Incorporation of gas/liquid equilibration control into a perifusion system to measure tissue-specific O ₂ concentration dependency of O ₂ consumption rate

Oxygen (O₂) regulates a multitude of cell functions, and many pathological states are linked to its delivery. We present an automated system for implementing rapid changes in dissolved gas composition in the inflow of a perifusion system that facilitates multiple assessments of tissue function. Features of the system include the ability to assess the effects of changes in both aqueous and dissolved components of the inflow media, and to collect fractions while measuring O₂ consumption rate (OCR) (in the face of changing dissolved O₂), facilitating the subsequent measurement of multiple classes of secreted compounds including metabolites, hormones, neurotransmitters, cell signals, and cytokines. We quantified OCR and lactate secretion rate (LSR) from retinal pigment epithelial (RPE) and INS-1 cells, and from primary tissues (retina, liver, and islets). Higher concentrations of extracellular O₂ were required for tissues than cells monolayers. Consistent with this observation, we found that OCR was not maximal at 21% O₂ for any tissue type we tested. That suggests 21% is too low to adequately provide O₂ for tissues in vitro. However, we found that at high levels of O₂, OCR in some tissues/cells rapidly decrease. LSR was reciprocally regulated relative to the O₂ dependency of OCR, except in tissue where high O₂ inhibits OCR. In summary, we describe a system that can control the concentration of extracellular O₂ and other gases. This instrument will allow researchers to investigate rapid effects of dissolved O₂ on metabolic activities of tissues and cells at O₂ concentrations optimal for the biological specimen.<b>NEW & NOTEWORTHY</b> Oxygen regulates a multitude of cell functions, and many diseases are linked to its delivery. In vitro experiments are critical for elucidating intracellular mechanisms. Whereas standard cell culture incubators allow exposure to varying oxygen concentrations over hours and days, systems enabling rapid and precise control of dissolved oxygen are not available. We present a system for implementing rapid changes in dissolved gas composition with a perifusion system that facilitates multiple assessments of tissue function.