Veres-Székely Apor, Szász Csenge, Pap Domonkos, Bokrossy Péter, Lenzinger Dorina, Visnovitz Tamás, Mihály Judith, Pálmai Marcell, Varga Zoltán, Őrfi László, Szabó Attila J, Vannay Ádám, Szebeni Beáta
Pediatric Center, Semmelweis University, 1083 Budapest, Hungary.
HUN-REN-SU Pediatrics and Nephrology Research Group, 1052 Budapest, Hungary.
Int J Mol Sci. 2025 Jun 11;26(12):5584. doi: 10.3390/ijms26125584.
Collective cell migration is crucial in various biological processes, including tumor progression and metastasis. The widely used scratch assay (wound healing assay) has limitations in throughput, reproducibility, and data analysis. To overcome these challenges, we previously developed the Transient Agarose Spot (TAS) assay, which enhanced assay precision and reproducibility. In this study, we present an improved microplate-based TAS assay. By using a microplate reader, we automated data acquisition, enabling the detection of cell migration in a 96-well plate format with greater throughput and accuracy. The new method applies Hoechst staining to label viable cells, providing a stable signal for kinetic analysis without compromising cell viability. We validated this approach with fluorophore-expressing cancer cells and demonstrated its ability to monitor dose-dependent effects of fetal bovine serum on cell migration. Additionally, we applied the microplate-based TAS assay to assess the anti-migratory effects of kinase inhibitors and mesenchymal stem cell-derived extracellular vesicles (EVs) on lung cancer cells. The assay accurately quantified migration inhibition and revealed the concentration-dependent effects of EVs, highlighting their potential as therapeutic agents. This microplate-based TAS assay provides a scalable, efficient, and cost-effective platform for high-throughput screening of cell migration and drug discovery, offering a robust alternative to traditional microscopy-based methods.
集体细胞迁移在包括肿瘤进展和转移在内的各种生物学过程中至关重要。广泛使用的划痕试验（伤口愈合试验）在通量、可重复性和数据分析方面存在局限性。为了克服这些挑战，我们之前开发了瞬时琼脂糖斑点（TAS）试验，该试验提高了试验的精度和可重复性。在本研究中，我们展示了一种改进的基于微孔板的TAS试验。通过使用微孔板读数器，我们实现了数据采集自动化，能够以更高的通量和准确性检测96孔板格式中的细胞迁移。新方法应用Hoechst染色标记活细胞，为动力学分析提供稳定信号，同时不影响细胞活力。我们用表达荧光团的癌细胞验证了这种方法，并证明了其监测胎牛血清对细胞迁移的剂量依赖性效应的能力。此外，我们应用基于微孔板的TAS试验评估激酶抑制剂和间充质干细胞衍生的细胞外囊泡（EVs）对肺癌细胞的抗迁移作用。该试验准确地量化了迁移抑制，并揭示了EVs的浓度依赖性效应，突出了它们作为治疗剂的潜力。这种基于微孔板的TAS试验为细胞迁移的高通量筛选和药物发现提供了一个可扩展、高效且经济高效的平台，为传统的基于显微镜的方法提供了一个强大的替代方案。
