Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 11/14/2016
Publication Date: N/A
Technical Abstract: Currently, American Type Culture Collection (ATCC) makes available two cell lines derived from the same lymphoblast-like suspension cell that have been confirmed by next-generation sequencing and RT-PCR to have either a single contaminate of BVDV2a (CRL-8037) or dual contaminates of both BVDV and BLV (CRL-2306). We cloned a cell line from ATCC stock that is free of BVDV and BLV (NADC-BL3-SF). Cell lines 8037 and 2306 and NADC-BL3-SF were evaluated by flow cytometry and transmission electron microscopy (TEM) to determine if contamination was associated with morphological differences. Flow cytometric analysis was conducted and forward and side scatter plots (scale 0-250 x 1,000) revealed two discrete populations of different sizes in CRL-8037 and NADC-BL3-SF cultures and one population with a range of different sized cells in CRL-2306 cultures. A minimum of 1 x 106 cells identified as small or large, respectively, were sorted into media and subjected to morphometric assessment by TEM. Sorted cells identified as small were measurably smaller in size as demonstrated by ellipse circumference (EC) measurement. Values for EC in the small and large cell populations for each cell line were: NADC-BL3-SF, 30.53 µm and 34.75; 8037, 31.49 and 37.79; and 2306, 35.26 and 37.98, respectively. Size was positively associated with increased levels of BLV based on staining using the BLV Mab gp51-G (IgG1). Cells sorted by intensity of BLV staining (BLV+ enriched) had the largest EC with a value of 41.08. Assessment of the small and large populations present in cultures of each cell line provided further evidence of morphological differences. Virus measuring 40-60 nm (consistent with the size of BVDV) was observed in membrane bound bodies by TEM in the BLV+ enriched cells from the 2306 cell line and cells from the smaller population of 8037 cultures. This data suggests contaminated cultures exhibit differences in cell morphology. Differences in cell morphology could be associated with differences in cell function suggesting that research conducted with contaminated cells could affect the outcome.