What is Cell Adhesion Assay?
The cell adhesion assay is designed to measure the ability of cells to attach to different surfaces or to each other. In a cell adhesion test, cells are cultured on a substrate coated with extracellular matrix proteins (such as fibronectin, collagen, or laminin). After a specified incubation period, unattached cells are washed off and the remaining cells are measured using crystal violet staining, microscopic counting, or optical density assays.
How is a cell adhesion assay performed?
Surface Preparation
Wells are coated with matrix proteins, such as fibronectin or collagen, to provide appropriate binding sites for cellular receptors. Usually, a solution is spread over the surface and incubated briefly to ensure proper adhesion.
Blocking
After coating, the surface is blocked with a mild protein solution like BSA to avoid non-specific binding. This ensures that differences between groups are due to actual adhesion and not random adsorption.
Cell Preparation
Cells are gently detached from the culture vessel and made into a uniform suspension to prevent clustering. Then, they are maintained in low-serum or serum-free medium to reduce effects of serum factors on adhesion.
Uniform Seeding
An equal number of cells is added to each well in order for any comparison between samples to be valid. Gentle mixing before seeding reduces heterogeneous sedimentation.
Short Incubation
The plate is incubated for a fixed time, usually short, to allow cells to attach themselves initially. Consistent temperature and CO₂ concentration, along with time, are some of the most important factors for reducing experimental variability.
Gentle Washing
Non-adherent cells are washed off by gentle washing after incubation. To avoid detachment of attached cells, uniform force should be applied and harsh flow must be avoided.
Measurement
The amount of adhered cells is measured using one of the simple methods, such as staining (reading optical density), fluorescence of live cells, or microscopic counting. Whichever method is selected will depend upon the available equipment and whether the measurement needs to be done with a live or fixed cell.
Controls
A non-coated well is used as a negative control, and a well with a strong coating or poly-D-lysine is used as a positive control. These controls make the interpretation of data easier and help to identify any procedural issues.
Quick Analysis
Background signals (blank) are subtracted, and the results are normalized against the positive control to obtain the adhesion percentage. This normalization makes comparisons between experiments more reliable.
Click For further information about Biocompatibility Testing
Factors Affecting Cell Adhesion
Biological Lock and Key
On the cell surface and surrounding areas, proteins act like locks and keys that bind together. When these pairs are compatible, adhesion occurs.
Calcium and Magnesium
The presence of calcium and magnesium ions assists in better “locking” of these molecules, while their deficiency weakens adhesion.
Substrate Quality
The type and coating of the surface under the cells (such as matrix proteins like fibronectin or collagen), its roughness/smoothness, and its hydrophilicity determine how easily cells adhere.
Health and Condition of the Cell
The health, growth stage, and activation or inactivation of intracellular pathways affect adhesion tendency. Cells in the proliferative stage generally adhere better.
Messengers and Surrounding Environment
Soluble factors like cytokines or growth factors, and basic conditions such as pH and temperature, can increase or decrease adhesion.
Force and Motion
Mild pressure or appropriate stretching can stabilize adhesion, but excessive force (such as harsh washing or rapid flow) can cause attached cells to detach.
Density of Adhesion Sites
The higher the number of attachment sites on the surface (e.g., thicker protein coating), the greater the likelihood and extent of adhesion.
Cell adhesion is the result of a combination of molecule pairing, ions, surface properties, cell condition, environmental conditions, and forces.
Importance of Cell Adhesion Testing
Cell adhesion testing is a key tool in cellular and molecular biology, designed to investigate the biological interactions of materials with cells. This test is especially important in evaluating the biocompatibility of medical materials, biomaterials, and engineered surfaces. As a fundamental process, cell adhesion plays a critical role in various cellular functions, including differentiation, proliferation, survival, and biochemical signaling. In fact, the ability of a material to facilitate cell adhesion is a key indicator of successful interaction between the material and the biological system.
The significance of this test lies in its ability to assess material behavior in a biological environment, especially in living tissues. For example, in the design of implants, materials that cannot establish proper adhesion with target cells (such as osteoblasts in bone implants) not only fail to integrate biologically but may also trigger adverse reactions like inflammation or fibrosis. Therefore, cell adhesion testing is crucial as an initial screening method for materials before in vivo testing.
At a more specialized level, this test enables the assessment of material surface effects, including chemical composition, surface energy, and topography on cell behavior. Materials with unsuitable surfaces may disrupt the formation of focal adhesions, which directly impacts cellular signaling and behavior. This assay also indirectly evaluates the potential of materials to facilitate more complex processes such as extracellular matrix formation and tissue integration.
Therefore, cell adhesion testing not only serves as a marker for biocompatibility but also acts as a valuable tool for optimizing the design of new materials with enhanced biological interactions. This is particularly important in tissue engineering and regenerative medicine, where advanced materials with specific properties are required. Ultimately, the results of this test can provide precise guidance for material selection in research and development stages as well as clinical approvals.
Click For more information about Cell Cytotoxicity Testing
Nikopharmed, as a specialized and reputable laboratory, conducts Cell Adhesion Testing in accordance with ISO 10993-5 standards. Utilizing advanced equipment, standardized culture environments, and a team of experienced cellular and molecular experts, Nikopharmed offers precise assessments of cell-material interactions with biomaterials and implants.