Miniaturization of an Anoikis assay using non-adhesive micromolded hydrogels

• Published in: Cytotechnology (Dordrecht) Vol. 56; no. 2; pp. 81 - 90
• Main Authors: Rago, Adam P., Napolitano, Anthony P., Dean, Dylan M., Chai, Peter R., Morgan, Jeffrey R.
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.02.2008; Springer; Springer Nature B.V
• Subjects: Adhesives; Anoikis; Apoptosis; Biochemistry; Biological and medical sciences; Biomedicine; Biotechnology; Cell division; Chemistry; Chemistry and Materials Science; Cytotoxicity; Endothelial cells; Epithelial cells; Extracellular matrix; Fibroblasts; Fundamental and applied biological sciences. Psychology; Hepatoma; Hydrogels; Image processing; Original Research Paper; Spheroids; Umbilical vein; St Louis Missouri; United States > US; Microtissue; Anoikis; Hydrogel; Microtumor; Micromold; Miniaturization; Adhesive
• ISSN: 0920-9069; 1573-0778
• DOI: 10.1007/s10616-007-9116-x

Anoikis is a specific form of apoptosis resulting from the loss of cellular attachment to extracellular matrix or other cells. Challenges in simulating these conditions in vitro make it difficult to generate a controlled, efficient assay to study anoikis. We developed a microscale method for analysis and quantification of anoikis using micromolded, non-adhesive hydrogels. These hydrogels allow for isolation and observation of single, unattached cells in an ordered array, and controlled distribution. Cell distributions resulting from multiple seeding densities were compared to a mathematical probability model. Normal human fibroblasts, human umbilical vein endothelial cells, and Mandin-Darby canine kidney epithelial cells were seeded at low densities of approximately one cell/well. Because the hydrogel is made of non-adhesive agarose, attachment was negligible. Survival was monitored using fluorescent microscopy, and quantified by image analysis. The attachment and proliferative potential of cells after being held in a non-adherent environment was assessed with a companion attachment assay. The data from both methods revealed that cells were able to survive much longer than expected without attachment. When tested with H35 rat hepatoma cells we showed that single cancer cells could grow into three-dimensional spheroids, demonstrating the utility of this method in understanding the role of anoikis in cancer.