A minichamber device for maintaining a constant carbon dioxide in ari atmosphere during prolonged culture of cells on the stage of an inverted microscope

Summary Construction details are described for a minichamber device that maintains a localized atmosphere of carbon dioxide in air over the stage of an innerted microscope. This device is easily constructed, from Plexiglas and its specifications can be adjusted to fit virtually any inverted microscopy. A flow of warm, humidified carbon dioxide in air gas mixture can be directed over a petri dish or unsealed culture flask to maintain the pH of bicarbonate-CO₂ buffered media. By this means, prolonged culture of cells directly on the microscope stage is made possible without occurrence of detrimental pH changes. If the microscope is fitted with an environmental control chamber to maintain temperature, cells can be maintained on the microscope stage for days, permitting frequent observation of cell growth and activity. Alternatively, continuous cine or video recordings can be made. For example, using this device, hamster and rhesus monkey embryos have been cultured for 2 to 5 d on an inverted microscope while continuous time-lapse recordings were made of cell division and differentiation and activity of cellular organelles. The work was supported by National Institutes of Health RR00167 to the Wisconsin Regional Primate Research Center and HD14765 to B.D.B., and Life Sciences University of Wisconsin-Madison.