A Method of Targeted Cell Isolation via Glass Surface Functionalization
A Method of Targeted Cell Isolation via Glass Surface Functionalization
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DOI：
10.3791/54315
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发表时间：
2016-09-01
影响因子：
1.2
通讯作者：
Imoukhuede, P. I.
中科院分区：
文献类型：
--
作者：
Ansari, Ali;Patel, Reema;Imoukhuede, P. I.
个体化医疗的采用和发展的限制因素之一是无法开发诊断工具来探测患者之间表达的个体细微差别。目前试图分离细胞以填充该小生境的方法导致生理表达的破坏，使得分离技术作为诊断工具无用。在该方案中，我们描述了用于细胞捕获和释放的表面的功能化和优化。该功能化表面将生物素化抗体与用氨基硅烷（APTES）、脱硫生物素和链霉亲和素功能化的玻璃表面整合。通过引入生物素促进细胞释放，允许由表面捕获的细胞的再收集和纯化。这种释放是通过靶向第二部分脱硫生物素来完成的，这导致了更加温和的释放模式。这种苛刻试剂和剪切力的减少减少了细胞表达的变化。功能化表面在单细胞混合物中捕获高达80%的细胞，并且已经证明在双细胞混合物中捕获50%。研究了该技术在异种移植和癌症分离研究中的应用。还描述了用于表面验证的定量技术，例如平板读数器和ImageJ分析。
One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.