Evaluation of 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic Acid (HEPES) as a Tissue Culture Buffer

The amino acid 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES) has been evaluated as a tissue culture buffer. HEPES has a molecular weight of 238.3, a pKa2 of 7.31 at 37°, a δpK/° of —0.014, exhibits no metal binding and is soluble to the extent of 2.25 M at 0°. HEPES (0.01 M) produced adequate buffering in all cell systems tested. HEPES was not toxic to primary monkey kidney, chick embryo fibro-blasts or puppy salivary gland cells, human embryonic lung, BHK-21/4, BS-C-1, Detroit 6-YT, GPS. HEp-2 KB, LM, MA-134, or RK-13 cells. The GPS cells were grown in suspension in HEPES and NaHCO3 buffered media. Although the population doubling times were not significantly different, a higher maximum cell density and viability were seen in the HEPES buffered system. HEPES had a minimal effect on the oxidation-reduction potential of well-poised media. HEPES had no effect on rubella virus titrations or on hemagglutination assays of polyoma or Sendai virus. The formulation of a HEPES buffered salt solution is presented.