Individual Tissue Culture System in a Disposable Capsule with Hypoxic Atmosphere

Abstract

In the present study, a novel system for the hypoxic culture of individual tissues was established for the subculturing of cell lines for research as well as for clinical culture of primary cells. To provide a hypoxic environment throughout the process of tissue handling and culture, we designed a clean bench with CO2 gas circulation and a hypoxic culture incubator containing disposable capsules. The bench top was covered with an acrylic chamber, and an atmosphere of 5.0% CO2-air was maintained using a sensor control. The cleanliness class of the chamber could easily be improved to 1 within 5 min of circulative filtration, even though it was found to be 105 before the unit was operated. Gas buffer solution (220 ml of 20 mM HEPES, 25 mM NaHCO3, pH 7.4) placed in a 500-ml plastic capsule in the unit stabilized the culture environment by functioning as a heat storage and gas pool. The inflow of air that occurred by the cap of the capsule was opened was excluded by the infusion of purging gas (5.0% CO2 and 95% N2); the O2 level returned to 2.0% within 4 min, after which the gas supply automatically switched to the culture gas (2.0% O2, 5.0% CO2, and 93% N2). If this purging process was omitted, restoration of the O2 level required 120 min, even though the inner volume was only 280 ml.