Continuous porcine cell lines developed from alveolar macrophages

Porcine monomyeloid cell lines were established following transfection of primary porcine alveolar macrophage cultures with plasmid pSV3neo, carrying genes for neomycin resistance and SV40 large T antigen. The parental clone 3D4 exhibited a relatively rapid doubling time (25.5 h), high plating efficiency and mixed phenotype with respect to growth on a solid support. Single cell cloning of the 3D4 parent resulted in establishment of several cell lines; three of them designated 3D4/2, 3D4/21 and 3D4/31 were selected for further characterization. All three clones supported the replication of vesicular stomatitis virus (VSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), swine poxvirus, African swine fever virus (ASFV), herpes simplex virus (HSV), parainfluenza virus, bovine adenovirus (BAV), vaccinia virus (VV), and porcine adenovirus (PAV). Under the conditions tested the cells did not support replication of porcine reproductive and respiratory syndrome virus (PRRSV). The swine myeloid character was confirmed for all three clones by fluorescence activated cell scanning (FACS) analysis using monoclonal antibodies 74-22-15 and DH59B, which recognize the pan-myeloid antigen cluster SWC3a. A subpopulation of each cell line was positive for nonspecific esterase activity and phagocytic activity to varying degrees depending on the media formulation. Cells from all three lines exhibited anchorage dependent growth when maintained in RPMI 1640 medium supplemented with 5–15% fetal bovine serum (FBS) and nonessential amino acids. Propagation in commercially formulated serum free media resulted in colony formation and growth in suspension. The addition of dimethyl sulfoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) to serum free media restored cell attachment. DMSO was also able to induce expression of CD14 monocyte marker in the 3D4/31 cell line maintained in FBS containing medium, as determined by FACS with monoclonal antibody CAM36A. Supplementation of RPMI medium with 10% porcine serum upregulated the expression of CD14 and induced expression of porcine macrophage markers recognized by antibodies 2B10 and 2G6 (Vet. Immunol. Immunopathol. 74 (2000) 163) in all three cell lines. The porcine myelomonocytic cell lines obtained may have a wide variety of applications in porcine virology and immunology.