Embedded Files
Introduction
Oxygen is a key regulator of cell survival and function and thus its level in cell culture medium must be adequately controlled.
During the bioreactor operation, cell culture medium circulates between the reactor and reservoir. Oxygen consumed by the cells is the scaffold is replenished by diffusive flux from air-liquid interface.
The concentrations of dissolved oxygen entering the tissue in the scaffold (inlet oxygen concentration) and exiting the tissue (outlet oxygen concentration) can be determined with oxygen sensors such as fiber optic oxygen sensors. Because it is technically difficult to measure oxygen concentration directly under the scaffold, it is instead measured in the reservoir well. This is a valid approach because oxygen transfer into cell culture medium through low-permeability polyurethane membrane can be neglected. Because of the oxygen gradients in the reservoir well, placement location of the oxygen sensor in the reservoir well must be carefully considered.
Oxygen Probes
Oxygen is detected using a 2-mm diameter optical fibers capped with ruthenium-based sensing layer (Presens, Germany - www.presens.de), where luminiscence of ruthenium molecules in an excited state is quenched by collisions with molecular oxygen. Phase modulation technique is used to evaluate luminiscence decay time. The probes have a very low drift over the course of several days and thus, do not require frequent recalibration. For this reason, they are highly suitable for long-term measurement of dissolved oxygen in cell culture systems.
A detail photograph of the custom-made oxygen sensor shows the ruthenium complex sensing layer attached to the tip of the probe. O-ring provides a seal between the connector and the optical fiber.
Photo: Karel Domansky
For mounting and connecting purposes, the custom-made oxygen probe is furnished with a connector and a union.
Photo: Karel Domansky
Custom lid holding oxygen probes
The custom lid of the perfused multiwell plate contains 24 ports centered on each well in the plate. Each port contains a connector and an O-ring to vertically position the oxygen probe. All probes are submerged to a short distance above the scaffold. Optical fibers coming vertically out from the lid are supported by a second platform ~10 cm above the lid. The second platform holds unions that connect the probes with the optical fibers coming from the OXY-4 meter (Presens, Germany). This arrangement facilitates adjustment of the probe depth and allows quick connections between the probes and the optical fibers.
Photo: Karel Domansky
Inside the lid, optical fibers with the ruthenium complex sensing layers are protruding a desired distance from the face of the lid. This distance determines the immersion depth of the probes in cell culture medium and the height above the scaffolds with cells.
Photo: Karel Domansky
Measurement setup
Perfused multiwell plate with seeded cells is placed inside an incubator. Using the unions on the optical fibers mounted to the upper platform of the lid, the probes are connected with optical fibers to a 4-channel measuring instrument (PreSens, Germany). Four-channel meter allows simultaneously to monitor, for example, inlet and outlet oxygen concentrations in two bioreactors and determine oxygen consumption rates of the cells in sample and control bioreactors. Technical specification about the instrument can be found here.
Photo: Karel Domansky
The plots below show measured concentration of dissolved oxygen in reservoir and reactor wells seeded with ~800 000 rat hepatocytes: (a) as a function of time post-seeding with a flow rate of 0.25 mL/min and a sampling rate of 0.2 measurements per minute; (b) as a function of flow rate (immediately following the time course measurement) with a sampling rate of 1 measurement per minute.
Note: Immediately after seeding, flow was initiated downward through the scaffold at 0.25 mL/min. After 8 hours the direction of the flow was reversed to dislodge dead cells and debris. Flow up throught the scaffold was maintained throughout the remaining culture period.
It can be seen that the inlet oxygen concentration in this experiment fluctuates modestly around ~145 µM. Although this concentration is higher than values reported for the human in vivo periportal (sinusoidal entry) region (84–91 µM), it is within arterial blood oxygen concentration ranges (104–146 µM) and below hyperoxic concentration levels. Oxygen concentration downstream of the tissue increases with increasing flow rate, from ~19 µM to ~125 µM. In human, the reported in vivo perivenous (sinusoidal exit) region dissolved free oxygen concentration is 42–49 µM. A tissue outlet concentration of ~50 µM is observed at a flow rate of 0.25 mL/ min, hence, this flow rate provides a reasonable approximation of physiological gradients. Gradients are steeper than those in vivo because culture medium does not contain hemoglobin, which serves as a depot of oxygen.
More information about oxygen measurements and modeling can be found in a journal article. [pdf]
Page updated
Report abuse