The most commonly used buffering system for mammalian cell cultures is a bicarbonate/CO2 system, which requires CO2 regulators and incubators to supply a constant level of CO2. As a replacement, Leibovitz developed a bicarbonate-free medium, L15, with relatively high levels of certain amino acids in the free base form. The most common method in use today for buffering mammalian cell cultures is the bicarbonate/CO2 system, which is based on the following equilibrium: CO2 + H2O HCO3 + H+ The use of bicarbonate is an attempt to mimic the buffering system of blood, but it has at least two major drawbacks. (1) The p/Q of bicarbonate is 6*1, which is far removed from the desired pH range of cell culture media (7-0-7-4). (2) Although bicarbonate is cheap, supplying a constant level of CO2 to cell cultures is definitely not, as it requires expensive CO2 incubators or fermentors.