Culture of Canine Hematopoietic Progenitor Cells in Methylcellulose Medium: A Reproducible Cloning Assay Cells in Methylcellulose Medium
*Pablo Gomez Ochoa, Juan Antonio Castillo, Manuel Gascon Perez, Javier Lucientes Curdi, Cristina Rodriguez Serrano, Jose Ignacio Arbea Sarasa
Peripheral blood in semisolid media achieves the study of the hematopoiesis and its pathology, moreover it gives a very relevant experimental model to evaluate blood-toxicity phenomenon. Nevertheless, these techniques have a limited using in dogs because of standardization difficulties, that were this assay objectives. First of all the selection of culture medium and later the using of recombinant human citoquines to quantify the stimulation produced, follows by sowing´s methodology, and colonies evaluation criteria, clusters and other growth pattern, that impedes the reproducibility of results.
Samples were taken from peripheral blood and bone marrow from 10 healthy dogs including these in the following procedure: isolation of mononuclear cells by centrifugation in a 1077 density gradient, 400G during 30 minutes, and washed three times with McCoy´s 5 A, to culture them, 150000 cells per plate were sown in standard culture medium with methylcellulosa, foetal bovine serum, 2 mercaptoethanol, L-glutamine and bovine pancreatic insulin -MethoCult de StemCell Tech-. Two options, adding different kind of growth factors (A and B) were performed in all the samples:
a. rhStem Cell Factor(50ng/ml), rh GM-CSF(10ng/ml), rh IL-3(10ng/ml), rh IL-6(20ng/ml), rh G-CSF(20ng/ml), rh EPO(3 units/ml).
b. The same as A but without EPO.
Incubation was made at 37°C in an environment of 5% CO2 and 100% relative humidity during 14 days. The reading with inverse microscope, evaluated the existence of colonies (more than 50 cells), clusters (between 10 and 50 cells), and the growth pattern of each plate.
In peripheral blood the next mean values were obtained for each 150000 cells sown.
Option (A): CFU-GM 5, CFU-E 2, Clusters 10. Growth sheet-shaped areas that included a big amount of haemoglobinized elements.
Option (B): CFU-GEMMk 1, CFU-GM 3, Clusters 12. Growth sheet-shaped areas of myeloid elements. Option (B) added with transferrin (200 mcg/ml) and CSF-G( 20ng/ml). There wasn't any growth of colonies, or clusters.
Results from samples taken from bone marrow were 10 times more than peripheral blood ones, but with relevant individual variations.
The methylcellulosa culture media added with foetal bovine serum, 2 mercaptoethanol, L-glutamine, and bovine pancreatic insulin, are an optimal support to evaluate hematopoietic progenitors both peripheral blood and bone marrow.
Human citoquines- Stem Cell Factor, CSF-GM, CSF-G, IL-3, IL-6, and EPO- get a stimulant growth effect in the in vitro canine hematopoiesis. However adding human transferrin, culture growth was annulled.
Using culture media with standardized levels of citoquines (MethoCult StemCell) attain the reproducibility of results that could be applied to canine hemopathies.