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Fungal Metabolite Screening Inside a Microfluidic Bioreactor

pH Measurements in Plant Cell Culture with pH-1 SMA HP5

L. Schmidt-Speicher1, C. Metzger2, F. Kinn1, B. Bailer3, Dr R. Ahrens1
1Karlsruhe Institute of Technology, Institute of Microstructure Technology, Eggenstein-Leopoldshafen, Germany
2Karlsruhe Institute of Technology, Botanical Institute, Karlsruhe, Germany
3Hochschule Biberach, Biberach, Germany

As sessile organisms, plants cannot easily circumvent stressors by relocating. To evaluate the quality and quantity of abiotic and biotic stresses experienced, plants have evolved complex signaling pathways. Oftentimes molecules belonging to potential pathogens are recognized on the level of the plants’ plasma membrane. Upon recognition, the signal is relayed from the outside of the cell into the cell, ultimately inducing a defense reaction, if possible [1]. In the Pathogen Triggered Immunity (PTI), a basal defense reaction, the signal is often transferred with the help of the secondary messenger Ca2+. The extracellular space contains a relatively high concentration of Ca2+, especially when juxtaposed to the concentration in the cytoplasm of the cell. To react to the presence of the pathogen, Ca2+ ions are transported into the cell to change the behavior of the cell with the help of transcription factors [2]. This import of Ca2+ occurs in combination with the transport of H+ across the plasma membrane [3]. The transport of protons results in a shift in the extracellular pH value that is easily quantifiable. The pH shift is a commonly used readout to assess whether plants mount a PTI reaction.

To screen multiple fungal metabolites for their immunoactivity in quick succession an easy and reliable system is needed to measure pH changes in the microfluidic bioreactor (MBR) developed by the KIT Institute of Microstructure Technology (IMT) in cooperation with the KIT Botanical Institute. Therefore, a retractable but sealable connector from the PreSens pH-1 SMA HP5 to the according sensor spot SP-HP5 in the MBR was designed and examined mechanically as well as in biologic experiments. The MBR is a two-chamber bioreactor that is used to cultivate plant cells (see Fig. 1A). A permeable membrane separates the cultivation- and the nutritional-flow-chamber. It was first designed by Tim Finkbeiner at the IMT [4] and then further developed. Cells can be brought into the cultivation-chamber via two openings at each end of the oval chamber. The openings are equipped with M5-threads to allow sealing them with suitable screws. MBRs can be connected to further MBRs via unidirectional flow. Thus, the cells remaining in the cultivation chamber, are continuously supplied with fresh medium and metabolic products from the previous MBRs while their own metabolic products are transported to the following MBRs and waste is carried off. The basic principle is to dissect the intercellular communication path to gain information on the involved processes and reactions via the cultivation of cells of one kind per MBR. The pH value is quantified via the pH-1 SMA HP5 while cultivating cells of the BY2 line of Nicotiana tabacum in the cultivation chamber of the MBRs. Substances whose immunoactivity is to be evaluated can be added to the medium supplied to the cells. Should an increase in the pH value be observable it can be interpreted as the first indication of an immunoactivity of the substance of interest.

Materials & Methods

Connection Setup and Mechanical Evaluation
Important criteria for the connection are sealing the growth chamber leakproof but removable and offering easy and intuitive handling. Furthermore, we aimed at a small building height of the connectors to keep the system suitable for observation under a microscope. According to these requirements four connector designs were manufactured. The connectors were tested in an MBR and their performance. The four connectors for the optical fiber (see Fig. 2) were manufactured from Polycarbonate (PC) as this is the MBR’s housing material. Two connectors were designed with a blind drilling while the other two connectors were drilled through and sealed afterwards. For the sealing a PC-foil, simple tape, and Polydimethylsiloxane (PDMS) were tested. This approach was used to determine, if the transparency – which was hampered in the blindly drilled connectors due to the scratch-marks from the drilling tool – affected the precision of the measurements. Furthermore, one connector of each kind was equipped with an additional groove to accommodate an extra sealing-ring as a second option if leaking occurred. First, the transparency of the manufactured connector screws was tested by measuring and comparing the transmission through the bottom of the blind-drilled hole as well as the sealing-foils using a Leitz Ergoplan microscope in combination with the Red Tide USB650 Fiber Optic Spectrometer from OceanInsight. Secondly, the manufactured connector-screws were tested regarding their sealing-capability. An MBR was sealed using the tested connector and hooked up to a syringe pump (Harvard Apparatus PHD Ultra). Then, deionized water (DI-water) was pumped through the MBR at a steady flow rate of 2 mL/min. The test was continued for 15 minutes to ensure that the sealing was fully tight and stayed dry. After leak-testing of the connectors the pH-1 SMA HP5 was connected to an MBR (Fig. 3 A). A pH sensor spot (SP-HP5) was placed on the side of the connector facing the inside of the MBR and the connector was screwed into the opening of the MBR. Finally, the optical fiber was placed in the according connector and three different pH-buffer-solutions (pH 6.00, pH 7.413 and, pH 8.00, Carl Roth) were used pumped through the system one at a time to evaluate the measuring performance of the sensor-connector. The measured data was compared to measurements of the buffer using a Mettler Toledo SevenCompact S220 as an external pH-sensor.

Cell Line and Cultivation Setup
For the tests on the BY2 cultivation a cell suspension culture of N. tabacum was cultivated in Murashige and Skoog medium (MS), containing 4.3 g/L Murashige-Skoog salts, 30 g/L saccharose, 200 mg/L KH2PO4, 100 mg/L Myo-Inositol, 1 mg/L Thiamine and 0.2 mg/L 2,4-dichlorophenoxyacetic acid. The cell culture was sub cultivated weekly by transferring 1.5 mL of seven days old cell suspension into 30 mL of fresh MS in a sterile Erlenmeyer flask and cultivated in the dark, at 26°C on an orbital shaker set to 150 rpm. To cultivate the cells inside of the MBR, 750 µL of 7 days old cell suspension was carefully pipetted into the cultivation chamber of the MBR and supplied with fresh MS medium using a peristaltic pump (see Fig. 3 B). The flow was organized in a unidirectional circular flow through the chip. The peristaltic pump operated to pump 134 µL/min through the tubing.

Measuring of Chitosan Induced pH Shift
Chitosan is known to induce an immune response in plant cells with a corresponding shift in extracellular pH. In this work, the BY2 cells cultivated in the MBR were treated with 25 µg/mL of chitosan (Sigma Aldrich Chemie GmbH, Germany) and a respective solvent control of 0.0025 % acetic acid. First, the setup was installed in a cleanbench as described above, with tubing connecting the MBR, the pump, and the MS medium vessel in a circular fashion. Then MS medium was pumped through the system at 134 µL/min and stopped just as it reaches the MBR the first time. 750 µL of 7 days old cell suspension was transferred into the cell chamber. The pump was started, and the chip was turned upright while being filled with MS to facilitate the expulsion of the remaining pockets of air. Once MS medium started to exit the chip’s outlet the chip was installed into the scaffolding in its final horizontal placement. Before starting the pH measurement, the sensor inside the chip was equilibrated for 90 minutes. Subsequently, the pH value was measured for 30 minutes to calculate the baseline pH. Then the chitosan or the acetic acid was added and the respective timepoint was defined as 0:00. After treatment, the pH was continuously measured every 30 seconds for two hours. For each treatment, a biological triplicate was created.

Results & Discussion

Mechanical Results
The two connector designs with the drilled-through opening for the optical fiber were covered with a PC-foil, PDMS and, 3M polyester tape 851 (“green tape”) to create a leak-proof boundary between the cultivation-chamber and the optical fiber outside the MBR. The PC-foil could not be bonded sufficiently in our experiments, but the 3M polyester tape 851 sealed the connector plug adequately. However, this tape-sealed-connector could not be used for pH-measurements due to the green color and low light transmission of the tape. The PDMS-sealed drilling-hole was sealed leakproof, too. Furthermore, the blindly drilled connectors both sealed the MBR sufficiently. There was no observable advantage for sealing the system in including an extra groove and additional sealing ring compared to the connector without those. The simple blindly drilled connector sealed the MBR sufficiently.
Regarding the transparency, the PDMS-sealed connector showed the lowest transmission-rate while the PC-foil-sealed connector transmitted about 95 % of the light (see Fig. 4). However, the low transmission of the PDMS might have resulted due to measurement setup-problems.

Next, the connector-sensor-setup was tested regarding the precision of pH measurements. As the pH-buffer-values by the manufacturers were accurate in comparison with the S220-sensor, the measurements were compared with the given values as a reference. The pH-values of the regarding buffer in the MBR were measured three times at the respective flow rate for ten minutes (at flow rate 0 mL/min and 2 mL/min) and for 45 minutes (at flow rate 0.2 mL/min). The box plots were generated for the combined databases of the three measurements (Fig. 5). The absolute deviation from the given buffer-value was determined. For all three conditions at all three pH-values the interquartile range is small ranging between 0.014 pH to 0.005 pH. Median and average are – besides the pH-evaluation in the 6-pH-buffer with no flow circling – almost the same. Thus, a symmetrical distribution of the measured values can be assumed.

However, if the different connectors are compared to a measurement in a glass beaker as a reference system, the measurements in the MBR show a clearly higher measuring of the same buffer than the measuring in the beaker (Fig. 6). This might result due to the fact, that the sensor is designed for glass reactors instead of measuring through polymers. Because no pattern can be derived from the usage of different connectors that would be in accordance with the different measured light-transmissions it is assumed that the different transparencies of the different connectors were not the main cause for the offset in the measurements.

Biological Experiments - Chitosan Induced pH-Shift
The microfluidic setup was installed as described previouly under a clean bench with an ambient temperature of 25°C. After equilibrating the sensor spot for 90 minutes the pH value was measured for 30 minutes before adding the Substance of Interest (SoI). Afterwards, the pH value was continuously observed for two hours with measuring points every 30 seconds.
The mean of the pH values measured 30 minutes before adding the SoI was considered the baseline pH and used to calculate the ΔpH of every time point(t):
ΔpH(Sol) = pH(t) - pHbaseline
To see whether chitosan induced a pH shift, the shift in pH induced by the solvent of chitosan needed to be deducted from the pH observed in the chitosan treatment:
ΔΔpHchitosan = ΔpHchitosan - ΔpHsolvent CTRL
The resulting data of ΔΔpHchitosan, ΔpHchitosan and ΔpHsolvent Control (CTRL) are all displayed in figure 7. All treatments were done in triplicates and the standard error of ΔpHchitosan and ΔpHsolventCTRL was noted. To calculate ΔΔpHchitosan the means of ΔpHchitosan and ΔpHsolventCTRL were deducted from one another. The significance of the differences between ΔpHchitosan and ΔpHsolventCTRL at different time points were calculated via the T Test with a significance level of p<0.05 and are displayed as a heatmap on the top of figure 7. The solvent CTRL showed a stark drop in pH starting at around 15 minutes after treatment and subsequently stayed at the lowered level. The chitosan treatment caused the pH level to slightly decrease and stabilize back to the baseline level. Considering the decrease in pH that was caused by the solvent, the ΔΔpHchitosan suggests that chitosan can induce a major increase in extracellular pH that is measurable with the pH-1 SMA HP5 system.

Conclusion

From the results of our experiments and different designs we concluded that the light transmission through the different connector designs did not seem to affect the measuring significantly. However, it seemed that the polymer-connectors showed a shift in the measured pH-value compared to a reference measurement through a glass beaker. Since glass is the appropriate reactor-material stated in the sensor guidelines there might be a special calibration to cope with the light refraction in glass that leads to a shift of the measured values when using a polymer. If this shift can be verified – maybe through more and differently set up tests – and would be accounted for the pH-1 SMA HP5 sensor with the according spots would be suitable to measure pH-values in our MBR.
In a previous experiment, we confirmed that 25 µg/mL of chitosan will cause an increase in extracellular pH in BY2 cells by measuring the pH with a benchtop pH meter that uses an electrode. Using the same treatment this work aims to prove that a combination of the MBR and the pH-1 SMA HP5 system can be used to observe the immune-response associated increase in extracellular pH. After starting the treatment, a significant increase in pH was observable after 15 minutes (see Fig. 7). This delay is explained by the time the treated MS needs to be pumped into the MBR. The pH shift observed indicates that the method can be used to screen for the immunoactivity of substances on plant cells. With the use of multiple channels and sensor spots, it will be possible to establish a high-throughput screening method. Furthermore, we investigated the development of nutritional gradients in the cultivation chamber of the MBR due to the oval shape of the chamber. Regarding this evaluation the analysis of the pH-value in a greater area, e.g., using the VisiSens system, would be interesting. However, the sensor-foils would have to be perforated for the nutritional flow to reach the cultivation chamber and a mounting process for the sensor-foils would have to be established.

Acknowledgement
We thank PreSens cordially for the opportunity to test their optical sensor setup for pH-measurements.

References
[1] Jones, Jonathan D. G.; Dangl, Jeffery L. (2006): The plant immune system. In: Nature 444 (7117), S. 323–329. DOI: 10.1038/nature05286.
[2] Lecourieux, David; Ranjeva, Raoul; Pugin, Alain (2006): Calcium in plant defence-signalling pathways. In: The New phytologist 171 (2), S. 249–269. DOI: 10.1111/j.1469-8137.2006.01777.x.
[3] Demidchik, Vadim; Shabala, Sergey; Isayenkov, Stanislav; Cuin, Tracey A.; Pottosin, Igor (2018): Calcium transport across plant membranes: mechanisms and functions. In: The New phytologist 220 (1), S. 49–69. DOI: 10.1111/nph.15266.
[4] Finkbeiner, Tim (2019): Entwicklung eines mikrofluidischen Bioreaktors für die Kultivierung von Pflanzenzellen. Dissertation. Karlsruher Institut für Technologie, Karlsruhe. Institut für Mikrostrukturtechnik.

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