Use of Tris(Hydroxymethyl)Aminomethane Buffers in Cultures of Diploid Human Fibroblasts. ∗

Summary

Tris-maleate and tris-citrate buffers were found to be exceedingly toxic to cultures of diploid human fibroblasts. Tris-HC1 buffers had relatively low toxicity, however; cultures continued to grow, although at reduced rates, at concentrations as high as 0.07 m in the presence of 7 mM bicarbonate. Small amounts of bicarbonate appeared to be essential for growth. A useful modification of Waymouth's medium was designed by titrating a somewhat reduced concentration of stock amino acids and vitamins to a pH of approximately 7.1 with NaHCO₃ and adjusting the final pH of the medium with tris-HC1 buffer to a pH of 7.3; the final concentration of bicarbonate was 7 mM, of tris, 25 mM and of serum, 10%. Excellent control of pH was obtained in closed systems in an atmosphere of air and in open systems in an atmosphere of approximately 2% CO₂; no toxicity has been observed with a dozen strains in continuous culture for periods of up to 6 months. Compared to Waymouth's medium (with 10% serum), growth in the tris-buffered modification was less predictable. Confluent monolayers were regularly obtained but they were seldom as thick as those fed with Waymouth's medium. With some batches of tris-buffered media, greatly improved growth was obtained by increasing the concentrations of magnesium and calcium. Tris-HCl buffers have also proven useful for controlling the pH of trypsinization media and of aged, unfed confluent monolayers; the latter could be maintained intact for at least 5 weeks when the pH was not permitted to fall below 7.0.