Zengel Pamela, Nguyen-Hoang Anna, Schildhammer Christoph, Zantl Roman, Kahl Valentin, Horn Elias
Department of Otorhinolaryngology, Head and Neck Surgery, Grosshadern Medical Centre, Ludwig-Maximilians-University of Munich, Munich, Germany.
BMC Cell Biol. 2011 May 18;12:21. doi: 10.1186/1471-2121-12-21.
Effective tools for measurement of chemotaxis are desirable since cell migration towards given stimuli plays a crucial role in tumour metastasis, angiogenesis, inflammation, and wound healing. As for now, the Boyden chamber assay is the longstanding "gold-standard" for in vitro chemotaxis measurements. However, support for live cell microscopy is weak, concentration gradients are rather steep and poorly defined, and chemotaxis cannot be distinguished from migration in a single experiment.
Here, we describe a novel all-in-one chamber system for long-term analysis of chemotaxis in vitro that improves upon many of the shortcomings of the Boyden chamber assay. This chemotaxis chamber was developed to provide high quality microscopy, linear concentration gradients, support for long-term assays, and observation of slowly migrating cells via video microscopy. AlexaFluor 488 dye was used to demonstrate the establishment, shape and time development of linear chemical gradients. Human fibrosarcoma cell line HT1080 and freshly isolated human umbilical vein endothelial cells (HUVEC) were used to assess chemotaxis towards 10% fetal calf serum (FCS) and FaDu cells' supernatant. Time-lapse video microscopy was conducted for 48 hours, and cell tracking and analysis was performed using ImageJ plugins. The results disclosed a linear steady-state gradient that was reached after approximately 8 hours and remained stable for at least 48 hours. Both cell types were chemotactically active and cell movement as well as cell-to-cell interaction was assessable.
Compared to the Boyden chamber assay, this innovative system allows for the generation of a stable gradient for a much longer time period as well as for the tracking of cell locomotion along this gradient and over long distances. Finally, random migration can be distinguished from primed and directed migration along chemotactic gradients in the same experiment, a feature, which can be qualified via cell morphology imaging.
由于细胞向特定刺激物的迁移在肿瘤转移、血管生成、炎症和伤口愈合中起着至关重要的作用，因此需要有效的趋化性测量工具。目前，博伊登小室试验是体外趋化性测量的长期“金标准”。然而，其对活细胞显微镜观察的支持较弱，浓度梯度相当陡峭且定义不明确，并且在单个实验中无法区分趋化性和迁移。
在此，我们描述了一种新型的一体化小室系统，用于体外趋化性的长期分析，该系统改进了博伊登小室试验的许多缺点。开发这种趋化性小室是为了提供高质量的显微镜观察、线性浓度梯度、对长期试验的支持以及通过视频显微镜观察缓慢迁移的细胞。使用AlexaFluor 488染料来证明线性化学梯度的建立、形状和时间演变。使用人纤维肉瘤细胞系HT1080和新鲜分离的人脐静脉内皮细胞（HUVEC）来评估对10%胎牛血清（FCS）和FaDu细胞上清液的趋化性。进行了48小时的延时视频显微镜观察，并使用ImageJ插件进行细胞跟踪和分析。结果显示，大约8小时后达到线性稳态梯度，并至少稳定48小时。两种细胞类型均具有趋化活性，并且可以评估细胞运动以及细胞间相互作用。
与博伊登小室试验相比，这种创新系统能够在更长的时间段内产生稳定的梯度，以及沿着该梯度并在长距离上跟踪细胞运动。最后，在同一实验中可以区分随机迁移与沿趋化梯度的引发迁移和定向迁移，这一特征可以通过细胞形态成像来鉴定。
