CO₂ CONTROL FOR DYNAMIC CELL CULTURE

CO₂ sensing and control independent of a CO₂ incubator

Be independent of the CO₂ Incubator

Control CO₂ of the media on the bench

Use the chip of your preference

Inline sensor can be added to any setup

Automated cell culture

Less manual work and more accuracy for your experiments

Plug-and-play platform

Beginner friendly pack with detailed user guide

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The importance of CO₂ control for cell culture

The human body maintains its pH through a buffering system based on bicarbonate and CO₂. In other words, CO_2,usually produced as waste from metabolism, reacts with H₂O to form carbonic acid, a weak acid. This reaction creates a dynamic equilibrium that shifts depending on the concentration of H⁺ to maintain the overall concentration of ions constant, as shown below. The amount of CO₂ and carbonate available to buffer the system is regulated by the lungs and kidneys according to the body’s needs.

In cell culture, the same principle is applied. Media usually contains a predefined concentration of carbonate and the CO₂ incubator maintains the internal atmosphere with a determined concentration of CO2, so the same dynamic equilibrium is formed. However, there is no automatic way to adjust the concentrations of the buffering components as the body does with the lungs and the kidney, so the cell culture media needs to be changed regularly to keep the pH within adequate ranges.

The importance of CO₂ control in dynamic cell culture

In dynamic cell cultures, that is, when cells are cultured under flow, the microenvironment parameters become more important to control because the small dimensions of the system make small changes in pH more impactful.

Thus, either the entire perfusion system is designed to fit inside the CO₂ incubator, or parts of the system (mainly the electronics) remain outside, and the chip with the cells is kept inside the incubator in a far-from-ideal configuration.

CO2 control in dynamic cell culture pack setup

With the current limitations in mind, we set out to assemble a pack to control CO₂ independently of the CO₂ incubator, so you can set it up wherever is more convenient for you. It also allows the system to be automated to improve the reproducibility of the results.

Our all-in-one pack guarantees good compatibility between the different instruments, allows you to start your experiment right away, is piloted by a single software, and can be used for other applications. We also provide continuous and full customer support for you to fulfill your experiment goals.

The pack contains:

OB1 MK4 pressure controller (Elveflow)
A flow sensor (MFS or BFS, Elveflow)
A pH/CO₂ sensor and flow cell for use in line in setup
Several Eppendorfs or Falcon reservoirs
Two 3/2 valves and a MUX Wire (Elveflow)
Tubing and luers
Microfluidic chips
Control and automation software (Elveflow)
User guide

This pack can also be combined with other microfluidic steps after the dynamic cell culture, for sequential injection of drugs for example.

Behavior of CO2 in cell culture media

Culturing cells inside a CO2 incubator set at 5% has become such a standard practice in biology labs. A report from Klein et al. [1], analyzing 810 papers, clearly illustrates this. Most cell culture parameters, such as temperature, CO2 and O2 concentration and pH, are often described but rarely characterized or measured during experiments.

Consequently, few realize that different cell culture media require different CO₂ levels to maintain the pH within acceptable ranges, as illustrated below. DMEM, for example, has 44 mM of NaHCO₃, requiring a higher concentration of CO₂ to achieve physiological pH [2].

ph ranges co2 control incubator — Theoretical pH ranges for different culture media in increasing CO2 levels in a 37°C humidified incubator. The lower and upper physiological range of pH is denoted by the red and green horizontal lines (pH 7.2-7.4). DMEM (44mM NaHCO3, orange line) is held at physiological pH at CO2 concentrations between 7.5% and 11%. Using DMEM in a 5% CO2 environment will result in a pH of 7.5 which although acceptable for most cell cultures is slightly outside the desired range of physiological pH. EMEM + Earle’s Balanced Salt Solution (BSS) (26mM NaHCO3, dark green line) is held at physiological pH with CO2 levels between 4.5% and 6.5%, whereas a much lower percentage of CO2 (near atmospheric levels) is required for EMEM + Hank’s BSS. (4mM NaHCO3, blue line). Reproduced from Cooper, Culture Collections, 2019 [3].

References

Klein, S.G., Alsolami, S.M., Steckbauer, A. et al. A prevalent neglect of environmental control in mammalian cell culture calls for best practices. Nat Biomed Eng 5, 787–792 (2021). https://doi.org/10.1038/s41551-021-00775-0
Michl, J., Park, K. C. & Swietach, P. Evidence-based guidelines for controlling pH in mammalian live-cell culture systems. Commun. Biol. 2019 21 2, 1–12 (2019).
Cooper, J. CO₂ concentration and pH control in the cell culture laboratory. Culture Collections, 2019. Accessed on 22nd November 2022.
Lu, C. & Verbridge, S. S. Microfluidic methods for molecular biology. Microfluid. Methods Mol. Biol. 1–376 (2016). doi:10.1007/978-3-319-30019-1

Customize your pack

This pack is highly customizable to fit your experiment specification and cell lines (mammalian, plant, primary…).

You can request additional pumping channels for the flow controller and flow rate sensors. Coriolis flow sensors can be chosen to replace the MFS to improve the flow control even more. Bubbles can be a problem for cells; the pack can contain a bubble remover to tackle this issue efficiently.

You can contact our experts to answer any questions about this pH control in the cell culture pack and how it can match your specifications.

– Check our other Packs for various applications –

How can we help your experiment

The packs applications are still under development, so we are not able to give you the tips or troubleshooting advice that we usually give in our user guides and application notes for the possible challenges you could face for a specific application experiment. That being said, we can always guarantee reliable and high-precision microfluidic flow control. Our microfluidic instruments are high-performance, versatile and user-friendly. Our experts will bring support and expertise during the setup and implementation of this sensor calibration pack.

Can a pack be customized

Yes! Our experts will establish which instruments are best suited for your application, such as the type of flow sensor or the number of flow controller channels you need to perform your experiment. Send us a message:

innovation@microfluidic.fr

Can I buy individual instruments?

You can order our instruments on the product section of our website.

Funding and Support

The Protomet, ALTERNATIVE, and LIFESAVER projects results helped develop this instrument pack, with funding from the European Union’s Horizon 2020 MSCA-ITN under grant agreement No 813873 (ProtoMet), the European Union under H2020-LC-GD-2020-3, grant agreement No. 101036702 (ALTERNATIVE) and the European Union under H2020-LC-GD-2020-3, grant agreement No. 101037090 (LIFESAVER).