Pericellular oxygen monitoring with integrated sensor chips for reproducible cell culture experiments

Abstract

Objectives: Here we present an application, in two tumour cell lines, based on the Sensing Cell Culture Flask system as a cell culture monitoring tool for pericellular oxygen sensing.

Materials and methods: T-47D (human breast cancer) and T98G (human brain cancer) cells were cultured either in atmospheric air or in a glove-box set at 4% oxygen, in both cases with 5% CO2 in the gas phase. Pericellular oxygen tension was measured with the help of an integrated sensor chip comprising oxygen sensor arrays.

Results: Obtained results illustrate variation of pericellular oxygen tension in attached cells covered by stagnant medium. Independent of incubation conditions, low pericellular oxygen concentration levels, usually associated with hypoxia, were found in dense cell cultures.

Conclusions: Respiration alone brought pericellular oxygen concentration down to levels which could activate hypoxia-sensing regulatory processes in cultures believed to be aerobic. Cells in culture believed to experience conditions of mild hypoxia may, in reality, experience severe hypoxia. This would lead to incorrect assumptions and suggests that pericellular oxygen concentration readings are of great importance to obtain reproducible results when dealing with hypoxic and normoxic (aerobic) incubation conditions. The Sensing Cell Culture Flask system allows continuous monitoring of pericellular oxygen concentration with outstanding long-term stability and no need for recalibration during cell culture experiments. The sensor is integrated into the flask bottom, thus in direct contact with attached cells. No additional equipment needs to be inserted into the flask during culturing. Transparency of the electrochemical sensor chip allows optical inspection of cells attached on top of the sensor.

Figure 1

Sensing Cell Culture Flask: the transparent sensor chip is embedded in a conventional tissue culture flask.

Figure 2

Detailed view of the Sensing Cell Culture Flask sensor chip embedded into the bottom of a conventional tissue culture (a). Micrograph of an Sensing Cell Culture Flask sensor chip before deposition of the hydrogel membrane (b). The small number and letters have been introduced into the metal layer of the sensor chip to simplify navigation during microscopy.

Figure 3

Number of T‐47D cells (top) and MCF ‐7 (bottom) as a function of time. Time zero here represents the time when all cells have attached, which was 6 h after seeding. The cell number is normalized to the amount of cells 6 h after seeding. The curves show results from chips with silicon nitride (●) and silicon oxide (▲) as top layer in comparison with the Nunclon™ Δ Surface (■) as control.

Figure 4

Pericellular oxygen level under conditions similar to a standard CO ₂ incubator, with 20% O ₂ and 5% CO ₂ inside the incubation atmosphere. Calibrated readings from the sensors on the chip surface where T‐47D cells (□) or T98G cells (○) are attached, readings from a Sensing Cell Culture Flask without cells were collected at the same time points (∇). The indicated disturbances are correlated with media shaking due to moving of the flask onto the microscope for photo capturing. Details about the regions A–D can be found in the text. 1% O₂ corresponds to approximately 10 μm dissolved oxygen in cell culture medium.

Figure 5

Pericellular oxygen level when sensor flasks were placed in an incubator with 4% O ₂ and 5% CO ₂. Two different starting cell densities of T‐47D cells 2 × 10⁶ cells (□) and 6 × 10⁶ cells (○) and 2 × 10⁶ T98G cells (∆) are included. The blank Sensing Cell Culture Flask without cells (∇) was placed into the incubator box 1 day prior to the flasks with cells and was already equilibrated at the start time point. The indicated disturbances are correlated with media shaking due to photo capturing on microscope. Details about the regions A–C can be found in the text. 1% O₂ corresponds to approximately 10 μm dissolved oxygen in cell culture medium.

Figure 6

Density of attached cells on the sensor chips, normalized to cells on the bottom of a 25‐cm ² flask beside the chip area at two different time points after seeding of cells. The images were taken during the oxygen measurements at 4% oxygen in the incubator, see Fig. 5. The cell numbers in brackets refer to the amount of cells seeded at time zero. The culture with 6 × 10⁶ T‐47D cells seeded was super‐confluent at 45 h. Therefore, no data could be taken from the microscopy images.

Figure 7

Microscopic pictures of the cells taken 45 h after seeding of cells. The flasks were incubated with 4% oxygen in the incubator atmosphere. The top row shows sections of the cells in the area of normal flask material. The bottom row shows part of the sensor chip respectively of the same flask as the one shown in the upper row. The cell numbers in brackets refer to the amount of cells seeded at time zero. The pictures are taken from the oxygen measurement experiment as represented in Fig. 5. In flasks with 6 × 10⁶ T‐47D cells seeded, the cells were super‐confluent and floating around.