Video microscopy and digital image processing applied to tissue engineering: Intracellular ion and cell adhesion measurements

[摘要] To provide a fundamental understanding of the structure/function relationship of cells and tissues, engineers have had to bridge the two worlds of engineering and of molecular and cell biology. Through this multidisciplinary environment, a new field of engineering has evolved, namely tissue engineering. Tissue engineering was applied to issues of vascular biology as well as tumor metastasis. More specifically, two distinct techniques were developed and implemented to provide quantitative information needed to obtain a fundamental understanding of the signal transduction process within vascular endothelial cells and the adhesion molecules involved in the heterotypic adherence between leukemia/lymphoma cells (LLC) and marrow stromal cells (MSC). A first generation digital imaging video microscopy system was developed to provide both quantitative and visual three dimensional information from individual vascular endothelial cells. With the combination of optical sectioning, video microscopy, digital image processing, deconvolution, fluorescence ratio imaging, and scientific visualization one is able to measure the dynamic changes in spatial distributions of intracellular ions. The technique was applied to pCa measurements in quiescent as well as ATP and shear stress stimulated endothelial cells. This technique permits high resolution visualization and quantification of three dimensional spatial distributions of ions as well as traditional temporal changes in ion concentration. In addition, a parallel flow detachment assay (PFDA) and a $sp{51}$Cr detachment assay, coupled with MoAb blocking experiments, were developed to quantify the attachment of LLC to confluent monolayers of MSC. The cell-cell interaction between LLC and MSC was chosen as a cell model to investigate adhesion properties as well as to characterize which adhesion molecules are involved in the apposition. Results from the temporal studies suggest the heterotypic adherence of LLC to MSC is a biphasic event and that the interactions occur very rapidly after the two cells come into contact. More specifically, the early phase of apposition solely involves VLA-4$alpha$/VCAM-1 interactions. The late phase proceeds despite the continuous presence of anti-VLA-4$alpha$ and is not affected by MoAbs to ICAM-1, LFA-1, CD44, VCAM-1, E-selectin, or L-selectin, suggesting the possible involvement of other adhesion molecules.

[发布机构] Rice University

[效力级别] Biomedical engineering