Growth of Human Embryonic Fibroblasts at Clonal Density: Concordance with Results from Mass Cultures JAMES R. SMITH AND KAREN I. BRAUNSCHWEIGER W.Alton Jones Cell Science Center, Old Barn Road, Lake Placid, New York 12946
ABSTRACT In the past, i t has been difficult to grow human diploid fibroblast cells a t clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1)population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures a t clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture. Normal human and chick fibroblast-like cells exhibit a limited proliferative capacity in culture while virus-transformed human cells and spontaneously and chemically transformed cells from other mammals seem to possess an unlimited ability to proliferate in culture (Hayflick, '77). The mechanisms underlying the long-term control of cell proliferation are unknown. An understanding of these mechanisms is of importance in many areas of biological and medical research. The use of clonal culture techniques as an analytical tool to study the finite in vitro proliferative potential of human diploid fibroblasts was introduced by Merz and Ross ('69). Since then other investigators have employed these techniques (Absher and Absher, '76; Martin et al., '74; Muggleton-Harris and Hayflick, '76; Smith and Hayflick, '74; Smith et al., '77, '78). However, the widespread use of clonal techniques in such studies has been hampered by the technical difficulties involved and a concern that human diploid cells in clonal culture might not proliferate at the same rate or to the same extent as in mass culture. Many of the technical problems associated with cloning normal diploid cells have been overcome by the development of new media specifically designed for optimum clonal growth (McKeehan et al., '77). The purpose of the present communication is to show that, under adequate environmental condiJ. CELL. PHYSIOL. (1979) 98: 597-602.
tions, the population doubling time, the percentage of non-dividing cells and other growth properties of WI-38 and IMR-90 (human embryonic lung fibroblasts) are not adversely affected by growth a t clonal densities. MATERIALS AND METHODS
The human embryonic lung fibroblast lines WI-38 and IMR-90 were used in these studies. WI-38 cultures were obtained from Doctor L. Hayflick (Oakland Childrens Hospital) and IMR-90 cultures were obtained from the Institute for Medical Research (Camden, New Jersey). Except as noted, mass cultures were maintained in MEM with Hanks' balanced salt solution (GIBCO), 10%fetal bovine serum (Reheis Chemical Co.) and 29 mM HEPES buffer (Sigma Chemical Co.). A. Determination ofpopulation doubling time Cultures were initiated 48 hours after the previous subculture. The cell layer was washed with Dulbecco's calcium magnesiumfree phosphate buffered saline (CMFPBS), and 0.5 ml of 0.25%pronase (Sigma Chemical Co.) in CMFPBS was added. When the cells had detached, they were suspended in complete medium, counted with a Model ZBI Coulter Counter, and seeded a t the appropriate density into a series of 60 mm dishes or 25 Received June 2, '78. Accepted a t . 2, '78.
597
598
JAMES R. SMITH AND KAREN I. BRAUNSCHWEIGER
cm2flasks. Except as noted, clonal density cultures (100cells/dish) were seeded in medium MCDB 104 (McKeehan et al., '77) supplemented with 5% FBS and maintained in 5% C0295%air at -98% relative humidity. Mass cultures (2.5 x lo5 cells/flask) were seeded (except as noted) in MEM with Hanks' salts, 10%FBS and 29 mM HEPES. In both clonal and mass cultures the medium was changed every two days. Cell number in clonal and mass cultures were determined in triplicate cultures every 24 hours for a period of five days. Clonal plates were fixed in 1%glutaraldehyde, stained with 0.5%crystal violet and the number of cells per dish determined by direct count under a dissecting microscope. In mass cultures suspensions of single cells were prepared by pronase treatment and the number of cells was determined by Coulter Counter. The population doubling times were derived from growth curves which had been fit to the experimental data by the least squares method. The growth of both clonal density and mass cultures was exponential for four days after seeding. Therefore, this time period was used to determine the population doubling times. B. Life-span of clonal and mass cultures Mass cultures maintained in MEM with Hanks' salts, 10% FBS, and 29 mM HEPES were subcultured weekly. At each subculturing, the total number of cells in the flask was determined, and 2.5 x lo5 cells were transferred to a new flask. The number of cells that had attached six hours after seeding was determined. For both clonal and mass cultures, the increase in cumulative population doublings a t each subculture was calculated based on the number of cells attached and the cell yield a t the time of the next subcultivation. Clonal cultures were initiated from mass cultures by seeding 100 cells into 60 mm dishes containing MCDB 104 with 5%FBS. At weekly intervals, three plates were fixed with 1% glutaraldehyde, stained with crystal violet and the number of cells per dish counted microscopically. Cells in parallel cultures were removed with pronase and reseeded a t 100 celldplate. Attachment a t six hours was determined by fixing, staining and counting the number of cells per plate. Mass cultures were considered to have achieved their proliferative limit when they did not exceed 5 x lo5 cells/flask in four
weeks. Clonal density cultures were considered to have reached their proliferative limit when they did not exceed 200 cells/plate in four weeks. RESULTS
To assess whether the growth of human diploid fibroblasts a t clonal density under our experimental conditions is equivalent to that in mass culture, we have compared several growth properties under both conditions: (1) population doubling time, (2) long-term proliferative potential, (3) percent attachment, and (4) percentage of cells capable of division. Figure 1 shows the results of a typical ex-
20
rI
a W
m
4z
-I -1 W
0 W
>
5-1 W
U
TIME (days) Fig. 1 Growth of clonal and mass cultures of WI-38 cells PDL 37. Mass cultures were inoculated with 2.5 X lo5 cellsff-25 flask on day 0 in BME ( 0 )or MCDB 104 (0). Clonal cultures were inoculated with 100 celld60 mm dish on day 0 in BME (A)or MCDB 104 (A). Each point is the average cell number from three culture vessels relative to the cell number on day 1.
CLONAL DENSITY CULTURE VERSUS MASS CULTURE
599
a t low density. The low density cultures were never allowed to grow t o a density of more than about 2,500 cells/60 mm dish. Figure 2 shows the cumulative population doublings achieved as a function of time for one such experiment. We found that the overall replication rate was initially higher in the clonal cultures than in mass cultures. This is probably due to the mass cultures being in a state of density dependent inhibition of division during part of each subcultivation period while the clonal cultures were not. The variability of such experiments is shown in table 2 which summarizes the results of several experiments using both WI-38 and IMR-90 cultures. In two experiments the total proliferative capacity of the low density cultures was 11 and 15 doubu o 20 40 60 80 lings less than the parallel mass cultures. TIME (days) However, in the other three experiments the Fig. 2 Cumulative population doublings as a function of time (days) for clonal ( A ) and mass ( 0 )cultures of WI- cultures kept a t low density went through 38 cells. Continuous clonal cultures were initiated from almost the same number of doublings as the mass cultures at PDL 40. mass cultures. Thus, there was no consistent adverse effect on in vitro life-span due to conperiment in which the population doubling tinuous low density culture. Finally we compared the percentage of cells times of clonal and mass cultures were compared. In this experiment clonal and mass cul- which failed to divide in clonal culture with tures were grown in both BME and MCDB the results obtained by Cristofalo and Sharf 104. MCDB 104 was designed for clonal (‘73) for mass cultures. These results are growth of human diploid fibroblasts and BME shown in figure 3. Our results for clonal culhad been widely used for mass culture of these tures agree with those obtained by Merz and cells. The growth rate was lower for mass cul- Ross (‘69) for clonal culture. In both cases the tures in MCDB 104 than BME. For clonal cul- clonal cultures resulted in a lower percentage tures, however, just the reverse was observed. of non-dividing cells than that obtained for Cell proliferation was essentially exponential mass cultures. This is probably due to the use from day 1to 4. Therefore, all subsequent pop- of different criteria for “non-dividing” cells in ulation doublings times were determined for mass and clonal cultures. In clonal cultures “non-dividing” cells were cells that had not this time period. Table 1summarizes the results from five ex- divided during one to two weeks, while in mass periments in which the population doubling culture cells that had not synthesized DNA in times were compared for clonal and mass cul- a 30 hour period were considered “non-dividtures. These experiments were performed at ing.” Since the interdivision time of a proporvarious population doubling levels (PDL). The population doubling times increase with inTABLE 1 creasing PDL in both clonal and mass culComparison of population doubling times in clonal and tures. In all cases the population doubling mass cultures of WI-38 cells times of clonal cultures are essentially the Population doubling time (hours) same as for mass cultures. PDL ’ Mass cultures Clonal cultures Even though the growth rate of WI-38 cells did not appear to be adversely affected by 24 21.6 24.7 growth a t clonal density, it was also important 36 19.0 22.1 31 22.5 21.4 to show that the long-term proliferative 45 23.9 24.1 ability was not drastically decreased by 51 30.8 29.0 growth a t clonal density. We compared the ’ Population doubling level of the mass cultures used to initiate number of population doublings achieved by for growth curves. cultures treated in the normal manner for cultures Population doubling time for mass culturea determined in BME mass culture and cultures kept continuously instead of MEM. ~
~~
~
~~
600
JAMES R. SMITH AND KAREN I. BRAUNSCHWEIGER TABLE 2
Total proliferatiue capacity (P.D.)
P.D. began
Clonal
Mass
21 28 42 40 45
65 43 57 73 61
64 54 72 72 62
IMR-90 IMR-90 IMR-90 WI-38 WI-38
I Population doubling at which continuous clonal cultures were initiated.
tion of these cells may be more than 30 hours this would result in a n underestimate in percent of cells which would eventually divide. The average percentage attachment measured six hours after inoculation was similar for mass (67%) and clonal (71%) cultures. Therefore the larger percentage of dividing cells in low density culture cannot be attributed to selection in favor of dividing cells due to a lower percentage attachment. DISCUSSION
m
m
I
0
I
I
I
80 100 PERCENT LIFE-SPAN COMPLETED
20
40
60
Fig. 3 Percentage of non-dividing WI-38 cells vs percent in vitro life-span completed. Open symbols from Cristofalo and Sharf ('73) for mass cultures. Solid squares (D) from Merz and Ross ('69) for clonal culture. The other solid symbols (V, A , 0 ) indicate percentages of cells which did not divide within two weeks in clonal cultures inoculated with 10-20 cells per 60 mm dish and incubated as described in MATERIALS AND METHODS (each symbol represents the results from a single subcultivation series of WI-38 cells).
When normal human fibroblast-like cells are plated a t clonal density a portion of the cells are capable of only a few divisions (Martin et al., '74; Smith and Hayflick, '74; Smith et al., '77, '781, while others can undergo a number of doublings approximating the doubling potential of the mass culture from which they were derived. The proportion of cells with low doubling potential increases with in vitro age of the parent culture. A major concern has been that the low doubling potential exhibited by some of these cells in clonal culture occurs because normal human fibroblasts are incapable of optimal growth a t clonal densities. We have shown previously (Smith et al., '77) that in clonal culture a substantial proportion of attached cells are unable to form clones of more than 16 cells (from 20%a t PDL 20 to 65% a t PDL 50 for WI-38 cells). If cells stopped proliferating after a few divisions in low density culture but not in mass culture, one would expect the population doubling time to be longer in low density culture than in mass culture. We have observed (in both mass and low density cultures) an increasing population doubling time with increasing PDL corresponding to the increase in low doubling potential colonies. The fact that the population doubling time increases to the same extent in clonal and mass cultures (table l), suggests that either, (1)the mass cultures contain the same proportion of cells with small doubling potential as the clonal cultures or ( 2 ) that the replication rate of the cells which are capable of extensive proliferation in clonal culture increases as a function of PDL to the exact extent needed to give the same doubling time for the population as a whole as observed in mass culture. We consider the first possibility to be more likely. The percentage of non-dividing cells in mass and clonal culture is a more direct test of the
CLONAL DENSITY CULTURE VERSUS MASS CULTURE
ability of human embryonic fibroblasts to proliferate under these conditions. We found, in agreement with Merz and Ross ('691, that the percentage of non-dividing cells was essentially the same in low density culture as found in mass culture (Cristofalo and Sharf, '73). Furthermore, we found that there was no consistent difference between the in vitro lifespan of IMR-90 and WI-38 cells grown in low density and mass culture. However, this is not a stringent test for the equivalence of clonal and mass cultures for two reasons. (1)The in vitro life-span of cultures of human embryonic fibroblasts in mass culture is quite variable (table 2 and Holliday et al., '77). (2) The in vitro life-span of a culture may be determined by a small proportion of cells with the greatest doubling potential. If this is the case then in initiating low density cultures with a 100 cell sample from mass cultures one would expect some of the low density cultures not to contain cells with the greatest doubling potential. These experiments do indicate however that any adverse effect on total proliferation potential due to low density growth cannot be very great. In conclusion, we find no evidence to support the idea that the low doubling potential observed for a proportion of cells in low density culture is an artifact of culture conditions. On the contrary, in three of the parameters studied where artifacts would be expected to cause differences between low density and mass cultures, the results were equivalent. Therefore, the results reported here support the idea that the behavior of individual cells and clones within a mass culture of human embryonic fibroblasts can be inferred from the behavior of the same cells grown a t clonal densities.
601
ACKNOWLEDGMENTS
We are grateful for the excellent assistance of Thomas Roberts in some aspects of this work. "his investigation was supported in part by the National Institute on Aging (Grant No. AG00338-01-Al) and the W. Alton Jones Foundation. LITERATURE CITED Absher, P. M., and R. G. Absher 1976 Clonal variation and aging of diploid fibroblasts. Cinematographic studies of cell pedigrees. Exp. Cell Res., 103: 247-255. Cristofalo, V. J., and B. B. Sharf 1973 Cellular senescence and DNA synthesis. Thymidine incorporation a s a measure of population age in human diploid cells. Exp. Cell Res., 76: 419-427. Hayflick, L. 1977 The cellular basis for biological aging. In: The Handbook of the Biology of Aging. C. E. Finch and L. Hayflick, eds. Van Nostrand Reinhold Co.,New York, pp. 159-186. Holliday, R., L. I. Huschtscha, G. M. Tarrant and T. B. L. Kirkwood 1977 Testing the commitment theory of cellular aging. Science., 198: 366-372. Martin, G.M., C. A. Sprague, T. H. Norwood and W. R. Pendergrass 1974 Clonal selection, attenuation and differentiation in an in uitro model of hyperplasia. Am. J. Pathol., 74: 137-154. McKeehan, W. L., K. A. McKeehan, S. L. Hammond and R. G. Ham 1977 Improved medium for clonal growth of human diploid fibroblasts a t low concentrations of serum protein. In Vitro., 13: 399-416. Merz, G . S.,and J. D. Ross 1969 Viability of human diploid cells as a function of in vitro age. J. Cell. Physiol., 74: 219-222. Muggleton-Harris, A. L., and L. Hayflick 1976 Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells. Exp. Cell Res., 103: 321-330. Smith, J. R., and L. Hayflick 1974 Variations in the lifespan of clones derived from human diploid fibroblasts cultures. J. Cell Biol., 62: 48-53. Smith, J. R., 0. Pereira-Smith and P. I. Good 1977 Colony size distribution as a measure of age in cultured human cells. A brief note. Mech Ageing Dev., 6: 283-286. Smith, J. R., 0. Pereira-Smith and E. L. Schneider 1978 Colony size distributions as a measure of in viuo and in uitro aging. Proc. Natl. Acad. Sci. (U.S.A.),75: 1353-1356.
ABSTRACT In the past, i t has been difficult to grow human diploid fibroblast cells a t clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1)population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures a t clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture. Normal human and chick fibroblast-like cells exhibit a limited proliferative capacity in culture while virus-transformed human cells and spontaneously and chemically transformed cells from other mammals seem to possess an unlimited ability to proliferate in culture (Hayflick, '77). The mechanisms underlying the long-term control of cell proliferation are unknown. An understanding of these mechanisms is of importance in many areas of biological and medical research. The use of clonal culture techniques as an analytical tool to study the finite in vitro proliferative potential of human diploid fibroblasts was introduced by Merz and Ross ('69). Since then other investigators have employed these techniques (Absher and Absher, '76; Martin et al., '74; Muggleton-Harris and Hayflick, '76; Smith and Hayflick, '74; Smith et al., '77, '78). However, the widespread use of clonal techniques in such studies has been hampered by the technical difficulties involved and a concern that human diploid cells in clonal culture might not proliferate at the same rate or to the same extent as in mass culture. Many of the technical problems associated with cloning normal diploid cells have been overcome by the development of new media specifically designed for optimum clonal growth (McKeehan et al., '77). The purpose of the present communication is to show that, under adequate environmental condiJ. CELL. PHYSIOL. (1979) 98: 597-602.
tions, the population doubling time, the percentage of non-dividing cells and other growth properties of WI-38 and IMR-90 (human embryonic lung fibroblasts) are not adversely affected by growth a t clonal densities. MATERIALS AND METHODS
The human embryonic lung fibroblast lines WI-38 and IMR-90 were used in these studies. WI-38 cultures were obtained from Doctor L. Hayflick (Oakland Childrens Hospital) and IMR-90 cultures were obtained from the Institute for Medical Research (Camden, New Jersey). Except as noted, mass cultures were maintained in MEM with Hanks' balanced salt solution (GIBCO), 10%fetal bovine serum (Reheis Chemical Co.) and 29 mM HEPES buffer (Sigma Chemical Co.). A. Determination ofpopulation doubling time Cultures were initiated 48 hours after the previous subculture. The cell layer was washed with Dulbecco's calcium magnesiumfree phosphate buffered saline (CMFPBS), and 0.5 ml of 0.25%pronase (Sigma Chemical Co.) in CMFPBS was added. When the cells had detached, they were suspended in complete medium, counted with a Model ZBI Coulter Counter, and seeded a t the appropriate density into a series of 60 mm dishes or 25 Received June 2, '78. Accepted a t . 2, '78.
597
598
JAMES R. SMITH AND KAREN I. BRAUNSCHWEIGER
cm2flasks. Except as noted, clonal density cultures (100cells/dish) were seeded in medium MCDB 104 (McKeehan et al., '77) supplemented with 5% FBS and maintained in 5% C0295%air at -98% relative humidity. Mass cultures (2.5 x lo5 cells/flask) were seeded (except as noted) in MEM with Hanks' salts, 10%FBS and 29 mM HEPES. In both clonal and mass cultures the medium was changed every two days. Cell number in clonal and mass cultures were determined in triplicate cultures every 24 hours for a period of five days. Clonal plates were fixed in 1%glutaraldehyde, stained with 0.5%crystal violet and the number of cells per dish determined by direct count under a dissecting microscope. In mass cultures suspensions of single cells were prepared by pronase treatment and the number of cells was determined by Coulter Counter. The population doubling times were derived from growth curves which had been fit to the experimental data by the least squares method. The growth of both clonal density and mass cultures was exponential for four days after seeding. Therefore, this time period was used to determine the population doubling times. B. Life-span of clonal and mass cultures Mass cultures maintained in MEM with Hanks' salts, 10% FBS, and 29 mM HEPES were subcultured weekly. At each subculturing, the total number of cells in the flask was determined, and 2.5 x lo5 cells were transferred to a new flask. The number of cells that had attached six hours after seeding was determined. For both clonal and mass cultures, the increase in cumulative population doublings a t each subculture was calculated based on the number of cells attached and the cell yield a t the time of the next subcultivation. Clonal cultures were initiated from mass cultures by seeding 100 cells into 60 mm dishes containing MCDB 104 with 5%FBS. At weekly intervals, three plates were fixed with 1% glutaraldehyde, stained with crystal violet and the number of cells per dish counted microscopically. Cells in parallel cultures were removed with pronase and reseeded a t 100 celldplate. Attachment a t six hours was determined by fixing, staining and counting the number of cells per plate. Mass cultures were considered to have achieved their proliferative limit when they did not exceed 5 x lo5 cells/flask in four
weeks. Clonal density cultures were considered to have reached their proliferative limit when they did not exceed 200 cells/plate in four weeks. RESULTS
To assess whether the growth of human diploid fibroblasts a t clonal density under our experimental conditions is equivalent to that in mass culture, we have compared several growth properties under both conditions: (1) population doubling time, (2) long-term proliferative potential, (3) percent attachment, and (4) percentage of cells capable of division. Figure 1 shows the results of a typical ex-
20
rI
a W
m
4z
-I -1 W
0 W
>
5-1 W
U
TIME (days) Fig. 1 Growth of clonal and mass cultures of WI-38 cells PDL 37. Mass cultures were inoculated with 2.5 X lo5 cellsff-25 flask on day 0 in BME ( 0 )or MCDB 104 (0). Clonal cultures were inoculated with 100 celld60 mm dish on day 0 in BME (A)or MCDB 104 (A). Each point is the average cell number from three culture vessels relative to the cell number on day 1.
CLONAL DENSITY CULTURE VERSUS MASS CULTURE
599
a t low density. The low density cultures were never allowed to grow t o a density of more than about 2,500 cells/60 mm dish. Figure 2 shows the cumulative population doublings achieved as a function of time for one such experiment. We found that the overall replication rate was initially higher in the clonal cultures than in mass cultures. This is probably due to the mass cultures being in a state of density dependent inhibition of division during part of each subcultivation period while the clonal cultures were not. The variability of such experiments is shown in table 2 which summarizes the results of several experiments using both WI-38 and IMR-90 cultures. In two experiments the total proliferative capacity of the low density cultures was 11 and 15 doubu o 20 40 60 80 lings less than the parallel mass cultures. TIME (days) However, in the other three experiments the Fig. 2 Cumulative population doublings as a function of time (days) for clonal ( A ) and mass ( 0 )cultures of WI- cultures kept a t low density went through 38 cells. Continuous clonal cultures were initiated from almost the same number of doublings as the mass cultures at PDL 40. mass cultures. Thus, there was no consistent adverse effect on in vitro life-span due to conperiment in which the population doubling tinuous low density culture. Finally we compared the percentage of cells times of clonal and mass cultures were compared. In this experiment clonal and mass cul- which failed to divide in clonal culture with tures were grown in both BME and MCDB the results obtained by Cristofalo and Sharf 104. MCDB 104 was designed for clonal (‘73) for mass cultures. These results are growth of human diploid fibroblasts and BME shown in figure 3. Our results for clonal culhad been widely used for mass culture of these tures agree with those obtained by Merz and cells. The growth rate was lower for mass cul- Ross (‘69) for clonal culture. In both cases the tures in MCDB 104 than BME. For clonal cul- clonal cultures resulted in a lower percentage tures, however, just the reverse was observed. of non-dividing cells than that obtained for Cell proliferation was essentially exponential mass cultures. This is probably due to the use from day 1to 4. Therefore, all subsequent pop- of different criteria for “non-dividing” cells in ulation doublings times were determined for mass and clonal cultures. In clonal cultures “non-dividing” cells were cells that had not this time period. Table 1summarizes the results from five ex- divided during one to two weeks, while in mass periments in which the population doubling culture cells that had not synthesized DNA in times were compared for clonal and mass cul- a 30 hour period were considered “non-dividtures. These experiments were performed at ing.” Since the interdivision time of a proporvarious population doubling levels (PDL). The population doubling times increase with inTABLE 1 creasing PDL in both clonal and mass culComparison of population doubling times in clonal and tures. In all cases the population doubling mass cultures of WI-38 cells times of clonal cultures are essentially the Population doubling time (hours) same as for mass cultures. PDL ’ Mass cultures Clonal cultures Even though the growth rate of WI-38 cells did not appear to be adversely affected by 24 21.6 24.7 growth a t clonal density, it was also important 36 19.0 22.1 31 22.5 21.4 to show that the long-term proliferative 45 23.9 24.1 ability was not drastically decreased by 51 30.8 29.0 growth a t clonal density. We compared the ’ Population doubling level of the mass cultures used to initiate number of population doublings achieved by for growth curves. cultures treated in the normal manner for cultures Population doubling time for mass culturea determined in BME mass culture and cultures kept continuously instead of MEM. ~
~~
~
~~
600
JAMES R. SMITH AND KAREN I. BRAUNSCHWEIGER TABLE 2
Total proliferatiue capacity (P.D.)
P.D. began
Clonal
Mass
21 28 42 40 45
65 43 57 73 61
64 54 72 72 62
IMR-90 IMR-90 IMR-90 WI-38 WI-38
I Population doubling at which continuous clonal cultures were initiated.
tion of these cells may be more than 30 hours this would result in a n underestimate in percent of cells which would eventually divide. The average percentage attachment measured six hours after inoculation was similar for mass (67%) and clonal (71%) cultures. Therefore the larger percentage of dividing cells in low density culture cannot be attributed to selection in favor of dividing cells due to a lower percentage attachment. DISCUSSION
m
m
I
0
I
I
I
80 100 PERCENT LIFE-SPAN COMPLETED
20
40
60
Fig. 3 Percentage of non-dividing WI-38 cells vs percent in vitro life-span completed. Open symbols from Cristofalo and Sharf ('73) for mass cultures. Solid squares (D) from Merz and Ross ('69) for clonal culture. The other solid symbols (V, A , 0 ) indicate percentages of cells which did not divide within two weeks in clonal cultures inoculated with 10-20 cells per 60 mm dish and incubated as described in MATERIALS AND METHODS (each symbol represents the results from a single subcultivation series of WI-38 cells).
When normal human fibroblast-like cells are plated a t clonal density a portion of the cells are capable of only a few divisions (Martin et al., '74; Smith and Hayflick, '74; Smith et al., '77, '781, while others can undergo a number of doublings approximating the doubling potential of the mass culture from which they were derived. The proportion of cells with low doubling potential increases with in vitro age of the parent culture. A major concern has been that the low doubling potential exhibited by some of these cells in clonal culture occurs because normal human fibroblasts are incapable of optimal growth a t clonal densities. We have shown previously (Smith et al., '77) that in clonal culture a substantial proportion of attached cells are unable to form clones of more than 16 cells (from 20%a t PDL 20 to 65% a t PDL 50 for WI-38 cells). If cells stopped proliferating after a few divisions in low density culture but not in mass culture, one would expect the population doubling time to be longer in low density culture than in mass culture. We have observed (in both mass and low density cultures) an increasing population doubling time with increasing PDL corresponding to the increase in low doubling potential colonies. The fact that the population doubling time increases to the same extent in clonal and mass cultures (table l), suggests that either, (1)the mass cultures contain the same proportion of cells with small doubling potential as the clonal cultures or ( 2 ) that the replication rate of the cells which are capable of extensive proliferation in clonal culture increases as a function of PDL to the exact extent needed to give the same doubling time for the population as a whole as observed in mass culture. We consider the first possibility to be more likely. The percentage of non-dividing cells in mass and clonal culture is a more direct test of the
CLONAL DENSITY CULTURE VERSUS MASS CULTURE
ability of human embryonic fibroblasts to proliferate under these conditions. We found, in agreement with Merz and Ross ('691, that the percentage of non-dividing cells was essentially the same in low density culture as found in mass culture (Cristofalo and Sharf, '73). Furthermore, we found that there was no consistent difference between the in vitro lifespan of IMR-90 and WI-38 cells grown in low density and mass culture. However, this is not a stringent test for the equivalence of clonal and mass cultures for two reasons. (1)The in vitro life-span of cultures of human embryonic fibroblasts in mass culture is quite variable (table 2 and Holliday et al., '77). (2) The in vitro life-span of a culture may be determined by a small proportion of cells with the greatest doubling potential. If this is the case then in initiating low density cultures with a 100 cell sample from mass cultures one would expect some of the low density cultures not to contain cells with the greatest doubling potential. These experiments do indicate however that any adverse effect on total proliferation potential due to low density growth cannot be very great. In conclusion, we find no evidence to support the idea that the low doubling potential observed for a proportion of cells in low density culture is an artifact of culture conditions. On the contrary, in three of the parameters studied where artifacts would be expected to cause differences between low density and mass cultures, the results were equivalent. Therefore, the results reported here support the idea that the behavior of individual cells and clones within a mass culture of human embryonic fibroblasts can be inferred from the behavior of the same cells grown a t clonal densities.
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ACKNOWLEDGMENTS
We are grateful for the excellent assistance of Thomas Roberts in some aspects of this work. "his investigation was supported in part by the National Institute on Aging (Grant No. AG00338-01-Al) and the W. Alton Jones Foundation. LITERATURE CITED Absher, P. M., and R. G. Absher 1976 Clonal variation and aging of diploid fibroblasts. Cinematographic studies of cell pedigrees. Exp. Cell Res., 103: 247-255. Cristofalo, V. J., and B. B. Sharf 1973 Cellular senescence and DNA synthesis. Thymidine incorporation a s a measure of population age in human diploid cells. Exp. Cell Res., 76: 419-427. Hayflick, L. 1977 The cellular basis for biological aging. In: The Handbook of the Biology of Aging. C. E. Finch and L. Hayflick, eds. Van Nostrand Reinhold Co.,New York, pp. 159-186. Holliday, R., L. I. Huschtscha, G. M. Tarrant and T. B. L. Kirkwood 1977 Testing the commitment theory of cellular aging. Science., 198: 366-372. Martin, G.M., C. A. Sprague, T. H. Norwood and W. R. Pendergrass 1974 Clonal selection, attenuation and differentiation in an in uitro model of hyperplasia. Am. J. Pathol., 74: 137-154. McKeehan, W. L., K. A. McKeehan, S. L. Hammond and R. G. Ham 1977 Improved medium for clonal growth of human diploid fibroblasts a t low concentrations of serum protein. In Vitro., 13: 399-416. Merz, G . S.,and J. D. Ross 1969 Viability of human diploid cells as a function of in vitro age. J. Cell. Physiol., 74: 219-222. Muggleton-Harris, A. L., and L. Hayflick 1976 Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells. Exp. Cell Res., 103: 321-330. Smith, J. R., and L. Hayflick 1974 Variations in the lifespan of clones derived from human diploid fibroblasts cultures. J. Cell Biol., 62: 48-53. Smith, J. R., 0. Pereira-Smith and P. I. Good 1977 Colony size distribution as a measure of age in cultured human cells. A brief note. Mech Ageing Dev., 6: 283-286. Smith, J. R., 0. Pereira-Smith and E. L. Schneider 1978 Colony size distributions as a measure of in viuo and in uitro aging. Proc. Natl. Acad. Sci. (U.S.A.),75: 1353-1356.
