A centrifugation cell adhesion assay for high‐throughput screening of biomaterial surfaces
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Abstract A quantitative analysis of cell adhesion is essential in understanding physiological phenomena and designing biomaterials, implant surfaces, and tissue‐engineering scaffolds. The most common cell adhesion assays used to evaluate biomaterial surfaces lack sensitivity and reproducibility and/or require specialized equipment and skill‐intensive operation. We describe a modified centrifugation cell adhesion assay that uses simple and convenient techniques with standard laboratory equipment and provides reliable, quantitative measurements of cell adhesion. This centrifugation assay applies controlled and uniform detachment forces to a large population of adherent cells, providing robust statistics for quantifying cell adhesion. The applicability of this system to the design and characterization of biomaterial surfaces is shown by evaluating cell adhesion on substrates using different coating proteins, cell types, seeding times, and relative centrifugal forces (RCF). Results verify that this centrifugation cell adhesion assay represents a simple, convenient, and standard method for high‐throughput characterization of a variety of biomaterial surfaces and conditions. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 328–333, 2003Keywords:
Biomaterial
Characterization
Biomaterial
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Coronary heart diseases result from the blockage of one of the coronary arteries, which nourishes the heart muscle. This process leads to ischaemia of a segment of the heart and death of the contractile tissue. As cardiac tissue is unable to regenerate itself, heart function is impaired. Tissue engineering (TE) is a field of science that integrates knowledge from biology, materials sciences, engineering and medicine to develop artificial, functional tissue constructs to replace defected tissues. In cardiac TE, contracting cells are seeded within supporting biomaterial scaffolds that provide them with the essential microenvironment for functional tissue assembly. Various strategies and methods for fabricating these scaffolds have been proposed and tested in the last decade, some of which combine multiple elements that altogether contribute to the formation of an improved functional tissue. This review summarises the unique properties of various composite biomaterial scaffolds and highlights their advantages over other pristine scaffolds for engineering functional three-dimensional cardiac patches.
Biomaterial
Cardiac muscle
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Protein Adsorption
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In order to study the biophysical forces involved in cell-substrate (or cell-cell) adhesion, it is necessary to measure the strength of adhesion. Two questions can be addressed using the centrifugal cell adhesion assay provided in this unit: what is the ligand-receptor affinity for cells adhering at 4 degrees C and what is the strength of the ATP-dependent processes that strengthen adhesion at 37 degrees C. In both cases, the strength of adhesion is measured as the resistance to disruption in the presence of a measured centrifugal force.
Centrifugal force
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ABSTRACT Adhesion of BHK cells to a variety of polymer surfaces carrying measured densities of hydroxyl and carboxyl groups was studied. The effects on cell adhesion of blocking hydroxyl groups by acetylation and carboxyl groups with diazomethane were measured. Hydroxyl groups were required for cell adhesion, though the very high surface densities of these groups diminished cell adhesion. The optimal surface density of OH groups for BHK adhesion was 2000 per 1× 10−11 cm2. Carboxyl groups slightly inhibit cell adhesion, since blocking of these groups by methylation increased adhesion. The role of oxidizing systems of cellular origin in conditioning of the substrate, in serum-free conditions, was demonstrated for leucocytes and BHK cells, in particular by the result that oxidizable substrates such as phytane and poly(l,2-butadiene) could be made suitable for cell adhesion by contact with cells.
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Objective:The adhesion of cells to cells and cells to stroma play very important role in the cell-cell information exchanges.Methods:The cell adhesion assay based on the 3 H-TdR incorporation assay was introduced. Firstly, coated cells were cultured to confluence in 96 well plate. After incubated with 3 H-TdR for 6h, the isotope labeled cells were added into plate wells and incubated for another 4 h. Then the un-adhered cells were removed by gently washing. The cpm was counted after cell harvest.Results:The cell adhesion assay based on the 3 H-TdR incorporation assay was very sensitive in the detection of cell adhesion. It could reflect the relative of cell to cell adhesion.Conclusion:The cell adhesion assay based on the 3 H-TdR incorporation assay is an useful method in the detection of cell to cell adhesion.
Cell counting
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The use of force spectroscopy to study the adhesion of living fibroblasts to their culture substrate was investigated. Both primary fibroblasts (PEMF) and a continuous cell line (3T3) were studied on quartz surfaces. Using a fibronectin-coated AFM cantilever, it was possible to detach a large proportion of the 3T3 cells from the quartz surfaces. Their adhesion to the quartz surface and the effects of topography on this adhesion could be quantified. Three parameters characteristic of the adhesion were measured: the maximum force of detachment, the work of adhesion, and the distance of detachment. Few PEMF cells were detached under the same experimental conditions. The potential and limitations of this method in measuring cell/surface interactions for adherent cells are discussed.
Force Spectroscopy
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