Carbon Dioxide Measurements Aren’t Sufficient to Assume Culture System Competence

This month, I would like to highlight why carbon dioxide (CO₂) measurement is not sufficient data to assume culture system competence. Embryo culture media in use today includes bicarbonate as the primary buffering agent to regulate media pH. The value range of pH stabilization is dependent on the partial pressure of carbon dioxide (CO₂) in an incubator. In this equilibrium, media pH is dynamic and changes with alterations to CO₂ gas levels. There is an inverse relationship between pH and CO₂, in that as the percentage of CO₂ increases the pH decreases and vice versa. The working CO₂ gas in your lab will affect your media pH including inaccuracies in the actual CO₂ percentage due to external environmental factors (frequent incubator use), equipment malfunction, or subtle differences made in your media prep technique.

A starting point to look at how incubators affect culture media pH performance is the CO₂ sensing equipment: thermal conductivity (TC) sensors and infrared (IR) sensors.

Thermal conductivity sensors regulate CO₂ levels by measuring the resistance of CO₂ gas to a reference gas, usually ambient room air. The sensor detects resistance changes caused by the input of CO₂ molecules and induces changes in flowrate to recover from a loss or excess CO₂. IR sensors use this physical quality to detect CO₂ gas, starting by passing IR light through the gaseous mix within the incubator. The IR sensor then uses a filter to isolate the wavelength specific to CO₂ hitting the sensor. Then calibrated circuitry calculates the difference between the light that strikes the sensor and the emitted source sample. For high CO₂ concentrations, more IR light is absorbed by the higher number of CO₂ gas molecules in the incubator sample leading to less IR light being detected.

Figure 2 depicts how pH monitoring data can reflect differences in CO₂ content within an incubator that a daily measurement reading of CO₂ percentage would miss. This is an excellent example of the differences in performance that can be expected between a TC sensor and an IR sensor. TC sensors in incubators with frequent door openings can lead to misleading CO₂ readings as their TC sensor drift over time. The actual pH value may be different than your pH expectations, as the air resistance that thermal conductivity sensors use to alter gas flow is affected by temperature and humidity. An IR sensor, by contrast, uses light absorption and is not dependent on temperature or humidity, making IR incubators a more stable culture environment in relation to CO₂ gas exchange.

For incubators with TC sensors, perform pH and temperature readings before any use or door openings, as the temperature and humidity will be the most stable. It is also critical to make sure that the water pan remains full, as changes in humidity from a dry water pan can affect the TC sensor’s accuracy. IR sensors will still need to be calibrated periodically (correctly) as they will drift over time, so even with an IR sensor; there are regular equipment maintenance and upkeep. For both types of the incubator, work habit observations are critical to determining performance. As lab staff open and close incubator doors the CO₂ levels often fluctuate throughout the day with the potential for a significant off-gas of CO₂ levels. This gas loss can be cut down if a front-loading incubator has plastic partitions or a Dutch door design for the interior. Even with a partition, CO₂ levels will need to recover quickly without an overshoot following door closings to maintain a stable pH range.  

Brief lid openings in benchtop incubators show minimal changes in pH, due primarily to the optimization of the embryo culture environment in the benchtop system complimented with the oil overlay of the media slowing changes in CO₂ content. However, even benchtop incubators need consistent pH data to determine the real culture environment, and a CO₂ measurement may be inaccurate. The pH profile in Figure 3 was generated from a routine pH test performed by a IVF clinic in a two-chamber, tri-mix, humidified benchtop Incubator.  The lab was unaware that there was a gas line blockage in the humidifying bottle tubing leading to the left chamber. This caused the pH to spike in the left chamber due to no gas flow. The blockage was discovered and was cleared at 7:30am the next morning, and the sample returned to regular stabilization thereafter. Compare this to the right side, which held a steady pH overnight.

Notice how it takes almost 12 hours to resolve the pH, which seems excessive-however, a maximum pH of nearly 7.8 was reached, which would be similar to out of the bottle conditions.  With a single CO₂ measurement for the day rather than performing pH monitoring, the effects of the gas line tubing would not be determined until the next time pH was taken, an investigation into poor embryo development occurred, or until the tubing was manually fixed/replaced.

The equipment used in your lab has a direct effect on your culture media, and understanding how the condition of your equipment affects pH is critical. Equally important, is not assuming your CO₂ concentrations are spot on or that they are recovering immediately from door openings. Monitoring pH allows you to understand how multiple factors are affecting your media pH truly. Every lab should replicate current culture media conditions while collecting pH values to determine whether equipment is functioning properly and if the observed pH range follows the lab’s predicted trends.