Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, Bari, Italy.
Tethis S.p.a., Milan, Italy.
PLoS One. 2024 Mar 8;19(3):e0297739. doi: 10.1371/journal.pone.0297739. eCollection 2024.
In recent years, the importance of isolating single cells from blood circulation for several applications, such as non-invasive tumour diagnosis, the monitoring of minimal residual disease, and the analysis of circulating fetal cells for prenatal diagnosis, urged the need to set up innovative methods. For such applications, different methods were developed. All show some weaknesses, especially a limited sensitivity, and specificity. Here we present a new method for isolating a single or a limited number of cells adhered to SBS slides (Tethis S.p.a.) (a glass slide coated with Nanostructured Titanium Dioxide) by Laser Capture Microdissection (LCM) and subsequent Whole Genome Amplification. SBS slides have been shown to have an optimal performance in immobilizing circulating tumour cells (CTCs) from early breast cancer patients. In this work, we spiked cancer cells in blood samples to mimic CTCs. By defining laser parameters to cut intact samples, we were able to isolate genetically intact single cells. We demonstrate that SBS slides are optimally suited for isolating cells using LCM and that this method provides high-quality DNA, ideal for gene-specific assays such as PCR and Sanger sequencing for mutation analysis.
近年来，从血液循环中分离单细胞对于多种应用的重要性不断增加，例如非侵入性肿瘤诊断、微小残留疾病监测以及产前诊断中循环胎儿细胞的分析，这促使人们需要建立创新的方法。为此，已经开发了不同的方法。所有方法都显示出一些弱点，特别是灵敏度和特异性有限。在这里，我们介绍了一种新的方法，用于通过激光捕获显微切割（LCM）和随后的全基因组扩增来分离粘附在 SBS 载玻片（Tethis S.p.a.）（涂有纳米结构二氧化钛的玻璃载玻片）上的单个或有限数量的细胞。SBS 载玻片已被证明在固定来自早期乳腺癌患者的循环肿瘤细胞（CTC）方面具有最佳性能。在这项工作中，我们在血液样本中混入癌细胞来模拟 CTC。通过定义切割完整样品的激光参数，我们能够分离出遗传完整的单细胞。我们证明 SBS 载玻片非常适合使用 LCM 分离细胞，并且该方法提供了高质量的 DNA，非常适合用于基因特异性测定，例如用于突变分析的 PCR 和 Sanger 测序。
