Design and development of microbioreactors for long-term cell culture in controlled oxygen microenvironments

Abstract

The ability to control the oxygen level to which cells are exposed in tissue culture experiments is crucial for many applications. Here, we design, develop and test a microbioreactor (MBR) for long-term cell culture studies with the capability to accurately control and continuously monitor the dissolved oxygen (DO) level in the cell microenvironment. In addition, the DO level can be controlled independently from other cues, such as the viscous shear-stress acting on the cells. We first analyze the transport of oxygen in the proposed device and determine the materials and dimensions that are compatible with uniform oxygen tension and low shear-stress at the cell level. The device is also designed to culture a statistically significant number of cells. We use fully transparent materials and the overall design of the device is compatible with live-cell imaging. The proposed system includes real-time read-out of actual DO levels, is simple to fabricate at low cost, and can be easily expanded to control the concentration of other microenvironmental solutes. We performed control experiments in the absence of cells to demonstrate that the MBR can be used to accurately modulate DO levels ranging from atmospheric level to 1%, both under no flow and perfusion conditions. We also demonstrate cancer cell attachment and viability within the MBR. The proposed MBR offers the unprecedented capability to perform on-line measurement and analysis of DO levels in the microenvironment of adherent cultures and to correlate them with various cellular responses.

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

“…The perfusion setting enhances delivery of nutrients throughout the scaffold and maintains constant oxygen levels, at values equivalent to 2D culture (≈ 21 %), for about 16 d. The positive difference of oxygen concentration between the inlet and outlet of the chamber for the first 16 d (∆O 2 Inlet-Outlet ≈ + 1.02 %) emphasises the metabolic consumption of O 2 by cultured cells. This result is consistent with those observed in other studies run for a shorter culture time (up to 7 d) (Abaci et al, 2012;Janssen et al, 2006a;Volkmer et al, 2012).…”

A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia

“…Here we define uniformity as all oxygen partial pressures within the channel are within 10% of the mean. Although there are many options for controlling oxygen levels in microdevices, the design using gas channels positioned above the cell channel 23,24 was adapted to offer the most uniform oxygen content. With these initial designs for the device, computational modeling of oxygen mass transport was used to determine the optimal dimensions and orientations of cell, migration, vascular, and gas channels.…”

“…Placing microdevices inside environment chambers with controlled gas flow can achieve chronic hypoxia 20,21 or cycles to model ischemia/reperfusion injury. 22 Temporal control of oxygen concentration also has been achieved with the inclusion of gas channels either overlying, 23,24 or underneath 25,26 the cell culture channels. Similarly, spatial control of oxygen concentration has been achieved with the inclusion of gas channels adjacent to [27][28][29] and overlying 30 the cell culture channels.…”

Methods to study the tumor microenvironment under controlled oxygen conditions

“…To control the oxygen concentration inside microfluidic platforms, several different mechanisms have been employed. The three most common methods (Figure 2) are (i) introducing oxygen scavenging chemicals into the platform [30, 31], (ii) controlling oxygen supply by implementing adjacent gas supply channels, e.g., in multilayer platforms [29, 32-39], and (iii) reducing oxygen supply by use of oxygen impermeable materials such as cyclic olefin copolymer (COC), polystyrene (PS), or poly(methyl methacrylate) (PMMA) [40-44]. A few other microfluidic approaches to control oxygen tension have been reported [42, 45-47], but they are challenging to fabricate or more cumbersome in operation.…”