Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level

Abstract: Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated.We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level.Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.