ESTABLISHMENT OF PORCINE MONOCYTE-LIKE CELL LINE

摘要

A monocyte-like cell line was established from peripheral blood sample of a healthy male pig in late 1999. Some cytological properties examined were presented. Leucocyte culture: The leucocytes fraction was obtained from blood sample by a gradient centrifugation with Lymphoprep (density: 1.077 g/ml, Nycomed, Pharma, AS). The mixed population of cells was suspended in the cell growth medium (CGM) at ca. 1,000,000 cells per ml and dispensed into glass flasks (culture dimension: 4 x 8 cm) at 10 ml to make stationary culture at 36.5C. The basal cell culture medium was a slight modification on the Dulbecco's modified Eagle medium. The further additives were 1 g of glucose, 2 g of galactose, 1 g of fructose, 0.2 g of HEPES, and twice amount of sodium pyruvate and N-acethyl-D(+)-glucosamine in 1,000 ml. In CGM, heat inactivated fetal bovine serum (FBS) was included at 10-20%. Antibiotics were added as usual. One day after the incubation, the primary cultures were rinsed with pre-warmed CGM to remove non-adhesive cells. After that ca. 30% of CGM was replaced with fresh one every 3-4 days during the incubation. At two weeks incubation, a conditioning medium collected from a canine monocyte-like cell culture (Kadoi's unpublished work) was partially added since it was filtered by a membrane filter (220 nm in pore size). Two days after the addition of the conditioning medium, several cell clones were started to grow actively. One of the clones, SW/K99, was serially passaged. After 6th passage level, the amount of FBS included in CGM was decreased at 10% since it was enough for cell growth.