Light-driven high-precision cell adhesion kinetics

Scientific reports, 2024

Cell adhesion experiments are important in tissue engineering and for testing new biologically active surfaces, prostheses, and medical devices. Additionally, the initial state of adhesion (referred to as nascent adhesion) plays a key role and is currently being intensively researched. A critical step in handling all adherent cell types is their dissociation from their substrates for further processing. Various cell dissociation methods and reagents are used in most tissue culture laboratories (here, cell dissociation from the culture surface, cell harvesting, and cell detachment are used interchangeably). Typically, the dissociated cells are re-adhered for specific measurements or applications. However, the impact of the choice of dissociation method on cell adhesion in subsequent measurements, especially when comparing the adhesivity of various surfaces, is not well clarified. In this study, we demonstrate that the application of a label-free optical sensor can precisely quantify the effect of cell dissociation methods on cell adhesivity, both at the single-cell and population levels. The optical measurements allow for high-resolution monitoring of cellular adhesion without interfering with the physiological state of the cells. We found that the choice of reagent significantly alters cell adhesion on various surfaces. Our results clearly demonstrate that biological conclusions about cellular adhesion when comparing various surfaces are highly dependent on the employed dissociation method. Neglecting the choice of cellular dissociation can lead to misleading conclusions when evaluating cell adhesion data from various sources and comparing the adhesivity of two different surfaces (i.e., determining which surface is more or less adhesive). The significance of studying cell adhesion has become essential in various fields, including cell biology, materials science, and surface chemistry. The study of cell adhesion can provide insights into fundamental principles of cell biology, such as the role of cell-surface interactions in cell differentiation and tissue development. By understanding how cells adhere to different materials, it becomes possible to develop new biologically active surfaces and medical devices 1,2 that can interact with cells in specific ways. These advancements are applied in the design of novel materials with both cell-repellent and cell-adhesive properties. Additionally, a thorough characterization of cancer cell adhesion can contribute to our understanding of factors that contribute to tumor progression and metastasis. A large number of cells are adherent and attach to the extracellular matrix components (ECM), which is a complex network of proteins and other molecules that surrounds cells and provides structural support, via integrin receptors. Integrins are expressed as heterodimers, their composition determines their ligand specificity 3,4. The tripeptide Arg-Gly-Asp (RGD) was identified as the minimal pattern required to trigger cell adhesion 5-7. Integrins form different types of adhesion complexes which are linked to the intracellular actin-cytoskeleton system through various adaptor molecules and thus enable mechanotransduction 8. Mechanotransduction plays a crucial role in regulating cellular functions, differentiation, and proliferation 9-11. In addition to integrins, cells can also use other adhesion molecules, such as cadherins or selectins, to adhere to other cells or the ECM. These molecules are involved in a range of biological processes, such as tissue development, immune response, and wound healing. Cell dissociation methods and reagents are widely used in tissue culture laboratories for handling adherent cell types, but their impact on subsequent measurements or tissue engineering is poorly understood. Cell dissociation

Journal of Receptors and Signal Transduction, 2009

Advances in Colloid and Interface Science

Cell-cell and cell-matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen-host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion.

Biotechnology Progress, 1994

A novel optical method was used for quantitative characterization and continuous measurement of both the adhesion and spreading of mammalian cells on inorganic surfaces. I t is based upon the effective refractive index change caused by cells when they adhere to a planar optical waveguide. We have applied this technique to measure the kinetics of the adhesion and spreading processes of baby hamster kidney (BHK) cells adhering to surfaces coated with fibronectin and under different culture conditions (PBS, medium, serum, EDTA). In comparison, hybridoma cells are only adsorbed to the surface and do not spread at all. Moreover, this technique also allows the mass of a n adsorbed protein adlayer to be determined very precisely and thus provides a valuable tool for screening suitable substrata as well as determining the influence of different culture conditions on cell adhesion and spreading. This sensitive test for substrate influence could be important in toxicity tests using adherent animal cells.

Biointerphases, 2016

Scientific reports, 2024

Selecting and isolating various cell types is a critical procedure in many applications, including immune therapy, regenerative medicine, and cancer research. Usually, these selection processes involve some labeling or another invasive step potentially affecting cellular functionality or damaging the cell. In the current proof of principle study, we first introduce an optical biosensor-based method capable of classification between healthy and numerous cancerous cell types in a label-free setup. We present high classification accuracy based on the monitored single-cell adhesion kinetic signals. We developed a high-throughput data processing pipeline to build a benchmark database of ~ 4500 single-cell adhesion measurements of a normal preosteoblast (MC3T3-E1) and various cancer (HeLa, LCLC-103H, MDA-MB-231, MCF-7) cell types. Several datasets were used with different cell-type selections to test the performance of deep learning-based classification models, reaching above 70-80% depending on the classification task. Beyond testing these models, we aimed to draw interpretable biological insights from their results; thus, we applied a deep neural network visualization method (grad-CAM) to reveal the basis on which these complex models made their decisions. Our proof-ofconcept work demonstrated the success of a deep neural network using merely label-free adhesion kinetic data to classify single mammalian cells into different cell types. We propose our method for label-free single-cell profiling and in vitro cancer research involving adhesion. The employed labelfree measurement is noninvasive and does not affect cellular functionality. Therefore, it could also be adapted for applications where the selected cells need further processing, such as immune therapy and regenerative medicine. During evolution, eukaryotes developed spatially separated organelles for performing various functions, and just like that, multicellular organisms arranged special cell types into tissues and organs in a highly organized manner. To form these complex structures, cells need to connect to each other and do so in a way that fulfills the needs of different tissue types 1. The various cellular connections are realized by four main cell adhesion molecules: the family of integrins, cadherins, immunoglobulins, and selectins. The predominant role in cell-matrix adhesion belongs to integrins. This heterodimeric transmembrane receptor anchors the cell to the extracellular matrix (ECM) by binding it to the actin cytoskeleton at the intracellular terminal. Integrins are not only capable of binding to the ECM, but exhibit signaling properties 2 as well. Each integrin comprises an alpha and beta subunit and shows a significant variance in their composition. There have been 18 alpha and 8 beta subunit variants described so far in mammals 3. This combinatoric diversity is a common phenomenon in molecular biology, resulting in the capability to realize innumerable functions with a limited number of molecular modules. It potentiates the many roles of integrins in seemingly distant biological processes, such as embryonic development, inflammatory responses, or

International journal of molecular sciences, 2015

Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artif...

Journal of cell communication and signaling, 2009

We have adapted the CyQuant(R) assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant(R) assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.

Scientific Reports, 2020

Single-cell adhesion force plays a crucial role in biological sciences, however its in-depth investigation is hindered by the extremely low throughput and the lack of temporal resolution of present techniques. While atomic force microcopy (AFM) based methods are capable of directly measuring the detachment force values between individual cells and a substrate, their throughput is limited to few cells per day, and cannot provide the kinetic evaluation of the adhesion force over the timescale of several hours. In this study a high spatial and temporal resolution resonant waveguide grating based label-free optical biosensor was combined with robotic fluidic force microscopy to monitor the adhesion of living cancer cells. In contrast to traditional fluidic force microscopy methods with a manipulation range in the order of 300–400 micrometers, the robotic device employed here can address single cells over mm-cm scale areas. This feature significantly increased measurement throughput, and...

Analytical and Bioanalytical Chemistry, 2021

The understanding of the initial cell adhesion to biomaterials is crucial for the survival of implants. The manifold possibilities to tailor an implant surface and the diverse requirements for different implant applications necessitate a timesaving and highly parallelized analytical methodology. Due to its intrinsic advantages (label-free, time-resolved, robust against temperature fluctuations, and particularly the multiplexing possibilities), single colour reflectometry (SCORE) is used for the first time to investigate cell adhesion to different extracellular matrix protein–coated surfaces. The excellent correlation between the novel SCORE technology and well-established reference methods proves that the results obtained by using this direct optical method are able to reflect the cell binding processes at the transducer surface. Additionally, the high time resolution of SCORE revealed the differences in the adhesion behaviour of the cells on the different extracellular matrix prote...