Introduction

Cell culturing is a core method in biological science and clinical research as well as in many biotechnological and biomedical engineering areas. Typically, mammalian cells are grown in a nutrient buffer in plastic culture flasks or multiwell plates, which are placed in a standard benchtop incubator. The incubator maintains the physiological conditions necessary for cell growth, such as sterility, proper temperature, pH, and osmotic pressure. Standard cell culturing is reliable and is used in the majority of cell culture-based experiments. However, it has limitations when dynamic processes of the cells have to be investigated. The cells can be taken out of the incubator for a brief observation and then returned to the incubator until the next observation, but this does not allow for continuous online monitoring of the cellular processes.

For the continuous real-time observation of cells, the incubator has to be: (i) enlarged to the size of a room to accommodate all observation instruments as well as the operator; (ii) modified to enclose the observation instrument and provide the instrument controls to the outside operator; or (iii) reduced to fit into the instrument; for example, on the stage of a microscope. The first option is usually chosen when running many parallel online experiments on a regular basis, but it requires a large investment and running costs. Furthermore, to allow the operator to breathe normally, the atmosphere is not enriched for CO₂, which requires special cell culture media and thus limits the range of cell types that can be investigated. Moreover, the operator working with the incubator might contaminate cell cultures. Enclosing the investigation instruments—typically a microscope—by the incubator is less costly and provides better sterility than a room incubator because it isolates the operator from the cell culture and the instrument. However, in this case, physical accessibility to the instrument is significantly reduced, and therefore it has to be equipped with remote controls and handling tools. Miniaturized cell culture incubators not only enable the growth and online observation of cells directly on the stage of an optical microscope, but they also provide the whole list of benefits intrinsic to microsystems, including low consumption of power and reagents as well as fast response due to the small mass and volume of the devices. In addition, this type of setup provides opportunities for parallel operation for high-throughput analysis and integration of multiple sensors for monitoring environmental parameters. Finally, the microsystems can be designed as single-use disposable devices due to the low costs, which are a consequence of the requirement for only a small amount of raw material and the potential for mass production using a batch-type fabrication (1).

The opportunities offered by microfabrication and microfluidic technologies in design and fabrication of miniaturized cell culture systems are being explored by researchers (2). One trend is the development of extremely small cell culture chambers intended for the study of single/few cell-related biological phenomena (3,4,5,6,7). Alternatively, microchambers are designed to provide and/or investigate effects of unique physiological cell culture conditions in terms of culture media composition, pressure, shear stress, and chemical and geometrical microenvironments (8,9,10,11,12,13,14,15,16,17).

Commercially available miniaturized cell culture systems are primarily developed for live-cell microscopy, sometimes including options for mechanical cell micromanipulations and electrophysiology. Typically, they consist of two parallel glass coverslips, defining the top and the bottom of the chamber, and a sealing spacer, providing the walls of the chamber (18). For long-term cultures, such systems contain inlets for continuous media perfusion, which provides fresh nutrients and removes metabolic wastes (19,20). The perfusion is typically driven by gravity or by peristaltic/ syringe pumps. The temperature required for cell growth is maintained by a heated airstream directed to the chamber, by heated water flow in the channels surrounding the chamber, or by integrated electric heaters consisting of resistance wires, thin films, or thermoelectric (Peltier elements), all controlled via an electronic feedback loop (20,21).

Several modifications of these setups exist, such as open chamber systems containing a cleaned oil layer on top of the culture media instead of a glass coverslip, which prevents fluid evaporation without hindering direct access to the cells by manipulation or probing tools (22,23). Some devices do not contain an integrated cell chamber but accommodate a standard cell culture dish and interface it with media perfusion and temperature control systems (Open Perfusion Microincubators; Harvard Apparatus, Holliston, MA, USA).