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Eradication of Mycoplasma contamination from cell lines of different origin by the 5-bromouracil-fluorochrome procedure
107
Cancer Letters, 46 (1989) 107- 112 Elsevier Scientific Publishers Ireland Ltd.
Eradication of Mycoplasma contamination from cell lines of different origin by the 5-bromouracil-fluorochrome procedure L. Calorini
and
S. Ruggieri
Institute ofGeneralPathology,
University ofFlorence,
u.Le G.B. Morgagni
50, L50134Norence
(Italy)
(Received 3 1 March 1988) (Revision received 10 January 1989) (Accepted 6 March 1989)
Summary Because mycoplasmas contaminate irreplaceable, established cell lines, seueral laboratories have been looking for effective procedures to eradicate the infection. This report presents the experience of our laboratory in using the 5-bromouracil-jluorochrome method developed by Marcus et al. [13] which turned out to be effective in eliminating Mycoplasma orale, M. hyorhinis and M. hominis from transformed cell lines of different origin. The metastatic potential of a highly metastatic murine fibroblastic cell line infected with M. orale was restored to its original level after the contamination was eliminated.
Keywords: mycoplasmas; 5-bromouracil-fluorochrome.
contamination;
Introduction Ever since mycoplasma contamination of cells in tissue culture was first reported in 1956 [18], cell biologists have been concerned that most established cell lines might be contaminated [3]. Procedures involving the use of antibiotics [l, 10,191, specific antisera Correspondence
to: S.
Ruggieri.
0304-3835/89/$03.50 0 1989 Elsevier Scientific Publishers Published and Printed in Ireland
active agents [14] or heat U71, surface treatment [ 1 l] have all been tried but have failed to yield reproducible results. Consequently, some investigators have decided to discard their mycoplasma-contaminated cells and to replace them, where possible, with new cultures. Others, instead, have chosen to ignore this contamination, arguing that mycoplasmas must be considered an unavoidable nuisance in working with tissue cultures. Moreover, since often there were no detectable cytopathic effects in mycoplasmainfected cultures, the mistaken notion arose that such contamination is devoid of biological influence. This assumption was disproved by the discovery that contaminated cells may radically change certain metabolic activities and biological properties [22]. It has thus become imperative for cell biologists to have available a reliable and relatively simple method for eliminating especially in mycoplasma contamination, those cases where contaminated cells cannot be replaced. The practice of decontaminating cultured cells by in vivo passage into nude mice [23] or histocompatible animals [12] is applicable only to tumorigenic cells. This procedure furthermore requires specialized animal rooms which are not within the reach of many laboratories. This report describes our experience with a simple procedure for eliminating infections by Ireland Ltd
108
different species of mycoplasmas from several, irreplaceable cell lines which were vital for pursuing our studies on the role of cell surface lipids in tumorigenicity, metastatic potential and cellular differentiation. Our model systems were the following: (a) Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (RSV) (B77-3T3 cells) [6]; (b) B77-AA6 cells, a high metastatic subclone isolated from B77 3T3 cells by growth in 0.6% agar [8]; (c) SR-Balb cells, a high metastatic line derived from a murine fibrosarcoma induced by the Schmidt-Ruppin strain of RSV [16]; (d) NB 41A3 cells, a clonal line of mouse neuroblastoma [Z] and (e) Friend erythroleukemic (FEL) cells (745A clone) [9]. We knew that mycoplasmas, due to their distinctive nutritional requirement for lipids [Zl], interfere with lipid metabolism of cells in tissue culture. Moreover, we found that spontaneous metastatic potential of B77-AA6 cells was lower than that originally reported for this cell type (81. The procedure we have explored was the 5-bromouracil-fluorochrome method proposed by Marcus et al. [13] which impressed us by its simplicity and rationale, namely, photosensitivity acquired only by the contaminating mycoplasmas due to the preferential incorporation into their DNA of 5bromouracil added to the growth media of Photosensitivity is contaminated cells. enhanced by binding the fluorochrome 33258 Hoechst to 5-bromouracil-containing DNA. Measurements of viability, cloning efficiency and doubling time have been used to detect possible toxic effects caused by the small amount of 5-bromouracil which has been shown to be incorporated into the cells (131. We also investigated whether eradication of contamination mycoplasma restored the spontaneous metastatic potential of B77-AA6 cells. Materials and methods
Culture conditions B77-3T3, B77-AA6, SR-Balb and NB 41A3
cells were cultivated in a monolayer with antibiotic-free Dulbecco’s modification of Eagle’s minimum essential medium (DMEM) (Flow Laboratories, Irvine, U.K.) supplemented with 5% fetal calf serum (FCS) (Flow Lab.). Friend erythroleukemic cells were grown in suspension culture with antibiotic-free RPM1 1640 medium (Flow Lab.) supplemented with 5% FCS (Flow Lab.). Cultures were maintained at 37OC in a humidified atmosphere of 5% CO, in air. Test for mycoplasma infection Mycoplasma contamination was tested by fluorescent Hoechst 33258 stain test [7] following the procedure described by MC Garrity et al. [15]. The direct culture test for mycoplasma detection was performed according to Barile [4]. Mycoplasma species were identified by the immunofluorescence technique [5]. The standard culture and the immunofluorescence technique were performed at the National Laboratory for Mycoplasmology, Institute of Microbiology, University of Padua. Procedure for mycoplasma eradication Cell cultures were first grown for 24 h in the appropriate medium and subsequently grown for 24 h in a fresh medium supplemented with 5-bromouracil (Sigma, St. Louis, MO) at a of 50 pg/ml. H-33258 concentration fluorochrome (Hoechst Italia, Milano, Italy) was then added to growth media at a concentration of 1 pg/ml, and cell cultures were further incubated for 30 min. The growth medium was partially discarded, leaving the cell layers covered by 2-3 mm of medium. Lids were replaced over the plates which were then illuminated for 30 min with a fluorescent lamp (20-W, Osram “Cool White” L2OW/20 tube, 59 cm long, spectral irradiation between 410 and 640 nm) at a distance of 7-8 cm. Cell cultures were washed with phosphate buffered solution (PBS) and then submitted three more times to the 5-bromouracil-fluorochrome-light treatment before the cells were harvested by trypsinizatiion. Following the recommenda-
109
tions reported in a paper by Marcus et al. [ 131, the cells were then diluted (150 cells/ml) in a medium containing low5M thymidine and 10e5 M 2-deoxycytidine and distributed, 1 ml per each 16-mm well, in a tissue culture cluster dish. Cells were grown for approximately 1 week, and the clones which resulted to be mycoplasma-free at the DNA fluorochrome stain test [7] were transferred into 30 mm Petri dishes where they were subcultivated for 4 weeks, Determination of cloning efficiency Cloning efficiency of B77-3T3, B77-AA6, SR-Balb and NB 41A3 cells was determined before and after treatment with the 5-bromprocedure by ouracil-fluorochrome-light growth in culture medium additionated with 0.3% w/v agar (Bacto Agar; Difco, Detroit, MI) . Determination of metastatic potential The B77-3T3, B77AA6 and SR-Balb cells, contaminated and uncontaminated, were harvested by mild trypsinization, washed twice in PBS and then suspended in FCS-free growth media at 25 x 105 cells/ml. Cells 500,000 were injected S.C. into the abdominal flank of 8-week-old female Balb/c mice (Charles River Italia, Calco, Italy). This is the minimal tumorigenic dose of B77-3T3, B77AA6 and SR-Balb cells which has been reported to produce palpable tumors in syngeneic animals after 3 weeks [8]. Upon the natural death of these animals, their lung metastases were examined with the aid of a dissecting microscope. Evaluation of potential metastatic was based on measurements of both incidence and number of metastases per animal. Results and discussion
Upon arrival at our laboratory, B77-3T3, B77-AA6, SR-Balb, FEL and NB 41A3 cell lines were found to be contaminated by mycoplasmas when tested by the DNA fluorochrome procedure [7]. Identification of
mycoplasma species revealed that the FEL cells were contaminated by M. hominis, B773T3, B77-AA6 and NB 41A3 cells by M. orale, and SR-Balb cells by M. hyorhinis. When the cell lines were treated by the 5 bromouracil-fluorochrome-light method [ 131, they were found to be free of mycoplasma contamination as checked by the DNA fluorochrome staining test (71 and by a direct culture method [4] immediately after treatment and 4 weeks later. Moreover, no contamination of mycoplasmas has been found in any of the treated cell lines after several months of growth in tissue cultures as shown in Fig. 1. Immediately after treatment with the 5-bromouracil-fluorochrome-light procedure [13], cells did not show any change of viability as tested by the Trypan blue exclusion test. Moreover, the treated cells showed values of cloning efficiency and doubling time not statistically different from those of the untreated cells (0.9O>P> 0.30, Student’s t-test), indicating that the 5-bromouracil-fluorochrome-light procedure did not cause major toxic effects on the cell lines under study. While the value of the spontaneous metastatic potential of contaminated B77-AA6 cells was less than that reported by Di Renzo and Bretti [8], it reached that value after eradication of mycoplasma contamination. The decontaminated SR-Balb cells gave a lower number of metastases per animal, but they exhibited an unchanged incidence of metastases (Table 1). Moreover, the median survival time of syngeneic mice injected with all the decontaminated cells was longer than that of animals injected with the mycoplasma contaminated cells. The difference in the median survival time between animals injected with contaminated and decontaminated B77-3T3, B77-AA6 and SR-Balb cells is reminiscent of the wasting syndrome reported for animals injected with mycoplasma-infected cells (201. We conclude by highly recommending the method of Marcus et al. [ 131 for eradicating mycoplasma contamination; it is a procedure that is both effective and easy to carry out as
Fig. 1. A, B77-AA6 cells infected by M. ode. Distinctive cytoplasmic fluorescence fluorescence [5]. B, B77-AA6 cells 8 weeks after from tbe 5-bromouracil-fluorochrome fluorescence can be seen [5]. Magnification 3400 x .
is seen in addition to nut :lear treatment (11). Only nut :lear
12/12
SR-BALB
12/12
3/8
. ,., ._.- ._.- _
-_- -_
_..- -..- ___,,_-_ -.-
-,__ --- -
--
--,-.- ---- ---
59 (45- 74)
48 (41-56)
6 (O-40) 16 (7-26)
49 (39-59)
Median survival time*
_____.
5/5
13/13
6/6
Incidence of tumor”
cells
-._--.--,
5/5
11/13
l/6
Incidence of metastasesb
Decontaminated
B77-3T3, B77-AA6 and SR-BALB cell lines.
3 (O-5)
No. of metastases per animak
No. of mice with tumor/no. of mice injected. No. of mice with metastases/no. of mice with tumors. Number of lung metastases per animal and range in parentheses. Median survival time in days and range in parentheses.
8/8
B77-AA6
2/8
Incidence of metastasesb
Incidence of turnoF
8/8
cells
Contaminated
B77-3T3
Cell line
Table1. Metastatic potential of contaminated and decontaminated
--.---_-_-
4 (l-8)
12 (O-71)
1 (O-l)
No. of metastases per animaF
-
95 (75- 115)
74 (55-93)
84 W-115)
Median survival time*
.-
._.
112 well as being within the reach of the average tissue culture laboratory. 10
Acknowledgements
We gratefully acknowledge the generous assistance received from Professor G .A. Meloni, Chairman of the Institute of Microbiology, University of Padua, during all phases of this report. This work has been supported by Consiglio Nazionale delle Ricerche (Special project: Oncologia), by Associazione Italiana per Ia Ricerca sul Cancro and by Minister0 della Pubblica Istruzione. References Audrey, G.N. and Chu, H.P. (1965) An in vitro comparison of the effect of some antibacterial, antifungal and antiprotozoa1 agents on various strains of Mycoplasma (pleuropneumonia-Ilke organisms: P.P.L.O.). J. Hyg., 63, l-23. Augusti-Tocco, G. and Sate, G. (1969) Establishment of of neurons from mouse functional clonal lines neuroblastoma. Proc. Natl. Acad. Sci. USA, 64, 311315. Barile, M.F., Hopps, H.E. and Grabowski, M. (1978) Incidence and sources of the Mycoplasma contamination. In: Mycoplasma Infection of Cell Culture, pp. 35-46. Editors: G.J. MC Garrity, D.G. Murphy and W.W. Nichols. Plenum Press, New York. Barile, M.F. (1979) Mycoplasmas-tissue cell interaction. In: The Mycoplasmas II, pp. 425-474. Editors: J.G. Tully and R.F. Whitcomb. Academic Press, New York. Bartle, M.F. and Grabowski, M.W. (1983) Detection and identification of mycoplasmas in infected cell cultures by direct immunofluorescent staining. Methods Mycoplasmol. 2,173-181. Bishop, J.M., Weiss, S.R., Oppermann, H., Hackett, P., Quintrell, N., Chen, L.S., Levintow, L. and Varmus, H.E. (1978) The strategy of retrovirus gene expression. In: Avian RNA Tumor Viruses, pp. 181-189. Editors: S. Barlati and C. De Giuli Morghen, Piccin, Padua. Chen, T.R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res., 104.255-262. Di Renzo, M.F. and Bretti, S. (1982) Characterization of stable spontaneous metastatic variant lines of the RSVtransfoimed mouse fibroblasts. Int. J. Cancer, 30, 751757. Friend, C., Patuleia, M.C. and De Harven, E. (1981) Erythrocitic maturation in vitro of murine (Friend) virus
11
12
13
14
15
16
17
18
19
20
21 22
23
induced leukemic cells. Natl. Cancer Inst. Monogr., 22, 505-522. Gurney Jr., T., Woolf, M.J., Abplanalp, L.J., McKittrick, N.H., Diets, J.N. and Cole, B.R. (1981) Elimination of Mycoplesma hyorhinis from four cell lines. In Vitro, 17, 993-996. Hayflick, L. (1960) Decontaminating tissue cultures infected with pleuropneumonia-like organisms. Nature, 18.783-784. Lombardo, M.J., and Lanks, K.W. (1982) Elimination of M. hyorhinis from murine neuroblastoma cell lines by in vivo passage. In Vitro, 18,251-253. Marcus, M., Uri. L., Nattenberg, A., Rottem, S. and Markowitz, 0. (1980) Selective killing of mycoplasmas from contaminated mammalian cells in cell cultures. Nature, 285,659-661. Mardh, P-A. (1975) Elimination of mycoplasmas from cell cultures with sodium polyanethol sulphonate. Nature, 254, 515-516. McGarrity, G.J. Steiner, T. and Vanaman, V. (1983) Detection of mycoplasmal infection of cell cultures by DNA fluorochrome staining. Methods Mycoplasmol., 2, 183190. Parsons, S.J., Riley, S.C., Mullen, E.E., Brock, E.J., Benjamin, D.C., Kuehl, W.M. and Parsons, J.T. (1979) Immune response to the scr gene product in mice bearing tumors induced by injection of avian sarcoma virus-transformed mouse cells. J. Viral., 32,40-46. Pollock, M.E. and Kenny, G.E. (1963) Mammalian cell cultures contaminated with the PPLO 111.Elimination of the PPLO with specific antiserum. Proc. Sot. Exp. Biol. Med., 112,176-181. Robinson, L.B., Wichelhausen, R.H. and Roizman, M. (1956) Contamination of human cell cultures by pleuropneumonia-like organisms. Science, 124, 11471148. Schmidt, J. and Erfle, V. (1984) Elimination of mycoplasmas from cell cultures and establishment of the mycoplasma-free cell lines. Exp. Cell Res., 152, 565570. Shin, S. and van Diggelen, O.P. (1978) Phenotypic interactions in mammalian cell lines after mycoplasma infection. In: Mycoplasma Infection of Cell Culture, pp. 191-212. Editors: G.J. MC Garrity, D.G. Murphy and W.W. Nichols. Plenum Press, New York. Smith, P.F. (1968) The lipids of Mycoplasma. Adv. Lipid Res., 6,69-105. Stanbridge, E.J. and Doersen, C-J. (1978) Some effect that mycoplasmas have upon their infected host. In: Mycoplasma Infection of Cell Culture, pp. 119-134. Editors: G.J. McGarrity, D.G. Murphy and W.W. Nichols, Plenum Press, New York. van Diggelen, O.P., Shin, S. and Phillips, D.M. (1977) Reduction in cellular tumorigenicity after mycoplasma infection and elimination of mycoplasma from infected cultures by passage in nude mice. Cancer Res., 37, 26802687.
Cancer Letters, 46 (1989) 107- 112 Elsevier Scientific Publishers Ireland Ltd.
Eradication of Mycoplasma contamination from cell lines of different origin by the 5-bromouracil-fluorochrome procedure L. Calorini
and
S. Ruggieri
Institute ofGeneralPathology,
University ofFlorence,
u.Le G.B. Morgagni
50, L50134Norence
(Italy)
(Received 3 1 March 1988) (Revision received 10 January 1989) (Accepted 6 March 1989)
Summary Because mycoplasmas contaminate irreplaceable, established cell lines, seueral laboratories have been looking for effective procedures to eradicate the infection. This report presents the experience of our laboratory in using the 5-bromouracil-jluorochrome method developed by Marcus et al. [13] which turned out to be effective in eliminating Mycoplasma orale, M. hyorhinis and M. hominis from transformed cell lines of different origin. The metastatic potential of a highly metastatic murine fibroblastic cell line infected with M. orale was restored to its original level after the contamination was eliminated.
Keywords: mycoplasmas; 5-bromouracil-fluorochrome.
contamination;
Introduction Ever since mycoplasma contamination of cells in tissue culture was first reported in 1956 [18], cell biologists have been concerned that most established cell lines might be contaminated [3]. Procedures involving the use of antibiotics [l, 10,191, specific antisera Correspondence
to: S.
Ruggieri.
0304-3835/89/$03.50 0 1989 Elsevier Scientific Publishers Published and Printed in Ireland
active agents [14] or heat U71, surface treatment [ 1 l] have all been tried but have failed to yield reproducible results. Consequently, some investigators have decided to discard their mycoplasma-contaminated cells and to replace them, where possible, with new cultures. Others, instead, have chosen to ignore this contamination, arguing that mycoplasmas must be considered an unavoidable nuisance in working with tissue cultures. Moreover, since often there were no detectable cytopathic effects in mycoplasmainfected cultures, the mistaken notion arose that such contamination is devoid of biological influence. This assumption was disproved by the discovery that contaminated cells may radically change certain metabolic activities and biological properties [22]. It has thus become imperative for cell biologists to have available a reliable and relatively simple method for eliminating especially in mycoplasma contamination, those cases where contaminated cells cannot be replaced. The practice of decontaminating cultured cells by in vivo passage into nude mice [23] or histocompatible animals [12] is applicable only to tumorigenic cells. This procedure furthermore requires specialized animal rooms which are not within the reach of many laboratories. This report describes our experience with a simple procedure for eliminating infections by Ireland Ltd
108
different species of mycoplasmas from several, irreplaceable cell lines which were vital for pursuing our studies on the role of cell surface lipids in tumorigenicity, metastatic potential and cellular differentiation. Our model systems were the following: (a) Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (RSV) (B77-3T3 cells) [6]; (b) B77-AA6 cells, a high metastatic subclone isolated from B77 3T3 cells by growth in 0.6% agar [8]; (c) SR-Balb cells, a high metastatic line derived from a murine fibrosarcoma induced by the Schmidt-Ruppin strain of RSV [16]; (d) NB 41A3 cells, a clonal line of mouse neuroblastoma [Z] and (e) Friend erythroleukemic (FEL) cells (745A clone) [9]. We knew that mycoplasmas, due to their distinctive nutritional requirement for lipids [Zl], interfere with lipid metabolism of cells in tissue culture. Moreover, we found that spontaneous metastatic potential of B77-AA6 cells was lower than that originally reported for this cell type (81. The procedure we have explored was the 5-bromouracil-fluorochrome method proposed by Marcus et al. [13] which impressed us by its simplicity and rationale, namely, photosensitivity acquired only by the contaminating mycoplasmas due to the preferential incorporation into their DNA of 5bromouracil added to the growth media of Photosensitivity is contaminated cells. enhanced by binding the fluorochrome 33258 Hoechst to 5-bromouracil-containing DNA. Measurements of viability, cloning efficiency and doubling time have been used to detect possible toxic effects caused by the small amount of 5-bromouracil which has been shown to be incorporated into the cells (131. We also investigated whether eradication of contamination mycoplasma restored the spontaneous metastatic potential of B77-AA6 cells. Materials and methods
Culture conditions B77-3T3, B77-AA6, SR-Balb and NB 41A3
cells were cultivated in a monolayer with antibiotic-free Dulbecco’s modification of Eagle’s minimum essential medium (DMEM) (Flow Laboratories, Irvine, U.K.) supplemented with 5% fetal calf serum (FCS) (Flow Lab.). Friend erythroleukemic cells were grown in suspension culture with antibiotic-free RPM1 1640 medium (Flow Lab.) supplemented with 5% FCS (Flow Lab.). Cultures were maintained at 37OC in a humidified atmosphere of 5% CO, in air. Test for mycoplasma infection Mycoplasma contamination was tested by fluorescent Hoechst 33258 stain test [7] following the procedure described by MC Garrity et al. [15]. The direct culture test for mycoplasma detection was performed according to Barile [4]. Mycoplasma species were identified by the immunofluorescence technique [5]. The standard culture and the immunofluorescence technique were performed at the National Laboratory for Mycoplasmology, Institute of Microbiology, University of Padua. Procedure for mycoplasma eradication Cell cultures were first grown for 24 h in the appropriate medium and subsequently grown for 24 h in a fresh medium supplemented with 5-bromouracil (Sigma, St. Louis, MO) at a of 50 pg/ml. H-33258 concentration fluorochrome (Hoechst Italia, Milano, Italy) was then added to growth media at a concentration of 1 pg/ml, and cell cultures were further incubated for 30 min. The growth medium was partially discarded, leaving the cell layers covered by 2-3 mm of medium. Lids were replaced over the plates which were then illuminated for 30 min with a fluorescent lamp (20-W, Osram “Cool White” L2OW/20 tube, 59 cm long, spectral irradiation between 410 and 640 nm) at a distance of 7-8 cm. Cell cultures were washed with phosphate buffered solution (PBS) and then submitted three more times to the 5-bromouracil-fluorochrome-light treatment before the cells were harvested by trypsinizatiion. Following the recommenda-
109
tions reported in a paper by Marcus et al. [ 131, the cells were then diluted (150 cells/ml) in a medium containing low5M thymidine and 10e5 M 2-deoxycytidine and distributed, 1 ml per each 16-mm well, in a tissue culture cluster dish. Cells were grown for approximately 1 week, and the clones which resulted to be mycoplasma-free at the DNA fluorochrome stain test [7] were transferred into 30 mm Petri dishes where they were subcultivated for 4 weeks, Determination of cloning efficiency Cloning efficiency of B77-3T3, B77-AA6, SR-Balb and NB 41A3 cells was determined before and after treatment with the 5-bromprocedure by ouracil-fluorochrome-light growth in culture medium additionated with 0.3% w/v agar (Bacto Agar; Difco, Detroit, MI) . Determination of metastatic potential The B77-3T3, B77AA6 and SR-Balb cells, contaminated and uncontaminated, were harvested by mild trypsinization, washed twice in PBS and then suspended in FCS-free growth media at 25 x 105 cells/ml. Cells 500,000 were injected S.C. into the abdominal flank of 8-week-old female Balb/c mice (Charles River Italia, Calco, Italy). This is the minimal tumorigenic dose of B77-3T3, B77AA6 and SR-Balb cells which has been reported to produce palpable tumors in syngeneic animals after 3 weeks [8]. Upon the natural death of these animals, their lung metastases were examined with the aid of a dissecting microscope. Evaluation of potential metastatic was based on measurements of both incidence and number of metastases per animal. Results and discussion
Upon arrival at our laboratory, B77-3T3, B77-AA6, SR-Balb, FEL and NB 41A3 cell lines were found to be contaminated by mycoplasmas when tested by the DNA fluorochrome procedure [7]. Identification of
mycoplasma species revealed that the FEL cells were contaminated by M. hominis, B773T3, B77-AA6 and NB 41A3 cells by M. orale, and SR-Balb cells by M. hyorhinis. When the cell lines were treated by the 5 bromouracil-fluorochrome-light method [ 131, they were found to be free of mycoplasma contamination as checked by the DNA fluorochrome staining test (71 and by a direct culture method [4] immediately after treatment and 4 weeks later. Moreover, no contamination of mycoplasmas has been found in any of the treated cell lines after several months of growth in tissue cultures as shown in Fig. 1. Immediately after treatment with the 5-bromouracil-fluorochrome-light procedure [13], cells did not show any change of viability as tested by the Trypan blue exclusion test. Moreover, the treated cells showed values of cloning efficiency and doubling time not statistically different from those of the untreated cells (0.9O>P> 0.30, Student’s t-test), indicating that the 5-bromouracil-fluorochrome-light procedure did not cause major toxic effects on the cell lines under study. While the value of the spontaneous metastatic potential of contaminated B77-AA6 cells was less than that reported by Di Renzo and Bretti [8], it reached that value after eradication of mycoplasma contamination. The decontaminated SR-Balb cells gave a lower number of metastases per animal, but they exhibited an unchanged incidence of metastases (Table 1). Moreover, the median survival time of syngeneic mice injected with all the decontaminated cells was longer than that of animals injected with the mycoplasma contaminated cells. The difference in the median survival time between animals injected with contaminated and decontaminated B77-3T3, B77-AA6 and SR-Balb cells is reminiscent of the wasting syndrome reported for animals injected with mycoplasma-infected cells (201. We conclude by highly recommending the method of Marcus et al. [ 131 for eradicating mycoplasma contamination; it is a procedure that is both effective and easy to carry out as
Fig. 1. A, B77-AA6 cells infected by M. ode. Distinctive cytoplasmic fluorescence fluorescence [5]. B, B77-AA6 cells 8 weeks after from tbe 5-bromouracil-fluorochrome fluorescence can be seen [5]. Magnification 3400 x .
is seen in addition to nut :lear treatment (11). Only nut :lear
12/12
SR-BALB
12/12
3/8
. ,., ._.- ._.- _
-_- -_
_..- -..- ___,,_-_ -.-
-,__ --- -
--
--,-.- ---- ---
59 (45- 74)
48 (41-56)
6 (O-40) 16 (7-26)
49 (39-59)
Median survival time*
_____.
5/5
13/13
6/6
Incidence of tumor”
cells
-._--.--,
5/5
11/13
l/6
Incidence of metastasesb
Decontaminated
B77-3T3, B77-AA6 and SR-BALB cell lines.
3 (O-5)
No. of metastases per animak
No. of mice with tumor/no. of mice injected. No. of mice with metastases/no. of mice with tumors. Number of lung metastases per animal and range in parentheses. Median survival time in days and range in parentheses.
8/8
B77-AA6
2/8
Incidence of metastasesb
Incidence of turnoF
8/8
cells
Contaminated
B77-3T3
Cell line
Table1. Metastatic potential of contaminated and decontaminated
--.---_-_-
4 (l-8)
12 (O-71)
1 (O-l)
No. of metastases per animaF
-
95 (75- 115)
74 (55-93)
84 W-115)
Median survival time*
.-
._.
112 well as being within the reach of the average tissue culture laboratory. 10
Acknowledgements
We gratefully acknowledge the generous assistance received from Professor G .A. Meloni, Chairman of the Institute of Microbiology, University of Padua, during all phases of this report. This work has been supported by Consiglio Nazionale delle Ricerche (Special project: Oncologia), by Associazione Italiana per Ia Ricerca sul Cancro and by Minister0 della Pubblica Istruzione. References Audrey, G.N. and Chu, H.P. (1965) An in vitro comparison of the effect of some antibacterial, antifungal and antiprotozoa1 agents on various strains of Mycoplasma (pleuropneumonia-Ilke organisms: P.P.L.O.). J. Hyg., 63, l-23. Augusti-Tocco, G. and Sate, G. (1969) Establishment of of neurons from mouse functional clonal lines neuroblastoma. Proc. Natl. Acad. Sci. USA, 64, 311315. Barile, M.F., Hopps, H.E. and Grabowski, M. (1978) Incidence and sources of the Mycoplasma contamination. In: Mycoplasma Infection of Cell Culture, pp. 35-46. Editors: G.J. MC Garrity, D.G. Murphy and W.W. Nichols. Plenum Press, New York. Barile, M.F. (1979) Mycoplasmas-tissue cell interaction. In: The Mycoplasmas II, pp. 425-474. Editors: J.G. Tully and R.F. Whitcomb. Academic Press, New York. Bartle, M.F. and Grabowski, M.W. (1983) Detection and identification of mycoplasmas in infected cell cultures by direct immunofluorescent staining. Methods Mycoplasmol. 2,173-181. Bishop, J.M., Weiss, S.R., Oppermann, H., Hackett, P., Quintrell, N., Chen, L.S., Levintow, L. and Varmus, H.E. (1978) The strategy of retrovirus gene expression. In: Avian RNA Tumor Viruses, pp. 181-189. Editors: S. Barlati and C. De Giuli Morghen, Piccin, Padua. Chen, T.R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res., 104.255-262. Di Renzo, M.F. and Bretti, S. (1982) Characterization of stable spontaneous metastatic variant lines of the RSVtransfoimed mouse fibroblasts. Int. J. Cancer, 30, 751757. Friend, C., Patuleia, M.C. and De Harven, E. (1981) Erythrocitic maturation in vitro of murine (Friend) virus
11
12
13
14
15
16
17
18
19
20
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