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Maintenance of differentiation in primary cultures of avian tendon cells
Prinfed in Sweden Copyrighf @I 1976 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental
MAINTENANCE
Cell Research
102 (1976) 63-7 1
OF DIFFERENTIATION
CULTURES
OF AVIAN
R. SCHWARZ,
IN PRIMARY
TENDON
L. COLARUSSO
CELLS
and P. DOTY
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, USA
SUMMARY When avian tendon cells are transferred from an in vivo environment to an in vitro environment, they lose their ability to synthesize large amounts of collagen relative to other cell proteins and thereby forgo the hallmark of their differentiated state. This research has systematically investigated the role of various components in the standard cell culture medium in order to decipher why it is an inadequate environment for maintaining differentiated function. The results show that serum levels in excess of 0.5 % can be detrimental to a high production of collagen synthesis. The magnitude of this serum effect was also found to be a function of cell density. High cell densities, apparently acting through an increase in the CO* concentration, reverse the inhibitory effect of serum. In addition, if the lactate ion concentration is raised to 30 mM (the highest concentration tested), the level of collagen synthesis relative to total cellular protein is restored to its initial level of 30%. Thus, the major differentiated function of avian tendon cells can be retained in cell culture, Moreover, the results appear to implicate high cell density as the normal stabilizing factor in maintaining differentiation in ovo. A high concentration of cells tend to switch the cell metabolism to one which is more anaerobic, thereby favoring high collagen synthesis. Reduction in the cell division rate, which also occurs at high cell densities, does not appear to effect the ratio of collagen to other cell proteins synthesized.
One of the most basic and persistant problems in biology lies in understanding the causes of stability of the differentiated state. A clarification of these causes would yield an improved understanding of differentiation, of commitment to development when this stability is acquired, and of malignant growth, when this stability is lost. While cells can maintain their differentiated function for long periods of time in vivo, transfer to an in vitro environment usually leads to a rapid loss of specialized synthesis [ 1,2,3]. Although some cell types do continue to make differentiated state products in cell culture, the effect of environment on the ability of cells to maintain their differentiated state is generally negative. Our aim, therefore, is to elucidate the j-761808
role that the environment plays in stabilizing the differentiated state in one specific case. A differentiated cell in vivo synthesizes specific products, and each product represents a certain percentage of the total production. Therefore, a cell culture is assumed to remain differentiated if it continues to synthesize the same percentages of its principal proteins relative to its total protein synthesis, as it did before being transferred to cell culture. We have used primary avian tendon (PAT) cells in our experiments for the following reasons. (1) As previously shown by Dehm & Prockop [4], whole tendons or freshly dissociated cells commit approx. 30% of their protein synthesis towards the production of the protein collagen. (2) Exp CdRes
102 (1976)
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Schwarz,
Colarusso
and Doty
These cells are easily isolated from the tendon matrix, yielding large numbers of cells with only a minimal amount of disruption and cell death. (3) Since tendon consists almost exclusively of fibroblasts, a uniform cell population can be obtained without the need for selective procedures. (4) PAT cells grow well in cell culture under a wide variety of conditions. (5) Collagen, the major cell product, can be assayed relatively easily. Another advantage of using PAT cells is that the synthesis of collagen has been well studied in cell culture for over ten years [5-IO]. While earlier experiments have advanced our understanding of the relationship between the growth of fibroblasts in culture and the synthesis of collagen, the effect of specific components in the culture medium on maintenance of differentiation has been largely neglected. This can be seen in a recent finding of Peterkofsky [ 1 l] who took cells from the frontal bones of chick embryos, known to devote 60 % of their total protein synthesis in ovo to the synthesis of collagen [12], and passaged them 3 times in cell culture. As a consequence, the relative collagen synthesis during exponential growth in these cells decreased to only 3 %. In addition, long-term culturing of several tibroblast cell lines reduces the relative collagen synthesis still further [9, 13, 141. Therefore, it appears that typical cell culture environments are not conducive to the maintenance of collagen synthesis. In this research, we have attempted to discover the basic causes for this loss of differentiation. We have limited our observations to a one week period of cell growth, since one observes within this period both the major adjustment to cell culture and the slow down in exponential growth as the cells reach confluency. The results show that a suitable environment can indeed be created for the maintenance of differentiation while ExpCellRes
102 (1976)
growth continues in culture. Consequently, it appears that further understanding of the environmental influences on a cell can lead to a clarification of the mechanisms used by the cell to maintain differentiation. MATERIALS
AND METHODS
Cell culture and media Cells were grown in tightly capped small (25 cm’, No. 3013) Falcon flasks. Initial cell number, medium changing, amount of medium/flask, and percentage of fetal calf serum used are described in the figure captions. At times cells were allowed to pre-attach in medium without serum before growth in serum medium, and this is also specified. The media used were as follows: Modified Krebs II [4]. FlOP 1151: made from nremixed powder (GlBCo) containing in addition 0.61 M Hepks pH 714 (N-21 hydroxyethyl piperazine-N-2-ethanesulfonic acid, Calbiochem) and saturated with 5 % CO,+air. F12P [16]: made from premixed powder (GlBCo) and saturated with 5% CO,+air. F12LPro: made from individual chemicals with concentrations and methods as described by Ham [ 161 excent that oroline is reduced to a third of its standard value; saturated with 5 % CO,+air. F12 [4]: made from individual chemicals with concentrations and methods as described by Ham [ 161 excent that NaCl and NaHCO, are resoectivelv 3.2 a/l and 7.5 g/l. The medium was bubbled with CO, until the pH was 7.0. All water was deionized, then glass distilled. The fetal calf serum employed was deactivated at 56°C for f h. (Colorado Serum Co.; Miles Lab.) All media contained streptomycin sulfate, 0.1 g/l and penicillin, 105 U/l and were filtered through 0.22 pm filters (Millipore). In most growth experiments, F12 was used in preference to FlO because it was found that F12 enhanced the growth of PAT cells in medium without serum.
Isolation of cells The procedure adopted was a slight modification of that described by Dehm & Prockop [4]. In general, 16-day embryos were used (cells from 14- and 15-day embryos have given similar results). The basic modification was the addition of 3 % fetal calf serum to the dissociation medium. This slowed the rate of dissociation of single cells from the tendon but results in a lower percentage of cell death from isolation (less than 5%). This isolation procedure produces reproducible results with respect to the number of cells/embryo. The yield varies, however, with the age of the embryo, and this has been used to calculate an approximate in vivo generation time (assuming that the isolation procedure is equally effective with embryos of different ages). The vield (cellslembrvo) from three different arres of embryos (three.preparations each) was found To be: 14 day, 7.2+0.4x 106; 15 day, 9.W 1.0x 106; 16 day, 13.3f0.4~ 106. From these numbers the generation time in vivo is estimated to be 55 h.
Maintenance
Fig. 1. Abscissa: cell no. ~10s; ordinate: (left) % [SHjproline incorporated into collagen; (right) corrected % collagen. The effects of cell number on the collagen synthesis in F12P with 3% fetal calf serum. Cells at varying concentrations were incubated for 2 h at 37°C in plastic tubes (No. 2025, Falcon) containing 2 ml F12P, 3% serum and 100 @Zi [3Hlproline. The ordinate shows (lef) the percentage of incorporated [sH]proline which is sensitive to collagenase; (right) this value corrected for the 5.2 ratio [12, 211 of proline content in collagen to the other cellular proteins. The correction is based on the method used by Peterkofsky [ 121.
of differentiation
in primary
cultures
appeared to yield more collagenase was used [ 171. The purification [17] was similar to previously published procedures [18]. The purified collagenase showed some general proteolytic activity [ 191 at very high collagenase concentrations. However, over a wide range of collagenase concentrations, all the collagen in our samples was degraded with no detectable general protease activity [17]. The collagen assay using collagenase was based on that described by Peterkofsky & Diegelmann [20]. The modifications introduced yielded a faster and easier method when applied to cell culture. An incubation mixture was made up of the following solutions: 0.8 ml of buffer “A” (0.1 M NaC!, 0.01 M Tes [N-Tris (hydroxymethyl) methyl-2-ammo-ethanesulfonic acid, Calbiochem] pH 7.2,O.OOl CaCl,, 1.0% (w/v) proline; 0.4 ml of 0.5 M Tes pH 7.2; 0.1 ml of 1 M HCl; and 0.5 ml of sample cells in 0.25 M NaOH which has been syringed four or five times through a 22 gauge, lt” needle). The incubation mix 0.25 ml/assay, was pipetted into three tubes (A, B and C). Collagenase (1OA of the appropriate concentration was added to the second tube (B), and pronase (50X, 1 mg/ml in buffer A) was added to the third (C). The tubes were incubated at 37°C for 1 h, and the reaction was stopped by the
Cell counting Two methods were used: a Coulter counter and a photographic method using a microlocator slide. Unless otherwise noted, the cell number was that obtained from the Coulter counter. In the absence of serum, cells attached firmly to the flask and were easily destroyed if removed. Therefore in these cases, counting was done photographically. A microlocator slide (Lynn Apparatus Co.) was attached to the underside of the flask and in this way a specific area could be reproducibly examined for growth. At least two specific squares/flask were selected and used throughout the experiment. To make a count, a square was photographed and the number of cells counted. Each square was approx. 114000 of the total area of the flask (25 cm*). For a given time point, the counts from all squares were averaged.
Labeling
of cells
In a one-week experiment, seven identical bottles were prepared. Each day the medium was poured off from one bottle. If the pulsing medium was different from the growth medium, the flask was rinsed with the new medium. Pulsing medium (l-2 ml) containing 10-100 &i of 2: 3’rHlproline (New England Nuclear) was added to the flask, and incubation was carried out for 3 h. The cell culture was then made 0.25 M in NaOH. After at least 10 min, the contents of the flask was vigorously pipetted several times with a Pasteur pipette and then transferred to plastic tubes and kept at 4°C until assayed.
Collagen
assay
The collagenase was isolated from CI. histolyticum (American Type Culture No. 8034). A subclone which
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Fig. 2. Abscissa: % fetal calf serum; ordinate: (lefr) % [3H]proline incorporated into collagen; (right) corrected % collagen. The effect of serum on the collagen synthesis of cells at low cell density in 0, modified Krebs II; 0, FlOP; and A, F12P. Cells in modified Krebs II and FlOP were incubated in 1 ml at a concentration of 6 x 105 cells/ml in plastic tubes for 3 h at 3PC with 100 @i [sHbroline and the appropriate percentage of fetal calf serum. Cells in F12P were incubated in 2 ml at a concentration of 3 x 105cells/ml in plastic flasks for 3 h at 37°C with 100 &i [sH]proline and the appropriate percentage of fetal calf serum. The results shown in F12P are not strictly comparable due to the lower cell density. The value for cells in media without serum is occasionally lower than expected; the usual result is that cells without added serum synthesize the same percentage of collagen in modified Krebs II, FlO, and F12. Exp Cell
Res 102 (1976)
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Schwarz,
Colarusso
and Doty
locl
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Fig. 3. Abscissa: molarity of NaHCO,; ordinate: (lefi) % [3H]proline incorporated into collagen; (right) corrected % collagen. Effect of increasing the CO* concentration on the percentage of collagen synthesis of cells in F12, 3% serum. The CO* concentration was varied by changing the NaHCO, concentration in F12 and adjusting the pH to 7.0 with CO,. The NaCl was reduced accordingly to keep the Na+ concentration constant. For two highest values tested the Na+ concentration exceeded what could be compensated for by not adding any NaCl. Cells in normal F12P were diluted 1: 15 into the appropriate media containing 3 % serum and 100 &i [3Hlproline to yield a cell concentration of 6~ 106cells/ ml. The total volume of 4 ml was placed in a 4 ml glass vial, tightly capped, and incubated at 37°C for 2 h.
addition of 1 ml, 10% TCA. The samples were filtered through 0.45 pm filters (Millipore) and rinsed three times with 5 ml of 2% TCA. The dried filters were counted in a liquid scintillation counter (Beckman LS250). Assays were carried out in duplicate and averaged. The counts were analysed as follows: tube A, total counts incorporated; tube B, non-collagen counts; tube C, background counts in non-protein material. Percentage [3Hlproline in collagen= [(A-c)-(B-c)]/(A-c)=(A-B)/(A-c)
RESULTS The effect of serum and cell density on colldgen synthesis in early cell culture
When PAT cells were placed in cell culture under standard conditions, we observed a rapid loss in their ability to synthesize a high percentage of collagen. This result conflicts with the results of Dehm & Prockop [4], who had not observed a loss in the relative proportion of collagen synthesis during the initial 2 h after transfer. Therefore, our first experiments were designed Exp CellRes
102 (1976)
to determine which elements in our cell system were causing rapid loss of differentiation. Although the media Dehm & Prockop employed differed from ours (modified Krebs II, minimal essential medium and modified Krebs II with 10% serum versus F12 with 3 % serum), the most obvious difference was in cell density. They used a relatively high cell density of 3.2 to 4.8~ 106 cells/ml compared with 0.6~ 106 cells/ml in our case. Therefore, in order to explore the effect of cell density, cells were incubated at various concentrations for 2 h in F12 containing 3 % serum with C3H]proline, and the percentage of collagen synthesized relative to total protein was determined. It can be seen in fig. 1, that relative collagen production rises from 4.5 % to 29 % as cell density increases from 0.2 to 9.2~ 106 cells/ml. The interpolated value at the cell densities used by Dehm & Prockop is 23-27%, in good agreement with their values (25-35 %). Using the same short-term cultures, we investigated the decline in the percentage of collagen synthesis at low cell density. The influence of serum concentration was then determined by measuring the percentage of collagen synthesis over the range of 0 to 15% fetal calf serum in three media. The results are shown in fig. 2. Serum does indeed progressively diminish the collagen production although the character of the effect varies with medium: the effect is initially more pronounced in growth media than in modified Krebs II media which is essentially a buffered salt solution. While PAT cells are sensitive to cell density in their ability to reverse the negative action of serum, the mechanism by which one cell communicates its presence to another cell is unclear. The specific ability of the cell to detect cell density may play a more general role in the initiation and stabilization of differentiated function than in just reversing the action of serum. There-
of differentiation
Maintenance
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in transfer. Consequently, the role of CO2 concentration was tested on cells at low 70 30 densities in 3% serum by increasing the h 25 60 NaHCO, concentration of the F12 medium 20 and titrating the medium with CO, to pH M I5 7.0, keeping the Na+ concentration con40 IO stant. The result was a striking increase in 30 75 relative collagen production, from 8 to 37 % 20 5 as shown in fig. 3. Therefore, increasing the 10 25 CO, concentration mimicks the effect of 0 3 4 5 6 high cell concentration. The action of CO, Fig. 4. Abscissa: time (days); ordinare: (left) % of appears to be specific since raising the rela[3Hlproline incorporated into collagen; (right) cortive 0, or NS concentration has a slightly rected % collagen. negative effect on the percentage of colCollagen synthesis at different fetal calf serum concentrations over a one week period. The various con- lagen made (results not shown [ 171). ditions for each experiment were: To summarize: cells during the first few Media hours in cell culture are strongly influenced Cell no. vol/flask %Serum (X 10-B) (ml) Type by high serum concentration and respond at
as 135 40
0 0.5 0.5 3 5 15
(7 (0) (A) (X) (0) (0)
1.8 A-: 0:6 0.6 0.6
10 5 20 20 :i
Fl2P F12P F12P F12 F12 F12
In all experiments the media was changed every other day. Cells in 0.5 % serum were pulsed in duplicate; the variations between flasks are shown. To allow easier comparison, the two 0.5 % serum cases, inoculated at widely differing initial cell densities/ml, are shown in heavy lines.
fore, how the cells detect cell density was explored further. Cell density sensing apparently did not rely on the physical touching of the cells. Changing the shape of the incubation vessel (from a conical test tube to a flat flask), which radically affects the degree of aggregation, did not alter the effect. The hypothesis that cell density is sensed through the accumulation of a stable excreted molecule was also tested and proved negative. Medium from cells grown at high density could not induce cells at low densities, in the presence of 3 % serum, to make a high percentage of collagen. Nevertheless, this approach was pursued in order to determine whether carbon-dioxide, a possible stable secreted molecule was lost
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kig. 5. Abscissa: time (days); ordinate: normalized cell count/flask (X lo-“). Growth curves for cells in X, Fl2P with 0%; 0, 0.5%; and 0, 3% serum. The counts are normalized to the cell count on the first day. The initial inoculum were:0%,1.8~108;0.5%, 1.2~10~;3%,1.2XlO~.Inthe two media with serum, the cells were allowed to attach in medium without serum (F12P, 10 ml) for 30 min, and then changed to medium with serum (20 ml). The cell counts were by microlocator slide technique (see Methods) except for the initial inoculum which is determined by a Coulter counter. The media was changed every 3 days and all experiments were stopped short of confluency due to the difficulties of counting cells which are tightly packed together. The approximate generation times after the initial lag in growth were: O%, 5 days; 0.5%. 1 day; 3%, 1 day. Exp Cell
Res 102 (1976)
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Schwarz, Colarusso and Doty
0
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Fig. 6. Abscissa: time (days); or&are: (left) % of [3H]proline incorporated into collagen; (right) corrected % collagen. The effect on the collagen synthesis of PAT cells on adding sodium lactate at different concentrations (mmoles): l ,O; 0, 10; A, 20; 0,30. In both (a) F12[4] and (b) F12P, the initial inoculum is 1.2~ 108 cells/flask and the media is changed on day no. 3. The serum concentration in both cases is 0.5 %. Cells are first allowed to attach to the flask in F12P without serum for 30 min before being changed to the appropriate growth media. In F12P there is 20 ml/flask and in F12[4] there is 60 ml/flask.
low cell density, by reducing the relative production of collagen to other proteins. Cells appear to overcome the effects of serum at high density by the action of increased concentration of CO, on their metabolism. The effect of serum on collagen synthesis in one week cell culture Having established conditions necessary to maintain the level of collagen synthesis for the first 2-3 h we proceeded to extend the time period to one week. The longer time scale restricted the range of acceptable experimental conditions because we were now interested in both growth and the ability of the cells to stay differentiated. PAT cells were cultured under a variety of conditions which allowed cell propagation, and the percentage of collagen synthesis was measured. One variable that could be easily manipulated was serum concentration. Cell density could be altered only over a relatively narrow range, in order to maintain longterm growth, and, within this range, it had only a minimal effect on the percentage of collagen synthesis. In fig. 4 is summarized the results of a series of typical one-week experiments. ExpCellRes
102 (1976)
The main results dealing with the effect of serum concentration, fall into two classes: low serum [OS% or no serum (to be discussed below)] and high serum [3 %, 5 %, or 15%]. In the high serum case, there was an initial loss in the percentage of collagen synthesis. This has already been seen in fig. 2. The decline occurs as the cells adapt to cell culture. As the cells divide and the cell density increases, the percentage of collagen synthesis improves and even surpasses the initial value. This post-adjustment increase with increasing cell density may be similar to that observed in fig. 1, where the negative effect of high serum was reversed by high cell density. If this is the case, then for experiments where the cells are attached, the cell density/flask. becomes more important than cell density/ml. In low serum, a much higher initial level in the percentage of collagen synthesis is seen, but this level declines with time. As the cell density/flask increases, the drop in relative collagen synthesis levels off. It should also be noted that in both high and low serum concentrations, cells display approximately the same relative collagen synthesis at confluency. The effect of changing cell density over the range where one still observes cell division is also shown in fig. 4. These experiments are done in 0.5 % serum, but the effect is similar at other serum concentrations. Increasing cell density has a small, positive, effect on the percentage of collagen synthesis. Experiments carried out in non-serum medium were more relevant because, under these conditions, the decrease in the percentage of collagen synthesis over the oneweek period could not be attributed to the effects of serum. Since the results for cells in 0% serum F12P and 0.5% serum F12P were almost superimposable one can conclude that low serum concentrations do not influence the relative production of col-
Maintenance
lagen. However, low concentrations of serum did radically change the generation time. The growth profiles of PAT cells in F12P containing O%, OS%, 3% serum are shown in fig. 5. In the two media containing serum, the cells were first allowed to attach in non-serum F12P for 30 min [22]. Cell attachment proceeded rapidly without serum, and, in this way, growth rates can be compared which are independent of attachment rates since these vary with serum concentration. The results were normalized to zero time and showed that media containing serum greatly enhanced growth and reduced the initial lag when compared with the non-serum medium. The results shown in figs 4 and 5 imply that relative collagen synthesis and rate of cell division are independent variables, affected by serum in different ways. Although cell culture conditions used in fig. 4 were slightly different from those used in fig. 5, these changes had no effect on the percentage of collagen synthesis (results not shown [17]). These one-week experiments show that the effect of serum on relative collagen synthesis can be removed by going to low serum concentrations (0.5%); that the decrease in collagen synthesis which occurs in the first 3 days is not primarily a consequence of a change in the growth rate; and that there is still an inhibitory factor present or an inducer absent, or present in insufficient quantities, to allow collagen synthesis to continue at its original extent. The maintenance of high level collagen synthesis
No matter what the course of relative collagen synthesis in the earlier stage of cell culture, PAT cells generally showed a small increase in the percentage of collagen synthesis as the cells reached confluency. This increase might be due to the excretion and accumulation of lactate by cells as they reach confluency [6, 231. If this is the case,
of differentiation
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raising the lactate concentration would help to stabilize the percentage of collagen synthesis at earlier times. It is possible that lactate ions compensate for the low cell density by artificially raising the concentration of an excreted metabolite. Moreover, since lactic acid is a product of anaerobic metabolism and since high COZ concentrations usually induce anaerobic metabolism, the combination could be complementary in their ability to increase the percentage of collagen synthesis. To test this hypothesis, two media were used with sodium lactate added (the salt is preferentially used since the acid reduces growth [6,24]): a high COZ, F12 [4] medium (F12 medium where the NaHCO, has been increased) and a normal medium, F12P; both containing 0.5% fetal calf serum. The results of adding various amounts of sodium lactate to these two media are shown in fig. 6. Successively higher concentrations of sodium lactate restore most of the decrease in relative collagen synthesis. In the F12 [4] medium, the initial level of 30% is still present after one week. Moreover, the higher CO, tension in the F12 [4] medium appears to enhance the relative amount of collagen synthesis especially as confluency is approached. Thus, increasing the lactate ion concentration to at least 30 mM, contributes decisively to the maintenance of a high level of collagen synthesis by tendon cells in culture. DISCUSSION The use of PAT cells has made possible a detailed study of collagen production in the transition from in ovo to in vitro environments. This period has been little studied due to the rather unstable condition brought on by a radical change in the cellular environment. There are rapid changes in generation time, population type, morEm CeNRes
102 (1976)
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Schwarz, Colarusso and Doty
phology and synthetic activity as the cells recover from being isolated, adapt to the in vitro environment, and select for the fastest growing cell type. Since PAT cells are easy to isolate, are of one cell type, adapt quickly to cell culture, and are similar from batch to batch, we were able to study this transition in a manner that appears to enlarge our understanding of how cells maintain differentiation in an in ovo environment. We have found how changes in the in vitro environment can be made in order to allow the cells to function normally with respect to synthesis of the same relative amount of the major protein product as they do in ovo. While the system is complex, the major changes in the medium needed to optimize conditions for collagen synthesis are simple. Serum, although still necessary for moderate growth, had to be greatly reduced in concentration (0.5 %). The concentration of lactate ions, normally excreted by the cells, had to be increased (30 mM). Besides stabilizing the percentage of collagen synthesis, relationships have emerged in the experiments that help to clarify cell regulation. They fall into two categories: (1) the relationship between growth (rate of cell division) and the percentage of collagen synthesis; and (2) the relationship between the percentage of collagen synthesis and anaerobic metabolism. An important question is whether cell division is compatible with differentiation: or, alternatively, is differentiation stimulated when growth is inhibited [25]. With PAT cells, growth appears to be compatible with a high percentage collagen synthesis both in ovo and in vitro. The cells in ovo appear to be capable of a relatively rapid generation time of 55 h while maintaining the ability to make a high percentage of collagen (see Methods: cell isolation). We have shown that wide variation between growth Exp CellRes
IO2 (1976)
in low serum and in non-serum media need not affect the percentage of collagen synthesis in cell culture. Thus, there does not appear to be a firm relationship between the relative level of collagen synthesized and the rate of cell division. Hence, an understanding of our observations must lie in a different direction. The hypothesis which best fits our data is that a switch to more anaerobic conditions triggers an increase in the level of collagen synthesis. Evidence comes from the observations that CO, causes semi-anaerobic cultures to increase their level of collagen synthesis under high serum conditions; and that lactate ions, a product of anaerobic metabolism, also promote higher relative collagen production. These may not be independent events but may be linked by a switch in the metabolism of the cell. Inhibiting this switch, as shown in Langness & Udenfriend [26] with L-929 cells, reduces lactate synthesis and reduces the activity of prolyl hydroxylase, thereby further relating collagen synthesis stimulation to anaerobic conditions. If it is assumed that confluent fibroblasts are in a semi-anaerobic state, as exemplified by the increase in the ratio of lactic acid excreted to glucose taken up [27], one can theorize as to what the effects of cell culture variation would be on PAT cells. Basically, PAT cells are sensitive, not to growth, but to cell density, and this is due to the accumulation of COZ. This increased level of COZ changes the cellular metabolism to one that is more anaerobic and results in lactic acid excretion. The cells are then stimulated by the increased lactate ion concentration to raise their level of collagen relative to total protein synthesis. It follows that cells would maintain a high percentage of collagen synthesis in cell culture if it were not for the restraints of growing
Maintenance cells in a flask and the resulting dilution of excreted lactate. Cells growing in one plane on the bottom of a flask would more closely resemble the geometry of a tendon if one could roll them up into a tight cylinder. Not having this low surface area configuration, the volume of the medium has to be increased, causing a reduction in the concentration of lactate ions produced by the cells. Without added lactate ions, the relative level of collagen synthesis falls in cell culture to about 10%. In the high serum cases, this situation is coupled with the serum inhibition of high levels of collagen synthesis, and this inhibition is cancelled by the semi-anaerobic conditions of high cell density. Although speculative, this theory provides a framework within which the complexities of cellular action can be further probed. This view of the behavior of cells need not be restricted to those in culture. It can hold for the stability of a differentiated cell within a tendon as well, and perhaps for other cells in their native environment. In the case of tendons, the concentration of metabolic products would be expected to remain high since the structure of a tendon prevents rapid exchange: cells are lined up between long rows of collagen fibers and capillaries are scarce [28]. Therefore, cells within a tendon may exist in a semianaerobic environment, and the subsequent excretion of lactic acid may stabilize the level of relative collagen synthesis. This viewpoint shows how the characteristics observed in cell culture can be functional in ovo. R. S. would like to thank Dr Helga Doty for her active encouragement of this research and Dr Ricardo Block for many rewarding discussions. Also, John Wozney for his enthusiastic help in the preparation of the collagenase.
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REFERENCES 1. Davidson, E H, Adv genet 12 (1964) 143. 2. Eazle. H. Science 148 (1%5) 42. 3. Green, H & Todaro, G J, ‘Ann rev microbial 21 (1967) 573. 4. Dehm, P & Prockop, D J, Biochim biophys acta 240 (1971) 358. 5. Green, H & Goldberg, B , Nature 200 (1%3) 1097. 6. -Ibid 204 (1964) 347. 7. - Proc natl acad sci US 53 (1965) 1360. 8. Green, H, Goldberg, B & Todaro, G J, Nature 212 (1%6) 631. 9. Peterkofsky, B, Arch biochem biophys 152 (1972) 318. 10. Bomstein, P, Ann rev biochem 43 (1974) 567. 11. Peterkofskv, B, Biochem bionhvs _ _ res commun 49 (1972) i34i. 12. Diegelmann, R F & Peterkofsky, B, Dev biol 28 (1972) 443. 13. Green, H & Meuth. M. Cell 3 (1974) 127. 14. Lang&s, U & Udenfriend, S; Proc natl acad sci us 71 (1974) 50. 15. Ham, R G, Exp cell res 29 (1%3) 515. 16. - Proc natl acad sci US 53 (1%5) 288. 17. Schwarz, R, Ph.D thesis,‘ Harvard University (1975). 18. Seifter, S & Harper, E, Methods in enzymology (ed G E Perlman & L Lorand) vol. 19, p. 613. Academic Press, New York (1970). 19. Miyoshi, M & Rosenbloom, J, Conn tissue res 2 (1974) 77. 20. Peterkofsky, B & Diegelmann, R, Biochemistry 10 (1971) 988. 21. Bomstein, P, von der Mark, K, Wyke, A W, Erlich, H P & Monson, J M, J biol them 247 (1972) 2808. 22. Temin, H M, Pierson, Jr R W & Dulak, N C, Growth nutrition and metabolism of cells in culture (ed G H Rothblat & V J Cristofalo) pp 4-l. Academic Press, New York (1972). 23. Comstock, J P 8z Udenfriend, S, Proc natl acad sci US 66 (1970) 552. 24. Rubin, H, Growth control in cell culture (ed G E W Wolstenholme & J Knight) pp 127-149. Churchill Livingstone, London (1971). 25. Rutter, W J, Pictet, R L & Morris, P W, Ann rev biochem 42 (1973) 601. 26. Langness, U & Udenfriend, S, Biology of fibroblast (ed E Kulonen & J Pikkarainen) pp. 373-377. Academic Press, New York (1973). 27. Bissell, M J, Hatie, C & Rubin, H, J natl cancer inst 49 (1972) 555. 28. Bloom, W & Fawcett, D W, A textbook of histology, 8th edn, pp 105-11, 268. Saunders, Philadelphia, Pa (1%2). Received January 13, 1976 Accepted May 24, 1976
This research was supported by USPHS HD-01229.
Exp
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Res 102 (1976)
Experimental
MAINTENANCE
Cell Research
102 (1976) 63-7 1
OF DIFFERENTIATION
CULTURES
OF AVIAN
R. SCHWARZ,
IN PRIMARY
TENDON
L. COLARUSSO
CELLS
and P. DOTY
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, USA
SUMMARY When avian tendon cells are transferred from an in vivo environment to an in vitro environment, they lose their ability to synthesize large amounts of collagen relative to other cell proteins and thereby forgo the hallmark of their differentiated state. This research has systematically investigated the role of various components in the standard cell culture medium in order to decipher why it is an inadequate environment for maintaining differentiated function. The results show that serum levels in excess of 0.5 % can be detrimental to a high production of collagen synthesis. The magnitude of this serum effect was also found to be a function of cell density. High cell densities, apparently acting through an increase in the CO* concentration, reverse the inhibitory effect of serum. In addition, if the lactate ion concentration is raised to 30 mM (the highest concentration tested), the level of collagen synthesis relative to total cellular protein is restored to its initial level of 30%. Thus, the major differentiated function of avian tendon cells can be retained in cell culture, Moreover, the results appear to implicate high cell density as the normal stabilizing factor in maintaining differentiation in ovo. A high concentration of cells tend to switch the cell metabolism to one which is more anaerobic, thereby favoring high collagen synthesis. Reduction in the cell division rate, which also occurs at high cell densities, does not appear to effect the ratio of collagen to other cell proteins synthesized.
One of the most basic and persistant problems in biology lies in understanding the causes of stability of the differentiated state. A clarification of these causes would yield an improved understanding of differentiation, of commitment to development when this stability is acquired, and of malignant growth, when this stability is lost. While cells can maintain their differentiated function for long periods of time in vivo, transfer to an in vitro environment usually leads to a rapid loss of specialized synthesis [ 1,2,3]. Although some cell types do continue to make differentiated state products in cell culture, the effect of environment on the ability of cells to maintain their differentiated state is generally negative. Our aim, therefore, is to elucidate the j-761808
role that the environment plays in stabilizing the differentiated state in one specific case. A differentiated cell in vivo synthesizes specific products, and each product represents a certain percentage of the total production. Therefore, a cell culture is assumed to remain differentiated if it continues to synthesize the same percentages of its principal proteins relative to its total protein synthesis, as it did before being transferred to cell culture. We have used primary avian tendon (PAT) cells in our experiments for the following reasons. (1) As previously shown by Dehm & Prockop [4], whole tendons or freshly dissociated cells commit approx. 30% of their protein synthesis towards the production of the protein collagen. (2) Exp CdRes
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These cells are easily isolated from the tendon matrix, yielding large numbers of cells with only a minimal amount of disruption and cell death. (3) Since tendon consists almost exclusively of fibroblasts, a uniform cell population can be obtained without the need for selective procedures. (4) PAT cells grow well in cell culture under a wide variety of conditions. (5) Collagen, the major cell product, can be assayed relatively easily. Another advantage of using PAT cells is that the synthesis of collagen has been well studied in cell culture for over ten years [5-IO]. While earlier experiments have advanced our understanding of the relationship between the growth of fibroblasts in culture and the synthesis of collagen, the effect of specific components in the culture medium on maintenance of differentiation has been largely neglected. This can be seen in a recent finding of Peterkofsky [ 1 l] who took cells from the frontal bones of chick embryos, known to devote 60 % of their total protein synthesis in ovo to the synthesis of collagen [12], and passaged them 3 times in cell culture. As a consequence, the relative collagen synthesis during exponential growth in these cells decreased to only 3 %. In addition, long-term culturing of several tibroblast cell lines reduces the relative collagen synthesis still further [9, 13, 141. Therefore, it appears that typical cell culture environments are not conducive to the maintenance of collagen synthesis. In this research, we have attempted to discover the basic causes for this loss of differentiation. We have limited our observations to a one week period of cell growth, since one observes within this period both the major adjustment to cell culture and the slow down in exponential growth as the cells reach confluency. The results show that a suitable environment can indeed be created for the maintenance of differentiation while ExpCellRes
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growth continues in culture. Consequently, it appears that further understanding of the environmental influences on a cell can lead to a clarification of the mechanisms used by the cell to maintain differentiation. MATERIALS
AND METHODS
Cell culture and media Cells were grown in tightly capped small (25 cm’, No. 3013) Falcon flasks. Initial cell number, medium changing, amount of medium/flask, and percentage of fetal calf serum used are described in the figure captions. At times cells were allowed to pre-attach in medium without serum before growth in serum medium, and this is also specified. The media used were as follows: Modified Krebs II [4]. FlOP 1151: made from nremixed powder (GlBCo) containing in addition 0.61 M Hepks pH 714 (N-21 hydroxyethyl piperazine-N-2-ethanesulfonic acid, Calbiochem) and saturated with 5 % CO,+air. F12P [16]: made from premixed powder (GlBCo) and saturated with 5% CO,+air. F12LPro: made from individual chemicals with concentrations and methods as described by Ham [ 161 excent that oroline is reduced to a third of its standard value; saturated with 5 % CO,+air. F12 [4]: made from individual chemicals with concentrations and methods as described by Ham [ 161 excent that NaCl and NaHCO, are resoectivelv 3.2 a/l and 7.5 g/l. The medium was bubbled with CO, until the pH was 7.0. All water was deionized, then glass distilled. The fetal calf serum employed was deactivated at 56°C for f h. (Colorado Serum Co.; Miles Lab.) All media contained streptomycin sulfate, 0.1 g/l and penicillin, 105 U/l and were filtered through 0.22 pm filters (Millipore). In most growth experiments, F12 was used in preference to FlO because it was found that F12 enhanced the growth of PAT cells in medium without serum.
Isolation of cells The procedure adopted was a slight modification of that described by Dehm & Prockop [4]. In general, 16-day embryos were used (cells from 14- and 15-day embryos have given similar results). The basic modification was the addition of 3 % fetal calf serum to the dissociation medium. This slowed the rate of dissociation of single cells from the tendon but results in a lower percentage of cell death from isolation (less than 5%). This isolation procedure produces reproducible results with respect to the number of cells/embryo. The yield varies, however, with the age of the embryo, and this has been used to calculate an approximate in vivo generation time (assuming that the isolation procedure is equally effective with embryos of different ages). The vield (cellslembrvo) from three different arres of embryos (three.preparations each) was found To be: 14 day, 7.2+0.4x 106; 15 day, 9.W 1.0x 106; 16 day, 13.3f0.4~ 106. From these numbers the generation time in vivo is estimated to be 55 h.
Maintenance
Fig. 1. Abscissa: cell no. ~10s; ordinate: (left) % [SHjproline incorporated into collagen; (right) corrected % collagen. The effects of cell number on the collagen synthesis in F12P with 3% fetal calf serum. Cells at varying concentrations were incubated for 2 h at 37°C in plastic tubes (No. 2025, Falcon) containing 2 ml F12P, 3% serum and 100 @Zi [3Hlproline. The ordinate shows (lef) the percentage of incorporated [sH]proline which is sensitive to collagenase; (right) this value corrected for the 5.2 ratio [12, 211 of proline content in collagen to the other cellular proteins. The correction is based on the method used by Peterkofsky [ 121.
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appeared to yield more collagenase was used [ 171. The purification [17] was similar to previously published procedures [18]. The purified collagenase showed some general proteolytic activity [ 191 at very high collagenase concentrations. However, over a wide range of collagenase concentrations, all the collagen in our samples was degraded with no detectable general protease activity [17]. The collagen assay using collagenase was based on that described by Peterkofsky & Diegelmann [20]. The modifications introduced yielded a faster and easier method when applied to cell culture. An incubation mixture was made up of the following solutions: 0.8 ml of buffer “A” (0.1 M NaC!, 0.01 M Tes [N-Tris (hydroxymethyl) methyl-2-ammo-ethanesulfonic acid, Calbiochem] pH 7.2,O.OOl CaCl,, 1.0% (w/v) proline; 0.4 ml of 0.5 M Tes pH 7.2; 0.1 ml of 1 M HCl; and 0.5 ml of sample cells in 0.25 M NaOH which has been syringed four or five times through a 22 gauge, lt” needle). The incubation mix 0.25 ml/assay, was pipetted into three tubes (A, B and C). Collagenase (1OA of the appropriate concentration was added to the second tube (B), and pronase (50X, 1 mg/ml in buffer A) was added to the third (C). The tubes were incubated at 37°C for 1 h, and the reaction was stopped by the
Cell counting Two methods were used: a Coulter counter and a photographic method using a microlocator slide. Unless otherwise noted, the cell number was that obtained from the Coulter counter. In the absence of serum, cells attached firmly to the flask and were easily destroyed if removed. Therefore in these cases, counting was done photographically. A microlocator slide (Lynn Apparatus Co.) was attached to the underside of the flask and in this way a specific area could be reproducibly examined for growth. At least two specific squares/flask were selected and used throughout the experiment. To make a count, a square was photographed and the number of cells counted. Each square was approx. 114000 of the total area of the flask (25 cm*). For a given time point, the counts from all squares were averaged.
Labeling
of cells
In a one-week experiment, seven identical bottles were prepared. Each day the medium was poured off from one bottle. If the pulsing medium was different from the growth medium, the flask was rinsed with the new medium. Pulsing medium (l-2 ml) containing 10-100 &i of 2: 3’rHlproline (New England Nuclear) was added to the flask, and incubation was carried out for 3 h. The cell culture was then made 0.25 M in NaOH. After at least 10 min, the contents of the flask was vigorously pipetted several times with a Pasteur pipette and then transferred to plastic tubes and kept at 4°C until assayed.
Collagen
assay
The collagenase was isolated from CI. histolyticum (American Type Culture No. 8034). A subclone which
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Fig. 2. Abscissa: % fetal calf serum; ordinate: (lefr) % [3H]proline incorporated into collagen; (right) corrected % collagen. The effect of serum on the collagen synthesis of cells at low cell density in 0, modified Krebs II; 0, FlOP; and A, F12P. Cells in modified Krebs II and FlOP were incubated in 1 ml at a concentration of 6 x 105 cells/ml in plastic tubes for 3 h at 3PC with 100 @i [sHbroline and the appropriate percentage of fetal calf serum. Cells in F12P were incubated in 2 ml at a concentration of 3 x 105cells/ml in plastic flasks for 3 h at 37°C with 100 &i [sH]proline and the appropriate percentage of fetal calf serum. The results shown in F12P are not strictly comparable due to the lower cell density. The value for cells in media without serum is occasionally lower than expected; the usual result is that cells without added serum synthesize the same percentage of collagen in modified Krebs II, FlO, and F12. Exp Cell
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Fig. 3. Abscissa: molarity of NaHCO,; ordinate: (lefi) % [3H]proline incorporated into collagen; (right) corrected % collagen. Effect of increasing the CO* concentration on the percentage of collagen synthesis of cells in F12, 3% serum. The CO* concentration was varied by changing the NaHCO, concentration in F12 and adjusting the pH to 7.0 with CO,. The NaCl was reduced accordingly to keep the Na+ concentration constant. For two highest values tested the Na+ concentration exceeded what could be compensated for by not adding any NaCl. Cells in normal F12P were diluted 1: 15 into the appropriate media containing 3 % serum and 100 &i [3Hlproline to yield a cell concentration of 6~ 106cells/ ml. The total volume of 4 ml was placed in a 4 ml glass vial, tightly capped, and incubated at 37°C for 2 h.
addition of 1 ml, 10% TCA. The samples were filtered through 0.45 pm filters (Millipore) and rinsed three times with 5 ml of 2% TCA. The dried filters were counted in a liquid scintillation counter (Beckman LS250). Assays were carried out in duplicate and averaged. The counts were analysed as follows: tube A, total counts incorporated; tube B, non-collagen counts; tube C, background counts in non-protein material. Percentage [3Hlproline in collagen= [(A-c)-(B-c)]/(A-c)=(A-B)/(A-c)
RESULTS The effect of serum and cell density on colldgen synthesis in early cell culture
When PAT cells were placed in cell culture under standard conditions, we observed a rapid loss in their ability to synthesize a high percentage of collagen. This result conflicts with the results of Dehm & Prockop [4], who had not observed a loss in the relative proportion of collagen synthesis during the initial 2 h after transfer. Therefore, our first experiments were designed Exp CellRes
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to determine which elements in our cell system were causing rapid loss of differentiation. Although the media Dehm & Prockop employed differed from ours (modified Krebs II, minimal essential medium and modified Krebs II with 10% serum versus F12 with 3 % serum), the most obvious difference was in cell density. They used a relatively high cell density of 3.2 to 4.8~ 106 cells/ml compared with 0.6~ 106 cells/ml in our case. Therefore, in order to explore the effect of cell density, cells were incubated at various concentrations for 2 h in F12 containing 3 % serum with C3H]proline, and the percentage of collagen synthesized relative to total protein was determined. It can be seen in fig. 1, that relative collagen production rises from 4.5 % to 29 % as cell density increases from 0.2 to 9.2~ 106 cells/ml. The interpolated value at the cell densities used by Dehm & Prockop is 23-27%, in good agreement with their values (25-35 %). Using the same short-term cultures, we investigated the decline in the percentage of collagen synthesis at low cell density. The influence of serum concentration was then determined by measuring the percentage of collagen synthesis over the range of 0 to 15% fetal calf serum in three media. The results are shown in fig. 2. Serum does indeed progressively diminish the collagen production although the character of the effect varies with medium: the effect is initially more pronounced in growth media than in modified Krebs II media which is essentially a buffered salt solution. While PAT cells are sensitive to cell density in their ability to reverse the negative action of serum, the mechanism by which one cell communicates its presence to another cell is unclear. The specific ability of the cell to detect cell density may play a more general role in the initiation and stabilization of differentiated function than in just reversing the action of serum. There-
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in transfer. Consequently, the role of CO2 concentration was tested on cells at low 70 30 densities in 3% serum by increasing the h 25 60 NaHCO, concentration of the F12 medium 20 and titrating the medium with CO, to pH M I5 7.0, keeping the Na+ concentration con40 IO stant. The result was a striking increase in 30 75 relative collagen production, from 8 to 37 % 20 5 as shown in fig. 3. Therefore, increasing the 10 25 CO, concentration mimicks the effect of 0 3 4 5 6 high cell concentration. The action of CO, Fig. 4. Abscissa: time (days); ordinare: (left) % of appears to be specific since raising the rela[3Hlproline incorporated into collagen; (right) cortive 0, or NS concentration has a slightly rected % collagen. negative effect on the percentage of colCollagen synthesis at different fetal calf serum concentrations over a one week period. The various con- lagen made (results not shown [ 171). ditions for each experiment were: To summarize: cells during the first few Media hours in cell culture are strongly influenced Cell no. vol/flask %Serum (X 10-B) (ml) Type by high serum concentration and respond at
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In all experiments the media was changed every other day. Cells in 0.5 % serum were pulsed in duplicate; the variations between flasks are shown. To allow easier comparison, the two 0.5 % serum cases, inoculated at widely differing initial cell densities/ml, are shown in heavy lines.
fore, how the cells detect cell density was explored further. Cell density sensing apparently did not rely on the physical touching of the cells. Changing the shape of the incubation vessel (from a conical test tube to a flat flask), which radically affects the degree of aggregation, did not alter the effect. The hypothesis that cell density is sensed through the accumulation of a stable excreted molecule was also tested and proved negative. Medium from cells grown at high density could not induce cells at low densities, in the presence of 3 % serum, to make a high percentage of collagen. Nevertheless, this approach was pursued in order to determine whether carbon-dioxide, a possible stable secreted molecule was lost
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kig. 5. Abscissa: time (days); ordinate: normalized cell count/flask (X lo-“). Growth curves for cells in X, Fl2P with 0%; 0, 0.5%; and 0, 3% serum. The counts are normalized to the cell count on the first day. The initial inoculum were:0%,1.8~108;0.5%, 1.2~10~;3%,1.2XlO~.Inthe two media with serum, the cells were allowed to attach in medium without serum (F12P, 10 ml) for 30 min, and then changed to medium with serum (20 ml). The cell counts were by microlocator slide technique (see Methods) except for the initial inoculum which is determined by a Coulter counter. The media was changed every 3 days and all experiments were stopped short of confluency due to the difficulties of counting cells which are tightly packed together. The approximate generation times after the initial lag in growth were: O%, 5 days; 0.5%. 1 day; 3%, 1 day. Exp Cell
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Fig. 6. Abscissa: time (days); or&are: (left) % of [3H]proline incorporated into collagen; (right) corrected % collagen. The effect on the collagen synthesis of PAT cells on adding sodium lactate at different concentrations (mmoles): l ,O; 0, 10; A, 20; 0,30. In both (a) F12[4] and (b) F12P, the initial inoculum is 1.2~ 108 cells/flask and the media is changed on day no. 3. The serum concentration in both cases is 0.5 %. Cells are first allowed to attach to the flask in F12P without serum for 30 min before being changed to the appropriate growth media. In F12P there is 20 ml/flask and in F12[4] there is 60 ml/flask.
low cell density, by reducing the relative production of collagen to other proteins. Cells appear to overcome the effects of serum at high density by the action of increased concentration of CO, on their metabolism. The effect of serum on collagen synthesis in one week cell culture Having established conditions necessary to maintain the level of collagen synthesis for the first 2-3 h we proceeded to extend the time period to one week. The longer time scale restricted the range of acceptable experimental conditions because we were now interested in both growth and the ability of the cells to stay differentiated. PAT cells were cultured under a variety of conditions which allowed cell propagation, and the percentage of collagen synthesis was measured. One variable that could be easily manipulated was serum concentration. Cell density could be altered only over a relatively narrow range, in order to maintain longterm growth, and, within this range, it had only a minimal effect on the percentage of collagen synthesis. In fig. 4 is summarized the results of a series of typical one-week experiments. ExpCellRes
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The main results dealing with the effect of serum concentration, fall into two classes: low serum [OS% or no serum (to be discussed below)] and high serum [3 %, 5 %, or 15%]. In the high serum case, there was an initial loss in the percentage of collagen synthesis. This has already been seen in fig. 2. The decline occurs as the cells adapt to cell culture. As the cells divide and the cell density increases, the percentage of collagen synthesis improves and even surpasses the initial value. This post-adjustment increase with increasing cell density may be similar to that observed in fig. 1, where the negative effect of high serum was reversed by high cell density. If this is the case, then for experiments where the cells are attached, the cell density/flask. becomes more important than cell density/ml. In low serum, a much higher initial level in the percentage of collagen synthesis is seen, but this level declines with time. As the cell density/flask increases, the drop in relative collagen synthesis levels off. It should also be noted that in both high and low serum concentrations, cells display approximately the same relative collagen synthesis at confluency. The effect of changing cell density over the range where one still observes cell division is also shown in fig. 4. These experiments are done in 0.5 % serum, but the effect is similar at other serum concentrations. Increasing cell density has a small, positive, effect on the percentage of collagen synthesis. Experiments carried out in non-serum medium were more relevant because, under these conditions, the decrease in the percentage of collagen synthesis over the oneweek period could not be attributed to the effects of serum. Since the results for cells in 0% serum F12P and 0.5% serum F12P were almost superimposable one can conclude that low serum concentrations do not influence the relative production of col-
Maintenance
lagen. However, low concentrations of serum did radically change the generation time. The growth profiles of PAT cells in F12P containing O%, OS%, 3% serum are shown in fig. 5. In the two media containing serum, the cells were first allowed to attach in non-serum F12P for 30 min [22]. Cell attachment proceeded rapidly without serum, and, in this way, growth rates can be compared which are independent of attachment rates since these vary with serum concentration. The results were normalized to zero time and showed that media containing serum greatly enhanced growth and reduced the initial lag when compared with the non-serum medium. The results shown in figs 4 and 5 imply that relative collagen synthesis and rate of cell division are independent variables, affected by serum in different ways. Although cell culture conditions used in fig. 4 were slightly different from those used in fig. 5, these changes had no effect on the percentage of collagen synthesis (results not shown [17]). These one-week experiments show that the effect of serum on relative collagen synthesis can be removed by going to low serum concentrations (0.5%); that the decrease in collagen synthesis which occurs in the first 3 days is not primarily a consequence of a change in the growth rate; and that there is still an inhibitory factor present or an inducer absent, or present in insufficient quantities, to allow collagen synthesis to continue at its original extent. The maintenance of high level collagen synthesis
No matter what the course of relative collagen synthesis in the earlier stage of cell culture, PAT cells generally showed a small increase in the percentage of collagen synthesis as the cells reached confluency. This increase might be due to the excretion and accumulation of lactate by cells as they reach confluency [6, 231. If this is the case,
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raising the lactate concentration would help to stabilize the percentage of collagen synthesis at earlier times. It is possible that lactate ions compensate for the low cell density by artificially raising the concentration of an excreted metabolite. Moreover, since lactic acid is a product of anaerobic metabolism and since high COZ concentrations usually induce anaerobic metabolism, the combination could be complementary in their ability to increase the percentage of collagen synthesis. To test this hypothesis, two media were used with sodium lactate added (the salt is preferentially used since the acid reduces growth [6,24]): a high COZ, F12 [4] medium (F12 medium where the NaHCO, has been increased) and a normal medium, F12P; both containing 0.5% fetal calf serum. The results of adding various amounts of sodium lactate to these two media are shown in fig. 6. Successively higher concentrations of sodium lactate restore most of the decrease in relative collagen synthesis. In the F12 [4] medium, the initial level of 30% is still present after one week. Moreover, the higher CO, tension in the F12 [4] medium appears to enhance the relative amount of collagen synthesis especially as confluency is approached. Thus, increasing the lactate ion concentration to at least 30 mM, contributes decisively to the maintenance of a high level of collagen synthesis by tendon cells in culture. DISCUSSION The use of PAT cells has made possible a detailed study of collagen production in the transition from in ovo to in vitro environments. This period has been little studied due to the rather unstable condition brought on by a radical change in the cellular environment. There are rapid changes in generation time, population type, morEm CeNRes
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phology and synthetic activity as the cells recover from being isolated, adapt to the in vitro environment, and select for the fastest growing cell type. Since PAT cells are easy to isolate, are of one cell type, adapt quickly to cell culture, and are similar from batch to batch, we were able to study this transition in a manner that appears to enlarge our understanding of how cells maintain differentiation in an in ovo environment. We have found how changes in the in vitro environment can be made in order to allow the cells to function normally with respect to synthesis of the same relative amount of the major protein product as they do in ovo. While the system is complex, the major changes in the medium needed to optimize conditions for collagen synthesis are simple. Serum, although still necessary for moderate growth, had to be greatly reduced in concentration (0.5 %). The concentration of lactate ions, normally excreted by the cells, had to be increased (30 mM). Besides stabilizing the percentage of collagen synthesis, relationships have emerged in the experiments that help to clarify cell regulation. They fall into two categories: (1) the relationship between growth (rate of cell division) and the percentage of collagen synthesis; and (2) the relationship between the percentage of collagen synthesis and anaerobic metabolism. An important question is whether cell division is compatible with differentiation: or, alternatively, is differentiation stimulated when growth is inhibited [25]. With PAT cells, growth appears to be compatible with a high percentage collagen synthesis both in ovo and in vitro. The cells in ovo appear to be capable of a relatively rapid generation time of 55 h while maintaining the ability to make a high percentage of collagen (see Methods: cell isolation). We have shown that wide variation between growth Exp CellRes
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in low serum and in non-serum media need not affect the percentage of collagen synthesis in cell culture. Thus, there does not appear to be a firm relationship between the relative level of collagen synthesized and the rate of cell division. Hence, an understanding of our observations must lie in a different direction. The hypothesis which best fits our data is that a switch to more anaerobic conditions triggers an increase in the level of collagen synthesis. Evidence comes from the observations that CO, causes semi-anaerobic cultures to increase their level of collagen synthesis under high serum conditions; and that lactate ions, a product of anaerobic metabolism, also promote higher relative collagen production. These may not be independent events but may be linked by a switch in the metabolism of the cell. Inhibiting this switch, as shown in Langness & Udenfriend [26] with L-929 cells, reduces lactate synthesis and reduces the activity of prolyl hydroxylase, thereby further relating collagen synthesis stimulation to anaerobic conditions. If it is assumed that confluent fibroblasts are in a semi-anaerobic state, as exemplified by the increase in the ratio of lactic acid excreted to glucose taken up [27], one can theorize as to what the effects of cell culture variation would be on PAT cells. Basically, PAT cells are sensitive, not to growth, but to cell density, and this is due to the accumulation of COZ. This increased level of COZ changes the cellular metabolism to one that is more anaerobic and results in lactic acid excretion. The cells are then stimulated by the increased lactate ion concentration to raise their level of collagen relative to total protein synthesis. It follows that cells would maintain a high percentage of collagen synthesis in cell culture if it were not for the restraints of growing
Maintenance cells in a flask and the resulting dilution of excreted lactate. Cells growing in one plane on the bottom of a flask would more closely resemble the geometry of a tendon if one could roll them up into a tight cylinder. Not having this low surface area configuration, the volume of the medium has to be increased, causing a reduction in the concentration of lactate ions produced by the cells. Without added lactate ions, the relative level of collagen synthesis falls in cell culture to about 10%. In the high serum cases, this situation is coupled with the serum inhibition of high levels of collagen synthesis, and this inhibition is cancelled by the semi-anaerobic conditions of high cell density. Although speculative, this theory provides a framework within which the complexities of cellular action can be further probed. This view of the behavior of cells need not be restricted to those in culture. It can hold for the stability of a differentiated cell within a tendon as well, and perhaps for other cells in their native environment. In the case of tendons, the concentration of metabolic products would be expected to remain high since the structure of a tendon prevents rapid exchange: cells are lined up between long rows of collagen fibers and capillaries are scarce [28]. Therefore, cells within a tendon may exist in a semianaerobic environment, and the subsequent excretion of lactic acid may stabilize the level of relative collagen synthesis. This viewpoint shows how the characteristics observed in cell culture can be functional in ovo. R. S. would like to thank Dr Helga Doty for her active encouragement of this research and Dr Ricardo Block for many rewarding discussions. Also, John Wozney for his enthusiastic help in the preparation of the collagenase.
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REFERENCES 1. Davidson, E H, Adv genet 12 (1964) 143. 2. Eazle. H. Science 148 (1%5) 42. 3. Green, H & Todaro, G J, ‘Ann rev microbial 21 (1967) 573. 4. Dehm, P & Prockop, D J, Biochim biophys acta 240 (1971) 358. 5. Green, H & Goldberg, B , Nature 200 (1%3) 1097. 6. -Ibid 204 (1964) 347. 7. - Proc natl acad sci US 53 (1965) 1360. 8. Green, H, Goldberg, B & Todaro, G J, Nature 212 (1%6) 631. 9. Peterkofsky, B, Arch biochem biophys 152 (1972) 318. 10. Bomstein, P, Ann rev biochem 43 (1974) 567. 11. Peterkofskv, B, Biochem bionhvs _ _ res commun 49 (1972) i34i. 12. Diegelmann, R F & Peterkofsky, B, Dev biol 28 (1972) 443. 13. Green, H & Meuth. M. Cell 3 (1974) 127. 14. Lang&s, U & Udenfriend, S; Proc natl acad sci us 71 (1974) 50. 15. Ham, R G, Exp cell res 29 (1%3) 515. 16. - Proc natl acad sci US 53 (1%5) 288. 17. Schwarz, R, Ph.D thesis,‘ Harvard University (1975). 18. Seifter, S & Harper, E, Methods in enzymology (ed G E Perlman & L Lorand) vol. 19, p. 613. Academic Press, New York (1970). 19. Miyoshi, M & Rosenbloom, J, Conn tissue res 2 (1974) 77. 20. Peterkofsky, B & Diegelmann, R, Biochemistry 10 (1971) 988. 21. Bomstein, P, von der Mark, K, Wyke, A W, Erlich, H P & Monson, J M, J biol them 247 (1972) 2808. 22. Temin, H M, Pierson, Jr R W & Dulak, N C, Growth nutrition and metabolism of cells in culture (ed G H Rothblat & V J Cristofalo) pp 4-l. Academic Press, New York (1972). 23. Comstock, J P 8z Udenfriend, S, Proc natl acad sci US 66 (1970) 552. 24. Rubin, H, Growth control in cell culture (ed G E W Wolstenholme & J Knight) pp 127-149. Churchill Livingstone, London (1971). 25. Rutter, W J, Pictet, R L & Morris, P W, Ann rev biochem 42 (1973) 601. 26. Langness, U & Udenfriend, S, Biology of fibroblast (ed E Kulonen & J Pikkarainen) pp. 373-377. Academic Press, New York (1973). 27. Bissell, M J, Hatie, C & Rubin, H, J natl cancer inst 49 (1972) 555. 28. Bloom, W & Fawcett, D W, A textbook of histology, 8th edn, pp 105-11, 268. Saunders, Philadelphia, Pa (1%2). Received January 13, 1976 Accepted May 24, 1976
This research was supported by USPHS HD-01229.
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