Department of Molecular Pharmacology, Physiology and Biotechnology, Center for Biomedical Engineering, Brown University, G-B393, Biomedical Center, 171 Meeting Street, Providence, RI, 02912, USA.
Cytotechnology. 2008 Feb;56(2):81-90. doi: 10.1007/s10616-007-9116-x. Epub 2007 Dec 22.
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.
细胞失巢凋亡是一种细胞失去与细胞外基质或其他细胞的附着而导致的特定形式的细胞凋亡。由于体外模拟这些条件存在挑战，因此很难生成一种可控且高效的方法来研究细胞失巢凋亡。我们开发了一种使用微成型非粘附水凝胶分析和定量细胞失巢凋亡的微尺度方法。这些水凝胶允许对单个未附着的细胞进行分离和有序排列观察，并可进行受控的细胞分布。将通过多种播种密度获得的细胞分布与数学概率模型进行了比较。将正常人成纤维细胞、人脐静脉内皮细胞和曼丁达犬肾上皮细胞以约每孔一个细胞的低密度播种到水凝胶中。由于水凝胶由非粘附琼脂糖制成，因此细胞的附着可以忽略不计。使用荧光显微镜监测细胞的存活情况，并通过图像分析进行定量。使用配套的附着测定法评估了细胞在非附着环境中保持一段时间后的附着和增殖潜力。两种方法的数据均表明，细胞在没有附着的情况下能够存活比预期长得多的时间。在用 H35 大鼠肝癌细胞进行测试时，我们证明单个癌细胞可以生长成三维球体，证明了该方法在理解细胞失巢凋亡在癌症中的作用方面的实用性。
