IN VITI~O Vo]. I1, No. 6, 1975
USE OF THE LOCAL ANESTHETIC LIDOCAINE FOR CELL HARVESTING AND SUBCULTIVATION MICHEL RABINOVITCH AND MARY JO DzSTEFANO Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, New York 10016 SUMMARY
Cell suspensions from monolayer cultures of 3T3, SV-3T3, BHK, BS-C-1, or macrophages, were prepared by brief exposure of the cultures to the amide anesthetic lidocaine. Cells were subcultivated six times without marked impairment of plating efficiency. The method should be applicable to physiological, biochemical, or immunological studies in which exposure of cells to proteolytic enzymes is to be avoided. Trypsin was first introduced by Rous and Jones (1) f~r the preparation and replating of cell suspensions from tissue cultures and has been widely applied since the 1950s (2). However, the enzyme should not be used in analytical or immunological studies requiring the intact composition of the cell surface. Proteolysis may also affect surface receptors for viruses, hormones, or drugs, and has been reported to in. fluence cell growth (3). Because other methods of detachment, such as divalent cation chelators or low temperature, are only useful for certain cell types, there is still scope for alternatives to proteoIytic enzymes. Cationic anesthetics reversibly inhibit cell-to-substrate adhesion and spreading (4, 5) and the~ studies have led to the development of lidocaine as an agent for cell harvesting and subcultivation. ~ MATERIALS AND METHODS
Lidocaine hydrochloride (Xylocaine) was a gift from Astra Pharmaceutical Co., Worcester, Mass. Tissue culture media were purchased from Grand Island Biological Co. Cells were grown in 60-mm Falcon tissue culture dishes in bicarDonate-buffered Dulbecco's modified Eagle medium enriched with 10% fetal bovine serum. Stocks of 3T3, SV-3T3, BHK, and BS-C-1 cells were kindly provided by Drs. H. Meiss and C. Basilico, from the Department of Pathology at this School of Medicine. Normal mouse peritoneal The results in this paper have been presented at the 14th Annual Meeting of the American Society for Cell Biology (6).
macrophages were maintained in culture for 2 to 4 days. Medium was changed every 3rd day. RESULTS
Cell detachment with lidocaine. Both subconfluent and confluent cultures were used. Medium was aspirated from the cultures, 1 ml of Dulbecco's modified Eagle medium enriched with 10% fetal bovine serum containing 12 mM lidocaine was added, and the monolayers were continuously observed with an inverted microscope. After 5 to 15 rain at room temperature, the cells either rounded up and detached singly (the usual case with subconfluent cultures) or started to come off in sheets (usually true for confluent cultures). Nearly all cells could then be dislodged by two to four jets of medium from a syringe. Cell clumps were broken by retreated pipetting. All of the cell lines used, as well as cultivated macrophages, could be harvested with this procedure. Optimal concentrations of lidocaine needed for detachment of other cell lines may have to be determined. Control cells in the absence of lidocaine did not detach, with the exception of SV-3T3, which were only partially removed by vigorous pipetting or syringing. Experiments with cultures of 3T3 indicated that cell removal was easier in medium containing 10% fetal bovine serum than in the absence of serum. Detachment was also obtained when lidocaine was dissolved in R P M I medium 1640, medium 199, or Eagle's minimum essential medium, all containing 10% fetal bovine serum. Lidocaine was less effective in Dulbecco's phos-
379
380
RABINOVITCII AND ])~:STEFANO
1)hate-buffered saline containing calcium and magnesium. No consistent advantage of detachment at 37~ over that at 22~ was observed. Replating of cells detached with lidocaine. Cell suspensions from lidocaine-treated cultures were either washed by centrifugation in drug-free medium or diluted 100-fold and plated. In both cases the cells reattached and spread in a few hours, with the exception of mouse macrophages which reattached and spread within 30 min. Serial transfers were performed using the dilution method and thus the cells were exposed to a maximum of 0.12 m.~i lidocaine until their medium Was changed. Stocks of 3T3, SV-3T3, and B I I K Cultures were successively split by treatment with 12 mM lidocaine six times over a period of 22 days. 3T3 cell cultures were split l:10 and SV-3T3 and B I I K were split 1:20. After the 6th passage with lidocaine treatment, (;ells '~vere detached a 7th time with the drug, cell numbers were counted in a hemoeytometer and, after appropriate dilution, 100 cells were plated. Cultures were fixed, stained, and the colonies counted 6 to 9 days later. Plating effieieneies were 58% for BHK, 41% for 3T3, and 39% for SV-3T3 (averages of eight plates each). in another experiment we atteml)ted to determine whether lidocaine continuously present in the culture medium would alter the plating eflMeney of 3T3 cells. A 3T3 stock was split with the anesthetic. Eleven days after l)lating 200 cells, plating efficiency was found to be 38% for the control (initially containing a maximum of 7 ~M lidoeaine) and 34 and 33%, respectively, for cultures grown in the presence of 0.2 or 0.5 m.~ lidocaine.
effects of lidocaine on cell adhesion and spreading (4, 5) suggests that the proposed method, involving exposure to relatively high concentrations of lidocaine for short periods of time, will not cause irreversible changes in membrane structure or confo~znation. Furthermore, cultures of nerve tissues could be treated with 0.34 mM lidocaine for 15 days without detectable mort)hological changes (l 1), and derInal fibroblasts will grow from skin biopsies taken from sites injected with 1% (34 raM) lidocaine (12). However, lidoeaine has also been reported to inhibit macromolecular synthesis (13, 14), in exI~riments where reversibility of the effect was not studied. The proposed method may be useful in studies in which cell suspensiorLs have to be obtained from monolayers without trypsinization. IIowever, before it can be used routinely for subcultivation, additional information about cellular changes th,~t may result from repeated or chronic exposure to the drug is required. We have found that other anesthetics, such as procaine, tetracaine, and fluphenazine, are too toxic for cell harvesting (unpublished). However, a new generation of cell-detaching drugs should be considered and developed. Such drugs should have an optimal oetanol-water partition coefficient (15) and a short half-life in tissue culture medium through either spontaneous degradation or catabolism by cellular or serum enzymes. 1.
2. DISCUSSION
Amphipathie anesthetics such a~s lidocaine interact reversibly with hydrophobic domains of lipids or proteins in cell membranes (7). Although the plasma membrane may be the initial target of the drug, metabolic effects mediated by mitochondria (8) and effects on Ca ~+ transport or on contractile microfilaments cannot be excluded (5). In order to understand the cell-detaching activity of lidocaine, information is needed on rates of uptake and efftux of the drug in mammalian cells; on rates of degradation by cellular or serum enzymes (9, 10); and on possible adaptive enzymatic respouses of cells to the presence of the drug. The reversibility of the inhibitory
3.
4.
5.
6.
REFERENCES Rous, P., and F. S. Jones. 1916. A method for obtaining suspensions of living cells from the fixed tissues, and for the plating out of individual cells. J. Exp. Med. 23: 549-555. Parker, I(. C. 1961. Melhods of Ti,~sue Culture. IIarper & Row, New York, p. 120. Burger, M. M. 1971. The significance of surface structure changes for growth control under crowded conditions. In: G. E. W. Wolstenholme and J. Knight (Eds.), Growlh Control in Cell Cullures. Ciba Foundation Symposium. Churchill Livingstone, Edinburgh, pp. 45-63. Rabinovitch, M., and M. J. l)eStefano. 1974. Macrophage spreading in vitro. III. The effect of metabolic inhibitors, anesthetics and other drugs on spreading induced by subtilisin. Exp. Cell Res. 88: 153-162. Rabinovitch, M., and M. J. DeStefano. 1(,}75. Cell to substrate adhesion and spreading: inhibition by cationic anesthetics. J. Cell. Physiol, 85: 189-194. Rabinovitch, M., and M. J. DeStefano. 1974. Use of the local anesthetic lidocaine for
LIDOCAINE FOR CELL DETACHMENT
7. 8.
9.
10.
11.
harvesting and subcultivation of cells. J. Cell Biol. 63: 278a. Seeman, P. 1972. Tile membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24: 583-655. Fink, B. R., G. E. Kenny, andW. E. Simpson. 1969. I)epression (if oxygen uptake in cell cultures by volatile, barbiturate and local anesthetics. Anesthesiology 30: 150-155. Geddes, I. C. 1971. The metabolism of local anesthetics as determined by the use of ~4C and all labeled material. Laval Med. 42: 668-679. Akerman, S. B. A., S. B. Ross, and A. Telc. 1973. The enzymatic hydrolysis of a series of aminoacylanilides in relation to their nerve blocking effect and toxicity. Acta Pharmacol. Toxicol. (Kbh.) 32: 88-96. Model, P. G., M. B. Bornstein, S. M. Crain,
12. 13. 14.
15.
381
and G. D. Pappas. 1971. An electron microscopic study of the development of synapses in cultured fetal mouse cerebrum continuously exposed to xylocaine. J. Cell Biol. 49: 362-371. Cooper, J. T., and S. Goldstein. 1973. Skin biopsy and successful fibroblast culture. Lancet 2: 673. Metcalf, S. M. 1971. Cell culture as a test system for toxicity. J. Pharm: Pharmacol. 23: 817-823. Schmidt, R. M., H. S. Rosenkranz, and J. F. Ryan. 1971. Inhibition of DNA synthesis by lidocaine and procaine. Experientia 27: 261-262. lIansch, C., and J. M. Clayton. 1973. Lipophilic character and bioloKical activity of drugs. II. The parabolic case. J. Pharm. Sci. 62 : 1-21.
This work was supported by grants AI 10969 and CA 15565 from the N~tional hL~tiiutcs of Health and by grant IN-14-0 from the American Ca.ncer Society. The authors thank Dr. H. Meiss for advice.
USE OF THE LOCAL ANESTHETIC LIDOCAINE FOR CELL HARVESTING AND SUBCULTIVATION MICHEL RABINOVITCH AND MARY JO DzSTEFANO Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, New York 10016 SUMMARY
Cell suspensions from monolayer cultures of 3T3, SV-3T3, BHK, BS-C-1, or macrophages, were prepared by brief exposure of the cultures to the amide anesthetic lidocaine. Cells were subcultivated six times without marked impairment of plating efficiency. The method should be applicable to physiological, biochemical, or immunological studies in which exposure of cells to proteolytic enzymes is to be avoided. Trypsin was first introduced by Rous and Jones (1) f~r the preparation and replating of cell suspensions from tissue cultures and has been widely applied since the 1950s (2). However, the enzyme should not be used in analytical or immunological studies requiring the intact composition of the cell surface. Proteolysis may also affect surface receptors for viruses, hormones, or drugs, and has been reported to in. fluence cell growth (3). Because other methods of detachment, such as divalent cation chelators or low temperature, are only useful for certain cell types, there is still scope for alternatives to proteoIytic enzymes. Cationic anesthetics reversibly inhibit cell-to-substrate adhesion and spreading (4, 5) and the~ studies have led to the development of lidocaine as an agent for cell harvesting and subcultivation. ~ MATERIALS AND METHODS
Lidocaine hydrochloride (Xylocaine) was a gift from Astra Pharmaceutical Co., Worcester, Mass. Tissue culture media were purchased from Grand Island Biological Co. Cells were grown in 60-mm Falcon tissue culture dishes in bicarDonate-buffered Dulbecco's modified Eagle medium enriched with 10% fetal bovine serum. Stocks of 3T3, SV-3T3, BHK, and BS-C-1 cells were kindly provided by Drs. H. Meiss and C. Basilico, from the Department of Pathology at this School of Medicine. Normal mouse peritoneal The results in this paper have been presented at the 14th Annual Meeting of the American Society for Cell Biology (6).
macrophages were maintained in culture for 2 to 4 days. Medium was changed every 3rd day. RESULTS
Cell detachment with lidocaine. Both subconfluent and confluent cultures were used. Medium was aspirated from the cultures, 1 ml of Dulbecco's modified Eagle medium enriched with 10% fetal bovine serum containing 12 mM lidocaine was added, and the monolayers were continuously observed with an inverted microscope. After 5 to 15 rain at room temperature, the cells either rounded up and detached singly (the usual case with subconfluent cultures) or started to come off in sheets (usually true for confluent cultures). Nearly all cells could then be dislodged by two to four jets of medium from a syringe. Cell clumps were broken by retreated pipetting. All of the cell lines used, as well as cultivated macrophages, could be harvested with this procedure. Optimal concentrations of lidocaine needed for detachment of other cell lines may have to be determined. Control cells in the absence of lidocaine did not detach, with the exception of SV-3T3, which were only partially removed by vigorous pipetting or syringing. Experiments with cultures of 3T3 indicated that cell removal was easier in medium containing 10% fetal bovine serum than in the absence of serum. Detachment was also obtained when lidocaine was dissolved in R P M I medium 1640, medium 199, or Eagle's minimum essential medium, all containing 10% fetal bovine serum. Lidocaine was less effective in Dulbecco's phos-
379
380
RABINOVITCII AND ])~:STEFANO
1)hate-buffered saline containing calcium and magnesium. No consistent advantage of detachment at 37~ over that at 22~ was observed. Replating of cells detached with lidocaine. Cell suspensions from lidocaine-treated cultures were either washed by centrifugation in drug-free medium or diluted 100-fold and plated. In both cases the cells reattached and spread in a few hours, with the exception of mouse macrophages which reattached and spread within 30 min. Serial transfers were performed using the dilution method and thus the cells were exposed to a maximum of 0.12 m.~i lidocaine until their medium Was changed. Stocks of 3T3, SV-3T3, and B I I K Cultures were successively split by treatment with 12 mM lidocaine six times over a period of 22 days. 3T3 cell cultures were split l:10 and SV-3T3 and B I I K were split 1:20. After the 6th passage with lidocaine treatment, (;ells '~vere detached a 7th time with the drug, cell numbers were counted in a hemoeytometer and, after appropriate dilution, 100 cells were plated. Cultures were fixed, stained, and the colonies counted 6 to 9 days later. Plating effieieneies were 58% for BHK, 41% for 3T3, and 39% for SV-3T3 (averages of eight plates each). in another experiment we atteml)ted to determine whether lidocaine continuously present in the culture medium would alter the plating eflMeney of 3T3 cells. A 3T3 stock was split with the anesthetic. Eleven days after l)lating 200 cells, plating efficiency was found to be 38% for the control (initially containing a maximum of 7 ~M lidoeaine) and 34 and 33%, respectively, for cultures grown in the presence of 0.2 or 0.5 m.~ lidocaine.
effects of lidocaine on cell adhesion and spreading (4, 5) suggests that the proposed method, involving exposure to relatively high concentrations of lidocaine for short periods of time, will not cause irreversible changes in membrane structure or confo~znation. Furthermore, cultures of nerve tissues could be treated with 0.34 mM lidocaine for 15 days without detectable mort)hological changes (l 1), and derInal fibroblasts will grow from skin biopsies taken from sites injected with 1% (34 raM) lidocaine (12). However, lidoeaine has also been reported to inhibit macromolecular synthesis (13, 14), in exI~riments where reversibility of the effect was not studied. The proposed method may be useful in studies in which cell suspensiorLs have to be obtained from monolayers without trypsinization. IIowever, before it can be used routinely for subcultivation, additional information about cellular changes th,~t may result from repeated or chronic exposure to the drug is required. We have found that other anesthetics, such as procaine, tetracaine, and fluphenazine, are too toxic for cell harvesting (unpublished). However, a new generation of cell-detaching drugs should be considered and developed. Such drugs should have an optimal oetanol-water partition coefficient (15) and a short half-life in tissue culture medium through either spontaneous degradation or catabolism by cellular or serum enzymes. 1.
2. DISCUSSION
Amphipathie anesthetics such a~s lidocaine interact reversibly with hydrophobic domains of lipids or proteins in cell membranes (7). Although the plasma membrane may be the initial target of the drug, metabolic effects mediated by mitochondria (8) and effects on Ca ~+ transport or on contractile microfilaments cannot be excluded (5). In order to understand the cell-detaching activity of lidocaine, information is needed on rates of uptake and efftux of the drug in mammalian cells; on rates of degradation by cellular or serum enzymes (9, 10); and on possible adaptive enzymatic respouses of cells to the presence of the drug. The reversibility of the inhibitory
3.
4.
5.
6.
REFERENCES Rous, P., and F. S. Jones. 1916. A method for obtaining suspensions of living cells from the fixed tissues, and for the plating out of individual cells. J. Exp. Med. 23: 549-555. Parker, I(. C. 1961. Melhods of Ti,~sue Culture. IIarper & Row, New York, p. 120. Burger, M. M. 1971. The significance of surface structure changes for growth control under crowded conditions. In: G. E. W. Wolstenholme and J. Knight (Eds.), Growlh Control in Cell Cullures. Ciba Foundation Symposium. Churchill Livingstone, Edinburgh, pp. 45-63. Rabinovitch, M., and M. J. l)eStefano. 1974. Macrophage spreading in vitro. III. The effect of metabolic inhibitors, anesthetics and other drugs on spreading induced by subtilisin. Exp. Cell Res. 88: 153-162. Rabinovitch, M., and M. J. DeStefano. 1(,}75. Cell to substrate adhesion and spreading: inhibition by cationic anesthetics. J. Cell. Physiol, 85: 189-194. Rabinovitch, M., and M. J. DeStefano. 1974. Use of the local anesthetic lidocaine for
LIDOCAINE FOR CELL DETACHMENT
7. 8.
9.
10.
11.
harvesting and subcultivation of cells. J. Cell Biol. 63: 278a. Seeman, P. 1972. Tile membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24: 583-655. Fink, B. R., G. E. Kenny, andW. E. Simpson. 1969. I)epression (if oxygen uptake in cell cultures by volatile, barbiturate and local anesthetics. Anesthesiology 30: 150-155. Geddes, I. C. 1971. The metabolism of local anesthetics as determined by the use of ~4C and all labeled material. Laval Med. 42: 668-679. Akerman, S. B. A., S. B. Ross, and A. Telc. 1973. The enzymatic hydrolysis of a series of aminoacylanilides in relation to their nerve blocking effect and toxicity. Acta Pharmacol. Toxicol. (Kbh.) 32: 88-96. Model, P. G., M. B. Bornstein, S. M. Crain,
12. 13. 14.
15.
381
and G. D. Pappas. 1971. An electron microscopic study of the development of synapses in cultured fetal mouse cerebrum continuously exposed to xylocaine. J. Cell Biol. 49: 362-371. Cooper, J. T., and S. Goldstein. 1973. Skin biopsy and successful fibroblast culture. Lancet 2: 673. Metcalf, S. M. 1971. Cell culture as a test system for toxicity. J. Pharm: Pharmacol. 23: 817-823. Schmidt, R. M., H. S. Rosenkranz, and J. F. Ryan. 1971. Inhibition of DNA synthesis by lidocaine and procaine. Experientia 27: 261-262. lIansch, C., and J. M. Clayton. 1973. Lipophilic character and bioloKical activity of drugs. II. The parabolic case. J. Pharm. Sci. 62 : 1-21.
This work was supported by grants AI 10969 and CA 15565 from the N~tional hL~tiiutcs of Health and by grant IN-14-0 from the American Ca.ncer Society. The authors thank Dr. H. Meiss for advice.
