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Quantitative colonial growth of mammalian cells in fibrin cells
Experimental
Ceil
Research
34, 595-611
(1964)
595
PRELIMINARY QUANTITATIVE
NOTES
COLONIAL GROWTH OF MAMMALIAN CELLS IN FIBRIN CELLS
Pharmakologisches
R.
SCHINDLER
Institut
der
Received
Universitiit
September
Bern,
Switzerland
13,1963’
Th e co1onial plating method as developed by Puck et al. [5] has become an outstanding tool in studies which require the determination of survival and multiplication of single isolated mammalian cells in culture under various conditions. This method, however, is not applicable to certain cell lines such as murine lymphoblasts (line L-5178) [3] and neoplastic mast cells (line P-815) [2,7]. The cells of these lines are characterized by their inability to attach to the glass surfaces of culture vessels. Therefore, a semisolid medium is required for the culture of colonies from single isolated cells. Agar, however, in concentrations (0.25 to 0.3 per cent) sufficient to produce a gel, as used by Puck et al. [5]. was found to be toxic to these cells even in the form of a purified preparation (Difco, “purified agar”) or after being washed according to the procedure of Dulbecco and Vogt [I]. Cultures of murine lymphoblasts [3] and neoplastic mast cells [2,7] are of especial interest for several reasons. First, these cell lines exhibit unusually short generation times in vitro of 8 to 15 hr [3,4, 61. ‘Because of this rapid cell reproduction in culture, growth of single cells into macroscopic colonies would be less time-consuming than with other cell lines. Furthermore, since these cells do not attach to glass surfaces, they offer many advantages with respect to simplicity of culture techniques. It seemed desirable, therefore, to modify the quantitative plating technique described by Puck in order to permit its application to these cell lines. With the method presented in this communication growth of single cells into colonies is obtained using a gel prepared by coagulation of a fibrinogen solution. Cultures in vi&o of a transplantable murine mast cell tumor (cell line P-815-X2) were used [2, 41. The general culture techniques and the medium have been described previously [7]. Additional components of the culture medium (solution A) were: L-asparagine (0.1 mM), L-serine (0.1 m&f) and Tris buffer (3 ml of 2 per cent tris(hydroxymethyl)-aminomethane in 0.15 N hydrochloric acid per 100 ml of medium); furthermore, citrovorum factor was replaced by folic acid (10 mg/l). In addition to this medium the following solutions were used: Solution B: 800 mg NaCl, 50 mg KCl, and 25 mg sodium citrate (Na,C,H,O, + 5) H,O) per 100 ml of water; Solution C (fibrinogen solution): 250 mg of bovine fibrinogen (obtained from Poviet, Amsterdam) are added to 50 ml of solution B. After standing for 2 hr, the fibrinogen is 1 Revised
version
received
April
9, 1964. Experimental
Cell
Research
34
R. Schinder dissolved by agitation of the suspension during 15 min with a magnetic stirrer. The undissolved material is removed by centrifugation, and the resulting clear fibrinogen solution is sterilized by irradiation with ultraviolet light. The mercury lamp used as the source of ultraviolet light was standardized with a suspension of E. coli. The dose of ultraviolet light used for sterilization of the fibrinogen solution
Fig. 1.-Colonies obtained from single cellsin a fibrin gel.
caused a reduction of viable microorganisms by a factor of approximately IO’. During irradiation the fibrinogen solution was agitated with a magnetic stirrer in order to provide an equal distribution of the radiation dose to all parts of the solution. Solution D (“cloning medium”): The composition of the cloning medium is the same as that of solution A, except for the following alterations: addition of thrombine (0.12 units per 100 ml); concentration of CaCl,: 0.6 m&Z. In order to avoid the formation of air bubbles within the fibrin clot, solution D is incubated at 37” for 12 to 24 hr prior to its use. Experimental procedure: A suspension of cells in culture is diluted with solution Experimental
Cell Research
31
Colonial
growth
of mammalian
cells
D in order to obtain a cell density of approximately 5 cells per ml. Of this highly diluted cell suspension, 12 ml are mixed in a culture tube (capacity 16 ml) with 3 ml of solution C and incubated at 37°C. Within a few min a transparent clot has farmed which keeps the suspended cells in place. 5 to 6 days later the individual cells have grown into clearly visible colonies, as illustrated in Fig. I. TABLE
I. Colony
yields
from isolated
Experiment No. 1 2 3 4 5 Average
P-815-X2
Cellsincubated (according to hemocytometer
counts) 180 180 180 180 180 180
cells in a fibrin
gel.
Colonies obtained 162 207 188 180 199 187.2
Standard variation
Il7.2
The cloning efficiency (number of colonies produced by 100 cells) was determined in 5 independent experiments. In each experiment, the number of cells per ml in a suspension culture was counted accurately in a hemocytometer, and the cell suspension was diluted appropriately in order to obtain a cell density of 5.0 cells per ml. Of this diluted cell suspension, three culture tubes containing 60 cells each were prepared as described above. The results of these experiments are presented in Table I and indicate that an average colony yield of 104 per cent (standard variation: k 9,5 per cent) was obtained. In two preliminary experiments, the method was also applied successfully, although with lower cloning efficiencies (11 and 13 per cent, respectively), to single cells of two strains of adult human skin fibroblasts. These were obtained from biopsy specimens and were grown initially in plasma clots, while at the time of the cloning experiments they were maintained as monolayer cultures. (For these fibroblasts, an enriched medium containing as additional components 1 mM pyruvate, 0.1 mM each of L-alanine, L-aspartic acid, L-glutamic acid, glycine, L-hydroxyproline and L-proline, 0.03 mM tbymidine, 0.03 mM hypoxanthine and 15 per cent fetal bovine serum instead of adult horse serum was used.) If the cell density is reduced to about two neoplastic mast cells per ml, the individual colonies may be easily separated, after removal of the fibrin gel into a petri dish, by cutting the clot into small pieces containing one colony each. From such colonies new cultures were obtained, the cells of which represented the progeny of a single cell. When subjected to a second cloning procedure, such genetically pure cultures gave rise to oolonies of markedly more uniform size and density than the original P-815-X2 culture. Experimental
Cell
Research
34
R. Schindler If the horse serum used was dialyzed prior to its addition to the cloning medium, the cloning efficiency was approximately equal to that obtained with undialyzed serum; also, in place of horse serum, fetal bovine serum (obtained from Microbiological Associates) was used with equally good results. No special nutritional requirements beyond those which were satisfied by the medium described could be demonstrated. The method described is very convenient in that no perfusion of the incubator with a CO,-Air mixture is required, since, in contrast to the petri dishes used in conventional plating methods, the screw caps of the culture tubes provide an airtight seal. Obviously, very little or no oxygen is required by the cells for rapid colonial growth. Because of the short generation time (IO to 12 hr) of the P-815 cells in culture, it takes only 5 to 6 days until the colonies arising from the individual cells are clearly visible and can be counted. The procedure described may therefore be useful in studies related to genetics, radiobiology and other aspects of mammalian cells in culture in which counts of “viable cells” by cloning methods are required. Summary-A procedure of cloning mammalian cells in culture is presented which is based on the use of fibrin gels. The application of this method to a cell line (P815-X2), the cells of which do not attach to glass surfaces and therefore cannot be cloned by conventional methods, is described. This work was supported by the Schweizerische Nationalfonds zur FBrderung der Forschung. In addition the author wishes to express his gratitude to Prof. W. Wilbrandt for his encouragement and interest and for helpful suggestions during the course of this work. Also, the expert technical assistance of Miss L. Hamseier is gratefully acknowledged.
wissenschaftlichen
REFERENCES 1. 2. 3. 4. 5. 6. 7.
DULBECCO, DUNN, T. FISCHER, GREEN, J. PUCK, T. SCHINDLER, SCHINDLER,
Experimental
R. and VOGT, B. and POTTER,
G. A., Ann,
Y. Acad.
34
Sci.
Med. Cancer
99, 167 (1954). Inst.
18, 587 (1957).
76, 673 (1958). P. and DAY, M., Biochem. Pharmacol. 3, 190 (1960). T., MARCUS, P. I. and CIECIURA, S. J., J. Expff Med. 103, 273 (1956), R., Helv. Physiol. Pharmacol. Acta 18, C 60 (1960). R., DAY, M. and FISCHER, G. A., Cancer Res. 19, 47 (1959).
Cell Research
N.
M., J. Exptl M., J. Natl
Ceil
Research
34, 595-611
(1964)
595
PRELIMINARY QUANTITATIVE
NOTES
COLONIAL GROWTH OF MAMMALIAN CELLS IN FIBRIN CELLS
Pharmakologisches
R.
SCHINDLER
Institut
der
Received
Universitiit
September
Bern,
Switzerland
13,1963’
Th e co1onial plating method as developed by Puck et al. [5] has become an outstanding tool in studies which require the determination of survival and multiplication of single isolated mammalian cells in culture under various conditions. This method, however, is not applicable to certain cell lines such as murine lymphoblasts (line L-5178) [3] and neoplastic mast cells (line P-815) [2,7]. The cells of these lines are characterized by their inability to attach to the glass surfaces of culture vessels. Therefore, a semisolid medium is required for the culture of colonies from single isolated cells. Agar, however, in concentrations (0.25 to 0.3 per cent) sufficient to produce a gel, as used by Puck et al. [5]. was found to be toxic to these cells even in the form of a purified preparation (Difco, “purified agar”) or after being washed according to the procedure of Dulbecco and Vogt [I]. Cultures of murine lymphoblasts [3] and neoplastic mast cells [2,7] are of especial interest for several reasons. First, these cell lines exhibit unusually short generation times in vitro of 8 to 15 hr [3,4, 61. ‘Because of this rapid cell reproduction in culture, growth of single cells into macroscopic colonies would be less time-consuming than with other cell lines. Furthermore, since these cells do not attach to glass surfaces, they offer many advantages with respect to simplicity of culture techniques. It seemed desirable, therefore, to modify the quantitative plating technique described by Puck in order to permit its application to these cell lines. With the method presented in this communication growth of single cells into colonies is obtained using a gel prepared by coagulation of a fibrinogen solution. Cultures in vi&o of a transplantable murine mast cell tumor (cell line P-815-X2) were used [2, 41. The general culture techniques and the medium have been described previously [7]. Additional components of the culture medium (solution A) were: L-asparagine (0.1 mM), L-serine (0.1 m&f) and Tris buffer (3 ml of 2 per cent tris(hydroxymethyl)-aminomethane in 0.15 N hydrochloric acid per 100 ml of medium); furthermore, citrovorum factor was replaced by folic acid (10 mg/l). In addition to this medium the following solutions were used: Solution B: 800 mg NaCl, 50 mg KCl, and 25 mg sodium citrate (Na,C,H,O, + 5) H,O) per 100 ml of water; Solution C (fibrinogen solution): 250 mg of bovine fibrinogen (obtained from Poviet, Amsterdam) are added to 50 ml of solution B. After standing for 2 hr, the fibrinogen is 1 Revised
version
received
April
9, 1964. Experimental
Cell
Research
34
R. Schinder dissolved by agitation of the suspension during 15 min with a magnetic stirrer. The undissolved material is removed by centrifugation, and the resulting clear fibrinogen solution is sterilized by irradiation with ultraviolet light. The mercury lamp used as the source of ultraviolet light was standardized with a suspension of E. coli. The dose of ultraviolet light used for sterilization of the fibrinogen solution
Fig. 1.-Colonies obtained from single cellsin a fibrin gel.
caused a reduction of viable microorganisms by a factor of approximately IO’. During irradiation the fibrinogen solution was agitated with a magnetic stirrer in order to provide an equal distribution of the radiation dose to all parts of the solution. Solution D (“cloning medium”): The composition of the cloning medium is the same as that of solution A, except for the following alterations: addition of thrombine (0.12 units per 100 ml); concentration of CaCl,: 0.6 m&Z. In order to avoid the formation of air bubbles within the fibrin clot, solution D is incubated at 37” for 12 to 24 hr prior to its use. Experimental procedure: A suspension of cells in culture is diluted with solution Experimental
Cell Research
31
Colonial
growth
of mammalian
cells
D in order to obtain a cell density of approximately 5 cells per ml. Of this highly diluted cell suspension, 12 ml are mixed in a culture tube (capacity 16 ml) with 3 ml of solution C and incubated at 37°C. Within a few min a transparent clot has farmed which keeps the suspended cells in place. 5 to 6 days later the individual cells have grown into clearly visible colonies, as illustrated in Fig. I. TABLE
I. Colony
yields
from isolated
Experiment No. 1 2 3 4 5 Average
P-815-X2
Cellsincubated (according to hemocytometer
counts) 180 180 180 180 180 180
cells in a fibrin
gel.
Colonies obtained 162 207 188 180 199 187.2
Standard variation
Il7.2
The cloning efficiency (number of colonies produced by 100 cells) was determined in 5 independent experiments. In each experiment, the number of cells per ml in a suspension culture was counted accurately in a hemocytometer, and the cell suspension was diluted appropriately in order to obtain a cell density of 5.0 cells per ml. Of this diluted cell suspension, three culture tubes containing 60 cells each were prepared as described above. The results of these experiments are presented in Table I and indicate that an average colony yield of 104 per cent (standard variation: k 9,5 per cent) was obtained. In two preliminary experiments, the method was also applied successfully, although with lower cloning efficiencies (11 and 13 per cent, respectively), to single cells of two strains of adult human skin fibroblasts. These were obtained from biopsy specimens and were grown initially in plasma clots, while at the time of the cloning experiments they were maintained as monolayer cultures. (For these fibroblasts, an enriched medium containing as additional components 1 mM pyruvate, 0.1 mM each of L-alanine, L-aspartic acid, L-glutamic acid, glycine, L-hydroxyproline and L-proline, 0.03 mM tbymidine, 0.03 mM hypoxanthine and 15 per cent fetal bovine serum instead of adult horse serum was used.) If the cell density is reduced to about two neoplastic mast cells per ml, the individual colonies may be easily separated, after removal of the fibrin gel into a petri dish, by cutting the clot into small pieces containing one colony each. From such colonies new cultures were obtained, the cells of which represented the progeny of a single cell. When subjected to a second cloning procedure, such genetically pure cultures gave rise to oolonies of markedly more uniform size and density than the original P-815-X2 culture. Experimental
Cell
Research
34
R. Schindler If the horse serum used was dialyzed prior to its addition to the cloning medium, the cloning efficiency was approximately equal to that obtained with undialyzed serum; also, in place of horse serum, fetal bovine serum (obtained from Microbiological Associates) was used with equally good results. No special nutritional requirements beyond those which were satisfied by the medium described could be demonstrated. The method described is very convenient in that no perfusion of the incubator with a CO,-Air mixture is required, since, in contrast to the petri dishes used in conventional plating methods, the screw caps of the culture tubes provide an airtight seal. Obviously, very little or no oxygen is required by the cells for rapid colonial growth. Because of the short generation time (IO to 12 hr) of the P-815 cells in culture, it takes only 5 to 6 days until the colonies arising from the individual cells are clearly visible and can be counted. The procedure described may therefore be useful in studies related to genetics, radiobiology and other aspects of mammalian cells in culture in which counts of “viable cells” by cloning methods are required. Summary-A procedure of cloning mammalian cells in culture is presented which is based on the use of fibrin gels. The application of this method to a cell line (P815-X2), the cells of which do not attach to glass surfaces and therefore cannot be cloned by conventional methods, is described. This work was supported by the Schweizerische Nationalfonds zur FBrderung der Forschung. In addition the author wishes to express his gratitude to Prof. W. Wilbrandt for his encouragement and interest and for helpful suggestions during the course of this work. Also, the expert technical assistance of Miss L. Hamseier is gratefully acknowledged.
wissenschaftlichen
REFERENCES 1. 2. 3. 4. 5. 6. 7.
DULBECCO, DUNN, T. FISCHER, GREEN, J. PUCK, T. SCHINDLER, SCHINDLER,
Experimental
R. and VOGT, B. and POTTER,
G. A., Ann,
Y. Acad.
34
Sci.
Med. Cancer
99, 167 (1954). Inst.
18, 587 (1957).
76, 673 (1958). P. and DAY, M., Biochem. Pharmacol. 3, 190 (1960). T., MARCUS, P. I. and CIECIURA, S. J., J. Expff Med. 103, 273 (1956), R., Helv. Physiol. Pharmacol. Acta 18, C 60 (1960). R., DAY, M. and FISCHER, G. A., Cancer Res. 19, 47 (1959).
Cell Research
N.
M., J. Exptl M., J. Natl