Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions

Abstract

DH82 Cell line has been utilized to grow Ehrlichia canis and RF/6A for drug evaluation in short-term cultures, as well as for replicating Anaplasma marginale. However, specific in vitro culture development conditions are unknown.

Several experiments were designed to solve inquiries of such procedure. Both cell lines were acquired from ATCC and put into culture. Na Pyruvate, NaHCO3 and fetal calf serum were used to enrich MEM culture media and incubated at 37 °C on 5% CO2 - air humid mixture atmosphere. First assays used 24 well plates and were focused on determination of a minimum initial cell concentration and cell density. In the former, averages of 62,500 cell/well for DH82 & 8,836 for RF/6A; were found. Later, experiments to identify a minimum cell density started with 5, 10, 20 & 40 cells/mm2, harvesting with a EDTA-Trypsin solution when confluence be reached. Growth indexes of 3,319.32, 1,956.70, 870.73 & 422.14 times and of 62.38, 63.51, 25.31 & 12.16 times, were respectively found for DH82 & RF/6A cell lines. For kinetics studies, 35 mm Ø sterile Petri dishes were used. Cultures were set with 20 cell/mm2 seed density for DH82 and 10 cell/mm2 for RF/6A. Dishes were randomly separated into two groups, with and without culture media periodical replacement. Maximum growth was observed at 336 and 315 h with 42.9 and 36.9 h doubling time in culture, respectively for DH82 & RF/6A cell lines. Generated data provided a model to define an in vitro growth pattern for future studies.