Medical University Graz, Core Facility Alternative Biomodels and Preclinical Imaging, Graz, Austria
Involved scientists: Prof. Beate Rinner, Thomas Hebesberger

Continuous pH measurements using a combination of 6-well cultivation plates with integrated pH Spots (PHSP5-PK7) were conducted. Two different cell lines, namely, Raji and MUG-Mel2 were used. Raji cells are cultivated in suspension in contrast to the adherent cultivation of MUG-Mel2. Different cell number were cultivated to observe different pH shifts during continuous pH measurements. Also, the possibility of simultaneous measurement of dissolved oxygen is presented.

pH and dissolved oxygen are highly important parameters for cell cultivation and crucial for cell growth and cell viability. With cell growth, medium acidification occurs which can be observed by the color change of the pH indicator phenol red which is added to the cell growth media. However, continuous monitoring of pH is of interest as information of growth rate and bioreactions can be obtained or media without phenol red have to be used.

In this experiment, two different cell lines were cultivated and pH and dissolved oxygen was monitored using PyroScience optical pH sensors. Raji cells were cultivated in RPMI media with 10% FBS whereas MUG Mel 2 are cultivated in RPMI Media with 10 % FBS and additional 2mM L-Glutamine. Both were cultivated at 37°C in the incubator with 5% CO2.

The sterile pH spots (Item No. PHSP5-PK7-CV, sterilized with 15 kGy beta radiation) were glued into the wells using two-components-epoxide resin. After curing of the glue, the sensor spots were calibrated using pH 4 and pH 10 buffers (Item No. PHCAL4 and PHCAL10) which were sterile filtered before use. Read out of the spots was perfomed by the 4-channel FireSting pro and optical fibers (Item No. SPFIB-BARE) which were introduced into the incubator to the 6-well plate. To fixate the optical fibers, a frame was 3D printed which allowed to fixate the fibers directly under the 6-well plate.

Raji Suspension Cells

The Raji B-Lymphocyte cells are cultivated as suspension cells. Different cell-numbers were used for cultivation in duplicates to obtain different pH acidification rates of the media. 2-wells were not treated with cells as reference. As seen in Fig. 2, the media acidification is depending on the cell-number in the well. The duplicates showed very good correlation. After the second change of media, the cells were let to dye in order to obtain data about the maximum pH shift which was measured to be 6.6. The spikes during the monitoring can be attributed to the removal of the well-plate from the 3D printed frame and therefore a measurement without the sensor spot on top of the optical fiber.

MUG-Mel2 Adherent Cells

Additionally, the adherent cell culture MUG-Mel2 was used for cultivation. This time, 2 oxygen spots (OXSP5) were included in 2 wells to obtain data about the oxygen content. The oxygen spots were also sterilized using beta-radiation. Calibration was performed before use at ambient air and with an oxygen free solution (sterile filtered Na2SO3 solution). The pH sensors were again calibrated using PHCAL4 and PHCAL10 which were sterile filtered prior use.

As the spots were glued on the bottom of the well, it was of high interest if the cells would adhere to the sensing surface. The surface of the oxygen sensor spots is highly hydrophobic and therefore no growth was expected. The pH sensor spot consists of a hydrogel polymer which has some degree of hydrophility on the surface. However, there was no growth visible on the top of both sensor spots. The cultivation of MUG-Mel2 was monitored over 10 days. 2 Wells were used without cells as reference. Every 3 days, the cells were trypsinized and suspended in new media which leads to the high pH change from pH < 7.0 back to the pH of the fresh media (7.3). All 4 wells show good correlation and no sensor spot was influenced by the cells or trypsin. The reference media shows a slight pH increase of 0.6 pH units over 10 days. This is a typical drift of the pH sensors at 37°C. Additionally, the oxygen content of 2 wells with MUG-Mel2 cells was monitored. After 100h, a high cell number was visible under the microscope indicating this high oxygen decrease. After 200h, the cell density was already higher than in normal cultivation as stress test. This high cell number let the oxygen decrease to around 65% air saturaton.

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