For the growth of photosynthetic organisms, supply of CO2 is essential. Experimental work on the uptake and utilisation of inorganic carbon, requires that CO2 concentrations can be adjusted and kept stable. Here we tested the suitability of a culture method that allows supply of CO2 to a cell suspension, without the need of a continuous external gas supply for experimental work with the diatom Phaeodactylum tricornutum. This approach utilizes buffers with different ratios of HCO3-/CO32- in one chamber of a two-tier vessel, releasing different amounts of CO2 to the gas phase of the vessel, which is shared with the cell culture in the other chamber of the vessel. We cultured P. tricornutum under three different CO2 concentrations, while monitoring cell density, CO2 concentration in the gas phase, and pH within the cultures. We found the method very useful for work with P. tricornutum and found that the method also allows the creation of CO2 deplete conditions. This culturing system, while not as precise as a chemostat culture with supply of gas mixtures containing CO2, is simple to use and offers the possibility to adjust CO2 growth conditions.