Upstream operations can benefit significantly from real-time, in-line testing of cell-culture conditions. Yet as Jake Boy (senior application scientist at Scientific Bioprocessing Inc., SBI) observed in his 5 November 2020 “Ask the Expert†webcast, traditional electrochemical probe sensors do not perform well in T-flasks, shake flasks, Petri dishes, and microfluidics devices. Boy described how fluorescence-based optical sensors can glean valuable data from small culture devices, assisting with research into cellular growth.
SBI’s Presentation
Boy began by highlighting successful applications of optical sensors in small culture devices. In one case, SBI worked with researchers at Virginia Commonwealth University to monitor rat chondrocyte growth in Petri dishes incubated with CO2. Optical sensors enabled real-time pH monitoring in those studies. SBI also partnered with German startup company aquila biolabs, adding optical sensors to its biomass monitoring systems for shake flasks. The teams tested that pairing by tracking the growth of Crabtree-positive Saccharomyces cerevisiae. Boy reflected that backscatter-intensity data from biomass sensors mapped out the microbe’s growth, including its characteristic shift from fermentative to purely respiratory metabolism. “But without dissolved oxygen (DO) sensing, we could not have predicted when that shift might happen or why.â€
Boy continued, “If you don’t know the level of oxygen that your cells are experiencing, you cannot be confident that they are healthy and genetically stable or that they are metabolizing appropriately.†He recalled a 1996 study (1) that used optical-sensor data to debunk the practice of leaving T-flask lids cracked open during incubation. That technique was presumed to permit gas flow into a flask. But sensor data proved otherwise. Researchers measured pH and DO in closed and partly opened T-flasks kept stationary during incubation. Both closure methods generated anoxic conditions after 60 hours of incubation.
Later studies confirmed those findings and showed that gentle agitation improves culture conditions, increasing oxygen mass transfer, maintaining physiological pH levels, and promoting cell growth. Researchers also determined that hypoxia drives genetic instability in cultured cells. Such lines of inquiry, Boy explained, reinforce that scientists must know rather than presume what conditions cells are experiencing. Optical sensors can provide such data.
SBI’s offerings facilitate sensor applications for small containers. A standard kit includes single-use pH and DO sensors, coaster-shaped readers, and interactive data-collection software. SBI sensors can measure pH levels between 6.0 and 8.0 and DO levels of 0–100%. Instruments provide up to 45 days of continuous monitoring or several months of intermittent monitoring. Unlike conventional sensors, those from SBI show low drift
(≤0.005 pH/min at 1-min scan intervals) and little susceptibility to photobleaching.
Optical sensors are ideal for noninvasive monitoring, Boy continued. SBI’s sensors are 5 mm wide and 0.3 mm tall, and they adhere strongly to vessel surfaces, ensuring minimal interference with culture media. SBI sensors transmit data wirelessly to readers, and they are designed for single use. Thus, the technologies pose no contamination risks.
Although SBI sensors and readers accommodate several containers, other solutions can facilitate sensing in challenging geometries. Among those are single-use fiber optic probes that measure pH, DO, and temperature. SBI also produces “star adapters†that attach sensor readers to curved walls. The company’s flow-through cells can take measurements in feeding and perfusion lines.
Boy added that increased control over a culture environment yields better data. SBI offers a device that rocks T-flasks during CO2 incubation. The company also has produced a shaker-flask deck that enables attachment of sensor readers to flasks.
Boy concluded by acknowledging strong demand and clear industry need for optical-sensor monitoring of other critical metabolites. SBI plans to unveil sensor solutions for glucose lactate in the second half of 2021.
Questions and Answers
Would replacing an electrochemical sensor with a fiber optic probe require any bioreactor redesign? SBI’s fiber optic probes bear the same threading as conventional probes, so they fit easily into most bioreactor systems.
How well do SBI sensors perform in viscous samples? They adhere strongly to vessels, so they maintain their integrity in viscous media. Neither media flow nor protein and metabolite aggregation diminish sensor performance.
How compatible are SBI shake-flask decks with commercially available shaker platforms? Shaker decks and flask holders can be outfitted to rest on existing systems. Those components are available for purchase.
Reference
1 Randers-Eichhorn L, et al. Noninvasive Oxygen Measurements and Mass Transfer Considerations in Tissue Culture Flasks. Biotechnol. Bioeng. 51(4) 1996:466–478; https://doi.org/10.1002/(sici)1097-0290(19960820)51:4%3C466::aid-bit10%3E3.0.co;2-g.