*Mª Luisa Fermín, Soledad Gaitán, Critina Fragío, Leticia Gonzalez, Luciana K. Ostronoff, Yaiza Forcada / Concepción Tejero
This in vitro study was carried out to define the best experimental conditions for producing canine neutrophils in long-term bone marrow culture (LTBMC), to determine the functional parameters of neutrophils obtained from peripheral blood and LTBMC and to ascertain whether these cells display physiological similarities. Our aim is to provide an experimental model enabling correlation between hemopoietic injury and neutrophil functionality.
Nine healthy spayed female Beagles between 2 and 3 years of age were used as marrow and blood donors. Bone marrow specimens were obtained from the iliac crest. LTBMC were established from bone marrow mononuclear cells (106 MNC/ml) cultured in 25-cm2 tissue-culture flasks and boosted after one week with 4 x 106 MNC. Various culture conditions were tested (fetal calf serum (FCS), horse serum (HS), hydrocortisone (HC) and different numbers of recharged cells) to obtain 60-70% of mature neutrophils. One week after recharging the cultures, supernatant cells were harvested to determine the number of neutrophils; O2- production with a continuous spectrophotometric assay of the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome-c; adenine nucleotides levels were measured by means of a bioluminescent assay method based on firefly luciferase. The statistical significance of differences was determined using Student's two-tailed t test.
End points studied in LTBMC culture production included confluence of stromal layers, cobblestone areas and quality and quantity of adherent cells. The non-adherent cells produced weekly were predominantly neutrophils (60-70%).
The best conditions for obtaining these cells were the following: 20% DS; 10-7 M HC; 107 mononuclear cells recharged with 4 x 106 mononuclear cells.
The capacity of culture-grown neutrophils to produce oxyradicals (mean value of 3.68 ± 0.26 nmol O2- /min/106 cells) was comparable to that from peripheral blood neutrophils (mean value of 5.09 ± 0.29 nmol O2- /min/106 cells). ATP-production and protein content were also similar in both populations of cells.
The authors demonstrate for the first time that canine neutrophils grown in cultures are able to produce oxyradicals, essential to enable them to carry out their physiological functions.
The experimental model described in this abstract permits correlation between hemopoietic injury and neutrophil functionality, which may be used as a preclinical model and may also have a diagnostic value in different canine pathological conditions.
These findings may help to improve future treatment strategies in cancer patients.