Growth of Fibroblast (CFU-F) And Macrophage Colonies in Liquid Cultures From Canine Peripheral Blood
*Pablo Gomez Ochoa, Juan Antonio Castillo Hernandez, Manuel Gascon Perez, Javier Lucientes Curdi, Cristina Rodriguez Serrano, Juan Jose Zarate Ramos
Growth of marrow-derived stromal cells in culture is well and extensively documented; nevertheless, knowledge about data from fibroblasts proliferation, coming from mononuclear cells culture of peripheral blood, is scarcely spread out.
Therefore, objectives of the work hereby described, included: demonstration of fibroblasts growth in peripheral blood culturing, searching for objective criteria for fibroblasts and macrophages quantification, and their application in the immune response evaluation.
Samples were taken, to carry out this task, from jugular vein of 5 healthy dogs and 2 leishmaniasic dogs, with conservative free heparin, separating mononuclear cells by centrifugation in a 1077 density gradient. Each sample was sown in triplicate -1000000 cells per plate- in McCoy´s 5a culture medium and 3 different options were performed, adding it:
1. 15% of heat inactivated foetal bovine serum (0.45 ml).
2. 5% of serum from same sample (0.15ml).
3. 5% of serum from another sample (0.15ml).
Incubation was performed in an environment of 5% CO2, at 37°C, and 100% relative humidity. After seven days, half culture medium was renewed.
An inverse microscopy was used for reading the culture at 10th and 14th days, evaluating the following parameters: 1- total growth (quantifying between 0- no growth- and 3 -all the plate covered by cells-), 2- fibroblasts/macrophages relationship and 3- fibroblastic confluence degree (CFU-F developing)
Samples from healthy dogs: a growth in macrophages and fibroblasts was achieved in all three culture plates, in a sheet-shaped distribution.
No differences were recorded in total growth between (B) and (C) options- level 2 after ten days and level 3 in the 14th day. (A) option, added with foetal bovine serum, only achieved level 1 of growth after 14 days of culturing. Fibroblasts/ macrophages relationship was 1:3 in all the samples and options. Confluence of fibroblasts, with colonies development (CFU-F) only was recorded in (B) and (C) options after 14 days of culturing.
Samples from leishmaniasic dogs: except for fibroblasts/ macrophages relationship (1:10) no differences were shown in the rest of growth parameters.
The culture in liquid medium of peripheral blood's mononuclear cells, has demonstrated itself as a very efficient method to obtain macrophages and fibroblasts.
Using of autologous, or other dog's serum, gives much better results than using foetal bovine serum, that is, macrophage and fibroblast population is able to cover all the plate (level 3 of growth), meanwhile fibroblasts/macrophages relationship was preserved.
In the dog's samples coming from infectious ones, a big rise in macrophage proliferation was recorded, that underwrites the utility of culture in liquid medium, as an outstanding pattern to evaluate cell-mediated immune response.