Correlating short-term Ca(2+) responses with long-term protein expression after activation of single T cells.

In order to elucidate the dynamics of cellular processes that are induced in context with intercellular communication, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed on the level of individual cells and correlated with each other. Here we present an approach that allows the initiation of cell-cell or cell-particle interactions and the analysis of cellular reactions within various regimes while the identity of each individual cell is preserved. It utilizes dielectrophoresis (DEP) and microfluidics in a lab-on-chip system. With high spatial and temporal precision we contacted single T cells with functionalized microbeads and monitored their immediate cytosolic Ca(2+) response. After this, the cells were released from the chip system and cultivated further. Expression of the activation marker molecule CD69 was analyzed the next day and correlated with the previously recorded Ca(2+) signal for each individual cell. We found a significant difference in the patterns of Ca(2+) traces between activated and non-activated cells, which shows that Ca(2+) signals in T cells can provide early information about a later reaction of the cell. Although T cells are non-excitable cells, we also observed irregular Ca(2+) transients upon exposure to the DEP field only. These Ca(2+) signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, we could identify experimental conditions that caused the least interference with the physiology of the cell.

T cell activation on a single-cell level in dielectrophoresis-based microfluidic devices.

The gentle and careful in vitro processing of live cells is essential in order to make them available to future therapeutic applications. We present a protocol for the activation of single-T cells based on the contact formation with individual anti-CD3/anti-CD28 presenting microbeads in a lab-on-chip environment. The chips consist of microfluidic channels and microelectrodes for performing dielectrophoretic manipulation employing a.c. electric fields. The dielectrophoretic guiding elements allow the assembly of cell-bead pairs while avoiding ill-defined physical contacts with their environment. After overnight cultivation of the manipulated cells, 77% of the bead-associated T cells expressed the activation marker molecule CD69. Physiological stress on the cells was shown to be mainly due to the single-cell cultivation and not to the manipulation in the chips. The same approach could be useful for the in vitro regulation of stem cell differentiation.