Dielectrophoresis (DEP) based Lab-on-a-chip devices for real-time monitoring of the activity of cytotoxic T-lymphocytes.

Cytotoxic T-lymphocytes (CTL) of cancer patients exhibit the ability to lyse tumor cells and this feature is of great importance for the control of the immune response against tumor onset and progression. Accordingly, the analysis of the cytolytic activity of isolated CTL populations against tumor cells is of great clinical impact, since the isolation of highly cytoxic CTL clones is a complex and long procedure needing of step by step validation of the enrichment achieved. In these experimental protocols, CTL are proposed for the development of tumor immunotherapy. Accordingly, protocols for immunotherapy have been developed for the treatment of melanomas, lymphoblastic leukemia . In this context, realtime monitoring of CTL activity in treated patients might retain prognostic importance. In respect to these issues, dielectrophoresis (DEP) based Lab-on-a-chip technology could represent a very appealing approach, since it allows the manipulation of large numbers of single cells or cell populations. We have employed two dielectrophoresis-based Lab-on-a-chip platforms for the real-time analysis of CTL-mediated cell lysis. One platform (the SmartSlide) displays parallel electrodes and generated cylinder-shaped cages within which CTL and target cells are entrapped. The second platform (the DEP Array) generates spherical DEP cages that can entrap clusters composed of several CTLs and a single (if present) target cell. We demonstrate that DEP-based devices can be used to manipulate CTL clustered to target cells with the aim to develop an efficient methodology to quantify CTL-mediated cytolysis and identification of CTL clusters or clones exhibiting high cytotoxic activity. Target tumor cells were labeled with calcein and their lysis evaluated within the DEP-based platforms. These are the advantages of the proposed approach: (a) no radioactive labeling is required; (b) an high numbers of target cells can be followed in parallel, obtaining statistical information (not possible with the Cr-51 release assay); (c) analysis at the single-cell level is feasible; (d) the protocol is fast and results are reached within 8-12 minutes; (d) the approach is suitable with the use of low numbers of target cells.