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Rapid screening of tissue culture cells for mycoplasmal contaminants
Journal
of Bio&cal
Standardization
1975 3, 181-184
Rapid screening of tissue culture cells for mycoplasmal contaminants.* A. J. Garvettt and Diane E. Reesont
Mycoplasmal contaminants alter the metabolism of uridine and uracil in tissue culture cells. A new technique for the detection of mycoplasma that exploits these changes has been modified to enable many samples to be monitored quickly and easily.
INTRODUCTION The hazards of mycoplasmal contamination of tissue culture cells are well-known and were reviewed recently by Stanbridge (1971), Fogh (1973) and Kenny (1973). Rapid detection of contaminants of cell substrates used for vaccine production is, of course, extremely important, but mycoplasmal infection is insidious and often discovered only by microbiological tests long after the tissue cells have become useless for their designated purpose (Rothblat, 1960; Paton, Jacobs & Perkins, 1965). Furthermore, some strains of mycoplasma do not grow on the media at present used for this detection (Fogh & Fogh, 1969 ; Markov, Bradvarova, Mintcheva, Petrov, Shishkov & Tsanev, 1969 ; Todaro, Aaronson & Rands, 1971). Levine (1972) and Perez, Kim, Gelbard & Djordjevic (1972) showed that mycoplasma increase levels of uridine phosphorylase in mammalian cell cultures and confer an ability to incorporate free nucleoside bases into RNA. Schneider, Stanbridge & Epstein (1974) exploited these two characteristics and demonstrated that the ratio of the specific activities of rH]uridine to pH]uracil incorporated into RNA was greatly reduced when cells were infected with mycoplasma. We describe in the present paper modifications of this method that allow the rapid determination of uridine and uracil incorporated into small samples of cells. * Received for publication 19 November 1974. f Division of Viral Products, National Institute for Biological Laboratories, Holly Hill, London NW 6RB, England.
Standards and Control,
Hampstead
181
A. J. GARRETT
MATERIALS
AND
AND
D.
E. REESON
METHODS
Cell cultures Cells were grown at 37 “C in 30-ml plastic flasks (Falcon Plastics Ltd) or in lo-ml flat-bottomed plastic tubes (Conway tubes, Turner Stayne Laboratories, Bishop Auckland, Co. Durham) containing 13-mm diameter glass coverslips in Eagle’s basal medium containing 15 mM-HEPES-NaOH buffer, 10% calf serum (Sera Services Ltd, Maidenhead, Berks) and 100 units/ml benzyl penicillin. The cells were seeded at concentrations sufficient to give 2040% coverage of the available surface when radioactive labelling was begun. Cell suspensions (1.0 ml) were added to each Conway tube. Incorporation of radioactive &dine and uracil [5-3H]uridine (27 Ci/mmole), [5-sH]uracil (24 Ci/mmole) and [2-14C]uracil (64 mCi/ mmole) were obtained from the Radiochemical Centre, Amersham, Bucks. Medium was aspirated from ,the cell cultures after 24 h growth and replaced with the same formulation containing RNA precursor(s) at 1 $X/ml. Radioactive labelling was terminated after 5 h or 18 h by washing the cell monolayer in the Conway tubes with cold phosphatebuffered saline (Dulbecco & Vogt, 1954) and adding 1 ml of cold 10% (w/v) trichloroacetic acid (TCA). The TCA was aspirated after 1 h at 4 “C, the cells were washed three times with cold 10% TCA, twice with ethanol and once with methanol. It was important to ensure that the alcohol washes removed all uracil that was non-specifically bound to the TCA-insoluble material. The discs were removed from the tubes, dried and counted in a Packard Spectrometer, model 3375, in scintillation fluid containing 4 g of 2,5benzene diphenyloxazole (PPO) and 100 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl) (POPOP)/litre of toluene. Cells grown in bottles were washed and removed from the plastic with pronase. They were then precipitated with TCA and extracted with perchloric acid as described by Schneider et al. (1974). The perchloric acid extracts were neutralized with KOH and samples of the supernatant fluid were counted in scintillation solution containing 8 g of PPO, 200 mg of POPOP and 600 ml of methanol/1400 ml of toluene. In those experiments using cells labelled simultaneously with rH]uridine and [14C]uracil, control cell cultures were labelled with 14C alone and used to measure the ‘spillover’ of 14C into the 3H counting channel. There was no overlap of 3H into the 14C counting channel. Mycoplasma strains Mycoplasma hyorhinis, M. arginini and Acholepkuma granularurn which were kindly provided by Dr Ruth Lemcke (Lister Institute of Preventive Medicine, Chelsea Bridge Road, London SWlW 8RH) were maintained in broth cultures. Tissue cells were examined no less than seven days after infection. Microbiological tests for mycoplasma used the agar and broth preparations of Hayflick (1965). RESULTS
AND
DISCUSSION
The results presented in Tables 1 and 2 confirm that measurement of the uptake of radioactive uridine and uracil in tissue cell cultures gives a rapid assessment of the presence or absence of mycoplasmal contaminants. Uridine : uracil ratios of less than 100 correlated with mycoplasmal contamination. Early detection of contaminants is 182
RAPID
SCREENING
FOR MYCOPLASMA
CONTAMINANTS
vitally important where large quantities of cells are employed, such as in vaccine production, and the methods we have described enable tissue culture cells to be screened more rapidly than has previously been possible. Results are obtained within 48 hours of plating the cells on coverslips. Very small quantities of cells (2 x 104/tube) can now be TABLE
Relative
1.
Mycoplasma contaminant
Cells MRC-.5* WI-38* WI-38 WI-38 WI-38 Hep-2 Hep-2 Hep-2 Vero
incorporations of [SH]uridine tissue cell cultures
None None None M. hyorhinis M. arginini None A. granularurn M. hyorhinis Unidentified
[3H]uracil
Uridine/ uracil
Fraction tested HCIOl HCIOl TCA TCA TCA TCA TCA TCA TCA
and
extract extract ppt ppt ppt ppt ppt ppt ppt
into
Microbiological test + + + + +
840 710 770 1 91 1023 63 2 1
* Cells were grown in 30-ml bottles ; other samples were grown in lo-ml flat-bottomed tubes. TABLE
2. Relative incorporations of [3H]uridine cell cultures Cells
Mycoplasma contaminant
3H/‘4C
MRC-5 WI-38 .Hep-2 Hep-2 ‘Vera
None None None M. hyorhinis Unidentified
952 602 1189 2 2
and [l’C]uracil
into tissue
Microbiological test
+ +
monitored, large numbers of samples can be handled easily because all washing steps are completed by aspiration and not by centrifugation, and measurement of the incorporation of r4C] uracil and rH]uridine into the same sample obviates the need to measure specific activities of RNA. However, in our experiments using rH]uracil and rH]uridine in separate samples (Table l), the great differences in the uridine : uracil ratios between contaminated and uncontaminated cultures made chemical measurement of RNA unnecessary. Schneider & Stanbridge (1975) have studied the simultaneous labelling of cells with [14C]uridine and PH] uracil; their procedure may be difficult to use when small amounts of uracil are incorporated and counts in the 3H counting channel are swamped by the overlap of 14C. These tests alone cannot identify contaminants, of course, but they do indicate potential dangers to the cell culturist. At present mycoplasmal strains can only be identified by cultural tests but, as some grow poorly or not at all in the standard broths and agars, Stanbridge & Schneider (1974) have proposed that the presence of mycoplasma is indicated by a characteristic alteration of the metabolism of uridine and uracil in tissue culture cells and by the appearance on polyacrylamide gel electrophoretograms of 23 S, and 16 S, ribosomal RNA species. 183
A. J. GARRETT
AND
D. E. REESON
Acknowledgement We thank Dr Eric Stanbridge work.
for supplying
us with information
from his unpublished
REFERENCES Dulbecco, R. & Vogt, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. Journal of Experimental Medicine 99, 167-182. Fogh, J. (1973). In Contamination in Tissue Culture Chapter 7, pp. 173-194 (Fogh, J., Ed.). New York and London: Academic Press. Fogh, J. & Fogh, H. (1969). Procedures for control of mycoplasma contamination of tissue cultures. Annals of the New York Academy of Sciences 172, 1S-30. Hayflick, L. (1965). Tissue cultures and mycoplasmas. Texas Reports on Biology and Medicine 23 (Suppl. l.), 285-303. Kenny, G. E. (1973). In Contamination in Tissue Culture Chapter 5, pp. 107-129 (Fogh, J., Ed.). New York and London: Academic Press. Levine, E. M. (1972). Mycoplasma contamination of animal cell cultures; a simple, rapid detection method. Experimental Cell Research 74, 99-109. Markov, G. G., Bradvarova, I., Mintcheva, A., Petrov, P., Shishkov, N. & Tsanev, R. G. (1969). Mycoplasma contamination of cell cultures. Interference with 38P-labelling pattern of RNA. Experimental Cell Research 57, 374-384. Paton, G. R., Jacobs, J. P. & Perkins, F. T. (1965). Chromosome changes in human diploid cell cultures infected with Mycoplasma. Nature 207, 43-45. Perez, A. G., Kim, J. H., Gelbard, A. S. & Djordjevic, B. (1972). Altered incorporation of nucleic acid precursors by mycoplasma-infected mammalian cells in culture. Experimental Cell Research 70, 301-310. Rothblat, G. H. (1960). PPLO contamination in tissue cultures. Annals of the New York Academy of Sciences 79, 430-432. Schneider, E. L., Stanbridge, E. J. & Epstein, C. J. (1974). Incorporation of [3H]uridine and [3H]uracil into RNA. A simple technique for the detection of mycoplasma contamination of cultured cells. Experimental Cell Research 84, 311-318. Stanbridge, E. (1971). Mycoplasmas and cell cultures. Bacteriological Reviews 35, 206-227. Stanbridge, E. J. & Schneider, E. L. (1974). Detection of mycoplasma contaminants in Congress on cell culture by biochemical methods. Bordeaux, France: International Mycoplasmas. Todaro, G. J., Aaronson, S. A. & Rands, E. (1971). Rapid detection of mycoplasma infected cell cultures. Experimental Cell Research 65, 256-257. Schneider, E. L. & Stanbridge, E. J. (1975). A simple biochemical technique for the detection of mycoplasma contamination of cultured cells. In Methods in Cell Biology (Prescott, D. M., Ed.) (In press).
184
of Bio&cal
Standardization
1975 3, 181-184
Rapid screening of tissue culture cells for mycoplasmal contaminants.* A. J. Garvettt and Diane E. Reesont
Mycoplasmal contaminants alter the metabolism of uridine and uracil in tissue culture cells. A new technique for the detection of mycoplasma that exploits these changes has been modified to enable many samples to be monitored quickly and easily.
INTRODUCTION The hazards of mycoplasmal contamination of tissue culture cells are well-known and were reviewed recently by Stanbridge (1971), Fogh (1973) and Kenny (1973). Rapid detection of contaminants of cell substrates used for vaccine production is, of course, extremely important, but mycoplasmal infection is insidious and often discovered only by microbiological tests long after the tissue cells have become useless for their designated purpose (Rothblat, 1960; Paton, Jacobs & Perkins, 1965). Furthermore, some strains of mycoplasma do not grow on the media at present used for this detection (Fogh & Fogh, 1969 ; Markov, Bradvarova, Mintcheva, Petrov, Shishkov & Tsanev, 1969 ; Todaro, Aaronson & Rands, 1971). Levine (1972) and Perez, Kim, Gelbard & Djordjevic (1972) showed that mycoplasma increase levels of uridine phosphorylase in mammalian cell cultures and confer an ability to incorporate free nucleoside bases into RNA. Schneider, Stanbridge & Epstein (1974) exploited these two characteristics and demonstrated that the ratio of the specific activities of rH]uridine to pH]uracil incorporated into RNA was greatly reduced when cells were infected with mycoplasma. We describe in the present paper modifications of this method that allow the rapid determination of uridine and uracil incorporated into small samples of cells. * Received for publication 19 November 1974. f Division of Viral Products, National Institute for Biological Laboratories, Holly Hill, London NW 6RB, England.
Standards and Control,
Hampstead
181
A. J. GARRETT
MATERIALS
AND
AND
D.
E. REESON
METHODS
Cell cultures Cells were grown at 37 “C in 30-ml plastic flasks (Falcon Plastics Ltd) or in lo-ml flat-bottomed plastic tubes (Conway tubes, Turner Stayne Laboratories, Bishop Auckland, Co. Durham) containing 13-mm diameter glass coverslips in Eagle’s basal medium containing 15 mM-HEPES-NaOH buffer, 10% calf serum (Sera Services Ltd, Maidenhead, Berks) and 100 units/ml benzyl penicillin. The cells were seeded at concentrations sufficient to give 2040% coverage of the available surface when radioactive labelling was begun. Cell suspensions (1.0 ml) were added to each Conway tube. Incorporation of radioactive &dine and uracil [5-3H]uridine (27 Ci/mmole), [5-sH]uracil (24 Ci/mmole) and [2-14C]uracil (64 mCi/ mmole) were obtained from the Radiochemical Centre, Amersham, Bucks. Medium was aspirated from ,the cell cultures after 24 h growth and replaced with the same formulation containing RNA precursor(s) at 1 $X/ml. Radioactive labelling was terminated after 5 h or 18 h by washing the cell monolayer in the Conway tubes with cold phosphatebuffered saline (Dulbecco & Vogt, 1954) and adding 1 ml of cold 10% (w/v) trichloroacetic acid (TCA). The TCA was aspirated after 1 h at 4 “C, the cells were washed three times with cold 10% TCA, twice with ethanol and once with methanol. It was important to ensure that the alcohol washes removed all uracil that was non-specifically bound to the TCA-insoluble material. The discs were removed from the tubes, dried and counted in a Packard Spectrometer, model 3375, in scintillation fluid containing 4 g of 2,5benzene diphenyloxazole (PPO) and 100 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl) (POPOP)/litre of toluene. Cells grown in bottles were washed and removed from the plastic with pronase. They were then precipitated with TCA and extracted with perchloric acid as described by Schneider et al. (1974). The perchloric acid extracts were neutralized with KOH and samples of the supernatant fluid were counted in scintillation solution containing 8 g of PPO, 200 mg of POPOP and 600 ml of methanol/1400 ml of toluene. In those experiments using cells labelled simultaneously with rH]uridine and [14C]uracil, control cell cultures were labelled with 14C alone and used to measure the ‘spillover’ of 14C into the 3H counting channel. There was no overlap of 3H into the 14C counting channel. Mycoplasma strains Mycoplasma hyorhinis, M. arginini and Acholepkuma granularurn which were kindly provided by Dr Ruth Lemcke (Lister Institute of Preventive Medicine, Chelsea Bridge Road, London SWlW 8RH) were maintained in broth cultures. Tissue cells were examined no less than seven days after infection. Microbiological tests for mycoplasma used the agar and broth preparations of Hayflick (1965). RESULTS
AND
DISCUSSION
The results presented in Tables 1 and 2 confirm that measurement of the uptake of radioactive uridine and uracil in tissue cell cultures gives a rapid assessment of the presence or absence of mycoplasmal contaminants. Uridine : uracil ratios of less than 100 correlated with mycoplasmal contamination. Early detection of contaminants is 182
RAPID
SCREENING
FOR MYCOPLASMA
CONTAMINANTS
vitally important where large quantities of cells are employed, such as in vaccine production, and the methods we have described enable tissue culture cells to be screened more rapidly than has previously been possible. Results are obtained within 48 hours of plating the cells on coverslips. Very small quantities of cells (2 x 104/tube) can now be TABLE
Relative
1.
Mycoplasma contaminant
Cells MRC-.5* WI-38* WI-38 WI-38 WI-38 Hep-2 Hep-2 Hep-2 Vero
incorporations of [SH]uridine tissue cell cultures
None None None M. hyorhinis M. arginini None A. granularurn M. hyorhinis Unidentified
[3H]uracil
Uridine/ uracil
Fraction tested HCIOl HCIOl TCA TCA TCA TCA TCA TCA TCA
and
extract extract ppt ppt ppt ppt ppt ppt ppt
into
Microbiological test + + + + +
840 710 770 1 91 1023 63 2 1
* Cells were grown in 30-ml bottles ; other samples were grown in lo-ml flat-bottomed tubes. TABLE
2. Relative incorporations of [3H]uridine cell cultures Cells
Mycoplasma contaminant
3H/‘4C
MRC-5 WI-38 .Hep-2 Hep-2 ‘Vera
None None None M. hyorhinis Unidentified
952 602 1189 2 2
and [l’C]uracil
into tissue
Microbiological test
+ +
monitored, large numbers of samples can be handled easily because all washing steps are completed by aspiration and not by centrifugation, and measurement of the incorporation of r4C] uracil and rH]uridine into the same sample obviates the need to measure specific activities of RNA. However, in our experiments using rH]uracil and rH]uridine in separate samples (Table l), the great differences in the uridine : uracil ratios between contaminated and uncontaminated cultures made chemical measurement of RNA unnecessary. Schneider & Stanbridge (1975) have studied the simultaneous labelling of cells with [14C]uridine and PH] uracil; their procedure may be difficult to use when small amounts of uracil are incorporated and counts in the 3H counting channel are swamped by the overlap of 14C. These tests alone cannot identify contaminants, of course, but they do indicate potential dangers to the cell culturist. At present mycoplasmal strains can only be identified by cultural tests but, as some grow poorly or not at all in the standard broths and agars, Stanbridge & Schneider (1974) have proposed that the presence of mycoplasma is indicated by a characteristic alteration of the metabolism of uridine and uracil in tissue culture cells and by the appearance on polyacrylamide gel electrophoretograms of 23 S, and 16 S, ribosomal RNA species. 183
A. J. GARRETT
AND
D. E. REESON
Acknowledgement We thank Dr Eric Stanbridge work.
for supplying
us with information
from his unpublished
REFERENCES Dulbecco, R. & Vogt, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. Journal of Experimental Medicine 99, 167-182. Fogh, J. (1973). In Contamination in Tissue Culture Chapter 7, pp. 173-194 (Fogh, J., Ed.). New York and London: Academic Press. Fogh, J. & Fogh, H. (1969). Procedures for control of mycoplasma contamination of tissue cultures. Annals of the New York Academy of Sciences 172, 1S-30. Hayflick, L. (1965). Tissue cultures and mycoplasmas. Texas Reports on Biology and Medicine 23 (Suppl. l.), 285-303. Kenny, G. E. (1973). In Contamination in Tissue Culture Chapter 5, pp. 107-129 (Fogh, J., Ed.). New York and London: Academic Press. Levine, E. M. (1972). Mycoplasma contamination of animal cell cultures; a simple, rapid detection method. Experimental Cell Research 74, 99-109. Markov, G. G., Bradvarova, I., Mintcheva, A., Petrov, P., Shishkov, N. & Tsanev, R. G. (1969). Mycoplasma contamination of cell cultures. Interference with 38P-labelling pattern of RNA. Experimental Cell Research 57, 374-384. Paton, G. R., Jacobs, J. P. & Perkins, F. T. (1965). Chromosome changes in human diploid cell cultures infected with Mycoplasma. Nature 207, 43-45. Perez, A. G., Kim, J. H., Gelbard, A. S. & Djordjevic, B. (1972). Altered incorporation of nucleic acid precursors by mycoplasma-infected mammalian cells in culture. Experimental Cell Research 70, 301-310. Rothblat, G. H. (1960). PPLO contamination in tissue cultures. Annals of the New York Academy of Sciences 79, 430-432. Schneider, E. L., Stanbridge, E. J. & Epstein, C. J. (1974). Incorporation of [3H]uridine and [3H]uracil into RNA. A simple technique for the detection of mycoplasma contamination of cultured cells. Experimental Cell Research 84, 311-318. Stanbridge, E. (1971). Mycoplasmas and cell cultures. Bacteriological Reviews 35, 206-227. Stanbridge, E. J. & Schneider, E. L. (1974). Detection of mycoplasma contaminants in Congress on cell culture by biochemical methods. Bordeaux, France: International Mycoplasmas. Todaro, G. J., Aaronson, S. A. & Rands, E. (1971). Rapid detection of mycoplasma infected cell cultures. Experimental Cell Research 65, 256-257. Schneider, E. L. & Stanbridge, E. J. (1975). A simple biochemical technique for the detection of mycoplasma contamination of cultured cells. In Methods in Cell Biology (Prescott, D. M., Ed.) (In press).
184