Abstract
The differences in behaviour between individual cells in a large population are often important, yet are masked in bulk analyses where only average parameters are measured. One unresolved question in the field of immunology is the extent to which important immunological phenomena such as immunodominance to cancer antigens correlates with the average activity of a population of antigen-specific T lymphocytes, or with the activity of individual "outlier" cells. Despite progress in single cell technologies, few platforms are available that can deliver time-resolved, functional analysis at single cell resolution, for these investigations. We have developed an accessible high-throughput platform to measure single T cell signalling in real time following time-controlled stimulation by live antigen presenting cells. The cell-trap array consists of thousands of individual microwells cast in an agarose block, which is biocompatible and permeable to nutrients. Single T cells are isolated in wells via passive sedimentation and size exclusion, achieving up to 90% occupancy. The device enables simultaneous activation of thousands of single CD8+ cells. Stimulation with soluble reagents (ionomycin, anti-CD3 antibodies) or antigen presenting cells leads to changes in intracellular calcium concentrations which were measured using calcium-chelating fluorophore dyes. The platform was used to demonstrate a range of activation profiles among individual cells of a cloned, antigen specific CD8+ T cell hybridoma in response to both nonspecific stimuli and specific, physiologically relevant antigen stimulation. The presence of two different activation profiles was demonstrated, together with rare outlier behaviour among cells that are essentially clonal.
Highlights
Microfluidics and single cell technologies have provided new tools to investigate time-resolved single cell activation.[1,2,3,4,5,6,7,8,9,10] Highthroughput approaches include devices where two cell populations are sequentially trapped to create cell–cell contacts, or aqueous droplets where two cells are co-encapsulated and thereby interact
Voldman and Dura reviewed the range of microfluidic tools for the study of immune cell interactions[2] and concluded that the open design of microwell arrays provides an easy method for single cell retrieval using micromanipulators
Limiting dilution sub-cloning was initially used on the lymphocytes population to select for cells that were highly sensitive to SIINFEKL (SL8) epitope, and sensitivity assays were periodically run to ensure the functionality of the cell model
Summary
Microfluidics and single cell technologies have provided new tools to investigate time-resolved single cell activation.[1,2,3,4,5,6,7,8,9,10] Highthroughput approaches include devices where two cell populations are sequentially trapped to create cell–cell contacts, or aqueous droplets where two cells are co-encapsulated and thereby interact. Amongst the various technologies that allow isolation and analysis of single cells, three high throughput methods are used for single cell pairing, namely hydrodynamic trapping in a flow (closed system),[11,12,13,14,15,16] sedimentation in micro-well arrays (open format)[17,18,19,20,21] and containment in microfluidic droplets.[22,23,24] Of these techniques, hydrodynamic traps and microwell arrays are the only techniques where the cell–cell contact time can be synchronised. T cell activation occurs within seconds[27] after encountering a target antigen, highlighting the need for a system that allows synchronized cell–cell contact with realtime recording of the first few minutes of interaction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
