Respiratory Physiology & Neurobiology 182 (2012) 47–52 Contents lists available at SciVerse ScienceDirect Respiratory Physiology & Neurobiology j our nal ho me p age: www.elsevier.com/locate/resphysiol A bioreactor for subjecting cultured cells to fast-rate intermittent hypoxia Theodora Tsapikouni a,b , Elena Garreta a,b,c , Esther Melo a,b,c , Daniel Navajas a,b,d , Ramon Farré a,b,c,∗ a Unitat de Biofísica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain b CIBER de Enfermedades Respiratorias, Bunyola, Spain c Institut d’Investigacions Biomèdiques August Pi Sunyer, Barcelona, Spain d Institut de Bioenginyeria de Catalunya, Barcelona, Spain a r t i c l e i n f o Article history: Accepted 2 January 2012 Keywords: Obstructive sleep apnea Hypoxia–reoxygenation Oxygen partial pressure a b s t r a c t High frequency intermittent hypoxia is one of the most relevant injurious stimuli experienced by patients with obstructive sleep apnea (OSA). Given that the conventional setting for culturing cells under intermit- tent hypoxia conditions is limited by long equilibration times, we designed a simple bioreactor capable of effectively subjecting cultured cells to controlled high-frequency hypoxic/normoxic stimuli. The biore- actor’s operation is based on exposing cells to a medium that is bubbled with the appropriate mixture of gases into two separate containers, and from there it is directed to the cell culture dish with the aid of two bidirectional peristaltic pumps. The device was tested on human alveolar epithelial cells (A549) and mouse melanoma cells (B16-F10), subjecting them to patterns of intermittent hypoxia (20 s at 5% O 2 and 50 s at 20% O 2 ), which realistically mimic OSA of up to severe intensity as defined by the apnea hypopnea index. The proposed bioreactor can be easily and inexpensively assembled and is of practical use for investigating the effects of high-rate changes in oxygen concentration in the cell culture medium. © 2012 Elsevier B.V. All rights reserved. 1. Introduction High frequency intermittent hypoxia is a significant biologi- cal challenge in obstructive sleep apnea (OSA). This syndrome, which is very prevalent in both adults and children (3–5% of population) (Lumeng and Chervin, 2008; Punjabi, 2008), is char- acterized by an abnormal increase in upper airway collapsibility during sleep (Farre et al., 2008a). As a result, OSA patients experi- ence repeated apneic events with a frequency of up to more than one per minute. These recurrent apneas induce severe periodic desaturations of oxygen in arterial blood and, consequently, acute intermittent hypoxia at tissue level (Almendros et al., 2010). There is ample evidence in the literature that intermittent hypoxia is a main determinant of the increased morbidity (cognitive, metabolic and cardiovascular) and mortality observed in OSA patients (Gozal and Kheirandish-Gozal, 2008; Kheirandish-Gozal et al., 2010b; Levy et al., 2009). Although the biological consequences of intermittent hypoxia have been thoroughly studied in animal and cell models of OSA (Farre et al., 2008b; Yuan et al., 2011), the information available on the effects of high frequency hypoxia–reoxygenation in cell stud- ∗ Corresponding author at: Unitat de Biofísica i Bioenginyeria, Facultat de Medic- ina, Casanova 143, 08036 Barcelona, Spain. Tel.: +34 93 402 4515; fax: +34 93 403 5278. E-mail address: rfarre@ub.edu (R. Farré). ies is scarce. The main reason for this lack of data is the difficulty in subjecting cultured cells to a pattern of intermittent hypoxia that realistically mimics OSA. Indeed, the conventional oxygen cham- ber setting, based on changing the O 2 content of the air, results in an excessively low rate of change of oxygen concentration at cell level because of the long time constant of gas diffusion from the air-medium interface to the cells cultured at the bottom of the cul- ture well (Allen et al., 2001). Some alternative methods have been proposed to circumvent this problem (Oppegard et al., 2010, 2009; Polinkovsky et al., 2009). The application of these methods has been limited, however, because they require very specific equip- ment, beyond the reach of most labs interested in investigating the effects of OSA-mimicking intermittent hypoxia on different kind of cells. To overcome the limitations imposed by the current experi- mental settings, we have designed and tested a readily available bioreactor for effectively subjecting cultured cells to oxygen partial pressures with controlled magnitude and time pattern. Our aim was specifically to simulate the high-frequency hypoxic stimulus char- acteristic of OSA. The bioreactor operates by exposing the cells, at accurately controlled intervals, to interchangeable media that have been previously equilibrated to the desired oxygen concentration. With our bioreactor prototype we achieved precise variations of oxygen concentration at cell culture level, from 20% to 5% in less than 10 s. Herein, we provide a detailed description of the proto- type, as well as the results of feasibility tests with different cell types. 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2012.01.001