- Email: [email protected]
Use of frozen chick and duck embryo cells for plaque assays of arboviruses and rickettsiae
Journal
of Biological
Standardization
1974 2, 329-332
Use of frozen chick and duck embryo cells for plaque assaysof arboviruses and rickettsiae” Albert T. McManus~, Darrell R. Parkert, Richard H. Kenyont, John P. Kondigt and Gerald A. Eddyt
Conditions for preparation, freezing, storing and plating of chick and duck embryo cell slurries are described. Comparisons of plaque assay sensitivity for fresh and previously frozen cells with Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis and Semliki Forest virus showed, for each virus, that the four classes of cells gave similar titres. Vesicular stomatis virus assayed equivalently in fresh and previously frozen chick cells, but was lower in duck embryo cells. Rickettsia rickettsii showed equal plaquing titre on chick and duck embryo cells.
INTRODUCTION Our experience with using eggs from individual flocks as sources for primary avian embryo tissue cultures has generally been satisfactory. However, on infrequent occasions in the past, our routine cell cultures have shown unpredicted changes in virus substrate usability. Investigations of these interruptions have revealed such causes as viral infection in the flock, physiological variation during flock moults and unexplained causes traceable only to the flock. We have investigated the possibility of preparing stocks of frozen primary avian embryo cells that could be used as interim substitutes for plaque assay substrates during times of flock difficulties. This report describes both the methodology used in our laboratory to prepare frozen chick and duck embryo cells and our experience with using these cells for viral and rickettaial plaque assays. * Received for publication 29 May 1974. t U.S. Army Medical Research Institute Md 21701, U.S.A.
of Infectious
Diseases,
Fort
Detrick,
Frederick, 329
A. T. MCMANUS
MATERIALS
ET
AND
AL.
METHODS
Preparation of cell stocks Chick and duck embryo slurries were prepared by similar methods. Nine-day chick embryo and 13-day duck embryos were used. After eggs were washed with 70% ethanol the air sac end was cut. Embryos were removed, decapitated and placed at room temperature in Puck’s saline A containing 100 units penicillin and 100 pg streptomycin/ml. Groups of 5@-60embryos were minced and embryo pieces washed by gravity sedimentation. The fluid was decanted and 0.25% trypsin in saline A was added at a ratio of 5 ml/chick embryo or 20 ml/duck embryo. The vessel content was then stirred at 37 “C for 30 min. This suspension was filtered twice through three layers of sterile gauze and added to centrifuge bottles. The filtrate was centrifuged at 200 g for 15 min at 4 “C and the supematant was removed by suction. Fifty ml of Medium 199 with Hanks salt supplemented with 10% foetal bovine serum (FBS), 10% dimethyl sulphoxide (Dougherty, 1962) and antibiotics were then added. The pellet was dispersed gently with a syringe and a 15-gauge needle. Cell counts and trypan blue exclusion viability counts were made. After counting, cell numbers were adjusted to 5-7 x 10’ viable cells/ml, packaged into glass ampoules and flame-sealed. Ampoules were then placed into a control rate freezer and cooled at 1 “C/mm to -50 “C. The batch was then placed directly into liquid nitrogen for storage. To recover and plate frozen cells, an ampoule was removed from the liquid nitrogen and thawed in a 37 “C water bath. The ampoule was opened and the cell suspension was immediately placed into 50 ml of cold (4 “C) Medium 199 with Hanks salts and 10% FBS. Cell and viability counts were made, and cells were diluted to 5-7 x 104viable cells/cm2 of desired cell surface. Plaque assays To compare the relative plaque assay sensitivities of frozen and non-frozen chick and duck embryo cells, we made a series of plaque assays on the four categories of cells. Frozen cells were recovered and plated as above. Fresh cells were prepared with the same trypsinization methods but were plated out directly in growth media into 75-cm vessels at 1.2 x lo6 cells/cm2 of growth area and incubated at 37 “C for 48 h prior to use. For virus assay, stock viruses from the Institute’s collection were diluted serially by lo-fold dilutions into cold Hanks balanced salt solution without bicarbonate supplemented with 20 rmw-Hepes, 2% inactivated FBS and antibiotics. Each virus was assayed from the same dilution vial on each of the four classes of cells. The viruses were assayed in duplicate. One ml of virus dilution was added per culture vessel. Flasks were incubated at 36 “C and rocked at 15-min intervals for 1 h. Overlay medium consisted of 1 part 2% Ion agar (Colab, Inc., Chicago Heights, Illinois, U.S.A.) to 1 part 2 X Eagles Basal Medium (pH 7.3) with 40 mM-Hepes and 26 mM-NaHCO,. A final concentration of 5% FBS was added. Cells were overlayed with 30 ml and incubated at 36 “C; 48 h later, 20 ml of Hanks balanced salt solution without phenol red but containing 1 : 10 000 neutral red and 5% FBS was added. Plaques were read 4 h after the addition of stain. Plaque assaysof Rickettsia rickettsii (Sheila Smith strain) were performed essentially by the methods of Weinberg, Stakebake & Gerone (1969) 25-cm2 vessels were used. Diluted rickettsiae were added at 0.1 ml/flask and were adsorbed at room temperature for 30 min. Cells were overlayed with 5 ml of Minimal Essential Medium with Earle’s balanced salts 330
PLAQUE
ASSAYS
USING
FROZEN
AVIAN
CELLS
containing a final concentration of 0.5% agarose (Marine Colloids, Rockland, Maine, U.S.A.). Inoculated flasks were incubated at 35 “C for 5 days. A second agar overlay containing 1 : 10 000 neutral red stain was then added to the flasks and the plaque counts were made the following day. RESULTS Cell yields Yield data are presented to facilitate intralaboratory comparison. For chick embryos, a lo-egg sample showed a mean embryo weight of l-8 g; after decapitation the mean weight was 0.85 g. The same size sample of duck eggs showed a mean embryo weight of 3.8 and 2.2 g, respectively. When 50 embryos were trypsinized as described above, the yield was 1.8 x 10s viable cells/chick embryo and 4.5 x 108viable cells/duck embryo. The packed cell volumes after centrifugation at 200 g for 15 min were 0.16 ml/chick embryo and O-45 ml/duck embryo. Cell freezing and recovery
Sampling of chick and duck frozen cell lots prepared as above has shown about 50% survival of the cell population. An examination of the cell losses at the various preparation stages indicated that the majority of losses were at the concentration and packaging steps. Viability counts on ampoules before and after freezing indicated little loss during freezing and thawing. At the plating concentrations described, monolayers suitable for plaque assays were ready (with a media change at day 5) by one week of incubation. We have found little variation in recovery of frozen cells for periods of up to 18 months of liquid nitrogen storage. Plaque assays
As shown in Table 1, plaque counts were generally comparable between chick and duck cells. The tested group A arboviruses agreed within O-5 log,,, for each strain. Vesicular stomatitis virus showed comparable values in fresh and frozen chick cells, but plaque counts were lower in duck cells. Plaque counts with R. rickettsii showed comparable results with fresh and frozen cells. 1. Viral and rickettsial*
TABLE
plaque assay comparisons
Cell type
EEE
Mean count (log,,)/mlt A WEE VEE SFV
Fresh chick Frozen chick Fresh duck Frozen duck
9.15 9.38 9.25 9.35
9.71 9.90 9.63 10.05
I
8.17 7.99 8.00 8.13
8.53 8-17 8.64 8.51
\ vsv 6.20 5.50 4.25 2.90
RMSF 6.69 6.57 6.78 6.76
* Viruses and Rickettsia: Group A, Eastern equine encephalitis (EEE). Western equine encephalitis (WEE). Venezuelan equine encephalitis (VEE). Semliki Forest virus (SFV). Rhabdovirus, Vesicular stomatitis virus (VSV). Rickettsia, R. rickettsii (Sheila Smith Strain) (RMSF). t Data are presented as the mean of two replicates.
331
A. T. MCMANUS
ET
AL.
DISCUSSION The plaque assaydata presented above were entirely consistent with our general experience. The lowered sensitivity of duck cells for VSV has been repeatedly confirmed with several independently passagedstocks of Indiana strain. Several of our colleagues have performed similar experiments with these and other viruses and found frozen cells acceptable as crisis substitutes for fresh cells in plaque neutralization assays and virus growth curve quantitations. Some investigators have requested and exclusively used the same batch of cells for extended periods. The availability of these stocks offers several advantages: they decrease the probability of work loss during periods of egg supply difficulties; extensive quality control and standardization are possible prior to critical requirements; and frozen stocks are transportable thus making them particularly suitable for laboratories which have no accessto a supply of acceptable eggs for primary avian cell cultures. In summary, we have found that primary chick and duck embryo cell slurries can be stored in liquid nitrogen for periods of up to 18 months. Experience has shown these cells to be acceptable under particular circumstances as substitutes for freshly prepared cells. Acknowledgements We thank Phebe W. Summers and Sheila Gaither for their help in the preparation of this
manuscript. REFERENCES Dougherty, R. N. (1962). Use of dimethyl sulphoxide for preservation of tissue culture cells by freezing. Nature, London 193, 550-552. Weinberg, J. R., Stakebake, J. R. 8z Gerone, P. J. (1969) Plaque assay for Rickettsia rickettsii. Journal of Bacteriology 98, 398-402.
332
of Biological
Standardization
1974 2, 329-332
Use of frozen chick and duck embryo cells for plaque assaysof arboviruses and rickettsiae” Albert T. McManus~, Darrell R. Parkert, Richard H. Kenyont, John P. Kondigt and Gerald A. Eddyt
Conditions for preparation, freezing, storing and plating of chick and duck embryo cell slurries are described. Comparisons of plaque assay sensitivity for fresh and previously frozen cells with Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis and Semliki Forest virus showed, for each virus, that the four classes of cells gave similar titres. Vesicular stomatis virus assayed equivalently in fresh and previously frozen chick cells, but was lower in duck embryo cells. Rickettsia rickettsii showed equal plaquing titre on chick and duck embryo cells.
INTRODUCTION Our experience with using eggs from individual flocks as sources for primary avian embryo tissue cultures has generally been satisfactory. However, on infrequent occasions in the past, our routine cell cultures have shown unpredicted changes in virus substrate usability. Investigations of these interruptions have revealed such causes as viral infection in the flock, physiological variation during flock moults and unexplained causes traceable only to the flock. We have investigated the possibility of preparing stocks of frozen primary avian embryo cells that could be used as interim substitutes for plaque assay substrates during times of flock difficulties. This report describes both the methodology used in our laboratory to prepare frozen chick and duck embryo cells and our experience with using these cells for viral and rickettaial plaque assays. * Received for publication 29 May 1974. t U.S. Army Medical Research Institute Md 21701, U.S.A.
of Infectious
Diseases,
Fort
Detrick,
Frederick, 329
A. T. MCMANUS
MATERIALS
ET
AND
AL.
METHODS
Preparation of cell stocks Chick and duck embryo slurries were prepared by similar methods. Nine-day chick embryo and 13-day duck embryos were used. After eggs were washed with 70% ethanol the air sac end was cut. Embryos were removed, decapitated and placed at room temperature in Puck’s saline A containing 100 units penicillin and 100 pg streptomycin/ml. Groups of 5@-60embryos were minced and embryo pieces washed by gravity sedimentation. The fluid was decanted and 0.25% trypsin in saline A was added at a ratio of 5 ml/chick embryo or 20 ml/duck embryo. The vessel content was then stirred at 37 “C for 30 min. This suspension was filtered twice through three layers of sterile gauze and added to centrifuge bottles. The filtrate was centrifuged at 200 g for 15 min at 4 “C and the supematant was removed by suction. Fifty ml of Medium 199 with Hanks salt supplemented with 10% foetal bovine serum (FBS), 10% dimethyl sulphoxide (Dougherty, 1962) and antibiotics were then added. The pellet was dispersed gently with a syringe and a 15-gauge needle. Cell counts and trypan blue exclusion viability counts were made. After counting, cell numbers were adjusted to 5-7 x 10’ viable cells/ml, packaged into glass ampoules and flame-sealed. Ampoules were then placed into a control rate freezer and cooled at 1 “C/mm to -50 “C. The batch was then placed directly into liquid nitrogen for storage. To recover and plate frozen cells, an ampoule was removed from the liquid nitrogen and thawed in a 37 “C water bath. The ampoule was opened and the cell suspension was immediately placed into 50 ml of cold (4 “C) Medium 199 with Hanks salts and 10% FBS. Cell and viability counts were made, and cells were diluted to 5-7 x 104viable cells/cm2 of desired cell surface. Plaque assays To compare the relative plaque assay sensitivities of frozen and non-frozen chick and duck embryo cells, we made a series of plaque assays on the four categories of cells. Frozen cells were recovered and plated as above. Fresh cells were prepared with the same trypsinization methods but were plated out directly in growth media into 75-cm vessels at 1.2 x lo6 cells/cm2 of growth area and incubated at 37 “C for 48 h prior to use. For virus assay, stock viruses from the Institute’s collection were diluted serially by lo-fold dilutions into cold Hanks balanced salt solution without bicarbonate supplemented with 20 rmw-Hepes, 2% inactivated FBS and antibiotics. Each virus was assayed from the same dilution vial on each of the four classes of cells. The viruses were assayed in duplicate. One ml of virus dilution was added per culture vessel. Flasks were incubated at 36 “C and rocked at 15-min intervals for 1 h. Overlay medium consisted of 1 part 2% Ion agar (Colab, Inc., Chicago Heights, Illinois, U.S.A.) to 1 part 2 X Eagles Basal Medium (pH 7.3) with 40 mM-Hepes and 26 mM-NaHCO,. A final concentration of 5% FBS was added. Cells were overlayed with 30 ml and incubated at 36 “C; 48 h later, 20 ml of Hanks balanced salt solution without phenol red but containing 1 : 10 000 neutral red and 5% FBS was added. Plaques were read 4 h after the addition of stain. Plaque assaysof Rickettsia rickettsii (Sheila Smith strain) were performed essentially by the methods of Weinberg, Stakebake & Gerone (1969) 25-cm2 vessels were used. Diluted rickettsiae were added at 0.1 ml/flask and were adsorbed at room temperature for 30 min. Cells were overlayed with 5 ml of Minimal Essential Medium with Earle’s balanced salts 330
PLAQUE
ASSAYS
USING
FROZEN
AVIAN
CELLS
containing a final concentration of 0.5% agarose (Marine Colloids, Rockland, Maine, U.S.A.). Inoculated flasks were incubated at 35 “C for 5 days. A second agar overlay containing 1 : 10 000 neutral red stain was then added to the flasks and the plaque counts were made the following day. RESULTS Cell yields Yield data are presented to facilitate intralaboratory comparison. For chick embryos, a lo-egg sample showed a mean embryo weight of l-8 g; after decapitation the mean weight was 0.85 g. The same size sample of duck eggs showed a mean embryo weight of 3.8 and 2.2 g, respectively. When 50 embryos were trypsinized as described above, the yield was 1.8 x 10s viable cells/chick embryo and 4.5 x 108viable cells/duck embryo. The packed cell volumes after centrifugation at 200 g for 15 min were 0.16 ml/chick embryo and O-45 ml/duck embryo. Cell freezing and recovery
Sampling of chick and duck frozen cell lots prepared as above has shown about 50% survival of the cell population. An examination of the cell losses at the various preparation stages indicated that the majority of losses were at the concentration and packaging steps. Viability counts on ampoules before and after freezing indicated little loss during freezing and thawing. At the plating concentrations described, monolayers suitable for plaque assays were ready (with a media change at day 5) by one week of incubation. We have found little variation in recovery of frozen cells for periods of up to 18 months of liquid nitrogen storage. Plaque assays
As shown in Table 1, plaque counts were generally comparable between chick and duck cells. The tested group A arboviruses agreed within O-5 log,,, for each strain. Vesicular stomatitis virus showed comparable values in fresh and frozen chick cells, but plaque counts were lower in duck cells. Plaque counts with R. rickettsii showed comparable results with fresh and frozen cells. 1. Viral and rickettsial*
TABLE
plaque assay comparisons
Cell type
EEE
Mean count (log,,)/mlt A WEE VEE SFV
Fresh chick Frozen chick Fresh duck Frozen duck
9.15 9.38 9.25 9.35
9.71 9.90 9.63 10.05
I
8.17 7.99 8.00 8.13
8.53 8-17 8.64 8.51
\ vsv 6.20 5.50 4.25 2.90
RMSF 6.69 6.57 6.78 6.76
* Viruses and Rickettsia: Group A, Eastern equine encephalitis (EEE). Western equine encephalitis (WEE). Venezuelan equine encephalitis (VEE). Semliki Forest virus (SFV). Rhabdovirus, Vesicular stomatitis virus (VSV). Rickettsia, R. rickettsii (Sheila Smith Strain) (RMSF). t Data are presented as the mean of two replicates.
331
A. T. MCMANUS
ET
AL.
DISCUSSION The plaque assaydata presented above were entirely consistent with our general experience. The lowered sensitivity of duck cells for VSV has been repeatedly confirmed with several independently passagedstocks of Indiana strain. Several of our colleagues have performed similar experiments with these and other viruses and found frozen cells acceptable as crisis substitutes for fresh cells in plaque neutralization assays and virus growth curve quantitations. Some investigators have requested and exclusively used the same batch of cells for extended periods. The availability of these stocks offers several advantages: they decrease the probability of work loss during periods of egg supply difficulties; extensive quality control and standardization are possible prior to critical requirements; and frozen stocks are transportable thus making them particularly suitable for laboratories which have no accessto a supply of acceptable eggs for primary avian cell cultures. In summary, we have found that primary chick and duck embryo cell slurries can be stored in liquid nitrogen for periods of up to 18 months. Experience has shown these cells to be acceptable under particular circumstances as substitutes for freshly prepared cells. Acknowledgements We thank Phebe W. Summers and Sheila Gaither for their help in the preparation of this
manuscript. REFERENCES Dougherty, R. N. (1962). Use of dimethyl sulphoxide for preservation of tissue culture cells by freezing. Nature, London 193, 550-552. Weinberg, J. R., Stakebake, J. R. 8z Gerone, P. J. (1969) Plaque assay for Rickettsia rickettsii. Journal of Bacteriology 98, 398-402.
332