Frozen Stored Murine Hybridoma Cells Can Be Used ... - Europe PMC

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1995, p. 3349–3351 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 12

Frozen Stored Murine Hybridoma Cells Can Be Used To Determine the Virulence of Listeria monocytogenes ARUN K. BHUNIA,* DAVID G. WESTBROOK, ROBERT STORY,

AND

MICHAEL G. JOHNSON

Food Safety and Hybridoma Laboratory, Department of Food Science, and University of Arkansas Biotechnology Center, University of Arkansas, Fayetteville, Arkansas 72704 Received 28 June 1995/Returned for modification 8 September 1995/Accepted 27 September 1995

Murine hybridoma cells, designated Ped-2E9, when stored up to 60 days at 2196&C or up to 48 days at 280&C, gave results equivalent to those for freshly grown murine hybridoma cells in an in vitro pathogenicity assay of Listeria species. Thus, laboratories do not need to have their own tissue culture facilities to maintain the hybridoma cells for the assay described. Listeria monocytogenes is a food-borne pathogen and causes listeriosis in immunocompromised humans (5, 9). Clinical manifestations of listeriosis include septicemia, meningitis, brain stem encephalitis, and liver abscess. L. monocytogenes also causes abortion and stillbirth in pregnant women. Animal and tissue culture models are widely used to study the virulence of Listeria spp. The carrageenan-treated immunocompromised murine model is the procedure approved by the Food and Drug Administration to determine the virulence of Listeria isolates (10). However, this procedure takes a long time (at least 3 days) for completion and requires an animal holding facility. Among the tissue culture models, professional phagocytes, such as J774 cells and peritoneal macrophages, and nonprofessional phagocytic cells, such as Caco-2 cells, are widely used (3, 6, 8). These cell lines provide qualitative rather than quantitative information on the virulence potential of Listeria isolates. In a previous study, we reported the use of myeloma and hybrid lymphocytic cell lines from both murine and human sources, which provided quantitative virulence information for a given Listeria sp. in just 6 h (2). In order to make the hybridoma cell model even more userfriendly for virulence tests, we investigated the use of frozen stored (at 280 and 21968C in liquid nitrogen [LN2]) hybridoma Ped-2E9 cells and tested these with both pathogenic and nonpathogenic Listeria spp. The results obtained are very promising and comparable to those with freshly grown hybridoma cells. L. monocytogenes Scott A (4b), ATCC 19116 (4c), and CHLR 587 (this untyped clinical isolate was kindly provided by Gordon Schutze, Children’s Hospital, Little Rock, Ark.), and L. innocua F4248 and L. welshimeri ATCC 35897 were used in this study. Listeria cultures were tested for hemolysin production on sheep blood agar prior to use in the hybridoma tissue culture assay. Murine hybridoma Ped-2E9 cells were originally developed in our laboratory by fusing myeloma (NS1) cells with spleen cells (1) and stored in an LN2 tank for future use. For hybridoma cell growth, Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, Ga.), hypoxanthine, and thymidine (DMEM-HT; Sigma) was used. Freezing hybridoma cells. One vial of LN2-stored hybridoma Ped-2E9 cells was rapidly thawed at 378C, and the cells

were allowed to grow in 10 ml of DMEM-HT in a humidified incubator maintained at 378C and 7% CO2. The Ped-2E9 cells were propagated in four T-150 tissue culture flasks (Costar, Cambridge, Mass.) in about 500 ml of DMEM-HT. The logphase (48 h) cells were collected from the flasks, and viable cells were counted, centrifuged at 300 3 g for 10 min, and resuspended with fresh DMEM-HT to obtain a cell population of 3 3 106/ml. The hybridoma cells were aliquoted (1 ml per vial) in several cryovials (13 by 80 mm, 4.5-ml tube; West Coast Scientific, Hayward, Calif.). A 0.075-ml amount of dimethyl sulfoxide (DMSO; Sigma) was added to each vial as a cryoprotectant, and vials were stored at 2808C in a freezer (7). About half of the vials were removed from the 2808C freezer after 12 h and placed in an LN2 tank, and the remaining half were left in the 2808C freezer until used. Reports indicate that hybridoma cells remain viable for more than 5 years when stored in LN2 and for 1 to 6 months when stored at 2808C in a freezer (4, 7). In this study we found that a few hybridoma cells died because of freezing and thawing after a maximum storage length of 60 days. LN2-stored cells showed a higher level of death (about 1 3 106 to 1.5 3 106/ml) than cells stored in a 2808C freezer, whose mortality levels ranged from 0.2 3 106 to 0.5 3 106/ml. From our previous study we found that a hybridoma cell population of about 2 3 106/ml provides the best virulence assay results (2). Therefore, hybridoma cell populations of about 3 3 106/ml per vial were used for freezing, assuming that about one-third of the cell population or about 106 would die because of freezing and thawing. That left a final viable cell population of about 2 3 106/ml per vial. Hybridoma cell assay. Several vials of hybridoma cells from a 2808C freezer or LN2 tank were retrieved and thawed rapidly (for 1 min) in a 378C water bath. Three milliliters of fresh DMEM-HT was added immediately to each vial, and hybridoma cells were harvested by centrifugation at 300 3 g for 10 min. The supernatant from each vial was discarded, and the hybridoma cell pellet was resuspended in 1 ml of DMEM-HT. Listeria cultures to be inoculated into Ped-2E9 cells were prepared essentially by the method described previously (2). Briefly, overnight-grown cultures of L. monocytogenes Scott A, ATCC 19116, and CHLR 587 were washed once with 20 mM phosphate-buffered saline (PBS), pH 7.0. A 0.1-ml amount of bacterial suspension (approximately 109 CFU/ml) was added to each properly marked hybridoma cell vial to obtain a multiplicity of exposure of 100 bacterial cells to 1 hybridoma cell. Listeria-inoculated hybridoma cells were incubated in a 378C bacteriological incubator for 6 h with occasional mixing. Con-

* Corresponding author. Mailing address: Food Microbiology and Immunochemistry Lab, Dept. of Food Science and Animal Industries, Alabama A & M University, P.O. Box 264, Huntsville (Normal), AL 35762. Phone: (205) 851-5445. Fax: (205) 851-5432. 3349

3350

NOTES

J. CLIN. MICROBIOL. TABLE 1. Viable cell counts and cytotoxicity assay results with hybridoma Ped-2E9 cellsa 16 days

Culture

Controle L. innocua F4248 L. welshimeri ATCC 35897 L. monocytogenes Scott A ATCC 19116 CHLR 587

32 days

60 days

Treatmentb

No. of cells (105) 6 SD (%)c

% LDHd

No. of cells (105) 6 SD (%)

% LDH


% LDH

21968C Fresh 21968C Fresh 21968C Fresh 21968C Fresh 21968C Fresh 21968C Fresh

18.5 6 1.8A,f (0) 25.3 6 9.8B (0) 14.8 6 1.6A (20) 23.8 6 1.8B (6) 18.1 6 3.5A (2) 27.2 6 0.8B (0) 0.5 6 0.1C (97) 0.9 6 0.1C (97) 0.6 6 0C (97) 1.5 6 0C (94) 0.6 6 0.1C (97) 0.3 6 0.1C (99)

45 13 47 24 47 4 83 93 96 93 95 95

14.9 6 2.7A (0) 22.5 6 3B (0) 16.5 6 0.5A (0) 19.9 6 0.6B (12) 14.5 6 1.3A (3) 24.9 6 0.7B (0) 0.8 6 0.1C (95) 3.5 6 0.4C (85) 1.3 6 0.4C (91) 0.9 6 0.1C (96) 2.2 6 0.3C (85) 1.1 6 0.1C (95)

40 17 54 46 41 20 94 97 91 80 95 82

19.1 6 0.3A (0) 22.7 6 1.2B (0) 18.6 6 1.1A (3) 20.8 6 0.4B (8) 13.2 6 0.4A (30) NTg 0.3 6 0.1C (99) 0.45 6 0.1C (98) 0.6 6 0.2C (97) 0.9 6 0.1C (96) 0.6 6 0.1C (97) NT

64 21 65 35 61 NT 76 85 92 NT 100 NT

Hybridoma Ped-2E9 cells were stored in LN2 (at 21968C) for 16, 32, or 60 days and then exposed to Listeria spp. for 6 h. Listeria strains were added to fresh hybridoma cells or hybridoma cells stored at 21968C at a ratio of 100:1. Viable cell counts after 6 h of exposure to Listeria spp. are presented as means 6 standard deviations. Numbers in parentheses represent percent dead cells compared with the dead cells of untreated controls. Counts were obtained from two separate experiments, and each was performed in duplicate. The average hybridoma cell count after 0 h was about 19.8 3 105/ml for LN2-stored cells and 25.0 3 105/ml for fresh hybridoma cells. d The percentages of LDH release represent the averages of two experiments performed on two separate occasions. The LDH released by Triton X-100 (1%)-treated hybridoma cells was considered 100% LDH activity. e The control contained hybridoma cells and PBS. f Means marked with the same letter (A, B, or C) are not significantly different (P , 0.05). g NT, not tested. a b c

trol hybridoma vials received either 0.1 ml of L. innocua or L. welshimeri or PBS. The virulence of Listeria spp. on hybridoma cells was measured by the trypan blue exclusion test or a lactate dehydrogenase (LDH) release assay (cytotoxicity assay). An aliquot of 0.1 ml of cell suspension was withdrawn from each vial at 0 and 6 h, and viable cell counts were performed by trypan blue (0.4%) staining (2). For LDH release assay, 0.2 ml of hybridoma cell suspension was withdrawn from each vial after 6 h of incubation and centrifuged at 300 3 g for 5 min and the supernatant was assayed for the release of cellular LDH enzyme from hybridoma cells according to the procedure described previously (2, 11). Virulence assays of Listeria spp. were carried out on days 16,

32, and 60 for LN2-stored hybridoma Ped-2E9 cells and on days 20, 32, and 48 for 2808C-freezer-stored cells. Results were compared with the results of virulence assays with laboratory-maintained fresh Ped-2E9 cells, which were run concurrently. L. monocytogenes killed about 97% of the cells stored for 16 days in LN2 in 6 h, whereas L. innocua and L. welshimeri caused only 2 to 20% hybridoma cell death (Table 1). Cytotoxicity assay results indicated that L. monocytogenes caused an 83 to 96% release of cellular LDH, whereas L. innocua and L. welshimeri caused only about a 47% release of LDH versus a 45% LDH release for controls. The viability counts and cytotoxicity assay results were compared with results with freshly

TABLE 2. Viable cell counts and cytotoxicity assay results with hybridoma Ped-2E9 cellsa 20 days Culture

Controle L. innocua F4248 L. welshimeri ATCC 35897 L. monocytogenes Scott A ATCC 19116 CHLR 587

Treatmentb

2808C Fresh 2808C Fresh 2808C Fresh 2808C Fresh 2808C Fresh 2808C Fresh

5

32 days 5

48 days

No. of cells (10 ) 6 SD (%)c

% LDHd

No. of cells (10 ) 6 SD (%)

% LDH


% LDH

27.8 6 2.8A,f (0) 18.7 6 2.6B (0) 24.4 6 2.4A (12) 13.7 6 0.8B (27) 24.2 6 0.2A (13) 17.9 6 0B (4) 2.1 6 0.4C (92) 0.6 6 0C (97) 2.9 6 0.7C (90) 1.8 6 0.2C (90) 3.5 6 0.5C (87) 1.8 6 0.3C (90)

30 11 40 4 35 20 87 69 87 86 100 90

25.0 6 0.9A (0) 23.6 6 0.8A (0) 22.3 6 2.1A (11) 22.95 6 1.1A (3) 23.9 6 1.4A (4) 19.6 6 0.5A (17) 0.5 6 0.5C (98) 0.1 6 0C (99) 1.2 6 0.1C (95) 0.1 6 0C (99) 1.5 6 0.1C (94) 0.4 6 0C (98)

40 12 43 19 35 92 81 77 93 83 75 82

26.8 6 1.4A (0) 20.3 6 1.3A (0) 22.6 6 0.8A (16) 20.9 6 0.3A (0) NTg NT 0.3 6 0.1C (99) 0.4 6 0C (98) 1.5 6 0.3C (94) 0.6 6 0.1C (97) NT NT

23 13 29 19 NT NT 85 89 90 84 NT NT

Hybridoma Ped-2E9 cells were stored at 2808C for 20, 32, or 48 days and then exposed to Listeria spp. for 6 h. Listeria strains were added to fresh or stored (at 2808C) hybridoma cells at a ratio of 100:1. c Viable counts of hybridoma cells are presented as means 6 standard deviations. Numbers in parentheses represent percent dead cells compared with dead cells in untreated controls. Counts are the averages of two separate experiments, and each was performed in duplicate. d LDH release was measured spectrophotometrically. e The control contained hybridoma cells and PBS. f Means marked with the same letter (A, B, or C) are not significantly different (P , 0.05). g NT, not tested. a b

VOL. 33, 1995

grown hybridoma cells. Percent viable hybridoma counts in the presence of Listeria spp. were very similar for freshly prepared and for LN2-stored cells. When cytotoxicity results were compared, fresh control hybridoma cells showed less LDH release than LN2-stored cells. This latter result was expected since control LN2-stored cells had higher numbers of dead cells, which increased the level of residual LDH activity. Virulence assays of Listeria spp. with 32- and 60-day-old LN2-stored hybridoma cells yielded results very similar to those for cells stored for 16 days (Table 1). Freezer (2808C)-stored hybridoma Ped-2E9 cells were also sensitive to L. monocytogenes infection (Table 2). Virulence assays conducted with hybridoma cells stored for 20 days showed that 87 to 100% of hybridoma cells were killed after 6 h of exposure to L. monocytogenes. Conversely, about 12 to 13% of cells died after exposure to L. innocua or L. welshimeri. Cytotoxicity assay results were also comparable to the results of the viable cell count assays. Similarly, hybridoma cells stored for 32 or 48 days were equally sensitive to pathogenic Listeria spp. Hybridoma cells stored in LN2 at either 21968C or 2808C did not show any difference in their viability patterns in the presence of Listeria spp. The above studies showed that hybridoma cells stored at 2808C are also sensitive to pathogenic Listeria spp. when compared with freshly grown hybridoma cells. Tissue culture assays may often be used as alternatives to animal model assays to rapidly identify the virulence potential of a given pathogenic microorganism. However, tissue culture is an expensive technique, requiring specialized equipment and growth media. In this study we used previously frozen hybridoma cells to assess the virulence of L. monocytogenes. We found that hybridoma cells frozen in LN2 or at 2808C could be used immediately without subculturing and these cells showed the same patterns of susceptibility to L. monocytogenes and less virulent Listeria spp. as fresh hybridoma cells. Also, virulence results can be obtained in 6 h, thus eliminating the need for on-site tissue culture facilities and other supplies usually required to maintain hybridoma cells. Some major advantages of using the hybridoma cell model over other tissue culture models for virulence tests are as follows. (i) Hybridoma cells grow rapidly (with a doubling time of approximately 15 h) and are easy to handle. (ii) Viable

NOTES

3351

hybridoma cells can be easily and unequivocally counted and differentiated from dead cells by trypan blue staining, with dead cells appearing dark blue and live cells remaining transparent. (iii) The virulence of L. monocytogenes isolates can be detected in 6 h. (iv) Frozen hybridoma cells described here can readily be used to test virulence of any suspected Listeria culture. This work was supported in part by funds from the USDA-NRI (92-37201-8175) Food Safety Competitive Grant Program and the USDA-CSRS Food Safety Consortium. REFERENCES 1. Bhunia, A. K., and M. G. Johnson. 1992. Monoclonal antibody-colony immunoblot method specific for isolation of Pediococcus acidilactici from foods and correlation with pediocin (bacteriocin) production. Appl. Environ. Microbiol. 58:2315–2320. 2. Bhunia, A. K., P. J. Steele, D. G. Westbrook, L. A. Bly, T. P. Maloney, and M. G. Johnson. 1994. A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources. Microb. Pathog. 16:99–110. 3. Cossart, P., and J. Mengaud. 1989. Listeria monocytogenes, a model system for the study of intracellular parasitism. Mol. Biol. Med. 6:463–474. 4. Doyle, A., and C. B. Morris. 1991. Maintenance of animal cells, p. 227–241. In B. E. Kirsop and A. Doyle (ed.), Maintenance of microorganisms and cultured cells. Academic Press, New York. 5. Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476–511. 6. Gaillard, J.-L., P. Berche, J. Mounier, S. Fichard, and P. Sansonetti. 1987. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. 55:2822– 2829. 7. Gustafson, B. 1989. Cryopreservation of hybridomas, p. 619–621. In J. W. Pollard and J. M. Walker (ed.), Animal cell culture. Humana Press, Clifton, N.J. 8. Pine, L., S. Kathariou, F. Quinn, V. George, J. D. Wenger, and R. E. Weaver. 1991. Cytopathogenic effects in enterocyte-like Caco-2 cells differentiate virulent from avirulent Listeria strains. J. Clin. Microbiol. 29:990–996. 9. Schuchat, A., B. Swaminathan, and C. V. Broome. 1991. Epidemiology of human listeriosis. Clin. Microbiol. Rev. 4:169–183. 10. Stelma, G. N., Jr., A. L. Reyes, J. T. Peeler, D. W. Francis, J. M. Hunt, P. L. Spaulding, C. H. Johnson, and J. Lovett. 1987. Pathogenicity test for Listeria monocytogenes using immunocompromised mice. J. Clin. Microbiol. 25:2085–2089. 11. Taffs, R., and M. Sitkovsky. 1993. Granule enzyme exocytosis assay for cytotoxic T lymphocyte activation, p. 3.16.5–3.16.6. In J. E. Colligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology. Wiley Interscience, New York.