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Updated: Jul 24, 2026
Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies
Published on: December 26, 2012
Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility
H M Weingartl1, M Sabara, J Pasick
1NCFAD, CFIA, 1015 Arlington St., Winnipeg, MB, Canada R3E 3M4. hweingartl@inspection.gc.ca
Abstract:
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.
Insights
New porcine monomyeloid cell lines were developed and characterized. These cell lines support the replication of numerous swine viruses, offering valuable tools for porcine virology and immunology research.
Area of Science:
- Veterinary Virology
- Cell Biology
- Immunology
Background:
- Primary porcine alveolar macrophage cultures are essential for studying swine viral infections.
- Establishing stable, characterized cell lines is crucial for reproducible research in porcine virology and immunology.
Purpose of the Study:
- To establish and characterize novel porcine monomyeloid cell lines.
- To assess the susceptibility of these cell lines to a broad range of swine viruses.
- To confirm the myeloid lineage and functional characteristics of the established cell lines.
Main Methods:
- Transfection of primary porcine alveolar macrophages with pSV3neo plasmid.
- Single-cell cloning to derive stable cell lines (3D4/2, 3D4/21, 3D4/31).
- Viral replication assays, fluorescence-activated cell scanning (FACS) for myeloid markers, esterase and phagocytic activity assays, and growth characteristic analysis under different media conditions.
Main Results:
- Three stable porcine monomyeloid cell lines (3D4/2, 3D4/21, 3D4/31) were successfully established.
- These cell lines supported the replication of a wide array of viruses including VSV, PRV, CSFV, SVDV, swine poxvirus, ASFV, HSV, parainfluenza virus, BAV, VV, and PAV, but not PRRSV.
- FACS analysis confirmed swine myeloid character (SWC3a), and cells exhibited varying degrees of esterase and phagocytic activity. Anchorage-dependent growth was observed in RPMI 1640 with FBS, while serum-free media allowed suspension growth, with attachment restored by DMSO or PMA. DMSO and porcine serum induced CD14 expression, and porcine serum upregulated macrophage markers.
Conclusions:
- The established porcine monomyeloid cell lines are robust and versatile tools for virological studies.
- These cell lines demonstrate susceptibility to a broad spectrum of swine viruses, excluding PRRSV.
- The characterized myeloid and macrophage markers indicate their utility in diverse applications within porcine virology and immunology.
