CO2 incubators are sealed, climate-controlled boxes used in life science laboratories to grow biological cell cultures. They are required to maintain the same conditions as inside the human body.
- 7.4 pH neutral
- 98.6°F (37°C)
- > 90% relative humidity
These three numbers create the optimal conditions for biological cell growth.
Why is additional CO2 needed in an incubator?
Humans are most comfortable at CO2 levels at or slightly above 400 ppm (0.04%) which raises the question, why would a CO2 incubator that is used to grow tissue cultures need CO2 levels of 5 - 10%?
In order to culture cells under optimum conditions, the media they grow in needs to stay at neutral pH (around pH 7). The H2O in the cells can be turned into a carbonic acid (H2CO3) buffer by adding additional CO2. The combination of H2O and CO2 results in bicarbonate (HCO3-) and H2CO3 which keeps the pH neutral, and therefore has been found to affect the growth of biological cells the least. Here's a short video that explains the chemical reaction.
In other words, by adding additional CO2 at the right level you prevent the pH inside the cells from becoming either alkaline or acidic, which both inhibit cell growth.
Maintaining CO2 Levels
CO2 levels inside a CO2 incubator are measured with accurate optical nondispersive infra-red (NDIR) sensors. There are two concerns when using an NDIR CO2 sensor:
- In high-altitude environments, the sensor readings must be adjusted to the change in barometric pressure.
- The sensor be guarded against high humidity. Too much water in the air can result in condensation inside the CO2 sensor which will render the sensor inoperative.
Maintaining Humidity Levels
While a water pan is often used to improve humidity, it creates potential problems with contamination and irregular humidity levels as the door is opened and closed. For that reason, an atomizer may be used to boost the humidity level along with a %RH sensor to control the humidity level.
Risks of contamination inside a CO2 incubator are primarily around cross contamination from fungi, viruses and bacteria. This can be controlled by sterilization between samples. It is important however, that sterilization with heat or superheated water not come into direct contact with the CO2 sensor. For this reason, the CO2 sensor should be integrated into the incubator in such a way that it is not impacted by the sterilization cycle. This can be solved by
- Gas sampling CO2 sensor (fixed or handheld) outside the incubator
- CO2 sensor with heat and moisture-resistant stainless-steel hood
Limitations of CO2 Incubators
A growing field of study is the limitations of CO2 incubators as they relate to oxygen levels. Common sense would tell us that since living tissue like human cell cultures thrive in 20.9% oxygen-filled air, that this should be the desired oxygen level inside a cell incubator. Since ambient air is 20.9% oxygen, CO2 incubators need not be sealed from outside air.
The problem is that different living cell culture grow at different oxygen levels. For example, while lung alveolar cells are exposed to 20.9% oxygen, the oxygen level in arterial blood falls to 10.5-13%. Many organs function normally at oxygen levels ranging from 2-8%. Logically, exposing every cell culture to the same 20.9% oxygen level would create a different environment than what they were exposed to inside the body, and hence, would change cell growth results.
The need to create anaerobic conditions for cell cultures includes a wide variety of applications including growing organ tissue parts, cancer diagnostics, genomic therapy, and virus and bacterium research for industrial, pharmaceutical and agricultural science.
To respond to this challenge, instead of using CO2 incubators scientists use sealed glove boxes with tri-gas CO2/O2/N2 incubation. The oxygen concentration is controlled by nitrogen and measured indirectly by measuring oxygen and carbon dioxide.
GasLab is working with several companies to integrate products like our 20% range CO2 and 25% range oxygen sensors that continuously sample the atmosphere inside an incubator, providing anaerobic conditions for optimal cell growth. In addition, we offer SST's Zirconia 25% oxygen sensor and will soon offer the MicroSENS IR 20% CO2 sensor, both designed to withstand the high heat needed for sterilization in cell culture incubators.
Photo by Shinryuu - Own work, Public Domain