lognormal distributions. The significance of differences in adhesion signals across various conditions was assessed using the non-parametric Kruskal–Wallis H-test, complemented by a Wilcoxon signed-rank test for paired comparisons. The significance of the findings was determined based on the p-values obtained from these analyses.
Results and Discussion
The experimental findings revealed substantial variations in the adhesion signals of HeLa cells across different surfaces and dissociation methods, with distinct patterns emerging based on the surface characteristics and dissociation protocols used. Notably, the choice of dissociation method exerted a significant impact on cell adhesion, particularly during the early stages of the adhesion process.
The study accentuated the crucial role of adhesion complexes in the initial attachment of cells to the extracellular matrix, emphasizing their transient nature and significant influence on cell adhesivity. These complexes, particularly submicron-sized dot-like structures, were pivotal in facilitating cell attachment during the early phases of adhesion. This underscores the need to consider the dynamic nature of adhesion complexes and their sensitivity to different cell dissociation methods.
Moreover, the differences in adhesion signals measured on various surfaces were found to be highly dependent on the cell harvesting technique employed. The variations observed in adhesion signals highlight the complexity of cell-substrate interactions and the sensitivity of adhesion processes to experimental conditions. This emphasizes the importance of careful interpretation of experimental data, especially when comparing adhesivity across different surfaces and dissociation methods.
The analysis of adhesion signal distributions on various surfaces and with different dissociation methods offered valuable insights into the heterogeneity of cell adhesion behavior. The observed differences in adhesion signals illuminated the diverse responses of HeLa cells to different experimental conditions, underlining the importance of considering the biological relevance of the surfaces and dissociation protocols in adhesion studies. This information is crucial for accurately interpreting experimental results and advancing our understanding of cellular behavior.
Conclusion
In conclusion, the study demonstrated the intricate interplay between cell dissociation methods, surface characteristics, and cell adhesivity, shedding light on the complexities of cell-substrate interactions. The results underscored the significance of selecting appropriate dissociation protocols in experimental studies to ensure accurate and meaningful interpretation of adhesion data.
By leveraging high-resolution optical sensors and advanced analytical techniques, the research provided valuable insights into the early stages of cell adhesion and the factors influencing adhesion dynamics. The study's findings have important implications for various fields, including cancer research and tissue engineering, where understanding cell adhesion processes is crucial.
Journal Reference
Kovács, K.D., Szittner, Z., Magyaródi, B. et al. (2024). Optical sensor reveals the hidden influence of cell dissociation on adhesion measurements. Scientific Reports 14, 11719. https://doi.org/10.1038/s41598-024-61485-6, https://www.nature.com/articles/s41598-024-61485-6
Article Revisions
- May 30 2024 - Title changed from "Optical Sensor Uncovers Cell Dissociation and Adhesion Dynamics " to "Sensing the Impact of Dissociation on Early Cell Adhesion"