IN VITRO Volume 15, No. 2, 1979 All rights reserved 9
E F F E C T S O F S O M E A N T I B I O T I C S ON T H E G R O W T H O F H U M A N DIPLOID SKIN FIBROBLASTS IN CELL CULTURE INGEBURG E. GOETZ, RANDI MOKLEBUST, ANDCATHERINE J. WARREN
Division of Neurosciences, City of Hope National Medical Center, Duarte, California 91010
SUMMARY
During serial subcultures 50/~g per ml gentamicin and penicillin (100 U per ml)-streptomycin ~100/~g per ml~ depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.
Key words: antibiotics; human skin fibroblasts; cell growth. INTRODUCTION periments to investigate this effect and present the Because of the constant threat of bacterial con- results in this paper. tamination in cell culture, antibiotics are added MATERIALS AND M E T H O D S routinely to the nutrient medium in many laboratories. The preferred antibiotic for many years Human skin diploid fibroblast cell lines were has been a mixture of penicillin G and streptomy- obtained either from the Genetic Mutant Cell Recin (pen-strep). During the last few years, genta- pository, Camden, New Jersey, or grown from micin has gained increasing acceptance because it skin biopsies in our laboratory according to the is effective against a wider range of bacteria than method outlined in the TCA Manual 17L All cell pen-strep and against some strains of mycoplasma lines were tested and found to be free from myco~l-3L Gentamicin, in addition, has a greater plasma contamination by a combination of biostability in tissue-culture medium than does pen- chemical and cultural methods performed by Irstrep (4), especially at 37 ~ C, the temperature vine Scientific Sales Co., Fountain Valley, Caliused for growing mammalian cells. fornia. Some of the cell lines were routinely grown In cell culture the recommended concentrations in the presence of 80 U per ml penicillin G and 80 are: penicillin G, 100 U per ml; streptomycin, 100 /~g per ml streptomycin. Before being used for the /~g per ml; and gentamicin, 100 t~g per ml. These reported studies all cell lines were grown through concentrations were not found to be noticeably four to five subcultures without antibiotics. toxic to Earle's L-929 cells or Eagle's KB cells by The nutrient medium employed was Eagle's Perlman (4). Byarugaba et al. t5~ reported that minimal essential medium with Earle's salts. In 6000 ~g/ml per ml penicillin G, 1250 ~g/ml addition it contained 0.1 mM nonessential amino streptomycin, or 1000 ~g per ml gentamicin per- acids, 1 mg per 1 biotin and 20% fetal bovine mitted unchanged growth of cultured human skin serum not heat-inactivated. The glutamine confibroblasts. Schafer et al. ~6) observed no toxicity centration was raised to 4 mM and the pH adwhen a variety of mammalian cells were grown justed to 7.2 to 7.4. Nonessential amino acids and through a large number of culture passages with biotin were not required by the cells but were in50/~g per ml gentamicin, or when cells were ex- cluded because the medium also was used for posed for a short time to several mg per ml. other purposes. Cell growth was considerably During the course of studies in our laboratory faster with 20% than with 10% serum. Cells were with human skin fibroblasts, we added 100 U per grown in plastic tissue-culture dishes at ml penicillin G and 100/~g per ml streptomycin, 37_+0.5 ~ C in a humidified gas of 5% CO2 in air. or 50 ~g per ml gentamicin to the culture medium Our routine culturing procedure consisted of and observed a decrease in the growth rate of the medium changes twice weekly and subculturing cells. Subsequently, we performed additional ex- when the cell layer was confluent, usually every 5 114
115
EFFECTS OF ANTIBIOTICS ON FIBROBLASTS to 7 days. Subculturing was done at room temperature using a buffered solution of 0.5% trypsin and 0.02% E D T A to disperse the cells. For the experiments with antibiotics the dispersed cells were suspended in medium without antibiotics and fresh dishes were plated at a concentration of 0.5 to 1.25 x 104 cells per cm ~, depending on the particular study. Antibiotics were added immediately from the concentrated stock solutions. Gentamicin was used to final concentrations of 25 to 400/ag per ml of medium and pen-strep to final concentrations of 50 to 100 U per ml penicillin and 50 to 100/ag per ml streptomycin. Antibiotics were mixed with the medium by agitation of the dish. The change in osmolarity was negligible because only 2.5 to 40 ~1 of stock solution was required per ml of medium. No antibiotics were added to control dishes. The above described procedure was used for each subsequent culture passage. To determine plating efficiency cells were dispersed and counted 1 day after being plated. Cell mitosis had not resumed at that time. Cell counts were made with a hemacytometer or with a Coulter counter. Samples of the cell suspensions were regularly examined microscopically to ensure that no cell clumps were present. Two dishes of cells were always grown for each antibiotic concentration and for the controls. At feedings and subculturing dishes were handled at random. Concentrated stock solutions for the nutrient medium, Hanks' saline without calcium and magnesium, trypsin-EDTA, and a solution containing 10,000 U per ml penicillin G sodium and 10,000 U per ml streptomycin sulfate were obtained from Grand Island Biological Co., Grand Island, New
York. Gentamicin reagent solution, l0 mg per ml was bought from Schering Corp., Kenilworth, New Jersey. Fetal bovine serum (not heat-inactivated) was purchased from Reheis Chemical Co., Phoenix, Arizona. Tissue culture dishes were obtained from either Falcon Plastics, Los Angeles, California, or Corning Scientific Products Division, Sunnyvale, California. Calculations
Accumulated number of cells: C A It, t = Cito~ x G F A ~t, G F A = GFitl~ x GFitz~ x . . . . .
GFtt~
CItn-l) C.4tt, l: Number of cells accumulated at time trt.
C~toj: Number of cells plated at time to. GF.41tnl: Accumulated growth factor at time tn.
GFum: Ratio of number of cells counted at time tn and number of cells plated at time t n - 1 . C': Number of cells counted. C: Number of cells plated.
Percent of loss of cells in medium with antibiotics: 100 ( a - b ) a
a: Cells per dish in medium without antibiotics. b: Cells per dish in medium with antibiotics.
TABLE
1
G AIN OR LOSS OF H UMAN S KIN F IBROBLASTS AFTER 6 TO62 D AYS IN ]V[EDIUM WITH G ENTAMICIN ORP E N I C I L L I N ~ TREPTOMYCIN AND R ECOVERY AFTER THE ANTIBIOTICS ARE OMITTED Percent Gain or Loss of Cells in Medium with Antibiotics at End of Growth Period a PenlU Iml)-strep(~ag/ml)
Gentamicin (~g/ml)
Cell Line
Growth Period
No.
275
1
2 3 4 5 6 7
days 0-6 6-12 12-18 18-25 25-33 33-47 47-62
Cells Plated at Beginning of Growth Period
50
cells Icm 2 x 104 1.0 1.0 1.0 1.0 1.0 0.5 0.5
+ 4.3 - 1.1 -14.2 -12.9 -11.2 -34.0 -32.4
50 Omitted alter 33 days
100
-31.8 + 2.5
+18.1 - 8.8 -19.5 -20.6 -25.3 -48.7 -53.9
100 Omitted after 33 days
104)
100 Omitted after 33 days
-50.4 + 2.2
- 0.5 - 3.0 -19.9 -20.0 -16.4 -19.5 -15.4
- 6.2 +27.8
a At the end of the growth period all cells were counted. From the average counts of cells in two dishes without antibiotics and in two dishes with a particular antibiotic concentration, percent loss or gain of cells was calculated for that growth period.
116
GOETZ, MOKLEBUST, AND WARREN RESULTS
] • 1070 I
L
One cell line was grown for 62 days through seven subcultures in medium with 50 or 100 ~g per ml gentamicin, 100 U per ml penicillin and 100 t~g per ml streptomycin, or without antibiotics. The effects of the antibiotics are shown in Table 1. During the first growth period with antibiotics, gentamicin, but not pen-strep, stimulated cell growth. In subsequent growth periods both antibiotics caused a loss in cell yield. In the second growth period this effect was very small and probably significant only for 100 gg per ml of gentamlcin. In growth periods 3 to 5, all concentrations tested caused considerable loss in cell yield, the magnitude of which remained approximately constant during this time. At the beginning of growth period 6, the cells in medium with antibiotics were split and grown with and without antibiotics to examine recovery of cell growth. Since there were insufficient ceils available in the cultures that had been grown with 100/~g per ml gentamicin, the plating density was decreased by one-half for all cultures, including the controls. In addition, the length of the growth period between subcultures was approximately doubled. This resuited in a further decreased cell yield in the cultures grown with gentamicin, whereas the effect of pen-strep remained approximately the same. In those dishes in which the antibiotics were omitted, the cell yield gradually returned to normal or above normal. Cells that previously had been grown in medium with pen-strep recovered more quickly than those previously grown with gentamicin. The cumulative increases in cells during this study are shown in Fig. 1. Compared to the controls the total loss of cells, after 62 days in
E
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LLI (J
LLI
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1 x I0 "t
~/~//6
r
--1
1 •
10 6
I x 105 ~ 0
.
10
.
.
20
.
.
.
30
.
.
.
40
.
.
50
~
60
--~ 70
DAYS IN MEDIUM WITH ANTIBIOTICS
FIG. 1. Effect of gentamicin and penicillin-streptomycin on growth of human skin fibroblasts during 62 days with regular subculturing. 9 - - 9 No antibiotics: x - - x , 100 ~g per ml gentamicin; A - - A , 50 ~g per ml gentamicin; 9 1 6 9 100 U per mi penicillin 13 and 100 ~g per ml streptomycin. medium with antibiotics, was 71.2% for 50 ~g per ml gentamiein, 87.9% for 100 ~g per ml gentamicin, and 60.6% for 100 U per ml penicillin and 100 gg per ml streptomycin. Fig. 1 shows that the control cells grew exponentially until day 47 after
TABLE 2 E FFECT OF GENTAMICIN ANDP ENICILLIN-STREPTOMYCIN ON FOUR D IFFERENT LINES OF HUMAN S KIN F IBROBLASTS Percent Loss of Cells in Medium with Antibiotics at End of Growfll Period a Cell Line
Growth Period
No.
967
days
Cells Plated at Beginning of Growth Period
Gentamicln i~g/rnl) 25
50
lOO
PenlU mll-strepl~g/ndl 200
400
50
IO0
cells/cm ~x l04
1 0-14 1.25 8.2 8.0 11.1 25.0 32.2 6.5 9.3 2 14-28 1.25 20.0 37.4 7.1 1061 1 0-14 1.25 3.1 6.9 13.2 22.9 42.6 0.0 4.2 2 14-28 1.25 19.7 36.9 5.8 1085 1 0-14 1.25 0,0 0.0 3.1 9.9 13.5 0.0 0.0 2 14-28 1.25 9.8 20.4 0.0 321 1 0-14 1.0 3.9 5.9 6.3 11.3 33.0 5.2 5.3 2 14-28 1.0 10.3 22.4 8.9 a At the end of the growth period all cells were counted. From the average count of cells in two dishes without antibiotics and in two dishes with a particular antibiotic concentration, percent loss of cells was calculated for that growth period.
EFFECTS OF ANTIBIOTICS ON FIBROBLASTS which cell growth declined. This may be a sign that the cells were approaching senescence at that time. The inhibitory effect of the antibiotics on growth of cultured human skin fibroblasts was confirmed with four additional cell lines by experiments of shorter duration (Table 2). In this study pen-strep was considerably less toxic than gentamicin. Loss of cell yield increased with the concentration of gentamicin and was already considerable at 200/~g per ml in two of the cell lines. Since the stimulatory effect of this antibiotic, which previously was observed at the end of the first growth period, did not occur in this study, an additional experiment was performed. Cells from another line were grown with 100 ~g per ml genta-
o
E
Q.. O3 4 J ..J
b..I (.3
] 17
micin, 100 U per ml penicillin and 100/~g per ml streptomycin, or no antibiotic for 14 days without subculturing; and counts were made 1, 3, 5, 7 and 14 days after the dishes were plated. The results in Fig. 2 show that the antibiotics had very little effect on cell growth until 5 days when gentamicin had clearly stimulated cell growth, whereas penstrep had caused a slight inhibition of growth. After 14 days gentamicin and pen-strep both had inhibited cell growth to approximately the same extent, the loss of cells being 19.2% and 18.1% of the controls, respectively. The plating efficiency of the examined cell lines ranged from 71% to 98% and was not affected by 25 to 400/~g per ml gentamicin or 50 to 100 U per ml penicillin and 50 to 100 #g per ml streptom y c i n . Examination of the cultures by phase microscopy did not reveal any differences between cells in medium with antibiotics and controls until 3 or 4 days after the cells had been plated into medium with the antibiotics. At that time less mitosis and more cell debris was observed in cultures with 400 #g per ml gentamicin than in those without antibiotics, but not in cultures with lower concentrations of gentamicin or with the concentrations of pen-strep tested. Fourteen days after plating, during which time the cells were fed every 3 or 4 days, but not subcultured, cells in medium without antibiotics had formed a dense layer. The cells were spread out and firmly attached to the culture dish. No morphological difference was observed in cells grown with 50 or 100 U per ml penicillin and 50 or 100/~g per ml streptomycin, or 50 or 100 #g per ml gentamicin. Cells in medium with 200 /~g per ml gentamicin showed some retraction from the dish. Cells in medium with 400/~g per ml gentamicin, in addition, contained many dark particles in the perinuclear area which were possibly enlarged lysosomes (Fig. 3 }. DISCUSSION
MEDIUM CHANGE
1
4
I
6
8
DAYS IN MEDIUM WITH
10
12
14
ANTIBIOTICS
FIG. 2. Effects of gentamicin and penicillinstreptomycin on growth of human skin fibroblasts during 14 days without subculturing. 9 - - 9 , No anttblo """ tics; x--~, 100/~g per ml gentam~cin; O - - O , 100 U per ml penicillin G and 100 ~g per ml streptomycin.
Our results show that 50 or 100/~g per ml gentamicin and 100 U per ml penicillin and 100 #g per ml streptomycin concentrations that are thought to be safe for cultured cells depress the growth of human skin fibroblasts. This effect appears only after one or two periods of logarithmic growth in the presence of the antibiotics and therefore was not observed by investigators who used shorter observation times (8). In our experiments gentamicin stimulated cell growth before it became inhibitory. This was also noticed by Litwin (9~ who grew human diploid lung fibroblasts for more than 100 days in the presence of gentamicin. The
118
GOETZ, MOKLEBUST, AND WARREN
FIG. 3. Effect of gentamicin on morphology of human skin fibroblasts. A, Cells grown 14 days without antibiotics; B, parallel culture of cells grown 14 days with 400 ~g per ml gentamicin showing retraction of cells from the surface and dark particles in the perinuclear area {arrows}. Phase contrast photomicrographs, x400.
EFFECTS OF ANTIBIOTICS ON FIBROBLASTS depression of cell growth that followed the stimulation was less than in our studies, but his controls were grown with 100 U per ml penicillin and 100/~g per ml streptomycin which, as shown by our results, interferes with cell growth. The same author found that 500 #g per ml gentamicin did not decrease reattachment of trypsinized cells. This was confirmed in our studies. The dark particles that we observed in the perinuclear area in cells maintained for 14 days in the presence of 400 #g per ml gentamicin were possibly enlarged lysosomes. This is suggested by the observation that gentamicin can accumulate in lysosomes, reaching a concentration of 250 times that of the culture fluid ~10). Streptomycin also accumulates in lysosomes, but only to a concentration of 20 times that of the culture fluid ~11 ~. Sharer e t al. (6) did not observe any morphological changes in human amnion or foreskin cells 3 to 5 days after 1000 to 6000 ~g per ml gentamicin had been added to the nutrient medium. Our results suggest that the observation time was too short. When the same cells were grown through 20 or more culture passages with 50 /~g per ml gentamicin, no changes in cell growth were observed. However, cell counts are not presented in the publication. Antibiotics are necessary when cell cultures are prepared from contaminated specimens or when a valuable contaminated culture has to be saved, but they should not be added routinely to the culture medium. Reasons given by Coriell (12) are that they encourage relaxing of aseptic techniques and development of resistant organisms or bacterial L-forms, as with penicillin. Some organisms are only suppressed by the antibiotic and may still cause biochemical changes in the culture medium or cells. In addition, our studies show that the antibiotics themselves can be harmful to the cells even at concentrations which are thought to be safe.
119
REFERENCES 1. Rudin, A., A. Healey, C.A. Phillips, D.W. Gump, and B. R. Frosyth. 1970. Antibacterial activity of gentamicin sulfate in tissue culture. Appl. Microbiol. 20: 989-990. 2. Braun, P., J. O. Klein, and E. H. Kass. 1970. Susceptibility of genital mycoplasmas to antimicrobial agents. Appl. Microbiol. 19: 62-70. 3. Perlman, D., S. B. Rahman, and J. B. Semar. 1967. Antibiotic control of mycoplasma in tissue culture. Appl. Microbiol. 15: 82-85. 4. Perlman, D. 1976. Some guidelines for selection of antibiotics for elimination of unwanted microbial contaminants in tissue cultures. Procedure 15143. TCA Manual 2: 383-386. 5. Byarugaba, W., H.W. Riidiger, T. KoskeWestphal, W. WShler, and E. Passarge. 1975. Toxicity of antibiotics on cultured human skin fibroblasts. Humangenetik 28: 263-267. 6. Schafer, T. W., A. Pascale, G. Shimonaski, and P. E. Came. 1972. Evaluation of gentamicin for use in virology and tissue culture. Appl. Microbiol. 23: 565-570. 7. Goetz, I. E. 1975. Growth of human skin fibroblasts from punch biopsies. Procedure 41523. TCA Manual 1: 13-15. 8. Fischer, A. B. 1975. Gentamicin as a bactericidal antibiotic in tissue culture. Med. Microbiol. Immunol. 161: 23-39. 9. Litwin, J. 1970. The effect of commercial and pure gentamicin on the growth of human diploid lung fibroblasts. Acta Pathol. Microbiol. Scand. Sec. B 78: 273-276. 10. Tulkens, P., and A. Trouet. 1974. Uptake and intracellular localization of kanamyein and gentamicin in the lysosomes of cultured fibroblasts. Soe. Beige de Biochimie, Reunion de Louvain-la-Neuve, pp. 1018-1019. 11. Tulkens, P., and A. Trouet. 1972. Uptake and intracellular localization of streptomycin in the lysosomes of cultured fibroblasts. Soc. Belge de Biochimie, Reunion de Strasbourg, pp. 623-624. 12. Corieli, L. L. 1973. Methods of prevention of bacterial, fungal, and other contaminations. In: Jorgen Fogh (Ed.), Contamination in Tissue Culture. Academic Press, London, pp. 29-49.
The authors gratefully acknowledge the technical assistance of Nestor Diaz.
E F F E C T S O F S O M E A N T I B I O T I C S ON T H E G R O W T H O F H U M A N DIPLOID SKIN FIBROBLASTS IN CELL CULTURE INGEBURG E. GOETZ, RANDI MOKLEBUST, ANDCATHERINE J. WARREN
Division of Neurosciences, City of Hope National Medical Center, Duarte, California 91010
SUMMARY
During serial subcultures 50/~g per ml gentamicin and penicillin (100 U per ml)-streptomycin ~100/~g per ml~ depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.
Key words: antibiotics; human skin fibroblasts; cell growth. INTRODUCTION periments to investigate this effect and present the Because of the constant threat of bacterial con- results in this paper. tamination in cell culture, antibiotics are added MATERIALS AND M E T H O D S routinely to the nutrient medium in many laboratories. The preferred antibiotic for many years Human skin diploid fibroblast cell lines were has been a mixture of penicillin G and streptomy- obtained either from the Genetic Mutant Cell Recin (pen-strep). During the last few years, genta- pository, Camden, New Jersey, or grown from micin has gained increasing acceptance because it skin biopsies in our laboratory according to the is effective against a wider range of bacteria than method outlined in the TCA Manual 17L All cell pen-strep and against some strains of mycoplasma lines were tested and found to be free from myco~l-3L Gentamicin, in addition, has a greater plasma contamination by a combination of biostability in tissue-culture medium than does pen- chemical and cultural methods performed by Irstrep (4), especially at 37 ~ C, the temperature vine Scientific Sales Co., Fountain Valley, Caliused for growing mammalian cells. fornia. Some of the cell lines were routinely grown In cell culture the recommended concentrations in the presence of 80 U per ml penicillin G and 80 are: penicillin G, 100 U per ml; streptomycin, 100 /~g per ml streptomycin. Before being used for the /~g per ml; and gentamicin, 100 t~g per ml. These reported studies all cell lines were grown through concentrations were not found to be noticeably four to five subcultures without antibiotics. toxic to Earle's L-929 cells or Eagle's KB cells by The nutrient medium employed was Eagle's Perlman (4). Byarugaba et al. t5~ reported that minimal essential medium with Earle's salts. In 6000 ~g/ml per ml penicillin G, 1250 ~g/ml addition it contained 0.1 mM nonessential amino streptomycin, or 1000 ~g per ml gentamicin per- acids, 1 mg per 1 biotin and 20% fetal bovine mitted unchanged growth of cultured human skin serum not heat-inactivated. The glutamine confibroblasts. Schafer et al. ~6) observed no toxicity centration was raised to 4 mM and the pH adwhen a variety of mammalian cells were grown justed to 7.2 to 7.4. Nonessential amino acids and through a large number of culture passages with biotin were not required by the cells but were in50/~g per ml gentamicin, or when cells were ex- cluded because the medium also was used for posed for a short time to several mg per ml. other purposes. Cell growth was considerably During the course of studies in our laboratory faster with 20% than with 10% serum. Cells were with human skin fibroblasts, we added 100 U per grown in plastic tissue-culture dishes at ml penicillin G and 100/~g per ml streptomycin, 37_+0.5 ~ C in a humidified gas of 5% CO2 in air. or 50 ~g per ml gentamicin to the culture medium Our routine culturing procedure consisted of and observed a decrease in the growth rate of the medium changes twice weekly and subculturing cells. Subsequently, we performed additional ex- when the cell layer was confluent, usually every 5 114
115
EFFECTS OF ANTIBIOTICS ON FIBROBLASTS to 7 days. Subculturing was done at room temperature using a buffered solution of 0.5% trypsin and 0.02% E D T A to disperse the cells. For the experiments with antibiotics the dispersed cells were suspended in medium without antibiotics and fresh dishes were plated at a concentration of 0.5 to 1.25 x 104 cells per cm ~, depending on the particular study. Antibiotics were added immediately from the concentrated stock solutions. Gentamicin was used to final concentrations of 25 to 400/ag per ml of medium and pen-strep to final concentrations of 50 to 100 U per ml penicillin and 50 to 100/ag per ml streptomycin. Antibiotics were mixed with the medium by agitation of the dish. The change in osmolarity was negligible because only 2.5 to 40 ~1 of stock solution was required per ml of medium. No antibiotics were added to control dishes. The above described procedure was used for each subsequent culture passage. To determine plating efficiency cells were dispersed and counted 1 day after being plated. Cell mitosis had not resumed at that time. Cell counts were made with a hemacytometer or with a Coulter counter. Samples of the cell suspensions were regularly examined microscopically to ensure that no cell clumps were present. Two dishes of cells were always grown for each antibiotic concentration and for the controls. At feedings and subculturing dishes were handled at random. Concentrated stock solutions for the nutrient medium, Hanks' saline without calcium and magnesium, trypsin-EDTA, and a solution containing 10,000 U per ml penicillin G sodium and 10,000 U per ml streptomycin sulfate were obtained from Grand Island Biological Co., Grand Island, New
York. Gentamicin reagent solution, l0 mg per ml was bought from Schering Corp., Kenilworth, New Jersey. Fetal bovine serum (not heat-inactivated) was purchased from Reheis Chemical Co., Phoenix, Arizona. Tissue culture dishes were obtained from either Falcon Plastics, Los Angeles, California, or Corning Scientific Products Division, Sunnyvale, California. Calculations
Accumulated number of cells: C A It, t = Cito~ x G F A ~t, G F A = GFitl~ x GFitz~ x . . . . .
GFtt~
CItn-l) C.4tt, l: Number of cells accumulated at time trt.
C~toj: Number of cells plated at time to. GF.41tnl: Accumulated growth factor at time tn.
GFum: Ratio of number of cells counted at time tn and number of cells plated at time t n - 1 . C': Number of cells counted. C: Number of cells plated.
Percent of loss of cells in medium with antibiotics: 100 ( a - b ) a
a: Cells per dish in medium without antibiotics. b: Cells per dish in medium with antibiotics.
TABLE
1
G AIN OR LOSS OF H UMAN S KIN F IBROBLASTS AFTER 6 TO62 D AYS IN ]V[EDIUM WITH G ENTAMICIN ORP E N I C I L L I N ~ TREPTOMYCIN AND R ECOVERY AFTER THE ANTIBIOTICS ARE OMITTED Percent Gain or Loss of Cells in Medium with Antibiotics at End of Growth Period a PenlU Iml)-strep(~ag/ml)
Gentamicin (~g/ml)
Cell Line
Growth Period
No.
275
1
2 3 4 5 6 7
days 0-6 6-12 12-18 18-25 25-33 33-47 47-62
Cells Plated at Beginning of Growth Period
50
cells Icm 2 x 104 1.0 1.0 1.0 1.0 1.0 0.5 0.5
+ 4.3 - 1.1 -14.2 -12.9 -11.2 -34.0 -32.4
50 Omitted alter 33 days
100
-31.8 + 2.5
+18.1 - 8.8 -19.5 -20.6 -25.3 -48.7 -53.9
100 Omitted after 33 days
104)
100 Omitted after 33 days
-50.4 + 2.2
- 0.5 - 3.0 -19.9 -20.0 -16.4 -19.5 -15.4
- 6.2 +27.8
a At the end of the growth period all cells were counted. From the average counts of cells in two dishes without antibiotics and in two dishes with a particular antibiotic concentration, percent loss or gain of cells was calculated for that growth period.
116
GOETZ, MOKLEBUST, AND WARREN RESULTS
] • 1070 I
L
One cell line was grown for 62 days through seven subcultures in medium with 50 or 100 ~g per ml gentamicin, 100 U per ml penicillin and 100 t~g per ml streptomycin, or without antibiotics. The effects of the antibiotics are shown in Table 1. During the first growth period with antibiotics, gentamicin, but not pen-strep, stimulated cell growth. In subsequent growth periods both antibiotics caused a loss in cell yield. In the second growth period this effect was very small and probably significant only for 100 gg per ml of gentamlcin. In growth periods 3 to 5, all concentrations tested caused considerable loss in cell yield, the magnitude of which remained approximately constant during this time. At the beginning of growth period 6, the cells in medium with antibiotics were split and grown with and without antibiotics to examine recovery of cell growth. Since there were insufficient ceils available in the cultures that had been grown with 100/~g per ml gentamicin, the plating density was decreased by one-half for all cultures, including the controls. In addition, the length of the growth period between subcultures was approximately doubled. This resuited in a further decreased cell yield in the cultures grown with gentamicin, whereas the effect of pen-strep remained approximately the same. In those dishes in which the antibiotics were omitted, the cell yield gradually returned to normal or above normal. Cells that previously had been grown in medium with pen-strep recovered more quickly than those previously grown with gentamicin. The cumulative increases in cells during this study are shown in Fig. 1. Compared to the controls the total loss of cells, after 62 days in
E
~
] x I0 B
U')
LLI (J
LLI
)
1 x I0 "t
~/~//6
r
--1
1 •
10 6
I x 105 ~ 0
.
10
.
.
20
.
.
.
30
.
.
.
40
.
.
50
~
60
--~ 70
DAYS IN MEDIUM WITH ANTIBIOTICS
FIG. 1. Effect of gentamicin and penicillin-streptomycin on growth of human skin fibroblasts during 62 days with regular subculturing. 9 - - 9 No antibiotics: x - - x , 100 ~g per ml gentamicin; A - - A , 50 ~g per ml gentamicin; 9 1 6 9 100 U per mi penicillin 13 and 100 ~g per ml streptomycin. medium with antibiotics, was 71.2% for 50 ~g per ml gentamiein, 87.9% for 100 ~g per ml gentamicin, and 60.6% for 100 U per ml penicillin and 100 gg per ml streptomycin. Fig. 1 shows that the control cells grew exponentially until day 47 after
TABLE 2 E FFECT OF GENTAMICIN ANDP ENICILLIN-STREPTOMYCIN ON FOUR D IFFERENT LINES OF HUMAN S KIN F IBROBLASTS Percent Loss of Cells in Medium with Antibiotics at End of Growfll Period a Cell Line
Growth Period
No.
967
days
Cells Plated at Beginning of Growth Period
Gentamicln i~g/rnl) 25
50
lOO
PenlU mll-strepl~g/ndl 200
400
50
IO0
cells/cm ~x l04
1 0-14 1.25 8.2 8.0 11.1 25.0 32.2 6.5 9.3 2 14-28 1.25 20.0 37.4 7.1 1061 1 0-14 1.25 3.1 6.9 13.2 22.9 42.6 0.0 4.2 2 14-28 1.25 19.7 36.9 5.8 1085 1 0-14 1.25 0,0 0.0 3.1 9.9 13.5 0.0 0.0 2 14-28 1.25 9.8 20.4 0.0 321 1 0-14 1.0 3.9 5.9 6.3 11.3 33.0 5.2 5.3 2 14-28 1.0 10.3 22.4 8.9 a At the end of the growth period all cells were counted. From the average count of cells in two dishes without antibiotics and in two dishes with a particular antibiotic concentration, percent loss of cells was calculated for that growth period.
EFFECTS OF ANTIBIOTICS ON FIBROBLASTS which cell growth declined. This may be a sign that the cells were approaching senescence at that time. The inhibitory effect of the antibiotics on growth of cultured human skin fibroblasts was confirmed with four additional cell lines by experiments of shorter duration (Table 2). In this study pen-strep was considerably less toxic than gentamicin. Loss of cell yield increased with the concentration of gentamicin and was already considerable at 200/~g per ml in two of the cell lines. Since the stimulatory effect of this antibiotic, which previously was observed at the end of the first growth period, did not occur in this study, an additional experiment was performed. Cells from another line were grown with 100 ~g per ml genta-
o
E
Q.. O3 4 J ..J
b..I (.3
] 17
micin, 100 U per ml penicillin and 100/~g per ml streptomycin, or no antibiotic for 14 days without subculturing; and counts were made 1, 3, 5, 7 and 14 days after the dishes were plated. The results in Fig. 2 show that the antibiotics had very little effect on cell growth until 5 days when gentamicin had clearly stimulated cell growth, whereas penstrep had caused a slight inhibition of growth. After 14 days gentamicin and pen-strep both had inhibited cell growth to approximately the same extent, the loss of cells being 19.2% and 18.1% of the controls, respectively. The plating efficiency of the examined cell lines ranged from 71% to 98% and was not affected by 25 to 400/~g per ml gentamicin or 50 to 100 U per ml penicillin and 50 to 100 #g per ml streptom y c i n . Examination of the cultures by phase microscopy did not reveal any differences between cells in medium with antibiotics and controls until 3 or 4 days after the cells had been plated into medium with the antibiotics. At that time less mitosis and more cell debris was observed in cultures with 400 #g per ml gentamicin than in those without antibiotics, but not in cultures with lower concentrations of gentamicin or with the concentrations of pen-strep tested. Fourteen days after plating, during which time the cells were fed every 3 or 4 days, but not subcultured, cells in medium without antibiotics had formed a dense layer. The cells were spread out and firmly attached to the culture dish. No morphological difference was observed in cells grown with 50 or 100 U per ml penicillin and 50 or 100/~g per ml streptomycin, or 50 or 100 #g per ml gentamicin. Cells in medium with 200 /~g per ml gentamicin showed some retraction from the dish. Cells in medium with 400/~g per ml gentamicin, in addition, contained many dark particles in the perinuclear area which were possibly enlarged lysosomes (Fig. 3 }. DISCUSSION
MEDIUM CHANGE
1
4
I
6
8
DAYS IN MEDIUM WITH
10
12
14
ANTIBIOTICS
FIG. 2. Effects of gentamicin and penicillinstreptomycin on growth of human skin fibroblasts during 14 days without subculturing. 9 - - 9 , No anttblo """ tics; x--~, 100/~g per ml gentam~cin; O - - O , 100 U per ml penicillin G and 100 ~g per ml streptomycin.
Our results show that 50 or 100/~g per ml gentamicin and 100 U per ml penicillin and 100 #g per ml streptomycin concentrations that are thought to be safe for cultured cells depress the growth of human skin fibroblasts. This effect appears only after one or two periods of logarithmic growth in the presence of the antibiotics and therefore was not observed by investigators who used shorter observation times (8). In our experiments gentamicin stimulated cell growth before it became inhibitory. This was also noticed by Litwin (9~ who grew human diploid lung fibroblasts for more than 100 days in the presence of gentamicin. The
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GOETZ, MOKLEBUST, AND WARREN
FIG. 3. Effect of gentamicin on morphology of human skin fibroblasts. A, Cells grown 14 days without antibiotics; B, parallel culture of cells grown 14 days with 400 ~g per ml gentamicin showing retraction of cells from the surface and dark particles in the perinuclear area {arrows}. Phase contrast photomicrographs, x400.
EFFECTS OF ANTIBIOTICS ON FIBROBLASTS depression of cell growth that followed the stimulation was less than in our studies, but his controls were grown with 100 U per ml penicillin and 100/~g per ml streptomycin which, as shown by our results, interferes with cell growth. The same author found that 500 #g per ml gentamicin did not decrease reattachment of trypsinized cells. This was confirmed in our studies. The dark particles that we observed in the perinuclear area in cells maintained for 14 days in the presence of 400 #g per ml gentamicin were possibly enlarged lysosomes. This is suggested by the observation that gentamicin can accumulate in lysosomes, reaching a concentration of 250 times that of the culture fluid ~10). Streptomycin also accumulates in lysosomes, but only to a concentration of 20 times that of the culture fluid ~11 ~. Sharer e t al. (6) did not observe any morphological changes in human amnion or foreskin cells 3 to 5 days after 1000 to 6000 ~g per ml gentamicin had been added to the nutrient medium. Our results suggest that the observation time was too short. When the same cells were grown through 20 or more culture passages with 50 /~g per ml gentamicin, no changes in cell growth were observed. However, cell counts are not presented in the publication. Antibiotics are necessary when cell cultures are prepared from contaminated specimens or when a valuable contaminated culture has to be saved, but they should not be added routinely to the culture medium. Reasons given by Coriell (12) are that they encourage relaxing of aseptic techniques and development of resistant organisms or bacterial L-forms, as with penicillin. Some organisms are only suppressed by the antibiotic and may still cause biochemical changes in the culture medium or cells. In addition, our studies show that the antibiotics themselves can be harmful to the cells even at concentrations which are thought to be safe.
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REFERENCES 1. Rudin, A., A. Healey, C.A. Phillips, D.W. Gump, and B. R. Frosyth. 1970. Antibacterial activity of gentamicin sulfate in tissue culture. Appl. Microbiol. 20: 989-990. 2. Braun, P., J. O. Klein, and E. H. Kass. 1970. Susceptibility of genital mycoplasmas to antimicrobial agents. Appl. Microbiol. 19: 62-70. 3. Perlman, D., S. B. Rahman, and J. B. Semar. 1967. Antibiotic control of mycoplasma in tissue culture. Appl. Microbiol. 15: 82-85. 4. Perlman, D. 1976. Some guidelines for selection of antibiotics for elimination of unwanted microbial contaminants in tissue cultures. Procedure 15143. TCA Manual 2: 383-386. 5. Byarugaba, W., H.W. Riidiger, T. KoskeWestphal, W. WShler, and E. Passarge. 1975. Toxicity of antibiotics on cultured human skin fibroblasts. Humangenetik 28: 263-267. 6. Schafer, T. W., A. Pascale, G. Shimonaski, and P. E. Came. 1972. Evaluation of gentamicin for use in virology and tissue culture. Appl. Microbiol. 23: 565-570. 7. Goetz, I. E. 1975. Growth of human skin fibroblasts from punch biopsies. Procedure 41523. TCA Manual 1: 13-15. 8. Fischer, A. B. 1975. Gentamicin as a bactericidal antibiotic in tissue culture. Med. Microbiol. Immunol. 161: 23-39. 9. Litwin, J. 1970. The effect of commercial and pure gentamicin on the growth of human diploid lung fibroblasts. Acta Pathol. Microbiol. Scand. Sec. B 78: 273-276. 10. Tulkens, P., and A. Trouet. 1974. Uptake and intracellular localization of kanamyein and gentamicin in the lysosomes of cultured fibroblasts. Soe. Beige de Biochimie, Reunion de Louvain-la-Neuve, pp. 1018-1019. 11. Tulkens, P., and A. Trouet. 1972. Uptake and intracellular localization of streptomycin in the lysosomes of cultured fibroblasts. Soc. Belge de Biochimie, Reunion de Strasbourg, pp. 623-624. 12. Corieli, L. L. 1973. Methods of prevention of bacterial, fungal, and other contaminations. In: Jorgen Fogh (Ed.), Contamination in Tissue Culture. Academic Press, London, pp. 29-49.
The authors gratefully acknowledge the technical assistance of Nestor Diaz.
