Growth Limitation of 3T3 Mouse Fibroblasts by Available Growth Surface Area and Medium Components CORNELIA R. THRASH 1 AND D E N N I S D. CUNNINGHAM Department of Medical Microbiology, College of Medicine, University of California, Irvine, California 92664
ABSTRACT Studies with untransformed fibroblasts demonstrate that growth of these cells in culture can be limited by the availability of both growth surface and medium components. Experiments using cells grown on coverslips, in which the only variable was available growth surface, indicate that when the medium to cell ratio is high, surface area is the principal factor limiting growth. At low medium to cell ratios, however, growth of cells is predominantly limited by medium components. The final number of cells per culture is almost directly proportional to available surface area when the culture medium is changed daily.
The growth of untransformed fibroblasts in culture stops at a density characteristic of the cell type and concentration of serum present in the culture medium. Although the events leading to this arrest of growth are not well understood, experiments have demonstrated that limitations of medium components is intimately involved (Kruse and Miedema, '65; Todaro et al., '67; Holley and Kiernan, '68; Dulbecco and Elkington, '73). There is also evidence that cell density is a critical factor which can regulate growth (Fisher and Yeh, '67; Stoker and Rubin, '67; Schutz and Mora, '68; Kolodny and Gross, '69; Clarke et al., '70; Dulbecco, '70; Martin and Rubin, '74), although it appears that cell-cell contacts are not directly involved in the inhibition of DNA synthesis and cell division (Martz and Steinberg, '72; Dulbecco and Elkington, '73; Stoker, '73; Holley, '74). Thus, "density-dependent" (Stoker and Rubin, '67) or "post-confluence" (Martz and Steinberg, '72) inhibition of growth better describes the phenomenon formerly known as "contact-inhibition'' of growth. Recently, to further clarify the conditions which limit growth of cells in dense culture, Dulbecco and Elkington ('73) plated fibroblastic or epithelial cells in a constant amount of medium in culture dishes of different sizes. They concluded that growth of epithelial cells is limited by the amount of surface available to them. In contrast, growth of fibroblastic Balb/c J . CELL.PHYSIOL..86. 301-310
3T3 cells appeared to be limited by the medium rather than available growth surface area. Since this result was at variance with our experience with Swiss mouse 3T3 fibroblasts, we repeated these experiments with both types of 3T3 cells, and in addition, designed new experiments using coverslips to investigate the role of available surface area and cell density on growth of these untransformed fibroblasts. By using the coverslip technique, we were able to design experiments in which the only variable was available growth surface. We found that with both Balb/c and Swiss 3T3 cells, availability of surface area (or cell density) is the principal factor limiting growth when the medium to cell ratio is high. However, at lower medium to cell ratios, growth is predominantly limited by medium components. MATERIALS A N D METHODS
Culturing of cells Swiss mouse 3T3 fibroblasts (clone 42) were obtained from Dr. George J. Todaro; Balb/c 3T3 cells were obtained from Dr. Dieter Paul and Dr. J. Elkington. They were grown as previously described in Dulbecco-Vogt modified Eagle's medium containing 100 units of penicillin, 100 pg of streptomycin per milliliter, plus the indicated amount of calf serum (Cunningham, Received Oct. 4, '74. Accepted Dec. 16, '74. 1 Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.
30 1
302
CORNELIA H THRASH AND DENNIS D CUNNINGHAM
' 7 2 ) . The growth medium contained 2.5 gins NaHCO:, per liter. Chowth of cells was carried out in an atmosphere of 5% CO2 in air to maintain the pH between 7.3 and 7.5. The cells were mycoplasma-free as routinely judged by the presence of nuclear and not cytoplasmic grains following au toradiography with (:jH) thymidine (Nardone et al.. '65). All experiments with iUalb/c 3T3 cells, except those noted in figure 2 were performed using cells obtained rrom Dr. Elkington.
Counting of cells 'The cells were rinsed with phosphatebuffered saline solution (PBS) and incubated for approximately ten minutes at 37 with 0.055% trypsin and 0.02 % EDTA in PBS. Aliquots of the suspended cells were diluted with PBS and counted in a Couiter counter. Similar results were obtained when cells were counted in a hemac ytometer chamber . L.
RESULTS
One method of examining the effect of available surface area on the number of cells present at saturation is to culture a given number o f cells in a constant amount of medium in tissue culture dishes of varied sizes. Using this technique, Dulbecco and Elkington ('73) found that the final number of untransformed Balb/c 3T3 and BHK 21 fibroblasts was related to the amount of medium present and not to the available growth surface area. They concluded from this that growth of these fibroblasts was h i t e d by medium components rather than available growth surface. We have repeated these experiments and concluded that growth of these cells is limited by both available surface area and medium components. Figure 1 shows a typical growth curve for the same Balb/c cells grown under the conditions used by Dulbecco and Elkington ('73). As can be seen, we obtained more cells in the dishes with greater surface area. Thus, surface area at least partly limited the growth of these cells. Further use of this technique a! various serum concentrations and seeding densities demonstrated that growth of riot only Balblc 3T3 cells (fig. 2a) but also Swiss mouse 3T3 cells (fig. 2c) was limited by availability of both the surface area and medium components. Figure 2b shows the
0
1
2
3
4
5
6
DAYS
Fig. 1 Growth curves of Balblc 3T3 cells. 1.0 1 0 5 Balb/c 3T3 cells (in 6.0 ml medium enriched with 10% calf serum) were seeded in Nunc plastic tissue culture dishes containing approximately 7 (U), 21 ( A - A), or 60 (CI) cm2 growth surface. The number of cells per dish was determined as described i n MATERIALS AND METHX
ODS.
theoretical slopes expected if cell growth were limited strictly by surface area (final number of cells directly proportional to dish size) or by medium components (same number of cells in all size dishes). As shown in figures 2a,c, intermediate slopes were obtained under nearly all of these culture conditions. This demonstrates that both surface area and medium components limited growth. In fact, within the range tested, the growth of Swiss 3T3 cells appeared to be limited mainly by surface area (fig. 2c). Growth of Balb/c 3T3 cells was also predominately limited by surface area, except in the largest dishes or when the serum to cell ratio was low (cells seeded at high density or cells grown in low serum). Growth of cells on coverslips The above results are potentially affected by variables other than surface area (for instance, medium depth). Since these variables might affect the number of cells at saturation, we designed experiments which eliminated all variables other than available growth surface area. This was accomplished by growing cells on glass coverslips in a constant amount and depth of medium. First, cells were seeded on glass coverslips at a given density. These coverslips were then transferred to bac-
30 3
LIMITATION OF FIBROBLAST GROWTH
1 -
1 -
I
i
r
I I
1
/ -J
!
P
I
7
21
61
SURFACE
AREA
(crn2)
Fig. 2 Effect of culture dish size on final number of cells at saturation. Cells were seeded 5.0 ( S O ) , in Nunc plastic tissue culture dishes in 6.0 ml medium containing 2.0 (M), 10 ( A- A ) , or 20 (A-A) percent calf serum and allowed to grow to a growth arrested state. The number of cells initially seeded is indicated by the heavy horizontal lines. After growth had stopped, as determined by microscopic examination, cells were counted 1 and 2 or 1 and 3 days later as described i n MATERIALS A N D METHODS. In all cases the cultures had reached a growth arrested state by this time. Subsequent increases in cell number, if any, were less than 10% per day. In several cases complete growth curves, like those shown in figure 1, or mitotic indices of cultures were also measured, determinations which confirmed that a growth arrested state had been achieved by the time the above measurements were made. Panel a, Balb/c 3T3 cells obtained from Dr.J. Elkington (dashed lines) and Dr. Dieter Paul (solid lines). Panel b, theoretical slopes expected if growth of cells were limited strictly by available surface area (M) or by medium limitation (H). Panel c, S w i s s 3T3 cells. All experiments were performed at least twice.
teriological grade culture dishes containing culture medium. These cells grew only on the glass coverslips and did not migrate or grow onto the culture dish. The number of cells per culture was therefore identical to the number of cells growing on the glass coverslips. To insure that these experiments were conducted on cultures which had reached their final saturation density, we measured cell number over a five day period. Typical growth curves, shown in figure 3, demonstrate that a growth-arrested state was achieved within four days in cultures of cells containing either 1, 2 , or 4 coverslips which were seeded at 0.2 x 1 0 5 cells per coverslip (panel a) or 0.8 X 1 0 - 5 cells per coverslip (panel b). In all experiments utilizing coverslips, the final number of cells at saturation was determined from similar growth curves. We first examined the effect of available surface area on the final number of cells
at saturation (fig. 4). These experiments were conducted so that available growth surface was the only variable. This was accomplished by placing different numbers of coverslips in identical bacteriological grade culture dishes containing equal volumes of medium. Cultures were set up so that the number of cells per coverslip was inversely proportional to the number of coverslips placed in the culture. Thus, each culture initially contained an equal number of cells. This insured that the initial medium to cell ratio was also constant. If final cell number were determined only by available growth surface area, the final number of cells per culture at saturation would be directly proportional to the number of coverslips per culture as shown by the upper dashed line in figure 4. On the other hand, if final cell number were limited only by medium components, the final number of cells per culture would be identical in all cultures as shown by the hori-
-
CORNELIA R . THRASH AND DENNlS D. CUNNINGHAM
I
'a
-I
b.
I\-
&--
r - - - - O
U
L 3
5
4
DAYS AFTER SEEDING
Fig. 3 Growth curves of Balblc 3T3 cells grown on coverslips. Eighteen millimeter round glass coverslips were seeded at 0.2 X 105 (panel a) or 0.8 X 1 0 5 (panel b) cells per coverslip in tissue culture dishes with medium containing 10% calf serum. After attachment of the cells, the coverslips wexe transferred to 50 mm bacteriological plastic Petri dishes with 6.0 ml medium containing 10% calf serum. No more than 0.1% of the cells ever migrated to the surface of the bacteriological dish. On the days indicated, the cells were removed from the coverslips and counted as described in MATERIALS A N D METHODS. One coverslip per culture, (O--O); two coverdips per culture, ( 0 4 ) ;or four coverslips per culture, ( A-A ).
,
-I
2
3
4
COVERSLIPS F'ER CULTURE Fig. 4 Relationship of available surface area to final cell number with the initial cell to medium ratio constant. Balblc 3T3 cells were seeded on coverslips as described in the legend to figure 3 so that the initial number of cells per culture was 0.8 x 1 0 5 , (M), or 1.6 X 105, (-0). The final cell density was determined by growth curves like those shown in figure - 3. Theoretical curves expected if only surface area. (O---O) or medium components ( C4)limited growth.
I
I
I
1
I
2
3
4
COVERSLIPS PER
CULTURE
Fig. 5 Relationship of available surface area to final cell number. Balblc 3T3 cells were seeded 0.4 ( 0 4 ) , 0.8 ( 0 4 ) and 1.6 at 0.2 (M), (X-X) X 105 cells per coverslip and treated as described in figure 3. The final cell density was determined from growth curves like those shown in figure 3. Theoretical curves if only surface area (S-0)or medium components ( C - W ) limited growth.
305
LIMITATION OF FIBROBLAST GROWTH
volume of medium was increased. For example, figure 4 shows a greater medium limitation in cultures initially containing 1.6 x 10.5 cells than in cultures containing 0.8 X 105 cells. Therefore, medium limitation increased as the cell to medium ratio increased. Experiments like those shown in figures 4 and 5 were also performed with Swiss 3T3 cells (not shown). While the results of these experiments were qualitatively similar to those using Balblc cells (figs. 4, 5) the absolute numbers of Swiss mouse 3T3 cells was considerably lower, presumably due to the lower saturation density charactersitic of the latter cell line.
zontal dashed line in figure 4. Actual data points in the figure (solid lines) reveal an intermediate condition reflecting growth limitation by both medium components and available growth surface area. This conclusion is based on experiments using two different initial numbers of cells per culture. Additional experiments, shown in figure 5, were carried out in which 1, 2, or 4 coverslips, containing the same number of cells per coverslip, were placed in identical bacteriological grade culture dishes containing equal volumes of medium. Four sets of these cultures were set up at different initial numbers of cells per coverslip. Within each set, the initial medium to cell ratio varied. The data in figure 5 show that, under these conditions, as well as when the ratio was constant (fig. 4), the final number of cells at saturation was limited by both available growth surface area and by medium components. The data in both figures 4 and 5 demonstrate that limitation of final cell number by medium components became more pronounced as the number of cells initially present per L '
Decay of medium components Since these experiments indicated that cell growth was limited by both available growth surface area and medium components, we further investigated the nature of this medium limitation. Because both serum and the defined medium contain thermolabile components, we first examined the effect of incubation of medium a t 37" in the absence of cells on its subse-
I
I
I
I
2
4
6
8
104
I
I
1
0
2
4
-
L
0
L
6
8
PREINCUBATION TIME (DAYS)
Fig. 6 Effect of preincubation of medium at 37O on growth of nonconfluent and maintenance of confluent Swiss 3T3 cells. Fresh medium containing 10% calf serum was placed in a 37O C& incubator. Aliquots were withdrawn at the times indicated on the abscissa and frozen. Panel a : 0.5 X 105 Swiss 3T3 cells were plated in 35 mm Falcon plastic tissue culture dishes. After attachment of cells, the medium was replaced with 2.0 ml of the preincubated medium. Cells on duplicate plates were counted as described in MATERIALS AND METHODS one (M), two (X-X), three (U), and four ( A - A ) days later. Panel b: 1.0 X lo5 Swiss 3T3 cells were plated in 35 mm Falcon plastic tissue culture dishes in 2.0 ml medium. After three days the cultures were growth arrested and had a final saturation density of 2.7 x 1 0 5 cells per dish. The medium then was replaced with 2.0 ml of the preincubated medium. The cells in duplicate plates were counted on the fifth (A-A) and eighth (0- - -0) days following the medium change.
306
CORNELlA R. THRASH AND DENNIS D. CUNNINGHAM
quent ability to support growth of nonconfluent cells and to maintain cell number of confluent 3T3 cells. Figure 6a demonstrates that preincubation of medium at 37" markedly decreased its ability to support the growth of 3'T3 cells. Cultures grown in medium which had been preincubated for eight days had a final cell number of about 54% of cultures grown in medium which had not been pre-incubated. Maintenance of confluent cultures, however, appeared to be relatively unaffected until the medium had been preincubated about eight days (fig. 6b). Thus, for growing cultures, the medium limitations observed were at least partly due to spontaneous decay of essential components. Furthermore, the partial medium limitations observed in figure 2 were probably overestimated by this medium decay, since the number of cells in the 7 cm2 dishes was determined at three to six days after seeding, while the number of cells in the 21 and 60 cm2 dishes was determined five to eight and seven to 12 days respectively after seeding. Thus, a greater decay of medium components probably occurred in the larger dishes, reducing its potential to support growth of these cells. Medium decay probably had a lesser effect on the coverslip experiments shown in figures 4 and 5 since cell numbers were determined for all cultures between days three and five. Alteration of medium by cells Further experiments indicated that the medium limitation observed in previous experiments (figs. 2 , 4, 5) was the result of alteration of the medium by cells as well as spontaneous medium decay. In these experiments, cells were cultured as in figure 4 with no medium change or were cultured with daily changes of fresh medium which had been kept at either 4 " or 37". Figure 7 indicates that significantly more cells were produced in cultures in which the culture medium was replaced daily with fresh medium which had been kept at 37" from day 0 than in cultures in which the medium remained unchanged. Thus, cellular utilization or inactivation of medium components was partially responsible for the medium dependence for growth of Balblc 3T3 cells. Furthermore, figure 7 demonstrates that the medium
1
.---
IL I
-
, L
2
i
3
!
-,A 4
COVERSLIPS PER CULTURE
Fig. 7 Relationship of available surface area to final number of cells per culture with daily medium changes. Balb/c 3T3 cells were seeded at 0.2, 0.4, and 0.8 X 105 cells per coverslip in cultures containing 4, 2, and 1 coverslip per culture r e spectively as described in the legend to figure 3. Growth-arrested cells were counted as described in MATERIALS AND METHODS on days 3, 4, and 5 and the values averaged. (A-A), medium replaced daily with fresh medium kept at 4 O from day 0; (M), medium replaced daily with fresh medium kept at 37O from day 0; (M), medium unchanged; (@--a),theoretical slope if surface area strictly limited growth; (C4),theoretical slope if medium components strictly limited growth.
limitation on the growth of Balb/c 3T3 cells can be accounted for by the combined effects of both the cellular alteration and spontaneous decay of medium components, since cells grown with daily changes of medium which had been maintained at 4" from day 0 showed no medium limitation. Thus, when medium components were not limiting, available surface area became the sole factor limiting growth and the final number of cells per culture was directly proportional to the available growth surface area. Additional experiments were performed with both Swiss and Balblc 3T3 cells in which culture medium was replaced daily with fresh medium containing 2-20% serum which had been kept at 4 " (fig. 8). Under these conditions, the final number of cells at saturation was almost directly proportional to the amount of surface available for growth. This further demonstrated that surface area became the sole factor
LIMITATION OF FIBROBLAST GROWTH BALB/C 3T3
SWISS 3T3 4
8
3
6
2
4
I
2
..f
I
0
x
1
1
.
1
1
1
1
12
32
3
5 3
9
24
O LL
6
16
W
LL
w
a
8
c n 3 _I
J
w
0
32
80
24
60
16
8 , L - -
I
2
3
4
I
2
3
4
COVERSLIPS PER CULTURE
Fig. 8 Relationship of available surface area to final number of Swiss and Balblc 3T3 cells with daily medium changes. Cells were seeded as described in the legend to figure 3 and the growth arrested cells counted as described in the legend to figure 7. Swiss 3T3 cells were seeded at 0.1, 0.2, and 0.4 X 105 cells per coverslip in cultures containing 4, 2, and 1 coverslip per culture respectively. Balb/c 3T3 cells were seeded as described in the legend to figure 7. The dashed lines represent the theoretical slopes expected if only surface area limited growth. Solid circles represent actual data points.
limiting growth when the medium was changed daily, indicating that the surface dependence shown in figure 7 was not peculiar to medium containing 10% serum. DISCUSSION
These studies demonstrate that the growth of untransformed Balblc and Swiss mouse 3T3 fibroblasts can be limited by available surface area and by the amount and concentration of medium components. Culture conditions can be manipulated (by changing the medium daily or by varying the cell to medium ratio) to alter the relative limitations by surface area and me-
307
dium components. Medium components become limiting for final cell number as the initial number of cells is increased in cultures receiving no medium change; surface area becomes limiting when there are fewer initial cells present per culture (figs. 2, 4, 5). In addition, when the medium is changed daily, the final number of cells at saturation is almost directly proportional to available growth surface area (fig. 8). However, figure 8 also shows that in cultures in which the greatest amount of growth occurred (four coverslips per dish in cultures containing six and twenty per cent serum) a slight medium dependence was observed. We did not determine whether more frequent medium changes might elininate this dependence on medium for growth. In the experiments reported here, the medium limitation of growth appears to involve at least two factors: a noncellular inactivation of growth-promoting activity and a cell-mediated alteration of the medium. Whether the cell-mediated alteration reflects utilization or inactivation of medium components or production of toxic or growth-inhibitory molecules by the cells cannot be distinguished by these experiments. The observation that available surface area can limit growth of fibroblasts is in concert with studies on neonatal rat heart cells and Swiss 3T3 and 3T6 cells by Kolodny and Gross ('69). Extensive growth of these cells occurred after the surface area available to them was increased; however, it is not clear whether a medium limitation also exists since the number of cells per cmz was not given for either the initial or final growth-arrested states. Recent evidence by Martin and Rubin ('74) further supports the existence of densitydependent growth controls. They found that chick embryo fibroblasts were capable of attaching and growing on bacteriological grade plastic culture dishes. In these dishes, cells migrated to form clumps which became growth-arrested after they achieved an average density of about 90 cells per clump. Transfer of the clumps to tissue culture grade plastic culture dishes with the medium in which they had become growth arrested resulted in migration of the cells out of the clumps. Subsequently, the cells re-entered a log
308
CORNELIA R. THRASH AND DENNIS D. CUNNINGHAM
phase of growth and became growth-arrested after they had achieved a confluent monolayer, demonstrating that the arrest of‘ growth in clumped cells was not due to medium limitation. They suggest that the density-dependent arrest of growth observed in the cell clumps might be relevant to growth control processes in vivo. Recent experiments by Dulbecco and Elkington (’73) like those shown in figure 2, indicate that the growth of untransformed Balb/c 3T3 and BHK 21 cells is restricted primarily by medium components rather than by available surface area. We observed limitation of growth by both surface area and by medium components using the same clone of Balb/c 3T3 cells. While the reasons for the differences between the two sets of data are not clear, likely explanations are that the medium used in our experiments (1 ) was altered less rapidly by the cells, or (2) contained different absolute or relative amounts of critical growth and maintenance factors. Since the final number of cells per culture is a n equilibrium between cell growth and detachment, a difference in the relative amounts of these factors might account for the differences in the results. In our experiments, no significant cell detachment occurred until after the final number of attached cells shown in the figures was achieved. This number remained constant for a variable period of time, after which cell detachment was noted with a concomitant decline in the total number of cells per culture (attached plus detached) (unpublished observations). It is unlikely that differences in the methods used to determine when a growth-arrested state was achieved could account for the discrepancies, since our results were identical using either mitotic indices (like Dulbecco and Elkington) or complete or partial growth curves as a measure of growth arrested state (unpublished observations). The mechanism by which available growth surface area limits the growth of cells is not clear. Evidence that it does not directly depend on cell-cell contacts has been discussed by several investigators (Stoker and Rubin, ’67; Martz and Steinberg, ’72; Dulbecco and Elkington, ’73; Stoker, ’73; Holley, ’74). Indeed, the operational limitation of final cell number by available growth surface area might ulti-
mately result from a limitation of medium components. For example, after growth to confluency, cells appear to have less access to medium components (Stoker and Rubin, ’67; Stoker, ’73) and they also take up certain nutrients from the growth medium at a reduced rate (Kohn, ’68; Cunningham and Pardee, ’69; Weber and Rubin, ’71; Sefton and Rubin, ’71). ACKNOWLEDGMENTS
This work was supported by U . S. Public Health Service Grant CA-12306. C. R. T. was supported by U . S. Public Health Service Training Grant (GM-02063) to the Department of Molecular Biology and Biochemistry. LITERATURE CITED Clarke, G. D., M . G. P. Stoker, A . Ludlow and M. Thornton 1970 Requirement of serum for DNA synthesis in BHK 21 cells: Effects of dens i t y , suspension, and virus transformation. Nature, 227: 798-801. Cunningham, D. D. 1972 Changes in phospholipid turnover following growth of 3T3 mouse cells to confluency. J. Biol. Chem., 247: 24642470. Cunningham, D. D., and A. B. Pardee 1969 Transport changes rapidly initiated by serum addition to “contact inhibited” 3T3 cells. Proc. Natl. Acad. Sci. (U.S.A.), 64: 1049-1056. Dulbecco, R. 1970 Topoinhibition and serum requirement of transformed and untransformed cells. Nature, 227: 802-806. Dulbecco, R., and J. Elkington 1973 Conditions limiting multiplication of fibroblastic and epithelial cells in dense cultures. Nature, 246: 197199. Fisher, H. W., and J. Yeh 1967 Contact inhibition in colony formation. Science, 155: 581-582. Holley, R. W. 1974 Serum factors and growth control. In: Control of Proliferation in Animal Cells. B. Clarkson and R. Baserga, eds. Cold Spring Harbor Press, N. Y., pp. 13-18. Holley, R. W., and J. A. Kiernan 1968 Contact inhibition of cell division i n 3T3 cells. Proc. Nat. Acad. Sci., 60: 300-304. Kohn, A . 1968 Thymidine metabolism and DNA synthesis in crowded monolayers of animal cells. Exp. Cell Res., 52: 161-172. Kolodny, G . M., and P. R. Gross 1969 A simple mechanical method for the efficient release of contact inhibition. Exp. Cell Res., 57: 4 2 3 4 3 2 . Kruse, P., and E. Miedema 1965 Production and characterization of multiple-layered population of animal cells. J. Cell Biol., 27: 273-279. Martin, G. R., and H. Rubin 1974 Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts. Exp. Cell Res., 85: 319333. Martz, E., and M . S. Steinberg 1972 The role of cell-cell contact in “contact” inhibition of cell division: A review and new evidence. J. Cell. Physiol., 79: 189-210.
LIMITATION OF FIBROBLAST GROWTH Nardone, R. M., J. Todd, P. Gonzales and E. V . Gaffney 1965 Nucleoside incorporation into strain L cells: Inhibition by pleuropneumonialike organisms. Science, 149: 1100-1101. Schutz, L . , a n d P. T. Mora 1968 The need for direct cell contact in “contact” inhibition of cell division in culture. J. Cell. Physiol., 71 : 1 4 . Sefton, B . M., and H. Rubin 1971 Stimulation of glucose transport in cultures of density-inhibited chick embryo cells. Proc. Natl. Acad. Sci. (U.S.A.), 68: 3154-3157. Stoker, M . G . P. 1973 Role of diffusion boundary layer in contact inhibition of growth. Nature, 246: 200-203.
309
Stoker, M. G. P., and H. Rubin 1967 Densitydependent inhibition of cell growth in cultures. Nature, 21 5: 171-172. Todaro, G. J., Y. Matsuya, S. Bloom, A. Robbins and H. Green 1967 Stimulation of RNA synthesis and cell division in resting cells by a factor present in serum. In: Growth Regulating Substances for Animal Cells i n Culture. V. Defendi and M. Stoker, eds. Wistar Inst. Press, Phila., pp. 87-101. Weber, M. J., and H. Rubin 1971 Uridine transport a n d RNA synthesis in growing and i n density-inhibited animal cells. J. Cell. Physiol., 77: 157-168.
ABSTRACT Studies with untransformed fibroblasts demonstrate that growth of these cells in culture can be limited by the availability of both growth surface and medium components. Experiments using cells grown on coverslips, in which the only variable was available growth surface, indicate that when the medium to cell ratio is high, surface area is the principal factor limiting growth. At low medium to cell ratios, however, growth of cells is predominantly limited by medium components. The final number of cells per culture is almost directly proportional to available surface area when the culture medium is changed daily.
The growth of untransformed fibroblasts in culture stops at a density characteristic of the cell type and concentration of serum present in the culture medium. Although the events leading to this arrest of growth are not well understood, experiments have demonstrated that limitations of medium components is intimately involved (Kruse and Miedema, '65; Todaro et al., '67; Holley and Kiernan, '68; Dulbecco and Elkington, '73). There is also evidence that cell density is a critical factor which can regulate growth (Fisher and Yeh, '67; Stoker and Rubin, '67; Schutz and Mora, '68; Kolodny and Gross, '69; Clarke et al., '70; Dulbecco, '70; Martin and Rubin, '74), although it appears that cell-cell contacts are not directly involved in the inhibition of DNA synthesis and cell division (Martz and Steinberg, '72; Dulbecco and Elkington, '73; Stoker, '73; Holley, '74). Thus, "density-dependent" (Stoker and Rubin, '67) or "post-confluence" (Martz and Steinberg, '72) inhibition of growth better describes the phenomenon formerly known as "contact-inhibition'' of growth. Recently, to further clarify the conditions which limit growth of cells in dense culture, Dulbecco and Elkington ('73) plated fibroblastic or epithelial cells in a constant amount of medium in culture dishes of different sizes. They concluded that growth of epithelial cells is limited by the amount of surface available to them. In contrast, growth of fibroblastic Balb/c J . CELL.PHYSIOL..86. 301-310
3T3 cells appeared to be limited by the medium rather than available growth surface area. Since this result was at variance with our experience with Swiss mouse 3T3 fibroblasts, we repeated these experiments with both types of 3T3 cells, and in addition, designed new experiments using coverslips to investigate the role of available surface area and cell density on growth of these untransformed fibroblasts. By using the coverslip technique, we were able to design experiments in which the only variable was available growth surface. We found that with both Balb/c and Swiss 3T3 cells, availability of surface area (or cell density) is the principal factor limiting growth when the medium to cell ratio is high. However, at lower medium to cell ratios, growth is predominantly limited by medium components. MATERIALS A N D METHODS
Culturing of cells Swiss mouse 3T3 fibroblasts (clone 42) were obtained from Dr. George J. Todaro; Balb/c 3T3 cells were obtained from Dr. Dieter Paul and Dr. J. Elkington. They were grown as previously described in Dulbecco-Vogt modified Eagle's medium containing 100 units of penicillin, 100 pg of streptomycin per milliliter, plus the indicated amount of calf serum (Cunningham, Received Oct. 4, '74. Accepted Dec. 16, '74. 1 Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.
30 1
302
CORNELIA H THRASH AND DENNIS D CUNNINGHAM
' 7 2 ) . The growth medium contained 2.5 gins NaHCO:, per liter. Chowth of cells was carried out in an atmosphere of 5% CO2 in air to maintain the pH between 7.3 and 7.5. The cells were mycoplasma-free as routinely judged by the presence of nuclear and not cytoplasmic grains following au toradiography with (:jH) thymidine (Nardone et al.. '65). All experiments with iUalb/c 3T3 cells, except those noted in figure 2 were performed using cells obtained rrom Dr. Elkington.
Counting of cells 'The cells were rinsed with phosphatebuffered saline solution (PBS) and incubated for approximately ten minutes at 37 with 0.055% trypsin and 0.02 % EDTA in PBS. Aliquots of the suspended cells were diluted with PBS and counted in a Couiter counter. Similar results were obtained when cells were counted in a hemac ytometer chamber . L.
RESULTS
One method of examining the effect of available surface area on the number of cells present at saturation is to culture a given number o f cells in a constant amount of medium in tissue culture dishes of varied sizes. Using this technique, Dulbecco and Elkington ('73) found that the final number of untransformed Balb/c 3T3 and BHK 21 fibroblasts was related to the amount of medium present and not to the available growth surface area. They concluded from this that growth of these fibroblasts was h i t e d by medium components rather than available growth surface. We have repeated these experiments and concluded that growth of these cells is limited by both available surface area and medium components. Figure 1 shows a typical growth curve for the same Balb/c cells grown under the conditions used by Dulbecco and Elkington ('73). As can be seen, we obtained more cells in the dishes with greater surface area. Thus, surface area at least partly limited the growth of these cells. Further use of this technique a! various serum concentrations and seeding densities demonstrated that growth of riot only Balblc 3T3 cells (fig. 2a) but also Swiss mouse 3T3 cells (fig. 2c) was limited by availability of both the surface area and medium components. Figure 2b shows the
0
1
2
3
4
5
6
DAYS
Fig. 1 Growth curves of Balblc 3T3 cells. 1.0 1 0 5 Balb/c 3T3 cells (in 6.0 ml medium enriched with 10% calf serum) were seeded in Nunc plastic tissue culture dishes containing approximately 7 (U), 21 ( A - A), or 60 (CI) cm2 growth surface. The number of cells per dish was determined as described i n MATERIALS AND METHX
ODS.
theoretical slopes expected if cell growth were limited strictly by surface area (final number of cells directly proportional to dish size) or by medium components (same number of cells in all size dishes). As shown in figures 2a,c, intermediate slopes were obtained under nearly all of these culture conditions. This demonstrates that both surface area and medium components limited growth. In fact, within the range tested, the growth of Swiss 3T3 cells appeared to be limited mainly by surface area (fig. 2c). Growth of Balb/c 3T3 cells was also predominately limited by surface area, except in the largest dishes or when the serum to cell ratio was low (cells seeded at high density or cells grown in low serum). Growth of cells on coverslips The above results are potentially affected by variables other than surface area (for instance, medium depth). Since these variables might affect the number of cells at saturation, we designed experiments which eliminated all variables other than available growth surface area. This was accomplished by growing cells on glass coverslips in a constant amount and depth of medium. First, cells were seeded on glass coverslips at a given density. These coverslips were then transferred to bac-
30 3
LIMITATION OF FIBROBLAST GROWTH
1 -
1 -
I
i
r
I I
1
/ -J
!
P
I
7
21
61
SURFACE
AREA
(crn2)
Fig. 2 Effect of culture dish size on final number of cells at saturation. Cells were seeded 5.0 ( S O ) , in Nunc plastic tissue culture dishes in 6.0 ml medium containing 2.0 (M), 10 ( A- A ) , or 20 (A-A) percent calf serum and allowed to grow to a growth arrested state. The number of cells initially seeded is indicated by the heavy horizontal lines. After growth had stopped, as determined by microscopic examination, cells were counted 1 and 2 or 1 and 3 days later as described i n MATERIALS A N D METHODS. In all cases the cultures had reached a growth arrested state by this time. Subsequent increases in cell number, if any, were less than 10% per day. In several cases complete growth curves, like those shown in figure 1, or mitotic indices of cultures were also measured, determinations which confirmed that a growth arrested state had been achieved by the time the above measurements were made. Panel a, Balb/c 3T3 cells obtained from Dr.J. Elkington (dashed lines) and Dr. Dieter Paul (solid lines). Panel b, theoretical slopes expected if growth of cells were limited strictly by available surface area (M) or by medium limitation (H). Panel c, S w i s s 3T3 cells. All experiments were performed at least twice.
teriological grade culture dishes containing culture medium. These cells grew only on the glass coverslips and did not migrate or grow onto the culture dish. The number of cells per culture was therefore identical to the number of cells growing on the glass coverslips. To insure that these experiments were conducted on cultures which had reached their final saturation density, we measured cell number over a five day period. Typical growth curves, shown in figure 3, demonstrate that a growth-arrested state was achieved within four days in cultures of cells containing either 1, 2 , or 4 coverslips which were seeded at 0.2 x 1 0 5 cells per coverslip (panel a) or 0.8 X 1 0 - 5 cells per coverslip (panel b). In all experiments utilizing coverslips, the final number of cells at saturation was determined from similar growth curves. We first examined the effect of available surface area on the final number of cells
at saturation (fig. 4). These experiments were conducted so that available growth surface was the only variable. This was accomplished by placing different numbers of coverslips in identical bacteriological grade culture dishes containing equal volumes of medium. Cultures were set up so that the number of cells per coverslip was inversely proportional to the number of coverslips placed in the culture. Thus, each culture initially contained an equal number of cells. This insured that the initial medium to cell ratio was also constant. If final cell number were determined only by available growth surface area, the final number of cells per culture at saturation would be directly proportional to the number of coverslips per culture as shown by the upper dashed line in figure 4. On the other hand, if final cell number were limited only by medium components, the final number of cells per culture would be identical in all cultures as shown by the hori-
-
CORNELIA R . THRASH AND DENNlS D. CUNNINGHAM
I
'a
-I
b.
I\-
&--
r - - - - O
U
L 3
5
4
DAYS AFTER SEEDING
Fig. 3 Growth curves of Balblc 3T3 cells grown on coverslips. Eighteen millimeter round glass coverslips were seeded at 0.2 X 105 (panel a) or 0.8 X 1 0 5 (panel b) cells per coverslip in tissue culture dishes with medium containing 10% calf serum. After attachment of the cells, the coverslips wexe transferred to 50 mm bacteriological plastic Petri dishes with 6.0 ml medium containing 10% calf serum. No more than 0.1% of the cells ever migrated to the surface of the bacteriological dish. On the days indicated, the cells were removed from the coverslips and counted as described in MATERIALS A N D METHODS. One coverslip per culture, (O--O); two coverdips per culture, ( 0 4 ) ;or four coverslips per culture, ( A-A ).
,
-I
2
3
4
COVERSLIPS F'ER CULTURE Fig. 4 Relationship of available surface area to final cell number with the initial cell to medium ratio constant. Balblc 3T3 cells were seeded on coverslips as described in the legend to figure 3 so that the initial number of cells per culture was 0.8 x 1 0 5 , (M), or 1.6 X 105, (-0). The final cell density was determined by growth curves like those shown in figure - 3. Theoretical curves expected if only surface area. (O---O) or medium components ( C4)limited growth.
I
I
I
1
I
2
3
4
COVERSLIPS PER
CULTURE
Fig. 5 Relationship of available surface area to final cell number. Balblc 3T3 cells were seeded 0.4 ( 0 4 ) , 0.8 ( 0 4 ) and 1.6 at 0.2 (M), (X-X) X 105 cells per coverslip and treated as described in figure 3. The final cell density was determined from growth curves like those shown in figure 3. Theoretical curves if only surface area (S-0)or medium components ( C - W ) limited growth.
305
LIMITATION OF FIBROBLAST GROWTH
volume of medium was increased. For example, figure 4 shows a greater medium limitation in cultures initially containing 1.6 x 10.5 cells than in cultures containing 0.8 X 105 cells. Therefore, medium limitation increased as the cell to medium ratio increased. Experiments like those shown in figures 4 and 5 were also performed with Swiss 3T3 cells (not shown). While the results of these experiments were qualitatively similar to those using Balblc cells (figs. 4, 5) the absolute numbers of Swiss mouse 3T3 cells was considerably lower, presumably due to the lower saturation density charactersitic of the latter cell line.
zontal dashed line in figure 4. Actual data points in the figure (solid lines) reveal an intermediate condition reflecting growth limitation by both medium components and available growth surface area. This conclusion is based on experiments using two different initial numbers of cells per culture. Additional experiments, shown in figure 5, were carried out in which 1, 2, or 4 coverslips, containing the same number of cells per coverslip, were placed in identical bacteriological grade culture dishes containing equal volumes of medium. Four sets of these cultures were set up at different initial numbers of cells per coverslip. Within each set, the initial medium to cell ratio varied. The data in figure 5 show that, under these conditions, as well as when the ratio was constant (fig. 4), the final number of cells at saturation was limited by both available growth surface area and by medium components. The data in both figures 4 and 5 demonstrate that limitation of final cell number by medium components became more pronounced as the number of cells initially present per L '
Decay of medium components Since these experiments indicated that cell growth was limited by both available growth surface area and medium components, we further investigated the nature of this medium limitation. Because both serum and the defined medium contain thermolabile components, we first examined the effect of incubation of medium a t 37" in the absence of cells on its subse-
I
I
I
I
2
4
6
8
104
I
I
1
0
2
4
-
L
0
L
6
8
PREINCUBATION TIME (DAYS)
Fig. 6 Effect of preincubation of medium at 37O on growth of nonconfluent and maintenance of confluent Swiss 3T3 cells. Fresh medium containing 10% calf serum was placed in a 37O C& incubator. Aliquots were withdrawn at the times indicated on the abscissa and frozen. Panel a : 0.5 X 105 Swiss 3T3 cells were plated in 35 mm Falcon plastic tissue culture dishes. After attachment of cells, the medium was replaced with 2.0 ml of the preincubated medium. Cells on duplicate plates were counted as described in MATERIALS AND METHODS one (M), two (X-X), three (U), and four ( A - A ) days later. Panel b: 1.0 X lo5 Swiss 3T3 cells were plated in 35 mm Falcon plastic tissue culture dishes in 2.0 ml medium. After three days the cultures were growth arrested and had a final saturation density of 2.7 x 1 0 5 cells per dish. The medium then was replaced with 2.0 ml of the preincubated medium. The cells in duplicate plates were counted on the fifth (A-A) and eighth (0- - -0) days following the medium change.
306
CORNELlA R. THRASH AND DENNIS D. CUNNINGHAM
quent ability to support growth of nonconfluent cells and to maintain cell number of confluent 3T3 cells. Figure 6a demonstrates that preincubation of medium at 37" markedly decreased its ability to support the growth of 3'T3 cells. Cultures grown in medium which had been preincubated for eight days had a final cell number of about 54% of cultures grown in medium which had not been pre-incubated. Maintenance of confluent cultures, however, appeared to be relatively unaffected until the medium had been preincubated about eight days (fig. 6b). Thus, for growing cultures, the medium limitations observed were at least partly due to spontaneous decay of essential components. Furthermore, the partial medium limitations observed in figure 2 were probably overestimated by this medium decay, since the number of cells in the 7 cm2 dishes was determined at three to six days after seeding, while the number of cells in the 21 and 60 cm2 dishes was determined five to eight and seven to 12 days respectively after seeding. Thus, a greater decay of medium components probably occurred in the larger dishes, reducing its potential to support growth of these cells. Medium decay probably had a lesser effect on the coverslip experiments shown in figures 4 and 5 since cell numbers were determined for all cultures between days three and five. Alteration of medium by cells Further experiments indicated that the medium limitation observed in previous experiments (figs. 2 , 4, 5) was the result of alteration of the medium by cells as well as spontaneous medium decay. In these experiments, cells were cultured as in figure 4 with no medium change or were cultured with daily changes of fresh medium which had been kept at either 4 " or 37". Figure 7 indicates that significantly more cells were produced in cultures in which the culture medium was replaced daily with fresh medium which had been kept at 37" from day 0 than in cultures in which the medium remained unchanged. Thus, cellular utilization or inactivation of medium components was partially responsible for the medium dependence for growth of Balblc 3T3 cells. Furthermore, figure 7 demonstrates that the medium
1
.---
IL I
-
, L
2
i
3
!
-,A 4
COVERSLIPS PER CULTURE
Fig. 7 Relationship of available surface area to final number of cells per culture with daily medium changes. Balb/c 3T3 cells were seeded at 0.2, 0.4, and 0.8 X 105 cells per coverslip in cultures containing 4, 2, and 1 coverslip per culture r e spectively as described in the legend to figure 3. Growth-arrested cells were counted as described in MATERIALS AND METHODS on days 3, 4, and 5 and the values averaged. (A-A), medium replaced daily with fresh medium kept at 4 O from day 0; (M), medium replaced daily with fresh medium kept at 37O from day 0; (M), medium unchanged; (@--a),theoretical slope if surface area strictly limited growth; (C4),theoretical slope if medium components strictly limited growth.
limitation on the growth of Balb/c 3T3 cells can be accounted for by the combined effects of both the cellular alteration and spontaneous decay of medium components, since cells grown with daily changes of medium which had been maintained at 4" from day 0 showed no medium limitation. Thus, when medium components were not limiting, available surface area became the sole factor limiting growth and the final number of cells per culture was directly proportional to the available growth surface area. Additional experiments were performed with both Swiss and Balblc 3T3 cells in which culture medium was replaced daily with fresh medium containing 2-20% serum which had been kept at 4 " (fig. 8). Under these conditions, the final number of cells at saturation was almost directly proportional to the amount of surface available for growth. This further demonstrated that surface area became the sole factor
LIMITATION OF FIBROBLAST GROWTH BALB/C 3T3
SWISS 3T3 4
8
3
6
2
4
I
2
..f
I
0
x
1
1
.
1
1
1
1
12
32
3
5 3
9
24
O LL
6
16
W
LL
w
a
8
c n 3 _I
J
w
0
32
80
24
60
16
8 , L - -
I
2
3
4
I
2
3
4
COVERSLIPS PER CULTURE
Fig. 8 Relationship of available surface area to final number of Swiss and Balblc 3T3 cells with daily medium changes. Cells were seeded as described in the legend to figure 3 and the growth arrested cells counted as described in the legend to figure 7. Swiss 3T3 cells were seeded at 0.1, 0.2, and 0.4 X 105 cells per coverslip in cultures containing 4, 2, and 1 coverslip per culture respectively. Balb/c 3T3 cells were seeded as described in the legend to figure 7. The dashed lines represent the theoretical slopes expected if only surface area limited growth. Solid circles represent actual data points.
limiting growth when the medium was changed daily, indicating that the surface dependence shown in figure 7 was not peculiar to medium containing 10% serum. DISCUSSION
These studies demonstrate that the growth of untransformed Balblc and Swiss mouse 3T3 fibroblasts can be limited by available surface area and by the amount and concentration of medium components. Culture conditions can be manipulated (by changing the medium daily or by varying the cell to medium ratio) to alter the relative limitations by surface area and me-
307
dium components. Medium components become limiting for final cell number as the initial number of cells is increased in cultures receiving no medium change; surface area becomes limiting when there are fewer initial cells present per culture (figs. 2, 4, 5). In addition, when the medium is changed daily, the final number of cells at saturation is almost directly proportional to available growth surface area (fig. 8). However, figure 8 also shows that in cultures in which the greatest amount of growth occurred (four coverslips per dish in cultures containing six and twenty per cent serum) a slight medium dependence was observed. We did not determine whether more frequent medium changes might elininate this dependence on medium for growth. In the experiments reported here, the medium limitation of growth appears to involve at least two factors: a noncellular inactivation of growth-promoting activity and a cell-mediated alteration of the medium. Whether the cell-mediated alteration reflects utilization or inactivation of medium components or production of toxic or growth-inhibitory molecules by the cells cannot be distinguished by these experiments. The observation that available surface area can limit growth of fibroblasts is in concert with studies on neonatal rat heart cells and Swiss 3T3 and 3T6 cells by Kolodny and Gross ('69). Extensive growth of these cells occurred after the surface area available to them was increased; however, it is not clear whether a medium limitation also exists since the number of cells per cmz was not given for either the initial or final growth-arrested states. Recent evidence by Martin and Rubin ('74) further supports the existence of densitydependent growth controls. They found that chick embryo fibroblasts were capable of attaching and growing on bacteriological grade plastic culture dishes. In these dishes, cells migrated to form clumps which became growth-arrested after they achieved an average density of about 90 cells per clump. Transfer of the clumps to tissue culture grade plastic culture dishes with the medium in which they had become growth arrested resulted in migration of the cells out of the clumps. Subsequently, the cells re-entered a log
308
CORNELIA R. THRASH AND DENNIS D. CUNNINGHAM
phase of growth and became growth-arrested after they had achieved a confluent monolayer, demonstrating that the arrest of‘ growth in clumped cells was not due to medium limitation. They suggest that the density-dependent arrest of growth observed in the cell clumps might be relevant to growth control processes in vivo. Recent experiments by Dulbecco and Elkington (’73) like those shown in figure 2, indicate that the growth of untransformed Balb/c 3T3 and BHK 21 cells is restricted primarily by medium components rather than by available surface area. We observed limitation of growth by both surface area and by medium components using the same clone of Balb/c 3T3 cells. While the reasons for the differences between the two sets of data are not clear, likely explanations are that the medium used in our experiments (1 ) was altered less rapidly by the cells, or (2) contained different absolute or relative amounts of critical growth and maintenance factors. Since the final number of cells per culture is a n equilibrium between cell growth and detachment, a difference in the relative amounts of these factors might account for the differences in the results. In our experiments, no significant cell detachment occurred until after the final number of attached cells shown in the figures was achieved. This number remained constant for a variable period of time, after which cell detachment was noted with a concomitant decline in the total number of cells per culture (attached plus detached) (unpublished observations). It is unlikely that differences in the methods used to determine when a growth-arrested state was achieved could account for the discrepancies, since our results were identical using either mitotic indices (like Dulbecco and Elkington) or complete or partial growth curves as a measure of growth arrested state (unpublished observations). The mechanism by which available growth surface area limits the growth of cells is not clear. Evidence that it does not directly depend on cell-cell contacts has been discussed by several investigators (Stoker and Rubin, ’67; Martz and Steinberg, ’72; Dulbecco and Elkington, ’73; Stoker, ’73; Holley, ’74). Indeed, the operational limitation of final cell number by available growth surface area might ulti-
mately result from a limitation of medium components. For example, after growth to confluency, cells appear to have less access to medium components (Stoker and Rubin, ’67; Stoker, ’73) and they also take up certain nutrients from the growth medium at a reduced rate (Kohn, ’68; Cunningham and Pardee, ’69; Weber and Rubin, ’71; Sefton and Rubin, ’71). ACKNOWLEDGMENTS
This work was supported by U . S. Public Health Service Grant CA-12306. C. R. T. was supported by U . S. Public Health Service Training Grant (GM-02063) to the Department of Molecular Biology and Biochemistry. LITERATURE CITED Clarke, G. D., M . G. P. Stoker, A . Ludlow and M. Thornton 1970 Requirement of serum for DNA synthesis in BHK 21 cells: Effects of dens i t y , suspension, and virus transformation. Nature, 227: 798-801. Cunningham, D. D. 1972 Changes in phospholipid turnover following growth of 3T3 mouse cells to confluency. J. Biol. Chem., 247: 24642470. Cunningham, D. D., and A. B. Pardee 1969 Transport changes rapidly initiated by serum addition to “contact inhibited” 3T3 cells. Proc. Natl. Acad. Sci. (U.S.A.), 64: 1049-1056. Dulbecco, R. 1970 Topoinhibition and serum requirement of transformed and untransformed cells. Nature, 227: 802-806. Dulbecco, R., and J. Elkington 1973 Conditions limiting multiplication of fibroblastic and epithelial cells in dense cultures. Nature, 246: 197199. Fisher, H. W., and J. Yeh 1967 Contact inhibition in colony formation. Science, 155: 581-582. Holley, R. W. 1974 Serum factors and growth control. In: Control of Proliferation in Animal Cells. B. Clarkson and R. Baserga, eds. Cold Spring Harbor Press, N. Y., pp. 13-18. Holley, R. W., and J. A. Kiernan 1968 Contact inhibition of cell division i n 3T3 cells. Proc. Nat. Acad. Sci., 60: 300-304. Kohn, A . 1968 Thymidine metabolism and DNA synthesis in crowded monolayers of animal cells. Exp. Cell Res., 52: 161-172. Kolodny, G . M., and P. R. Gross 1969 A simple mechanical method for the efficient release of contact inhibition. Exp. Cell Res., 57: 4 2 3 4 3 2 . Kruse, P., and E. Miedema 1965 Production and characterization of multiple-layered population of animal cells. J. Cell Biol., 27: 273-279. Martin, G. R., and H. Rubin 1974 Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts. Exp. Cell Res., 85: 319333. Martz, E., and M . S. Steinberg 1972 The role of cell-cell contact in “contact” inhibition of cell division: A review and new evidence. J. Cell. Physiol., 79: 189-210.
LIMITATION OF FIBROBLAST GROWTH Nardone, R. M., J. Todd, P. Gonzales and E. V . Gaffney 1965 Nucleoside incorporation into strain L cells: Inhibition by pleuropneumonialike organisms. Science, 149: 1100-1101. Schutz, L . , a n d P. T. Mora 1968 The need for direct cell contact in “contact” inhibition of cell division in culture. J. Cell. Physiol., 71 : 1 4 . Sefton, B . M., and H. Rubin 1971 Stimulation of glucose transport in cultures of density-inhibited chick embryo cells. Proc. Natl. Acad. Sci. (U.S.A.), 68: 3154-3157. Stoker, M . G . P. 1973 Role of diffusion boundary layer in contact inhibition of growth. Nature, 246: 200-203.
309
Stoker, M. G. P., and H. Rubin 1967 Densitydependent inhibition of cell growth in cultures. Nature, 21 5: 171-172. Todaro, G. J., Y. Matsuya, S. Bloom, A. Robbins and H. Green 1967 Stimulation of RNA synthesis and cell division in resting cells by a factor present in serum. In: Growth Regulating Substances for Animal Cells i n Culture. V. Defendi and M. Stoker, eds. Wistar Inst. Press, Phila., pp. 87-101. Weber, M. J., and H. Rubin 1971 Uridine transport a n d RNA synthesis in growing and i n density-inhibited animal cells. J. Cell. Physiol., 77: 157-168.
