Controlling O2 and CO2 level

Reasons to control O₂ and CO₂ level

Cells need oxygen for their respiration and CO2 to have the correct pH and carbonate concentration. The pH and carbonate concentration can be controlled by "CO2-independent media" that are media with buffers, i.e. mainly carbonate salts that are balanced to keep the concentrations and pH correct. Over time, however, it is better for the media with cells to be in equilibrium with air with the correct concentration of O₂ and CO₂.

Ways of achieving correct gas mixtures

There are two basic ways of controlling the gas mixture composition: flow control or pressure-volume control

Flow control

Commercial gas blender measure the flow of each gas and regulate the flow of each input gas to achieve a controlled composition of the output gas. This requires a constant overall flowrate and no changes in the pressure at the inlet or outlet of the device. These gas blenders are therefore well suited to control the gases in volumes that are at atmospheric pressure and in contact with the media (in order to establish diffusive equilibrium) where the cells are.

MCQ gas blender

Our gas blender can take up to 3 input gases with input pressures < 2 bar (optimal pressure is 1 bar) and output a gas flow at constant flow rate 0-500 ml/min at a constant pressure smaller than the input pressure. The controller settings is done in the computer interface. [Manual for MCQ gas blender]

Pressure - volume control

If we have a container with a manometer (pressure sensor + display), accurate valves and pressurized pure gases we can let in the gases successively to control the final composition. First let's assume that the gases are ideal

[math] P_i=N_i \frac{kT}{V} [/math]

and that the gas mixtures are ideal as well

[math] P_{tot}=\sum_i P_i [/math]

If we desire a CO2 concentration of 5 per cent, [math]x_{CO2}=0.05 [/math] (which is much larger than the athmospheric CO2 concentration of [math]4\cdot 10^{-4}[/math]) that means that

[math] x_{CO2}=\frac{N_{CO2}}{N_{CO2}+N_{air}}=\frac{P_{CO2}}{P_{CO2}+P_{air}}=\frac{P_{CO2}}{P_{tot}} [/math]

and that first one fills up the container with air to the pressure [math]P_{air}[/math] and then continue to fill the container with CO2 until the pressure reaches [math]P_{tot}=\frac{1}{1-x}P_{air}\approx 1.053 P_{air}[/math]

Controlling O₂, CO₂ level and pH in media inside microfluidic network

Constant, premixed media flow

• Flow media with correct composition through the microfluidic network over the cells.
• This flow should be as small as possible to reduce the shear forces on the cells and it must be large enough to assure that the cells do not change the media composition appreciably before it has passed all the cells.
• Preparation of correct gas concentration can be done as in the left hand of the figure below, but put media in instead of water. After bubbling the correct gas composition through the mediia it will be ready to be injected.
• Transfer fluid to a gas tight syringe and use syringe pump.

Variable gas concentration

Flow a gas mixture with a regulated composition (including 100% humidity) over a membrane that allows exchange of gas between gas and the media in the microfluidic device. The exchange will then be diffusion controlled and if the distances exceed a few hundred micrometers one needs to estimate the diffusive flow rates to assure that the exchange is fast enough. One realization of this is given in the figure below. If one wants to control O2 level independently one should use three gas bottles (O2, N2 and CO2) as input to the gas blender.

Gas contol in microfluidic device