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O2, pH and CO2 Monitoring in Microfluidic Mammalian Cell Culture
Cultivated Meat Bioprocess Optimization with SensorPlugs
Vasa Radonic, Ivana Podunavac, Mila Djisalov, Teodora Knezic, Ivana Gadjanski
BioSense Institute, Novi Sad, Serbia
We tested the efficacy of PreSens SensorPlugs for monitoring mammalian cell culturing processes in microfluidic bioreactors (MBs) with integrated sensors. MRC-5 fibroblasts (ATCC® CCL-171™) were used as a model of adherent mammalian cells instead of primary cell culture of animal-derived satellite cells/myoblasts. SensorPlugs showed good ability to monitor adherent mammalian cell culture parameters inside the MB over time.
The cell culture medium is the most significant cost driver for cultivated meat production. Optimizing the bioreactors' design and instrumentation, through additional monitoring features via sensors can help in decreasing these costs and achieving maximum cell production capacity per unit medium volume. Scale-down approaches have long been applied in bioprocessing to resolve scale-up problems. Miniaturized bioreactors have thrived as a tool to obtain process-relevant data during early stage process development. We applied this principle i.e., the 'lab-on-a-chip' (LoC) approach when developing a new generation of low-cost sensors for monitoring of various cell culture parameters such as biomass, ammonia and glutamine in cell culturing media. We used our custom-made microfluidic bioreactors complemented with our impedimetric sensor and an optical sensor in combination with the SensorPlugs for O2, CO2 and pH, provided by PreSens. The main idea of our project was to try to identify which processes are going on inside the microfluidic bioreactor, as well as to be able to quantify the effect on cell behavior, cell viability, and reagents effectiveness. We tested the efficacy of PreSens SensorPlugs for monitoring bacterial and mammalian cell cultures in microfluidic bioreactors. In addition, we performed a set of pilot experiments with human saliva, as a potential further expansion of the sensors’ application for taste/flavor evaluation of the final product of the cultivated meat bioprocess. Here our results for cell culture monitoring are shown.
Materials & Methods
The multilayered MB chips were manufactured using the transparent and biocompatible materials glass and PMMA. The top and the middle layer were manufactured in PMMA and the interconnecting layers were made using 3M double-sided adhesive tapes (3M™ GPT-020F, St. Paul, MN 55144-1000, Minneapolis, USA). The bottom layer of the chip for cell culture was made of glass where the impedance sensor was printed with the commercial Agfa-Gevaert N.V. nano silver ink with 15 % of Ag nanoparticles and the conductive ink using piezocontrolled inkjet printer Fuji Dimatix DMP-3000. The sensor was covered with a thin layer of SU-8 3000 Microchem resist. The top layer contained inlet/outlet holes whose diameters were adapted for pipetting the sample while filling the chip. Three holes with a diameter of 2.1 mm were made in the top layer for mounting the PreSens SensorPlugs. The sensors were in direct contact with the sample through the holes which enabled real-time measuring of the parameters in the sample. MRC-5 fibroblasts (ATCC® CCL-171™) were used as a model of an adherent cell type. All experiments with MRC-5 fibroblasts were done at the Institute of Oncology Institute of Vojvodina, Sremska Kamenica, Serbia. DMEM which contains 10% FBS was used as a cultivation medium.
Monitoring O2, CO2 and pH during Cultivation
Before any measurements were done, we performed biocompatibility testing of the used materials for MF chip fabrication. MRC-5 cells for cultivation were placed inside the MF chips at a concentration of 50.000 cell/mL. Fig. 1 shows the results of the cultivation of MRC-5 cells during 48 h, and a doubling time and growth curve was calculated inside the MF chip. It can be noticed that the cells adhered nicely to the glass without the need for additional coating agents. This type of cells settles faster and binds to the substrate, which is most pronounced in the first hour after inoculation. Total number of cells obtained after 48h cultivation was 262.500 cells/mL with a viability of 95.45 %.
Biomass concentration was measured using UV/VIS absorbance spectra recorded in the near-infra-red region using a process spectrometer and a fiber optical probe placed on the top of the microfluidic chip. Since viable cells are polarizable and act as dipoles, impedance measurement was combined with optical measurement to obtain information about the cell concentration and an estimation of cell viability.
In the proposed research, impedimetric and optic measurements of biomass concentration are combined with measurements of essential cell growth parameters: pH, O2 and CO2. The cell culturing was done in a static cell culture system, where cells can attach to the cell substrate in static conditions after seeding. The cells had optimal conditions for growth, but the media with nutrients, essential for progression of the culturing process is limited.
Measurements were done inside the microfluidic chip with different concentrations of live and dead cells. The live-cell parameters were monitored through several experiments and the means values were presented in the Fig. 2, while for the dead concentration the cells were killed after a certain cultivation time with DMSO. For this purpose, we prepared 6250 cell/mL MRC-5 cells for cultivation inside the MF chips. DMEM which contains 10% FBS was used as a cultivation medium. The pH, O2 and CO2 values were measured inside the media, and cells counted using cytometer, while the biomass was estimated using an integrated impedimetric sensor and optical spectroscopy in NIR region.
The initial pH value (when we seeded cells inside MF chip) at t = 0 min was about 7.4. After cultivation of cells, we observe the acidification of the culture, an increase in % of carbon dioxide, and a simultaneous decrease in % of oxygen during incubation. For the smaller concentration of the cells there were no significant changes in all parameters. As the concentration increased CO2 level rose exponentially, and O2 exponentially decreased. In case of the dead cells DMSO was added to the media which additionally influenced changes in all parameters, especially the pH. Also, significant differences in the measured concentrations of the gases over time could be observed.
Based on the presented results, we can conclude that the PreSens SensorPlugs are a convenient way of monitoring basic parameters (O2, pH, CO2) in the microfluidic cell culture. PreSens SensorPlugs present an important tool for scale-down approach analysis and process monitoring in cultivated bioprocess optimization. Future experiments will be directed toward continuous monitoring and optimization of culturing processes, examining the effects of cell activity inside the microbioreactor, as well as nutrients' effects on the cell growth factor. The research will be focused on finding the correlation between readings of pH, O2, and CO2 sensors with additional sensors (impedimetric, optical, and electrochemical) integrated into microfluidic bioreactors that we developed at the Institute for the measurement of conductivity of the medium, metabolite or biomass. The correlation between pH, O2, and CO2 with cell biomass and viability will be founded in future experiments.