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Biological effects of shock waves: Cell disruption, viability, and proliferation of L1210 cells exposed to shock waves in vitro
Ultrasound in Med. & Biol. Vol. 16, No. 6, pp. 587-594, 1990 Printed in the U.S.A.
0301-5629]90 $3.00 + .DO © 1990 Pergamon Press plc
OOriginal Contribution BIOLOGICAL EFFECTS OF SHOCK WAVES: CELL DISRUPTION, VIABILITY, AND PROLIFERATION OF L1210 CELLS EXPOSED TO SHOCK WAVES IN VITRO STEFAN GAMBIHLER, MICHAEL DELIUS a n d WALTER BRENDEL * Institute for Surgical Research, University of Munich, Munich, Federal Republic of Germany (Received 7 September 1989; in final form 25 February 1990) A b s t r a c t - - L l 2 1 0 cells were exposed in suspension to shock waves generated with a Dornier XL1 lithotripter. After 1000 discharges at 25 kV, the number of nondisrupted cells was 15% and the number of trypan blue excluding cells was 7% as compared to 100% in sham treated controls; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Overall proliferation of cells which were trypan blue negative after exposure exceeded 70% of the proliferation of sham treated controls, except after 1000 shocks at 25 kV, where proliferation was reduced to 42%. The latter reduction in proliferation was found to be due to a reduced growth for 24 h after exposure, with a return to normal proliferation during the following days. Limiting dilution analysis revealed that the reduced growth was mainly due to a transitory increase of the doubling time and not to a reduction of the number of proliferating cells. Cell disruption by shock waves was completely inhibited by exposing the cells at an elevated pressure of 101 atmospheres, pointing to the possible involvement of cavitation in the shock wave effect.
Key Words: Cavitation, Cell disruption, LI210 cells, Limiting dilution, Lithotripter, M T r assay, Shock waves, Trypan blue assay, Viability.
INTRODUCTION
Shock waves are single pressure pulses of microsecond duration with a peak pressure of several hundred atmospheres. They are used in medicine for the fragmentation of urinary and biliary calculi (Chaussy et al. 1980; Sauerbruch et al. 1986). As compared to ultrasound, little is known about biological effects of shock waves on cells in vitro. Similar to ultrasound, shock-wave lithotripters are known to produce acoustic cavitation (Coleman et al. 1987) and cavitation is considered to be a mechanism for stone disintegration and tissue damage caused by shock waves (Delius et al. 1988a; Delius et al. 1988c). In the present study, the biological effects of shock waves are characterized for L1210 mouse leukemia cells in vitro. Cell disruption and ability to exclude trypan blue (Tennant 1964) were examined and related to the electrical energy input at the lithotripter for shock waves generated at different operating voltages and discharge numbers. Proliferation was determined by 3-4,5-dimethyl-thiazol-2,5 diphenyl tetrazolium bromide (MTT) assay (Mosmann 1983; Carmichael et al. 1987a, 1987b), and by studying the time course of proliferation. Limiting dilution
Disruption of cells has long been known to be an effect of ultrasound in vitro (Clarke and Hill 1970; Kaufman et al. 1977; Fu et al. 1980; Ciaravino et al. 1981b; Dooley et al. 1983). In addition, it has been reported that a part of the remaining cells is rendered nonviable as determined by vital dye exclusion or colony formation, and cells surviving sonication with ultrasound show growth retardation and form smaller colonies (Kaufman et al. 1977; Miller et al. 1977; Kaufman and Miller 1978; Fu et al. 1980; Ciaravino et al. 198 lb). Ultrasound is known to generate cavitation, which is "any observable activity involving bubbles" (Crum 1982), and cavitation has been reported to be an important mechanism for the biological effects (Clarke and Hill 1970; Leake et al. 1980; Ciaravino et al. 1981a; Sacks et al. 1981; Vivino et al. 1985). Address correspondence to: S. Gambihler, Institute for Surgical Research, Klinikum Grosshadern, 8000 Muenchen 70, Federal Republic of Germany. Deceased. 587
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analysis (Lefkovits and Waldman 1979, 1984) was used to determine the plating efficiency. The role of cavitation for cell disruption was evaluated. MATERIALS AND METHODS
Cell cultures L 1210 mouse leukemia cells (kindly supplied by Dr. H. P. Kraemer, Behringwerke, Marburg, FRG) were grown as suspension culture in RPMI 1640 medium supplemented with 15% heat inactivated fetal calf serum, 2% sodium pyruvate, and 1% antibioticantimycotic solution (Gibco, Eggenstein, FRG). Shock waves The principle of electrohydraulic shock wave generation has been described earlier (Forssmann et al. 1977). Shock waves generated with an experimental Dornier lithotripter model XL1 were administered at a rate of 100 per min, electrodes were replaced after every 500 discharges. According to measurements from Mueller (Dornier Medizintechnik, Germering, FRG) with PVDF needle probes, shock waves generated at an operating voltage of 20 kV have a peak positive pressure of 84 MPa with a focal length and width of 22 mm and 5mm; due to focal shift maximum pressure is reached 5 mm behind the geometric focus of the ellipsoid along the longitudinal axis (Mueller 1989, in press). Exposure vials made of polypropylene (Interchem, Muenchen, FRG) had an inner diameter of 11.7 mm and a height of 47 mm. The vials were positioned so that the geometric focus of the ellipsoid, indicated by a laser system, was 10 mm above the bottom of the vial. The pressure field in the vials was not homogeneous due to shock wave focusing. The water in the lithotripter tank was degassed by a vacuum pump; oxygen concentration was 2-4 mg/L. Experimental procedures Samples of 2 X 10 6 log phase L1210 cells in 5.2 mL culture medium were transferred into vials and kept in the lithotripter tank maintained at 35-37°C. Every single experiment consisted of one or more samples exposed to shock waves and one sham treated control sample which was placed in the waterbath outside of the shock wave field. The effect of shock waves generated at different operating voltages and with different discharge numbers was related to the electrical energy input at the lithotripter, determined as W = ½C U 2 X d i s c h a r g e n u m b e r [kWs]
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where C is the capacitance of the capacitor (80 nF) and U is the operating voltage used. For experiments at elevated pressure, the vials were transferred into a steel chamber, which was filled with water of the lithotripter tank, closed under water and put under pressure by nitrogen. The shock waves entered the chamber through a 0.3 mm steel plate. Samples were exposed to shock waves at ambient pressure or at 101 atmospheres. Control samples were exposed to only ambient or elevated pressure.
Cell disruption and ability to exclude trypan blue The reliability of cell counting after exposure to shock waves was determined in triplicate by three different observers in a hemocytometer by counting morphologically intact cells which revealed well reproducible intra- and interobserver values (Table 1). The number of nondisrupted cells was counted in triplicate for each sample. The cells were scored for the ability to exclude trypan blue with equal amounts of cell suspension and trypan blue (2 g/L in 0.9% NaCI solution; Fluka, Buchs, Switzerland). After 3 rain at room temperature the number of stained and unstained cells was determined in a hemocytometer in triplicate for each sample. M T T assay 3-4,5-dimethyl-thiazol-2,5 diphenyl tetrazolium bromide (MTT) was obtained from Sigma (Taufkirchen, FRG). Cell numbers of sham treated controls and of samples exposed to shock waves, as determined by trypan blue assay, were adjusted to 9 X 10 3 trypan blue negative cells/mL. Into each well of 96well round bottom microtitre plates (Nunc, Roskilde, Denmark) 0.05 mL culture medium and 0.1 mL cell suspension were plated. Sixteen wells were plated per sample and incubated at 37°C in a humidified 5% CO2 atmosphere. After 72 h, 0.05 mL MTT (2.5 g/L
Table 1. N u m b e r o f n o n d i s r u p t e d L1210 cells after exposure to shock waves at 25 kV, as d e t e r m i n e d by three different observers. Nondisrupted cells [X 104/mL] Observer
Discharge number
B.S.
Control 100 250 500 I000
45.5 _+ 1.4 27.7 + 0.7 21.2 + 0.5 10.0 + 0.2 2.0 + 0.0
N.W. 42.7 25.1 21.0 10.7 4.3
+_ 1.1 _+ 2.3 + 1.9 _ 0.8 ___0.5
S.G. 42.6 25.9 21.4 9.8 3.3
Figures represent the mean (+SD) of three cell countings.
+ 0.3 _+ 1.1 + 1.7 _+0.2 + 0.3
Biological effects of shock waves: L 1210 cells • S. GAMBIHLER et al.
in 0.9% NaC1 solution) was added to each well. After a further incubation time o f 4 h the supernatant fluid was carefully removed and 0.1 m L dimethylsulfoxide (Merck, Darmstadt, FRG) was added to each well. Plates were agitated on a plate shaker and absorbance at 490 n m was measured within 5 min using a Dynatech platereader model M R 580. In control experiments cell suspensions exposed to 500 shock waves at 25 kV were centrifuged and the supernatant was added to cells not exposed to shock waves. Proliferation, as determined by M T T assay, was unchanged as c o m p a r e d to untreated control cells.
Time course of proliferation Cell numbers of sham treated controls and of samples exposed to shock waves at 25 kV, as determined by trypan blue assay, were adjusted to concentrations of 1 X 103 trypan blue negative cells/mL, transferred into 25 cm 2 culture flasks (Nunc) and incubated for 5 days. Every 24 h after seeding the number o f cells was determined with the trypan blue assay. The doubling time o f the cells was determined from the resulting growth curves.
Limiting dilution analysis Limiting dilution analysis (Lefkovits and Waldman 1979, 1984) was used to determine the plating efficiency. The frequency distribution of cells over the wells after plating of low numbers of cells into microtitre plates can be described by the Poisson distribution. W h e n low n u m b e r s o f L1210 ceils are plated at a given number of inoculated cells per well (u) and the plating efficiency (k) is defined as the ratio o f the average n u m b e r o f inoculated/proliferating cells, the fraction of wells without growth (Fo) is described as
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were plated at each cell concentration and incubated. After two weeks the fraction of wells without growth (Fo) was determined. The corresponding curves were calculated with the least square method and the plating efficiencies were taken from the slopes of the lines. The curves representing the sham treated controls and the samples exposed to shock waves were compared based on one-sided t test (Sachs 1973) at a 5% significance level. RESULTS
Cell disruption In sham treated controls, 92% + 11% of the 2 X 10 6 cells per vial were recovered; the recovery of the sham treated control cells of each experiment was set to 100% and the number of nondisrupted cells after exposure to shock waves was related to the control value of each experiment. With higher voltages and shock wave numbers, the number of recovered cells decreased progressively. In Fig. 1, the relative number of nondisrupted cells after treatment is related to the electrical energy input during shock wave application. Figure la combines points gained from treatments at the same voltages but different shock wave numbers; in Fig. lb, the same values are shown, but lines combine points gained from treatments with the same shock wave numbers but at different voltages.
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Fig. 1. Cell disruption. Number of nondisrupted cells (yaxis), given as percentage of recovered sham treated control cells, related to electrical energy input at the lithotripter (x-axis). The same data are given in both panels, (a) representing curves at different operating voltages and (b) showing curves at different discharge numbers. Points represent the mean of three separate experiments. Error bars show the standard deviation.
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Higher voltages at a similar electrical energy input (obtained by a lower discharge n u m b e r ) , caused greater cell disruption than lower voltages (Fig. la). Vice versa, a higher n u m b e r of shock waves was less effective in disrupting cells than a lower number of shock waves, when compared at a similar electrical energy input (obtained by a lower operating voltage; Fig. 1b). For example, after exposure of L 1210 cells to 2000 shocks generated with 20 kV, corresponding to an electrical energy input of 32 kWs, 37% __ 13% of the cells were still nondisrupted, whereas after exposure to 1000 shock waves at 25 kV, corresponding to an electrical energy input of 25 kWs, 15% +_ 1% of the cells were nondisrupted.
Trypan blue negative cells The n u m b e r o f trypan blue excluding cells was related to the total n u m b e r of recovered sham treated control cells, which was set to 100%. In sham treated controls, the ability to exclude trypan blue was 98% + 1%. In Fig. 2, the relative n u m b e r of trypan blue negative cells after treatment is related to the electrical energy input; Fig. 2a combines points gained from treatments at the same voltages but different shock wave numbers; in Fig. 2b the same values are shown but lines combine points from treatments with the same shock wave numbers but at different voltages.
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Table 2. Number of trypan blue positive L I210 cells after exposure to shock waves, given as percentage of recovered sham treated control cells. Trypan blue positive cells (%) Operating voltage
Discharge number
15 kV
20 kV
25 kV
Control 100 250 500 1000 2000
1_+ 1 5_+1 8_+3 12_+6 19 +_ 3 27 _+ 6
2_+ 2 7_+ 2 12-+ 2 16_+ 6 19 +_ 10 20 _+ 11
2-+- 1 12_+6 18_+7 11_+3 8 -+ 3 n.d.
Figures represent the mean (_+SD) of three experiments. n.d. = not determined.
The same trend was seen as with cell disruption--a more pronounced effect with increasing voltage than with increasing shock-wave number. For example, after exposure to 2000 shock waves at 20 kV (32 kWs), 17% ___6% of the cells were trypan blue negative, and this number decreased to 7% + 1% after 1000 shock waves at 25 kV (25 kWs).
Trypan blue positive cells The difference between Figs. 1 and 2 was caused by cells which were morphologically intact, but trypan blue positive. Their number was again related to the total n u m b e r of recovered sham treated control cells. It reached a m a x i m u m of 27% +_ 6% after treatment with 2000 shock waves at 15 kV (Table 2). If the n u m b e r of trypan blue cells was related to the n u m b e r of intact, trypan blue negative cells, the values did not show the trend of a greater effect of higher voltages at a similar electrical energy input. For example, the ratio was 0.50 + 0.20 after exposure to 2000 shocks at 20 kV (32 kWs) and was 0.51 _+ 0.11 after e x p o s u r e to 1000 shocks at 25 kV (25 kWs).
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Fig. 2. Trypan blue negative cells. Number of trypan blue excluding cells (y-axis), given as percentage of recovered sham treated control cells, related to electrical energy input at the lithotripter (x-axis). The same data are given in both panels, (a) representing curves at different operating voltages and (b) showingcurves at different discharge numbers. Points represent the mean of three separate experiments. Error bars show the standard deviation.
Proliferation of cells that had been trypan blue negative after exposure to shock waves was examined in 3 different sets o f experiments. In the first set, overall proliferation during the 3 days following exposure was determined by M T T assay (Fig. 3). Proliferation was 68% + 11% after exposure to 2000 shocks at 20 kV (32 kWs). This value was exceeded under all conditions with one exception: after exposure to 1000 shock waves at 25 kV (25 kWs) it was reduced to 42% -!-_ 19%. In the second set, proliferation was examined in more detail by studying the time course of proliferation o f cells exposed to shock waves at 25 kV (Fig. 4).
Biological effects o f s h o c k waves: L I 2 1 0 cells • S. GAMBIHLER et al.
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Role of cavitation
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Fig. 3. MTT assay. Overall proliferation of cells that had been trypan blue negative after exposure to shock waves (y-axis), given as percentage of the proliferation of recovered sham treated control cells, related to electrical energy input at the lithotripter (x-axis). The same data are given in both panels, (a) representing curves at different operating voltages and (b) showing curves at different discharge numbers. Points represent the mean of three separate experiments. Error bars show the standard deviation.
Cells treated with 100, 250 or 500 shock waves at 25 kV grew exponentially from the very beginning for 120 h with a doubling time o f 11 h, similar to sham treated control cells. Cells treated with 1000 shock waves at 25 kV showed reduced growth during the first 24 h after application of shock waves; during this period, the relative n u m b e r of cells increased only by a factor o f 2.2 as compared to a factor of 4.3 of sham treated control cells. After the first 24 h the cells returned to normal proliferation. The diminished growth during the first 24 h after exposure to 1000 shock waves at 25 kV could be due to either a reduced n u m b e r of proliferating cells (the result of Fig. 4 could be obtained if only 40% of the cells would proliferate after shock wave exposure), or to an increase in the doubling time of the cells. In the third experiment, we tested the plating efficiency of cells with the limiting dilution technique. Sham treated controls showed a plating efficiency of 96% (Fig. 5), while the plating efficiency o f cells exposed to 1000 shock waves at 25 kV was 77%, which is a small although significant decrease (p < 0.05 in one-sided t test). This plating efficiency was too high to account for the reduced proliferation which could only be explained by a plating efficiency in the range of 40% (Fig. 4), and therefore the doubling time must have been increased. Considering a plating efficiency of
The role o f cavitation for cell disruption was evaluated by exposing L 1210 cells to shock waves in a pressure chamber. T h e n u m b e r o f n o n d i s r u p t e d L1210 cells exposed to 500 shock waves at 30 kV at ambient pressure was 25% _+ 12% as compared to sham treated controls (Table 3). Cell disruption was absent when the cells were exposed to shock waves at a pressure elevated to 101 atmospheres. Treatment with pressure only showed no effect on cell disruption. DISCUSSION T h e p r i m a r y effects o f shock waves on suspended L1210 cells observed in this study were cell disruption, decreased ability to exclude trypan blue
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Fig. 4. Time course of proliferation. Relative number of cells that had been trypan blue negative after exposure to shock waves at 25 kV (y-axis), given for every 24 h after seeding (x-axis). Points represent the mean of three separate experiments. Error bars show the standard deviation. The dotted line represents a back extrapolation of the curve for the cells treated with 1000 shock waves at 25 kV, indicating that the reduced proliferation of cells exposed to 1000 shock waves could be explained by a plating efficiency of 40%.
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and a t r a n s i t o r y decrease in proliferation. Cell disruption by shock waves was absent when cells were exposed to shock waves at an elevated pressure, suggesting that cavitation is a major mechanism in producing cell disruption. Exposure o f L1210 cells to 100-2000 shock waves generated at operating voltages of 15-25 kV, corresponding to an electrical energy input of 0.9-32
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kWs caused cell disruption, which varied over a similar range as cell disruption caused by ultrasound (Clarke and Hill 1970; Kaufman et al. 1977; Fu et al. 1980; Ciaravino et al. 198 lb; Dooley et al. 1983). The effect of shock waves was related to the electrical energy input calculated as W = ½C U 2 (C = capacitance of the capacitor; U = operating voltage used) times the number of shock waves applied. This relation was recently suggested by Whelan and Finlayson (1988) for the quantitation of stone fracture by shock waves. In contrast to stone fracture, electrical energy input does not adequately describe the effect o f shock waves on cells in vitro; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Comparing kidney haemorrhages in dogs after exposure to 3000 shock waves generated with 20 kV and 40 nF (Delius et al. 1988c) with haemorrhages after exposure to 1500 shock waves generated with 20 kV and 90 nF (Delius et al. 1988b) suggested that less kidney damage might have occurred with the lower shock wave energy (by a lower capacitance) and an increased shock wave number; so a similar relation between electrical energy input and bioeffects as reported in this in vitro study might also occur in vivo. L 1210 cells which were nondisrupted after exposure to shock waves were only partly trypan blue negative. Thus, a second effect of shock waves was an alteration of the integrity o f cellular membranes. Again, electrical energy input does not sufficiently characterize the magnitude o f this effect o f shock waves. The majority of nondisrupted cells was able to exclude trypan blue. Only after exposure to 2000 shock waves at 20 kV (32 kWs) and after exposure to 1000 shock waves at 25 kV (25 kWs) the ratio of trypan blue positive/trypan blue negative cells was approximately 50% (Table 4). The absolute number T a b l e 4. R a t i o o f t r y p a n b l u e positive to t r y p a n blue negative L 1210 cells after e x p o s u r e to s h o c k waves.
Table 3. Number of nondisrupted L 1210 cells after exposure to 500 shock waves at 30 kV in a pressure chamber, given as a percentage of recovered sham treated control cells. Nondisrupted cells (%) Pressure in the pressure chamber Discharge number
1 atm.
101 arm.
Control 500
100 25.3 + 12.1
94.0 -+ 17.1 110.7 _+ 16.6
Figures represent the mean (_SD) of three experiments.
Trypan blue positive/trypan blue negative cells Operating voltage
Discharge number
15 kV
20 kV
25 kV
Control 100 250 500 1000 2000
0.01 + 0.01 0.06+0.01 0.09 -+ 0.03 0.15 _+ 0.06 0.25_+0.07 0.36 + 0.06
0.02 _+ 0.02 0.08_+0.02 0.15 -+ 0.02 0.21 _+ 0.05 0.37_+0.13 0.50 + 0.20
0.02 _+ 0.01 0.13_+0.05 0.28 -+ 0.08 0.28 _ 0.07 0.51 +0.11 n.d.
Figures represent the mean (_+SD) of three experiments. n.d. = not determined.
Biological effects of shock waves: L 1210 cells • S. GAMBIHLER et al.
o f cells which were not able to exclude trypan blue was reached after exposure to 2000 shock waves at 15 kV (18 kWs) and there was even a decrease in the absolute n u m b e r o f trypan blue positive cells at high discharge numbers at 25 kV. This decrease was not due to less damage, as indicated by the progressively increasing ratio of trypan blue positive/trypan blue negative cells (Table 4). After exposure to high discharge numbers at 25 kV, most cells were obviously completely disrupted. The third effect of shock waves was a reduced proliferation of cells exposed to 1000 shock waves at 25 kV. This exposure condition reduced growth during the first 24 h after exposure, while after this period the cells continued to proliferate at the same rate as sham treated controls. Limiting dilution analysis, originally introduced to immunology for frequency estimates of rare cells (Lefkovits and Waldman 1979, 1984), was used to determine the plating efficiency; for cells which were trypan blue negative after exposure to 1000 shock waves at 25 kV it was relatively high. This could not explain the reduced proliferation; the lack of growth of L 1210 cells after exposure to 1000 shock waves at 25 kV was due to an increased doubling time during the first 24 h after exposure. This is in contrast to Kaufman and Miller (1978), who suggested the lack of growth of V-79 cells during the first 24 h after sonication to be due to a balance of proliferation of attached viable cells and loss of nonviable cells into the medium. In the experiments where L1210 cells were exposed to shock waves in a pressure chamber, the m a x i m u m operating voltage available at the Dornier XL1 lithotripter was used, since it was expected that the shock waves are attenuated when they pass the steel membrane which was used to close the pressure chamber. Cell disruption after exposure to 500 shock waves at 30 kV at ambient pressure in the chamber was in the same range as compared to disruption after 500 shock waves at 25 kV w i t h o u t the pressure chamber. Cell disruption was c o m p l e t e l y absent when cells were exposed to shock waves at a pressure elevated to 101 atmospheres. This suggests that cavitation is an important mechanism for the effects of shock waves, as it is for the biological effects of ultrasound (Clarke and Hill 1970; Leake et al. 1980; Ciaravino et al. 1981a; Sacks et al. 1981: Vivino et al. 1985). In contrast to studies on the bioeffects of ultrasound, where the acoustic energy is directly stated, the shock wave effect was related to the electrical energy input for shock wave generation, as the available data on acoustic parameters of shock waves gen-
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erated with the Dornier XL1 lithotripter are still insufficient. We hope that data on the lithotripter sound field will be available in the near future, as they will allow to relate the biological effects o f shock waves to acoustic parameters. In summary, the effect of shock waves on L 1210 cells in vitro are similar to the effect of pulsed and continuous wave ultrasound. The electrical energy input for shock wave generation does not adequately describe their effect. The major mechanism of shock waves in generating these effects appears to be cavitation. Acknowledgements--This work was supported by the Award for the Advancement of European Science from the Koerber-Stiftung, Hamburg and Dornier Medizintechnik GmbH, Germering. We would like to express our appreciation to Mrs. B. Sonntag for her excellent technical assistance. The results are part of the medical thesis of S. Gambihler.
REFERENCES Carmichael, J.; Degraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47:936-942; 1987a. Carmichael, J.; Degraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity testing. Cancer Res. 47:943-946; 1987b. Chaussy, C.; Schmiedt, E.; Brendel, W. Extracorporally induced destruction of kidney stones by shock waves. Lancet ii: 12651268; 1980. Ciaravino, V.; Miller, M. W.; Carstensen, E. L. Pressure-mediated reduction of ultrasonically induced cell lysis. Radiat. Res. 88:209-213; 1981a. Ciaravino, V.; Miller, M. W.; Kaufman, G. E. The effect of I MHz ultrasound on the proliferation of synchronized chinese hamster V-79 cells. Ultrasound Med. Biol. 7:175-184; 1981b. Clarke, P. R.; Hill, C. R. Physical and chemical aspects of ultrasonic disruption of cells. J. Acoust. So. Am. 47:649-653; 1970. Coleman, A.; Saunders, J.; Crum, L.; Dyson, M. Acoustic cavitation generated by an extracorporeal shockwave lithotripter. Ultrasound Med. Biol. 13:69-76; 1987. Crum, L. Acoustic cavitation. Proceedings of the 1982 IEEE Ultrasonics Symposium. New York: IEEE; 1982:1-11. Delius, M.; Brendel, W.; Heine, G. A mechanism of gallstone destruction by extracorporeal shock waves. Naturwissenschaften 75:200-201; 1988a. Delius, M.; Enders, G.; Xuan, Z.; Liebich, H. G.; Brendel, W. Biological effects of shock waves: kidney damage by shock waves in dogs--dose dependence. Ultrasound Med. Biol. 14:117-122; 1988b. Delius, M.; Jordan, M.; Eizenhoefer, H.; Marlinghaus, E.; Heine, G. et al. Biological effects of shock waves: kidney haemorrhage by shock waves in dogs--administration rate dependence. Ultrasound Med. Biol. 14:689-694; 1988c. Dooley, D. A.; Child, S. Z.; Carstensen, E. L.; Miller, M. W. The effects of continuous wave and pulsed ultrasound on rat thymocytes in vitro. Ultrasound Med. Biol. 9:379-384; 1983. Forssmann, B.; Hepp, W.; Eisenberger, F.; Wanner, K. Eine Merhode zur beruehrungsfreien Zertruemmerung von Nierensteinen durch Stosswellen. Biomed. Tech. 22:164-168; 1977. Fu, Y. K.; Miller, M. W.; Lange, C. S.; GriflSths, T. D.; Kaufman, G. E. Ultrasound lethality to synchronous and asynchronous
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Chinese hamster V-79 cells. Ultrasound Med. Biol. 6:39-46; 1980. Kaufman, G. E.; Miller, M. W. Growth retardation in chinese hamster V-79 cells exposed to 1 MHz ultrasound. Ultrasound Med. Biol. 4:139-144; 1978. Kaufman, G. E.; Miller, M. W.; Gritfiths, T. D.; Ciaravino, V.; Carstensen, E. L. Lysis and viability of cultured mammalian cells exposed to 1 MHz ultrasound. Ultrasound Med. Biol. 3:21-25; 1977. Leake, E. S.; Wright, M. J.; Kremkau, F. W. Ultrasonic effects on alveolar macrophages in suspension. J. Clin. Ultrasound 8:465-472; 1980. Lefkovits, I.; Waldman, H., eds. Limiting dilution analysis of cells in the immune system. Cambridge: Cambridge University Press; 1979. Lefkovits, I.; Waldman, H. Limiting dilution analysis of the cells of immune system 1. The clonal basis of the immune response. Immunology Today 5:265-268; 1984. Miller, M. W.; Ciaravino, V.; Kaufman, G. E. Colony size and giant cell formation from mammalian cells exposed to 1 MHz ultrasound. Radiat. Res. 71:628-634; 1977. Mosmann, T. Rapid colorimetric assay for cellular growth and
Volume 16, Number 6, 1990 survival: application to proliferation and cytotoxicity assays. J. lmmunol. Methods 65:55-63; 1983. Mueller, M. Comparison of Dornier lithotripters. Physics in Lithotripsy. London, UK; 1989. Mueller, M. Comparison of Dornier lithotripters. Biomed. Tech. [in press]. Sachs, L., ed. Angewandte Statistik. Berlin: Springer-Verlag; 1973. Sacks, P. G.; Miller, M. W.; Sutherland, R. M. Influences of growth conditions and cell-cell contact on responses on tumor cells to ultrasound. Radiat. Res. 87:175-186; 1981. Sauerbruch, T.; Delius, M.; Paumgartner, G.; Holl, J.; Wess, O. et al. Fragmentation of gallstones by extracorporeal shock waves. N. Engl. J. Med. 314:818-822; 1986. Tennant, J. R. Evaluation of the trypan blue technique for determination of cell viability. Transplantation 2:685-694; 1964. Vivino, A. A.; Boraker, D. K.; Miller, D.; Nyborg, W. Stable cavitation at low ultrasonic intensities induces cell death and inhibits 3H-TdR incorporation by con-A-stimulated murine lymphocytes in vitro. Ultrasound Med. Biol. 11:751-759; 1985. Whelan, J. P.; Finlayson, B. An experimental model for the systematic investigation of stone fracture by extracorporeal shock wave lithotripsy. J. Urol. 140:395-400; 1988.
0301-5629]90 $3.00 + .DO © 1990 Pergamon Press plc
OOriginal Contribution BIOLOGICAL EFFECTS OF SHOCK WAVES: CELL DISRUPTION, VIABILITY, AND PROLIFERATION OF L1210 CELLS EXPOSED TO SHOCK WAVES IN VITRO STEFAN GAMBIHLER, MICHAEL DELIUS a n d WALTER BRENDEL * Institute for Surgical Research, University of Munich, Munich, Federal Republic of Germany (Received 7 September 1989; in final form 25 February 1990) A b s t r a c t - - L l 2 1 0 cells were exposed in suspension to shock waves generated with a Dornier XL1 lithotripter. After 1000 discharges at 25 kV, the number of nondisrupted cells was 15% and the number of trypan blue excluding cells was 7% as compared to 100% in sham treated controls; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Overall proliferation of cells which were trypan blue negative after exposure exceeded 70% of the proliferation of sham treated controls, except after 1000 shocks at 25 kV, where proliferation was reduced to 42%. The latter reduction in proliferation was found to be due to a reduced growth for 24 h after exposure, with a return to normal proliferation during the following days. Limiting dilution analysis revealed that the reduced growth was mainly due to a transitory increase of the doubling time and not to a reduction of the number of proliferating cells. Cell disruption by shock waves was completely inhibited by exposing the cells at an elevated pressure of 101 atmospheres, pointing to the possible involvement of cavitation in the shock wave effect.
Key Words: Cavitation, Cell disruption, LI210 cells, Limiting dilution, Lithotripter, M T r assay, Shock waves, Trypan blue assay, Viability.
INTRODUCTION
Shock waves are single pressure pulses of microsecond duration with a peak pressure of several hundred atmospheres. They are used in medicine for the fragmentation of urinary and biliary calculi (Chaussy et al. 1980; Sauerbruch et al. 1986). As compared to ultrasound, little is known about biological effects of shock waves on cells in vitro. Similar to ultrasound, shock-wave lithotripters are known to produce acoustic cavitation (Coleman et al. 1987) and cavitation is considered to be a mechanism for stone disintegration and tissue damage caused by shock waves (Delius et al. 1988a; Delius et al. 1988c). In the present study, the biological effects of shock waves are characterized for L1210 mouse leukemia cells in vitro. Cell disruption and ability to exclude trypan blue (Tennant 1964) were examined and related to the electrical energy input at the lithotripter for shock waves generated at different operating voltages and discharge numbers. Proliferation was determined by 3-4,5-dimethyl-thiazol-2,5 diphenyl tetrazolium bromide (MTT) assay (Mosmann 1983; Carmichael et al. 1987a, 1987b), and by studying the time course of proliferation. Limiting dilution
Disruption of cells has long been known to be an effect of ultrasound in vitro (Clarke and Hill 1970; Kaufman et al. 1977; Fu et al. 1980; Ciaravino et al. 1981b; Dooley et al. 1983). In addition, it has been reported that a part of the remaining cells is rendered nonviable as determined by vital dye exclusion or colony formation, and cells surviving sonication with ultrasound show growth retardation and form smaller colonies (Kaufman et al. 1977; Miller et al. 1977; Kaufman and Miller 1978; Fu et al. 1980; Ciaravino et al. 198 lb). Ultrasound is known to generate cavitation, which is "any observable activity involving bubbles" (Crum 1982), and cavitation has been reported to be an important mechanism for the biological effects (Clarke and Hill 1970; Leake et al. 1980; Ciaravino et al. 1981a; Sacks et al. 1981; Vivino et al. 1985). Address correspondence to: S. Gambihler, Institute for Surgical Research, Klinikum Grosshadern, 8000 Muenchen 70, Federal Republic of Germany. Deceased. 587
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analysis (Lefkovits and Waldman 1979, 1984) was used to determine the plating efficiency. The role of cavitation for cell disruption was evaluated. MATERIALS AND METHODS
Cell cultures L 1210 mouse leukemia cells (kindly supplied by Dr. H. P. Kraemer, Behringwerke, Marburg, FRG) were grown as suspension culture in RPMI 1640 medium supplemented with 15% heat inactivated fetal calf serum, 2% sodium pyruvate, and 1% antibioticantimycotic solution (Gibco, Eggenstein, FRG). Shock waves The principle of electrohydraulic shock wave generation has been described earlier (Forssmann et al. 1977). Shock waves generated with an experimental Dornier lithotripter model XL1 were administered at a rate of 100 per min, electrodes were replaced after every 500 discharges. According to measurements from Mueller (Dornier Medizintechnik, Germering, FRG) with PVDF needle probes, shock waves generated at an operating voltage of 20 kV have a peak positive pressure of 84 MPa with a focal length and width of 22 mm and 5mm; due to focal shift maximum pressure is reached 5 mm behind the geometric focus of the ellipsoid along the longitudinal axis (Mueller 1989, in press). Exposure vials made of polypropylene (Interchem, Muenchen, FRG) had an inner diameter of 11.7 mm and a height of 47 mm. The vials were positioned so that the geometric focus of the ellipsoid, indicated by a laser system, was 10 mm above the bottom of the vial. The pressure field in the vials was not homogeneous due to shock wave focusing. The water in the lithotripter tank was degassed by a vacuum pump; oxygen concentration was 2-4 mg/L. Experimental procedures Samples of 2 X 10 6 log phase L1210 cells in 5.2 mL culture medium were transferred into vials and kept in the lithotripter tank maintained at 35-37°C. Every single experiment consisted of one or more samples exposed to shock waves and one sham treated control sample which was placed in the waterbath outside of the shock wave field. The effect of shock waves generated at different operating voltages and with different discharge numbers was related to the electrical energy input at the lithotripter, determined as W = ½C U 2 X d i s c h a r g e n u m b e r [kWs]
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where C is the capacitance of the capacitor (80 nF) and U is the operating voltage used. For experiments at elevated pressure, the vials were transferred into a steel chamber, which was filled with water of the lithotripter tank, closed under water and put under pressure by nitrogen. The shock waves entered the chamber through a 0.3 mm steel plate. Samples were exposed to shock waves at ambient pressure or at 101 atmospheres. Control samples were exposed to only ambient or elevated pressure.
Cell disruption and ability to exclude trypan blue The reliability of cell counting after exposure to shock waves was determined in triplicate by three different observers in a hemocytometer by counting morphologically intact cells which revealed well reproducible intra- and interobserver values (Table 1). The number of nondisrupted cells was counted in triplicate for each sample. The cells were scored for the ability to exclude trypan blue with equal amounts of cell suspension and trypan blue (2 g/L in 0.9% NaCI solution; Fluka, Buchs, Switzerland). After 3 rain at room temperature the number of stained and unstained cells was determined in a hemocytometer in triplicate for each sample. M T T assay 3-4,5-dimethyl-thiazol-2,5 diphenyl tetrazolium bromide (MTT) was obtained from Sigma (Taufkirchen, FRG). Cell numbers of sham treated controls and of samples exposed to shock waves, as determined by trypan blue assay, were adjusted to 9 X 10 3 trypan blue negative cells/mL. Into each well of 96well round bottom microtitre plates (Nunc, Roskilde, Denmark) 0.05 mL culture medium and 0.1 mL cell suspension were plated. Sixteen wells were plated per sample and incubated at 37°C in a humidified 5% CO2 atmosphere. After 72 h, 0.05 mL MTT (2.5 g/L
Table 1. N u m b e r o f n o n d i s r u p t e d L1210 cells after exposure to shock waves at 25 kV, as d e t e r m i n e d by three different observers. Nondisrupted cells [X 104/mL] Observer
Discharge number
B.S.
Control 100 250 500 I000
45.5 _+ 1.4 27.7 + 0.7 21.2 + 0.5 10.0 + 0.2 2.0 + 0.0
N.W. 42.7 25.1 21.0 10.7 4.3
+_ 1.1 _+ 2.3 + 1.9 _ 0.8 ___0.5
S.G. 42.6 25.9 21.4 9.8 3.3
Figures represent the mean (+SD) of three cell countings.
+ 0.3 _+ 1.1 + 1.7 _+0.2 + 0.3
Biological effects of shock waves: L 1210 cells • S. GAMBIHLER et al.
in 0.9% NaC1 solution) was added to each well. After a further incubation time o f 4 h the supernatant fluid was carefully removed and 0.1 m L dimethylsulfoxide (Merck, Darmstadt, FRG) was added to each well. Plates were agitated on a plate shaker and absorbance at 490 n m was measured within 5 min using a Dynatech platereader model M R 580. In control experiments cell suspensions exposed to 500 shock waves at 25 kV were centrifuged and the supernatant was added to cells not exposed to shock waves. Proliferation, as determined by M T T assay, was unchanged as c o m p a r e d to untreated control cells.
Time course of proliferation Cell numbers of sham treated controls and of samples exposed to shock waves at 25 kV, as determined by trypan blue assay, were adjusted to concentrations of 1 X 103 trypan blue negative cells/mL, transferred into 25 cm 2 culture flasks (Nunc) and incubated for 5 days. Every 24 h after seeding the number o f cells was determined with the trypan blue assay. The doubling time o f the cells was determined from the resulting growth curves.
Limiting dilution analysis Limiting dilution analysis (Lefkovits and Waldman 1979, 1984) was used to determine the plating efficiency. The frequency distribution of cells over the wells after plating of low numbers of cells into microtitre plates can be described by the Poisson distribution. W h e n low n u m b e r s o f L1210 ceils are plated at a given number of inoculated cells per well (u) and the plating efficiency (k) is defined as the ratio o f the average n u m b e r o f inoculated/proliferating cells, the fraction of wells without growth (Fo) is described as
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were plated at each cell concentration and incubated. After two weeks the fraction of wells without growth (Fo) was determined. The corresponding curves were calculated with the least square method and the plating efficiencies were taken from the slopes of the lines. The curves representing the sham treated controls and the samples exposed to shock waves were compared based on one-sided t test (Sachs 1973) at a 5% significance level. RESULTS
Cell disruption In sham treated controls, 92% + 11% of the 2 X 10 6 cells per vial were recovered; the recovery of the sham treated control cells of each experiment was set to 100% and the number of nondisrupted cells after exposure to shock waves was related to the control value of each experiment. With higher voltages and shock wave numbers, the number of recovered cells decreased progressively. In Fig. 1, the relative number of nondisrupted cells after treatment is related to the electrical energy input during shock wave application. Figure la combines points gained from treatments at the same voltages but different shock wave numbers; in Fig. lb, the same values are shown, but lines combine points gained from treatments with the same shock wave numbers but at different voltages.
I00
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:
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where e is the base of the natural logarithm. When F0 is plotted against u in a semilog plot, the plating efficiency k is directly obtained from the slope of the resulting line. Assuming that all cells proliferate (k = 1), the above expression describes the dotted line in Fig. 5 with the slope - k = - 1 . Cell numbers of sham treated controls and of samples exposed to 1000 shock waves at 25 kV, as determined by trypan blue assay, were adjusted to concentrations of 5, 10, 20 and 40 trypan blue negative cells/mE One-tenth m L cell suspension and 0.05 m L medium were plated into microtitre plates, i.e., the average n u m b e r of inoculated cells per well (u) was ½, 1, 2, or 4 cells. One hundred ninety-two wells
0 Z
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Fig. 1. Cell disruption. Number of nondisrupted cells (yaxis), given as percentage of recovered sham treated control cells, related to electrical energy input at the lithotripter (x-axis). The same data are given in both panels, (a) representing curves at different operating voltages and (b) showing curves at different discharge numbers. Points represent the mean of three separate experiments. Error bars show the standard deviation.
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Higher voltages at a similar electrical energy input (obtained by a lower discharge n u m b e r ) , caused greater cell disruption than lower voltages (Fig. la). Vice versa, a higher n u m b e r of shock waves was less effective in disrupting cells than a lower number of shock waves, when compared at a similar electrical energy input (obtained by a lower operating voltage; Fig. 1b). For example, after exposure of L 1210 cells to 2000 shocks generated with 20 kV, corresponding to an electrical energy input of 32 kWs, 37% __ 13% of the cells were still nondisrupted, whereas after exposure to 1000 shock waves at 25 kV, corresponding to an electrical energy input of 25 kWs, 15% +_ 1% of the cells were nondisrupted.
Trypan blue negative cells The n u m b e r o f trypan blue excluding cells was related to the total n u m b e r of recovered sham treated control cells, which was set to 100%. In sham treated controls, the ability to exclude trypan blue was 98% + 1%. In Fig. 2, the relative n u m b e r of trypan blue negative cells after treatment is related to the electrical energy input; Fig. 2a combines points gained from treatments at the same voltages but different shock wave numbers; in Fig. 2b the same values are shown but lines combine points from treatments with the same shock wave numbers but at different voltages.
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Volume 16, N u m b e r 6, 1990
Table 2. Number of trypan blue positive L I210 cells after exposure to shock waves, given as percentage of recovered sham treated control cells. Trypan blue positive cells (%) Operating voltage
Discharge number
15 kV
20 kV
25 kV
Control 100 250 500 1000 2000
1_+ 1 5_+1 8_+3 12_+6 19 +_ 3 27 _+ 6
2_+ 2 7_+ 2 12-+ 2 16_+ 6 19 +_ 10 20 _+ 11
2-+- 1 12_+6 18_+7 11_+3 8 -+ 3 n.d.
Figures represent the mean (_+SD) of three experiments. n.d. = not determined.
The same trend was seen as with cell disruption--a more pronounced effect with increasing voltage than with increasing shock-wave number. For example, after exposure to 2000 shock waves at 20 kV (32 kWs), 17% ___6% of the cells were trypan blue negative, and this number decreased to 7% + 1% after 1000 shock waves at 25 kV (25 kWs).
Trypan blue positive cells The difference between Figs. 1 and 2 was caused by cells which were morphologically intact, but trypan blue positive. Their number was again related to the total n u m b e r of recovered sham treated control cells. It reached a m a x i m u m of 27% +_ 6% after treatment with 2000 shock waves at 15 kV (Table 2). If the n u m b e r of trypan blue cells was related to the n u m b e r of intact, trypan blue negative cells, the values did not show the trend of a greater effect of higher voltages at a similar electrical energy input. For example, the ratio was 0.50 + 0.20 after exposure to 2000 shocks at 20 kV (32 kWs) and was 0.51 _+ 0.11 after e x p o s u r e to 1000 shocks at 25 kV (25 kWs).
100 s h o c k s
i
t
Proliferation
I
i
10 1,5 20 25 30
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kWs
(a)
(b)
Fig. 2. Trypan blue negative cells. Number of trypan blue excluding cells (y-axis), given as percentage of recovered sham treated control cells, related to electrical energy input at the lithotripter (x-axis). The same data are given in both panels, (a) representing curves at different operating voltages and (b) showingcurves at different discharge numbers. Points represent the mean of three separate experiments. Error bars show the standard deviation.
Proliferation of cells that had been trypan blue negative after exposure to shock waves was examined in 3 different sets o f experiments. In the first set, overall proliferation during the 3 days following exposure was determined by M T T assay (Fig. 3). Proliferation was 68% + 11% after exposure to 2000 shocks at 20 kV (32 kWs). This value was exceeded under all conditions with one exception: after exposure to 1000 shock waves at 25 kV (25 kWs) it was reduced to 42% -!-_ 19%. In the second set, proliferation was examined in more detail by studying the time course of proliferation o f cells exposed to shock waves at 25 kV (Fig. 4).
Biological effects o f s h o c k waves: L I 2 1 0 cells • S. GAMBIHLER et al.
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77% and a 2.2-fold increase in cell number, the doubling time during the first day after treatment was calculated to be 18 h.
Role of cavitation
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Fig. 3. MTT assay. Overall proliferation of cells that had been trypan blue negative after exposure to shock waves (y-axis), given as percentage of the proliferation of recovered sham treated control cells, related to electrical energy input at the lithotripter (x-axis). The same data are given in both panels, (a) representing curves at different operating voltages and (b) showing curves at different discharge numbers. Points represent the mean of three separate experiments. Error bars show the standard deviation.
Cells treated with 100, 250 or 500 shock waves at 25 kV grew exponentially from the very beginning for 120 h with a doubling time o f 11 h, similar to sham treated control cells. Cells treated with 1000 shock waves at 25 kV showed reduced growth during the first 24 h after application of shock waves; during this period, the relative n u m b e r of cells increased only by a factor o f 2.2 as compared to a factor of 4.3 of sham treated control cells. After the first 24 h the cells returned to normal proliferation. The diminished growth during the first 24 h after exposure to 1000 shock waves at 25 kV could be due to either a reduced n u m b e r of proliferating cells (the result of Fig. 4 could be obtained if only 40% of the cells would proliferate after shock wave exposure), or to an increase in the doubling time of the cells. In the third experiment, we tested the plating efficiency of cells with the limiting dilution technique. Sham treated controls showed a plating efficiency of 96% (Fig. 5), while the plating efficiency o f cells exposed to 1000 shock waves at 25 kV was 77%, which is a small although significant decrease (p < 0.05 in one-sided t test). This plating efficiency was too high to account for the reduced proliferation which could only be explained by a plating efficiency in the range of 40% (Fig. 4), and therefore the doubling time must have been increased. Considering a plating efficiency of
The role o f cavitation for cell disruption was evaluated by exposing L 1210 cells to shock waves in a pressure chamber. T h e n u m b e r o f n o n d i s r u p t e d L1210 cells exposed to 500 shock waves at 30 kV at ambient pressure was 25% _+ 12% as compared to sham treated controls (Table 3). Cell disruption was absent when the cells were exposed to shock waves at a pressure elevated to 101 atmospheres. Treatment with pressure only showed no effect on cell disruption. DISCUSSION T h e p r i m a r y effects o f shock waves on suspended L1210 cells observed in this study were cell disruption, decreased ability to exclude trypan blue
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Ultrasound in Medicine and Biology
Average number of inoculated ceils per well (u) 0 ,~,
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and a t r a n s i t o r y decrease in proliferation. Cell disruption by shock waves was absent when cells were exposed to shock waves at an elevated pressure, suggesting that cavitation is a major mechanism in producing cell disruption. Exposure o f L1210 cells to 100-2000 shock waves generated at operating voltages of 15-25 kV, corresponding to an electrical energy input of 0.9-32
Volume 16, Number 6, 1990
kWs caused cell disruption, which varied over a similar range as cell disruption caused by ultrasound (Clarke and Hill 1970; Kaufman et al. 1977; Fu et al. 1980; Ciaravino et al. 198 lb; Dooley et al. 1983). The effect of shock waves was related to the electrical energy input calculated as W = ½C U 2 (C = capacitance of the capacitor; U = operating voltage used) times the number of shock waves applied. This relation was recently suggested by Whelan and Finlayson (1988) for the quantitation of stone fracture by shock waves. In contrast to stone fracture, electrical energy input does not adequately describe the effect o f shock waves on cells in vitro; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Comparing kidney haemorrhages in dogs after exposure to 3000 shock waves generated with 20 kV and 40 nF (Delius et al. 1988c) with haemorrhages after exposure to 1500 shock waves generated with 20 kV and 90 nF (Delius et al. 1988b) suggested that less kidney damage might have occurred with the lower shock wave energy (by a lower capacitance) and an increased shock wave number; so a similar relation between electrical energy input and bioeffects as reported in this in vitro study might also occur in vivo. L 1210 cells which were nondisrupted after exposure to shock waves were only partly trypan blue negative. Thus, a second effect of shock waves was an alteration of the integrity o f cellular membranes. Again, electrical energy input does not sufficiently characterize the magnitude o f this effect o f shock waves. The majority of nondisrupted cells was able to exclude trypan blue. Only after exposure to 2000 shock waves at 20 kV (32 kWs) and after exposure to 1000 shock waves at 25 kV (25 kWs) the ratio of trypan blue positive/trypan blue negative cells was approximately 50% (Table 4). The absolute number T a b l e 4. R a t i o o f t r y p a n b l u e positive to t r y p a n blue negative L 1210 cells after e x p o s u r e to s h o c k waves.
Table 3. Number of nondisrupted L 1210 cells after exposure to 500 shock waves at 30 kV in a pressure chamber, given as a percentage of recovered sham treated control cells. Nondisrupted cells (%) Pressure in the pressure chamber Discharge number
1 atm.
101 arm.
Control 500
100 25.3 + 12.1
94.0 -+ 17.1 110.7 _+ 16.6
Figures represent the mean (_SD) of three experiments.
Trypan blue positive/trypan blue negative cells Operating voltage
Discharge number
15 kV
20 kV
25 kV
Control 100 250 500 1000 2000
0.01 + 0.01 0.06+0.01 0.09 -+ 0.03 0.15 _+ 0.06 0.25_+0.07 0.36 + 0.06
0.02 _+ 0.02 0.08_+0.02 0.15 -+ 0.02 0.21 _+ 0.05 0.37_+0.13 0.50 + 0.20
0.02 _+ 0.01 0.13_+0.05 0.28 -+ 0.08 0.28 _ 0.07 0.51 +0.11 n.d.
Figures represent the mean (_+SD) of three experiments. n.d. = not determined.
Biological effects of shock waves: L 1210 cells • S. GAMBIHLER et al.
o f cells which were not able to exclude trypan blue was reached after exposure to 2000 shock waves at 15 kV (18 kWs) and there was even a decrease in the absolute n u m b e r o f trypan blue positive cells at high discharge numbers at 25 kV. This decrease was not due to less damage, as indicated by the progressively increasing ratio of trypan blue positive/trypan blue negative cells (Table 4). After exposure to high discharge numbers at 25 kV, most cells were obviously completely disrupted. The third effect of shock waves was a reduced proliferation of cells exposed to 1000 shock waves at 25 kV. This exposure condition reduced growth during the first 24 h after exposure, while after this period the cells continued to proliferate at the same rate as sham treated controls. Limiting dilution analysis, originally introduced to immunology for frequency estimates of rare cells (Lefkovits and Waldman 1979, 1984), was used to determine the plating efficiency; for cells which were trypan blue negative after exposure to 1000 shock waves at 25 kV it was relatively high. This could not explain the reduced proliferation; the lack of growth of L 1210 cells after exposure to 1000 shock waves at 25 kV was due to an increased doubling time during the first 24 h after exposure. This is in contrast to Kaufman and Miller (1978), who suggested the lack of growth of V-79 cells during the first 24 h after sonication to be due to a balance of proliferation of attached viable cells and loss of nonviable cells into the medium. In the experiments where L1210 cells were exposed to shock waves in a pressure chamber, the m a x i m u m operating voltage available at the Dornier XL1 lithotripter was used, since it was expected that the shock waves are attenuated when they pass the steel membrane which was used to close the pressure chamber. Cell disruption after exposure to 500 shock waves at 30 kV at ambient pressure in the chamber was in the same range as compared to disruption after 500 shock waves at 25 kV w i t h o u t the pressure chamber. Cell disruption was c o m p l e t e l y absent when cells were exposed to shock waves at a pressure elevated to 101 atmospheres. This suggests that cavitation is an important mechanism for the effects of shock waves, as it is for the biological effects of ultrasound (Clarke and Hill 1970; Leake et al. 1980; Ciaravino et al. 1981a; Sacks et al. 1981: Vivino et al. 1985). In contrast to studies on the bioeffects of ultrasound, where the acoustic energy is directly stated, the shock wave effect was related to the electrical energy input for shock wave generation, as the available data on acoustic parameters of shock waves gen-
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erated with the Dornier XL1 lithotripter are still insufficient. We hope that data on the lithotripter sound field will be available in the near future, as they will allow to relate the biological effects o f shock waves to acoustic parameters. In summary, the effect of shock waves on L 1210 cells in vitro are similar to the effect of pulsed and continuous wave ultrasound. The electrical energy input for shock wave generation does not adequately describe their effect. The major mechanism of shock waves in generating these effects appears to be cavitation. Acknowledgements--This work was supported by the Award for the Advancement of European Science from the Koerber-Stiftung, Hamburg and Dornier Medizintechnik GmbH, Germering. We would like to express our appreciation to Mrs. B. Sonntag for her excellent technical assistance. The results are part of the medical thesis of S. Gambihler.
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