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Introduction

Cell adhesion is the process of cells interacting with and attaching to a surface, a substrate or another cell. Adhesion is a highly regulated process, comprising dynamic cell - cell and cell - extracellular matrix (ECM) interactions. For mammalian cells, it is mediated by specific, complex structures such as selectins, integrins, syndecans, and cadherins, which determine the specificity of such interactions. Moreover, adhesion plays a significant role in cellular signaling processes by receiving and integrating internal as well as external signals from the environment.  Consequently, cell adhesion is of major importance in questions regarding tissue development, inflammation, immunity, cancer progression, and others. [1]

Today, there are many different techniques, each of them answering different questions regarding cell adhesion: How fast do cells attach to a surface? Do they detach or change their morphology/adhesion area because of a substance or physical stress? How good is their attachment? 

CAN-Spectroscopy is a technique, which allows answering all previously mentioned questions. In the following, different experimental approaches including sample data and analysis are shown. 

Attachment & detachment kinetics

Attachment and detachment kinetics are used to determine, how fast cells attach or detach. Toxic substances, ECMs, physical stress or cell viability influence the delicate process of cell adhesion. The CAN-Q Chip can be coated with various ECMs. Substances, e.g. toxins, in the cell culture media don’t interfere with CAN-Spectroscopy measurements. CAN-Spectroscopy is a powerful technique to study cell adhesion and morphological changes. 

Experiment

An attachment assay is an easy technical realization to quantify cell adhesion: Cell in are suspension, the suspension is pipetted onto the CAN-Q Chip during a measurement. The cells sediment to the surface of the CAN-Q Chip, where they first form an initial attachment and then start to spread out and reach for neighboring cells. The process of attachment is quantified on a phenotypic morphological level. If the adhesion process is perturbed, this becomes visible during an attachment experiment. 

Analysis

For analysis, the following parameters can be obtained from the CAN-Q Analyzer:

  • The number of cells attached on the CAN-Q Chip over time and the CAN-Q Chips coverage rate
  • The adhesion areas of all cells as distribution or as adhesion area over time for a single cell
  • The morphology of the adhesion areas (solidity, circularity, eccentricity and others). 

As example data, the previously described cell attachment assay was performed using human fibroblasts (cell line BJJ) on a fibronectin coated CAN-Q Chip. The cells were added after five minutes. The following video shows the raw data from the CAN-Q System. 

As example data, the previously described cell attachment assay was performed using human fibroblasts cell line BJJ on a fibronectin coated CAN-Q Chip. Cells were added after five minutes. The following video shows raw data from the CAN-Q System.

 

For analysis, first a cell detection algorithm was used to detect and track all cells attached on the CAN-Q Chip over time. The developed algorithm can separate cells even in a cluster. 

One a morphological level, several information can be obtained: As a first and simple quantification of cell adhesion kinetics, the adhesion area of every cell attached to the CAN-Q Chip is analyzed. In the following graphic the size distributions for different times from the above measurement are shown. Over time the average size of the adhesion area shifts from ~500 µm² to ~700 µm². 

Get this dataset

We offer the above shown data and the CAN-Q Analyzer as download. If you want to discovery the possibilities of CAN-Spectroscopy at your computer, please write an eMail to sales(at)venneos.com. 

Adhesion potential

But can spectroscopy is more than just an imaging technique. An analysis of the measured power spectral densities reveals additional information about the cell-chip contact.

One parameter, which is of highest interest to quantify cell adhesion potential, is the parameter r_J. In the physical model, r_J describes the horizontal electrical resistance of the cell culture media between the lower cell membrane and chip surface. If the cell membrane comes closer to the surface of the chip, r_J  is increased – it is basically like a cable: the thinner, the higher the resistance or in this case - the better the attachment. So r_J can be used as a measure for adhesion potential. 

 

Experiment

As a proof of principle, human fibroblasts were seeded on CAN-Q Chips with different ECM coating (Collagene I, Poly-L-Lysin, Fibronectin and Laminin). After 24h, CAN-Spectroscopy measurements were performed to obtain r_J. 

Analysis

For analysis, again a cell detection algorithm was first used to identify single cells on the chip. For every detected cell, the adhesion parameter was calculated in the whole adhesion area. For interpretation, the largest value found per adhesion area was used to calculate a distribution over all cells attached.  

 

BJJ Fibroblasts showed the same adhesion potential on fibronectin and on collagen I coated chips. For Poly-L-Lysin the adhesion was slightly increased, for laminin it was significantly increased.

Of course, the parameter r_J can be correlated with all other phenotypic information on a single cell level, providing more insights. Additionally, statistics on the r_J under a single cell can be applied, e.g. how homogenous is r_J in the adhesion area of the cell?