the tissue carrier within a perfusion culture container guarantees during transport of medium provision with always fresh nutrition and respiratory gas from all sides. At the same time it prevents an unphysiological accumulation of metabolic products and an overshoot of paracrine factors. To maintain for the whole culture period this controlled environment, the metabolized medium is collected in a separate waste bottle. In consequence, medium is not recirculated. Stabilization of pH during perfusion culture Normally cell culture experiments are performed in a CO2 incubator. Also perfusion culture experiments can be performed in such an atmosphere. However, a much better solution is the performance of perfusion culture experiments under atmospheric air on a laboratory table, since it facilitates the complete handling. However, in this case the culture medium has to be adjusted to atmospheric air. Keeping media in a 5% CO2 atmosphere within an incubator always a relatively high amount of NaHCO3 is contained to maintain a constant pH between 7.2 and 7.4. If such a formulated medium is used for perfusion culture outside a CO2 incubator, the pH will shift from the physiological range to much more alkaline values due to the low content of CO2 (0.3%) in atmospheric air. For that reason any medium used for perfusion culture outside a CO2 incubator has to be stabilized by reducing the NaHCO3 concentration and/or by adding biological buffers such as HEPES (GIBCO/Invitrogen, Karlsruhe, Germany) or BUFFER ALL (Sigma-Aldrich-Chemie, München, Germany). The necessary amount can be easily determined by admixing increasing amounts of biological buffer solution to an aliquot of medium. Then the medium must equilibrate over night on a thermo plate at 37 °C under atmospheric air. For example, application of 50 mmol/L HEPES or an equivalent of BUFFER ALL (ca. 1%) to IMDM (Iscove’s Modified Dulbecco’s Medium, GIBCO/Invitrogen) will maintain a constant pH of 7.4 throughout long term perfusion culture under atmospheric air on a laboratory table. Availability of oxygen in medium To obtain in a perfusion culture experiment a high saturation of O2 a selected medium such as IMDM has to be transported through a gas permeable silicone tube. The use of a silicone tube provides a large surface for gas exchange by diffusion due to a thin wall (1 mm), the small inner diameter (1 mm) and its extended length (1 m). For example, analysis of IMDM (3024 mg/L NaHCO3, 50 mmol/L HEPES) equilibrated against atmospheric air during