- Email: [email protected]
Studies on cartilage cells in vitro
Experimental
Cell Research
STUDIES II.
CHANGES
35, 337-348
(1964)
337
ON CARTILAGE
CELLS
IN
V1TRO
IN AGGREGATION AND IN CARTILAGE-FORMING ACTIVITY OF CELLS MAINTAINED IN MONOLAYER CULTURES Y. KURODA’
Department
of Zoology,
of Chicago,
University Received
July
Chicago,
Zll.,
U.S.A.
31, 1963
IN th e preceding paper, changes in cell morphology, growth characteristics, and production of matrix in populations of dissociated chondrocytes grown in monolayer cultures were described. The question of the nature of these changes was briefly raised and the need for further information on the functional traits of the monolayer-cultured cells stressed. It is known that freshly dissociated cartilage-forming cells are capable of forming compact aggregates and that the reconstructed tissue differentiates into typical cartilage [5, 7-91. Using these two criteria of histotypic effectiveness, the properties of monolayer grown chondrocytes were further examined. The present paper deals, therefore, primarily with (a) the effects of precultivation in monolayer on the capacity of chondrocytes to form aggregates; (b) with differentiation in aggregates of such cells; and finally, (c) with attempts to test the persistence of some of the changes occurring in monolayer-grown chondrocytes. MATERIALS
AND
METHODS
Methods for obtaining suspensions of viable cartilage cells from femora of 13-day chick embryos and composition of culture media were as previously described [5]. To obtain monolayer cultures of these cells, 20 ml aliquots of cell suspension (approximately 5 x IO4 cells per ml of standard culture medium) were dispensed into T-60 flasks. These were gassed with a 5 per cent CO, air mixture, and incubated at 37.5%. At different intervals the cultures were harvested as follows: The culture medium was removed, the culture rinsed three times with CMF solution, then incubated for 10 min in CMF, at 37.5”C under the gas mixture. CMF was then replaced with 0.5 per cent trypsin in CMF, the flasks were gassed and incubated for 10 min to cause detachment and dissociation of the cells. The resulting suspension was rinsed three times by centrifugation in CMF, 2 min at 2000 rpm; the pellets were dispersed into a 1 Present 33 Joancho,
address: Kitaku,
Dr. Y. Kuroda, Osaka, Japan.
Department
of Genetics,
Osaka
University,
Experimenlal
Medical
Cell
School,
Research
3.5
Y. Kuroda suspension of single cells in standard medium by flushing 10 to 15 times through a fine pipette. To obtain cell aggregates, the procedure of aggregation by rotation was used [5]. 3 ml aliquots of cell suspension in standard medium (approximately lo6 cells per ml medium) were distributed into 25 ml Erlenmeyer flasks and gassed with the CO, air mixture. The flasks were placed on a gyratory shaker rotating at a constant speed of 70 rpm, 3/4 inch diameter of rotation and a constant temperature of 38X, and examined after 24 or 48 hr. Aggregability of cells was assessed by counting and measuring the aggregates. Organ cultures of aggregates.-Some of the aggregates harvested after 24 or 48 hr were organ-cultured on nutrient agar to test their capacity for further differentiation. The nutrient agar was made up of one part of standard culture medium with double concentrations of horse serum 20 per cent and embryo extract 4 per cent, and one part of 2 per cent agar (Bacto-agar, Difco) dissolved in glucosol solution1 adjusted to pH 7.2. Equal quantities of the two parts were combined at a temperature of 50°C the mixture poured into small Petri dishes and allowed to gel. Aggregates were placed on the surface of the gel and the Petri dishes incubated at 37.5% in a container, gassed with CO, + air mixture, and maintained at saturation humidity; after 2 to 5 days, the cultures were fixed in Carnoy’s fixative, and prepared for histological examination. Mouse procartilage cells, used for commingling with monolayer modified chick chondrocytes, were obtained from limb-buds of 13-day mouse embryos. The tissue was treated with CMF solution for 10 min followed by a 15 min incubation with 1.0 per cent trypsin solution at 37.5”C under 5 per cent CO, air mixture. The rinsed tissue was dispersed into single cells in standard medium by flushing through the tip of a fine pipette. Chick chondrocytes precultured in monolayer for different periods were dispersed into single cells and commingled in varying proportions with the dissociated mouse procartilage cells. Aggregation procedure and cultivation on agar medium were as outlined above. RESULTS
Changes in aggregability
of monolayer-grown
chondrocytes
Size and number of aggregates.-Cartilage cells cultured without interference in monolayer for various lengths of time were redispersed, suspended in standard culture medium and their manner of aggregation was established by the standard procedure: rotation. Uniform conditions were maintained in all these tests. The aggregates formed in each flask in all series of tests were photographed and their size and number determined. Femoral chondrocytes freshly obtained from 13-day chick embryos, when tested by rotation for 24 hr, consistently formed one or two large aggregates with an average diameter of 1.15 mm. These spherical or oval aggregates 1 Exactly omitted. Experimental
the
same
composition
Cell Research
35
as Tyrode’s
solution
except
that
the sodium
bicarbonate
is
Aggregation
of cartilage
cells
339
had a relatively smooth surface and frequently showed evidence of fusion from smaller cell clusters (Fig. 1). Precultivation in monolayer resulted in striking changes in the aggregation patterns of the cells. Chondrocytes maintained in monolayer cultures for 1 day, then tested for aggregation formed several aggregates and a number of
__--I .m---.--M-.-- ---.--m---’ V’
‘Text-Fig. I.-Relationship between duration of precultivation in monolayer and the number and average diameter of the cell aggregates. Squares represent number of aggregates: dots represent average diameter of aggregates and the solid line indicates diameter distribution of aggregates from 3 x IO6 cells precultured in monolayer for varying days.
smaller cell clumps, ranging in diameter from 1.05 to 0.18 mm. Cells precultured in monolayer for 2 days formed 10 to 30 small aggregates and had a diameter range of 0.85 to 0.05 mm (Fig. 2). After 3 days precultivation in monolayer, a few aggregates 0.75 mm in diameter were formed, in addition to numerous smaller cell clusters. The average diameter of aggregates precultured for 5 days in monolayer fell to 0.09 mm (Fig. 3). This decline continued with further precultivation, however, at a lesser rate than initially (Fig. 4). From 15 days in monolayer on (Fig. 5), only a slight decline in size of aggregates was detectable, and subsequently the pattern became stabilized to homogeneous small clusters, up to 0.05 mm in diameter (Fig. 6). Experimental
Cell Research
35
Y. Kuroda
Fig.
l.-Aggregate
of
Fig.
2.-Aggregates
of a progeny
freshly
dissociated
of chondrocytes
chondrocytes cultured
from
in monolayer
femur
of 13.day
for 2 days.
chick
Fig.
3.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 5 days.
Fig. 4.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 9 days.
Fig.
5.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 15 days.
Fig.
6.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 29 days.
embryo.
This relationship between duration of precultivation in monolayer and the number and average diameter of the cell aggregates is shown in Text-Fig. 1. The maximum diameter of aggregates decreased gradually, whereas the minimum diameter did not vary noticeably throughout the series. It might be stressed here that the decline in the average diameter of aggregates was Experimenfal
Cell Research
35
Aggregation
of cartilage
cells
341
steepest after the first 2-3 days of precultivation in monolayer; i.e., at the time when increase in cell number in the monolayer cultures was absent or slow. Free and attached cells in aggregation
cultures
The above changes in aggregability of monolayer grown chondrocytes were accompanied by an increased tendency of the cells to become attached to the wall of the rotating flasks. This resulted in the formation of a ring-like rim of cells, on the glass at the level of the surface of the medium. Thus, aggregation cultures consisted of cell aggregates, free cells floating in the medium, and attached rim cells. It was of interest to determine the quantitative relations between the free and the rim cells relative to duration of precultivation of the cells in monolayer and to their aggregation patterns. 24-hr aggregation cultures were used. After removal of aggregates the free cells in the
Text-Fig. 2.-Number of attached rim cells and free cells concentration of 3 x lo8 cells previously cultured in monolayer
after 24 hr in gyrating for varying days.
flasks
in a
Text-Fig. S.--Size distribution of aggregates formed by each of commingled suspensions of chick chondrocytes maintained in monolayer cultures for 15 days and freshly dissociated mouse cartilage-forming cells. Dots represent average diameter of aggregates; line indicates diameter distribution of aggregates in cultures with a concentration of approximately 2 x loo cells per ml medium. Experimental
Cell Research
35
Y. Kuroda medium were counted. The rim cells were dissociated with trypsin and counted. The results (Text-Fig. 2) showed little change in the counts of free cells but sharply increasing numbers of rim cells; this increase was particularly striking after the first 3 days of precultivation in monolayer, i.e., coincident with the rapid decline in the size of aggregates in these cultures. Thus, as the number of preferentially mutually adhesive cells declined, the number of cells tending to become attached to glass increased. This increase in the number of rim cells may thus represent a surprisingly rapid adaptation of the monolayer-grown cells to attach to glass. Changes in chondrogenic
activity
of monolayer
grown
cells
Differentiation of aggregates.-24hr aggregates formed by chondrocytes freshly obtained from embryos consisted of procartilage with the round or oval cells embedded in a small amount of translucent matrix (Fig. 7). Each aggregate was surrounded by a few layers of flattened cells. When organcultured on nutrient agar for 3-5 days, the aggregates differentiated into typical hypertrophic cartilage (Fig. 8). Similarly, aggregates formed by chondrocytes precultured in monolayer for 1 day also consisted of procartilage capable of further typical differentiation. Thus, under these experimental conditions, cells capable of chondrogenic activity were able to differentiate histotypically. This point is important because of the decline in chondrogenesis in aggregates from monolayer cultured cells.
Fig.
7.--Section
of an aggregate
of freshly
Fig. S.-Section 3 days.
of an aggregate
of freshly
Fig. 9.-Section
of an aggregate
of a progeny
of chondrocytes
precultured
in monolayer
for 2 days.
of aggregates
of a progeny
of chondrocytes
precultured
in monolayer
for 4 days.
Fig.
lO.--Section
dissociated dissociated
Fig. Il.-Section of a concentrated drop maintained on agar media for 5 days.
of aggregates
Fig. 12.-Section on agar media
from
of a cell pellet for 5 days.
obtained
Fig. 13.--24.hr aggregates of freshly chondrocytes precultured for 15 days chick cells with light-staining nuclei.
chondrocytes. chondrocytes
precultured
12-day-monolayer
dissociated in monolayer
maintained
in monolayer cultures
which
on agar
media
for 6 days were
for
and
maintained
mouse limb bud cells commingled with chick (2 : 1). Mouse cells with dark-staining nuclei;
Fig. 14.-Section through a chimeric aggregate consisting of embryonic mouse limb buds commingled with chick chondrocytes precultured for 15 days in monolayer. Aggregates maintained on agar media for 3 days. Mouse cells (m) (darkly staining nuclei) embedded in a common matrix with chick cells (c) (light nuclei). Experimental
Cell
Research
35
343
cartilage cells
Experimental
Cell Iieseurch 35
Y. Kuroda 24-hr aggregates of cells precultured in monolayer for 2 days were in a precartilaginous stage. There was little matrix; it had a more opaque appearance; and the cells were more tightly packed (Fig. 9). After 3 days in monolayer cultures, the cells formed aggregates in which the amount of matrix was further reduced and its opaqueness increased. This change was even more pronounced in aggregates of cells precultured in monolayur for 4 days (Fig. 10). The cytoplasm in many of the cells had a fibrous, frequently vacuolated, appearance. The larger aggregates were surrounded by flattened cells while most of the smaller aggregates had no such covering. In organ cultures, scattered nodules of cartilage and pre-cartilage developed. 24-hr aggregates of chondrocytes precultured in monolayer for more than 5 days showed no identifiable matrix. For cultivation on nutrient agar these small aggregates were bunched and piled up. In organ cultures of aggregates obtained from cells precultured in monolayer for 6 to 9 days, the small aggregates fused into large nodules that contained areas of precartilage (Fig. 11). Cells aggregated after 9 or more days in monolayer did not difTABLE
I. Cartilage-forming
activity monolayer
Days in monolayer
Aggregates
0 1 2 3 4 5 6 9 12 15 22 29 44 59 72 100 128 + + -I-, Hypertrophic Experimental
Cell
of aggregates and of rim cellsprecultured for varying periods.
++ + -I+ !!I -
Organcultured aggregates +++ +++ ++ ++ + + -t
-
-
cartilage;
Research
35
+ + , procartilage;
-
+ , prechondral
Attached cell pellets organcultured
. .. . .. ++ ++ + + + stage;
- , no differentiation.
in
Aggregation
of cartilage
cells
345
ferentiate into any stage recognizable as chondrogenesis; after several days on nutrient agar masses of unidentified fibrous tissue mere present (Fig. 12). Results of the above tests are summarized in Table I. Differentiation of rim cells.-Fragments of the rim formed by cells attached to the flask wall in rotating cultures were removed and cultured on nutrient agar for 3 to 5 days. Their differentiation corresponded to that of aggregates from same cultures, and showed a comparable but more rapid decline in chondrogenic activity (Table I). Effect of freshly obtained cartilage-forming cells on aggregation of monolayer grown chondrocytes.-The behavior of monolayer modified cells in the presence of freshly dissociated cells was studied by using mixtures of chick and mouse cells. The differences in size and staining of cells from these two species [3-51 permitted identification in composite cell mixtures. Chondrocytes maintained continuously in monolayer cultures for 15 days were redispersed and commingled with freshly dissociated cartilage-forming cells from the limb bud mesoblasts of 13-day mouse embryos. The proportions used were: 3 : 0, 2 : 1, 1 : 1, 1 : 2, and 0 : 3 (chick : mouse). Approximate cell concentration in the composite suspensions was 2 x lo6 per ml medium. The cells were aggregated by rotation in gyrating flasks. The size distribution of aggregates formed by each of these cell combinations is represented in Text-Fig. 3. The monolayer-grown chick chondrocytes alone formed small aggregates with a relatively short range of size averaging 0.06 mm in diameter. Aggregates formed by freshly obtained mouse cartilage-forming cells alone had a considerably wider size distribution and an average diameter of 0.19 mm. In flasks with two parts of monolayer-grown cells and one part freshly obtained cells, large aggregates with a diameter of up to 0.79 mm formed. This maximum size of aggregates did not noticeably increase in the presence of higher proportions of freshly dissociated cells, but the average diameter of aggregates increased with the rise in proportion of mouse cells. Effect of cartilage-forming cells on differentiation in aggregates of monolayer modified chondrocytes.-As described above, aggregates formed by chondrocytes maintained continuously in monolayer for 15 days showed no histologically detectable evidence of chondrification; even when organ-cultured for several days on nutrient agar, they failed to differentiate into cartilage. It seemed of interest to examine the effect of cartilage-forming cells on those monolayer-modified cells. It was first found that the two kinds of cells could be associated: 24hr aggregates that formed in suspensions comprising monolayer modified chondrocytes and freshly obtained mouse cartilageforming cells (2 : 1) had a chimeric composition and consisted of interspersed Experimental
Cell Research
35
346
Y. Kuroda
chick and mouse cells easily distinguishable by their size and staining differences (Fig. 13). The larger aggregates contained also masses of mouse procartilage cells surrounded by interspersed chick and mouse cells. After cultivation on nutrient agar, these aggregates contained scattered chondrified nodules which consisted of interspersed chick and mouse cells (Fig. 14) in addition to mouse cartilage. 24-hr aggregates formed in suspensions comprising equal proportions of both cell kinds also were of chimeric composition. In organ cultures, many of these aggregates differentiated into cartilage, and many of the nodules consisted of both kinds of cells closely associated in a common cartilaginous matrix. M’hile there were always areas of mouse cartilage only there were no regions that consisted of chondrified chick cells. Organ cultures of aggregates formed in suspensions of one part chick with tlvo parts mouse cells differentiated into cartilage consisting predominantly of mouse cells with scattered chimeric areas. The significance of the effects of freshly dissociated cells on monolayermodified chondrocytes, \vas tested by intermingling the latter with cells from other than cartilage-forming tissues. Dissociated liver and kidney cells from newborn mice were commingled in equal proportions with the dispersed, monolayer-modified chondrocytes. In aggregates formed by rotation of such cultures, the cells of each kind tended to group separately and showed no such close intermingling as in the co-aggregates with cartilage-forming cells. There was no noticeable chondrogenesis in any of these cultures. It appears thus, that the ability to enter into close architectural association \vith the monolayer-grown chondrocytes and to elicit chondrification was restricted, in these series of tests, to the freshly dissociated cartilage-forming cells.
DISCUSSION
It was shown that chondrocytes maintained in monolayer cultures decline in their tendency to form aggregates and in their ability to differentiate into cartilage. The second of these findings confirms the results of Holtzer et al. [ 1 ] obtained with cartilage cells from another tissue. It is of interest that the decline in aggregability of the monolayer-grown cells was steepest during the first days of cultivation and that it preceded the decline in chondrogenic activity of the cells. Both changes coincided with the progressive reduction in the number of round cells and their transformation into fibroblastic forms [2]. Another facet in this course of events was the progressively increasing Experimental
Cell Research
35
Aggregation
of
CaFtdage
CdS
tendency of the monolayer-grown chondrocytes to attach to glass, rather than to each other or to remain in suspension. The progressive decline in aggregability of monolayer-cultured chondrocytes agrees with similar findings on other types of cells [J]. It was postulated that it reflects changes in the adhesive properties of cells consequent upon metabolic adaptation to in vitro conditions [‘L]. It is possible that, since mutual attachment of cells may be mediated by specific cell products functioning at the cell surface, the biogenesis of these products may be affected by the metabolic adjustments related to the persistence of cells in a dispersed state 161. The decline in chondrogenic activity in monolayer-grown cells may represent another aspect of the syndrome of functioning in a dispersed state. It may represent a diversion to other tasks of the biosynthetic machinery of the cells primarily towards proliferation but perhaps also towards elaboration of other, as yet unidentified differentiation products. Other interpretations are, of course, conceivable at present. Of suggestive interest is the finding that monolayer-modified chondrocytes, though by themselves unable to elaborate chondrihed matrix, appear to form cartilage when co-aggregated with freshly dissociated cartilage-forming cells. These findings call for certain obvious reservations. First, in the present experiments, cells maintained for 15 days in monolayer were used; whether cells from older monolayer cultures would react similarly is unknown since the relatively low frequency of detected “reversions” to chondrogenic activity suggests that the competence to react to the chondrogenic stimulus may decline further with longer cultivation in monolayer. Second, the close proximity in composite aggregates between the monolayer-derived cells and the cartilage-forming cells leaves open the possibility that the first were passively entrapped in the matrix elaborated by the latter. However, the presence of separate cartilage capsules around many of the monolayer-derived cells suggests formation of matrix by these cells and supports the possibility that “reversion” to chondrogenic activity may have indeed occurred. If so, then the question of mechanism arises, with reference to the nature of the decline and gain of chondrogenic competence, and of the causal conditions. It may be speculated that the freshly obtained, cartilage-forming cells provided the monolayer-modified cells with metabolites not present in the culture medium but essential for chondrogenesis; or that a transfer is involved of a more specific “primer” or “template” conducive to biosynthesis of chondrihable matrix but modified, dilated, or lost in the course of monolayer cultivation of the cells. The testing of such assumptions is feasible within the framework of available methodologies. 23-641804
Experimental
Cell Research
35
Y. Kuroda SUMMARY
1. Articular and epiphyseal cartilage cells, dissociated from femora of 13-day chick embryos were cultured in monolayer for various lengths of time. The cells were redispersed, tested for aggregability by rotation in flasks, and the aggregates examined for presence of cartilage. 2. Freshly dissociated chondrocytes always formed one or two giant aggregates. Cultivation in monolayer reduced progressively the tendency of cells to aggregate, resulting in smaller and less compact clusters. Decrease in size of aggregates was steep following the first days of cultivation, then levelled off. 3. Aggregates of freshly dissociated chondrocytes differentiated readily into cartilage. Cultivation in monolayer resulted in a decline in the chondrogenic activity of the cells. The steep decrease in aggregability of the monolayergrown cells preceded the marked decline in cartilage forming activity. After 9 days of growth in monolayer, the cell, when aggregated, displayed no cartilage forming activity. 4. Chondrocytes maintained in monolayer for 15 days and devoid of chondrogenic activity were coaggregated with freshly dissociated cartilageforming cells from mouse embryos. Chimeric aggregates, were formed and after further cultivation showed lumps of cartilage constructed by both kinds of cells. The significance of these results is briefly discussed. This work was carried out during the tenure by the author of an International Postdoctoral Research Fellowship of the National Institutes of Health. It was aided by Grant No. C-4272(to A. A. Moscona)from the National Cancer Institute, U.S.P.H.S. The author wishes to express his deepest appreciation to Professor A. A. Moscona, for his constant interest and advice throughout the course of this investigation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
HOLTZER, H., ABBOTT, J., LASH, J. and HOLTZER, S., Proc. Natl. KURODA, Y., Exptl Cell Res. 35, 326 (1964). MOSCONA, A., Proc. Nat!. Acad. Sei. 43, 184 (1957). in Developing Cell Systems and their Control. The Ronald __ Exvtl Cell Res. 22, 455 (1961). __ J. kellular Comp. Physibl. 60, Suppl. 1, 65 (1962). 86, 287 (1952). MOSCONA, A. and MOSCONA, H., J. Anat. TRINKHAUS, J. P. and GROVES, P. W., Proc. Natl Acad. Sci. 41, WEISS, P. and MOSCONA, A., J. Embryol. Exptl Morphol. 6, 238
Experimenfal
Cell
Research
35
Acad.
Sci.
46,
Press
Co.,
New
787 (1955). (1958).
1533
York,
(1960).
1960.
Cell Research
STUDIES II.
CHANGES
35, 337-348
(1964)
337
ON CARTILAGE
CELLS
IN
V1TRO
IN AGGREGATION AND IN CARTILAGE-FORMING ACTIVITY OF CELLS MAINTAINED IN MONOLAYER CULTURES Y. KURODA’
Department
of Zoology,
of Chicago,
University Received
July
Chicago,
Zll.,
U.S.A.
31, 1963
IN th e preceding paper, changes in cell morphology, growth characteristics, and production of matrix in populations of dissociated chondrocytes grown in monolayer cultures were described. The question of the nature of these changes was briefly raised and the need for further information on the functional traits of the monolayer-cultured cells stressed. It is known that freshly dissociated cartilage-forming cells are capable of forming compact aggregates and that the reconstructed tissue differentiates into typical cartilage [5, 7-91. Using these two criteria of histotypic effectiveness, the properties of monolayer grown chondrocytes were further examined. The present paper deals, therefore, primarily with (a) the effects of precultivation in monolayer on the capacity of chondrocytes to form aggregates; (b) with differentiation in aggregates of such cells; and finally, (c) with attempts to test the persistence of some of the changes occurring in monolayer-grown chondrocytes. MATERIALS
AND
METHODS
Methods for obtaining suspensions of viable cartilage cells from femora of 13-day chick embryos and composition of culture media were as previously described [5]. To obtain monolayer cultures of these cells, 20 ml aliquots of cell suspension (approximately 5 x IO4 cells per ml of standard culture medium) were dispensed into T-60 flasks. These were gassed with a 5 per cent CO, air mixture, and incubated at 37.5%. At different intervals the cultures were harvested as follows: The culture medium was removed, the culture rinsed three times with CMF solution, then incubated for 10 min in CMF, at 37.5”C under the gas mixture. CMF was then replaced with 0.5 per cent trypsin in CMF, the flasks were gassed and incubated for 10 min to cause detachment and dissociation of the cells. The resulting suspension was rinsed three times by centrifugation in CMF, 2 min at 2000 rpm; the pellets were dispersed into a 1 Present 33 Joancho,
address: Kitaku,
Dr. Y. Kuroda, Osaka, Japan.
Department
of Genetics,
Osaka
University,
Experimenlal
Medical
Cell
School,
Research
3.5
Y. Kuroda suspension of single cells in standard medium by flushing 10 to 15 times through a fine pipette. To obtain cell aggregates, the procedure of aggregation by rotation was used [5]. 3 ml aliquots of cell suspension in standard medium (approximately lo6 cells per ml medium) were distributed into 25 ml Erlenmeyer flasks and gassed with the CO, air mixture. The flasks were placed on a gyratory shaker rotating at a constant speed of 70 rpm, 3/4 inch diameter of rotation and a constant temperature of 38X, and examined after 24 or 48 hr. Aggregability of cells was assessed by counting and measuring the aggregates. Organ cultures of aggregates.-Some of the aggregates harvested after 24 or 48 hr were organ-cultured on nutrient agar to test their capacity for further differentiation. The nutrient agar was made up of one part of standard culture medium with double concentrations of horse serum 20 per cent and embryo extract 4 per cent, and one part of 2 per cent agar (Bacto-agar, Difco) dissolved in glucosol solution1 adjusted to pH 7.2. Equal quantities of the two parts were combined at a temperature of 50°C the mixture poured into small Petri dishes and allowed to gel. Aggregates were placed on the surface of the gel and the Petri dishes incubated at 37.5% in a container, gassed with CO, + air mixture, and maintained at saturation humidity; after 2 to 5 days, the cultures were fixed in Carnoy’s fixative, and prepared for histological examination. Mouse procartilage cells, used for commingling with monolayer modified chick chondrocytes, were obtained from limb-buds of 13-day mouse embryos. The tissue was treated with CMF solution for 10 min followed by a 15 min incubation with 1.0 per cent trypsin solution at 37.5”C under 5 per cent CO, air mixture. The rinsed tissue was dispersed into single cells in standard medium by flushing through the tip of a fine pipette. Chick chondrocytes precultured in monolayer for different periods were dispersed into single cells and commingled in varying proportions with the dissociated mouse procartilage cells. Aggregation procedure and cultivation on agar medium were as outlined above. RESULTS
Changes in aggregability
of monolayer-grown
chondrocytes
Size and number of aggregates.-Cartilage cells cultured without interference in monolayer for various lengths of time were redispersed, suspended in standard culture medium and their manner of aggregation was established by the standard procedure: rotation. Uniform conditions were maintained in all these tests. The aggregates formed in each flask in all series of tests were photographed and their size and number determined. Femoral chondrocytes freshly obtained from 13-day chick embryos, when tested by rotation for 24 hr, consistently formed one or two large aggregates with an average diameter of 1.15 mm. These spherical or oval aggregates 1 Exactly omitted. Experimental
the
same
composition
Cell Research
35
as Tyrode’s
solution
except
that
the sodium
bicarbonate
is
Aggregation
of cartilage
cells
339
had a relatively smooth surface and frequently showed evidence of fusion from smaller cell clusters (Fig. 1). Precultivation in monolayer resulted in striking changes in the aggregation patterns of the cells. Chondrocytes maintained in monolayer cultures for 1 day, then tested for aggregation formed several aggregates and a number of
__--I .m---.--M-.-- ---.--m---’ V’
‘Text-Fig. I.-Relationship between duration of precultivation in monolayer and the number and average diameter of the cell aggregates. Squares represent number of aggregates: dots represent average diameter of aggregates and the solid line indicates diameter distribution of aggregates from 3 x IO6 cells precultured in monolayer for varying days.
smaller cell clumps, ranging in diameter from 1.05 to 0.18 mm. Cells precultured in monolayer for 2 days formed 10 to 30 small aggregates and had a diameter range of 0.85 to 0.05 mm (Fig. 2). After 3 days precultivation in monolayer, a few aggregates 0.75 mm in diameter were formed, in addition to numerous smaller cell clusters. The average diameter of aggregates precultured for 5 days in monolayer fell to 0.09 mm (Fig. 3). This decline continued with further precultivation, however, at a lesser rate than initially (Fig. 4). From 15 days in monolayer on (Fig. 5), only a slight decline in size of aggregates was detectable, and subsequently the pattern became stabilized to homogeneous small clusters, up to 0.05 mm in diameter (Fig. 6). Experimental
Cell Research
35
Y. Kuroda
Fig.
l.-Aggregate
of
Fig.
2.-Aggregates
of a progeny
freshly
dissociated
of chondrocytes
chondrocytes cultured
from
in monolayer
femur
of 13.day
for 2 days.
chick
Fig.
3.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 5 days.
Fig. 4.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 9 days.
Fig.
5.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 15 days.
Fig.
6.-Aggregates
of a progeny
of chondrocytes
cultured
in monolayer
for 29 days.
embryo.
This relationship between duration of precultivation in monolayer and the number and average diameter of the cell aggregates is shown in Text-Fig. 1. The maximum diameter of aggregates decreased gradually, whereas the minimum diameter did not vary noticeably throughout the series. It might be stressed here that the decline in the average diameter of aggregates was Experimenfal
Cell Research
35
Aggregation
of cartilage
cells
341
steepest after the first 2-3 days of precultivation in monolayer; i.e., at the time when increase in cell number in the monolayer cultures was absent or slow. Free and attached cells in aggregation
cultures
The above changes in aggregability of monolayer grown chondrocytes were accompanied by an increased tendency of the cells to become attached to the wall of the rotating flasks. This resulted in the formation of a ring-like rim of cells, on the glass at the level of the surface of the medium. Thus, aggregation cultures consisted of cell aggregates, free cells floating in the medium, and attached rim cells. It was of interest to determine the quantitative relations between the free and the rim cells relative to duration of precultivation of the cells in monolayer and to their aggregation patterns. 24-hr aggregation cultures were used. After removal of aggregates the free cells in the
Text-Fig. 2.-Number of attached rim cells and free cells concentration of 3 x lo8 cells previously cultured in monolayer
after 24 hr in gyrating for varying days.
flasks
in a
Text-Fig. S.--Size distribution of aggregates formed by each of commingled suspensions of chick chondrocytes maintained in monolayer cultures for 15 days and freshly dissociated mouse cartilage-forming cells. Dots represent average diameter of aggregates; line indicates diameter distribution of aggregates in cultures with a concentration of approximately 2 x loo cells per ml medium. Experimental
Cell Research
35
Y. Kuroda medium were counted. The rim cells were dissociated with trypsin and counted. The results (Text-Fig. 2) showed little change in the counts of free cells but sharply increasing numbers of rim cells; this increase was particularly striking after the first 3 days of precultivation in monolayer, i.e., coincident with the rapid decline in the size of aggregates in these cultures. Thus, as the number of preferentially mutually adhesive cells declined, the number of cells tending to become attached to glass increased. This increase in the number of rim cells may thus represent a surprisingly rapid adaptation of the monolayer-grown cells to attach to glass. Changes in chondrogenic
activity
of monolayer
grown
cells
Differentiation of aggregates.-24hr aggregates formed by chondrocytes freshly obtained from embryos consisted of procartilage with the round or oval cells embedded in a small amount of translucent matrix (Fig. 7). Each aggregate was surrounded by a few layers of flattened cells. When organcultured on nutrient agar for 3-5 days, the aggregates differentiated into typical hypertrophic cartilage (Fig. 8). Similarly, aggregates formed by chondrocytes precultured in monolayer for 1 day also consisted of procartilage capable of further typical differentiation. Thus, under these experimental conditions, cells capable of chondrogenic activity were able to differentiate histotypically. This point is important because of the decline in chondrogenesis in aggregates from monolayer cultured cells.
Fig.
7.--Section
of an aggregate
of freshly
Fig. S.-Section 3 days.
of an aggregate
of freshly
Fig. 9.-Section
of an aggregate
of a progeny
of chondrocytes
precultured
in monolayer
for 2 days.
of aggregates
of a progeny
of chondrocytes
precultured
in monolayer
for 4 days.
Fig.
lO.--Section
dissociated dissociated
Fig. Il.-Section of a concentrated drop maintained on agar media for 5 days.
of aggregates
Fig. 12.-Section on agar media
from
of a cell pellet for 5 days.
obtained
Fig. 13.--24.hr aggregates of freshly chondrocytes precultured for 15 days chick cells with light-staining nuclei.
chondrocytes. chondrocytes
precultured
12-day-monolayer
dissociated in monolayer
maintained
in monolayer cultures
which
on agar
media
for 6 days were
for
and
maintained
mouse limb bud cells commingled with chick (2 : 1). Mouse cells with dark-staining nuclei;
Fig. 14.-Section through a chimeric aggregate consisting of embryonic mouse limb buds commingled with chick chondrocytes precultured for 15 days in monolayer. Aggregates maintained on agar media for 3 days. Mouse cells (m) (darkly staining nuclei) embedded in a common matrix with chick cells (c) (light nuclei). Experimental
Cell
Research
35
343
cartilage cells
Experimental
Cell Iieseurch 35
Y. Kuroda 24-hr aggregates of cells precultured in monolayer for 2 days were in a precartilaginous stage. There was little matrix; it had a more opaque appearance; and the cells were more tightly packed (Fig. 9). After 3 days in monolayer cultures, the cells formed aggregates in which the amount of matrix was further reduced and its opaqueness increased. This change was even more pronounced in aggregates of cells precultured in monolayur for 4 days (Fig. 10). The cytoplasm in many of the cells had a fibrous, frequently vacuolated, appearance. The larger aggregates were surrounded by flattened cells while most of the smaller aggregates had no such covering. In organ cultures, scattered nodules of cartilage and pre-cartilage developed. 24-hr aggregates of chondrocytes precultured in monolayer for more than 5 days showed no identifiable matrix. For cultivation on nutrient agar these small aggregates were bunched and piled up. In organ cultures of aggregates obtained from cells precultured in monolayer for 6 to 9 days, the small aggregates fused into large nodules that contained areas of precartilage (Fig. 11). Cells aggregated after 9 or more days in monolayer did not difTABLE
I. Cartilage-forming
activity monolayer
Days in monolayer
Aggregates
0 1 2 3 4 5 6 9 12 15 22 29 44 59 72 100 128 + + -I-, Hypertrophic Experimental
Cell
of aggregates and of rim cellsprecultured for varying periods.
++ + -I+ !!I -
Organcultured aggregates +++ +++ ++ ++ + + -t
-
-
cartilage;
Research
35
+ + , procartilage;
-
+ , prechondral
Attached cell pellets organcultured
. .. . .. ++ ++ + + + stage;
- , no differentiation.
in
Aggregation
of cartilage
cells
345
ferentiate into any stage recognizable as chondrogenesis; after several days on nutrient agar masses of unidentified fibrous tissue mere present (Fig. 12). Results of the above tests are summarized in Table I. Differentiation of rim cells.-Fragments of the rim formed by cells attached to the flask wall in rotating cultures were removed and cultured on nutrient agar for 3 to 5 days. Their differentiation corresponded to that of aggregates from same cultures, and showed a comparable but more rapid decline in chondrogenic activity (Table I). Effect of freshly obtained cartilage-forming cells on aggregation of monolayer grown chondrocytes.-The behavior of monolayer modified cells in the presence of freshly dissociated cells was studied by using mixtures of chick and mouse cells. The differences in size and staining of cells from these two species [3-51 permitted identification in composite cell mixtures. Chondrocytes maintained continuously in monolayer cultures for 15 days were redispersed and commingled with freshly dissociated cartilage-forming cells from the limb bud mesoblasts of 13-day mouse embryos. The proportions used were: 3 : 0, 2 : 1, 1 : 1, 1 : 2, and 0 : 3 (chick : mouse). Approximate cell concentration in the composite suspensions was 2 x lo6 per ml medium. The cells were aggregated by rotation in gyrating flasks. The size distribution of aggregates formed by each of these cell combinations is represented in Text-Fig. 3. The monolayer-grown chick chondrocytes alone formed small aggregates with a relatively short range of size averaging 0.06 mm in diameter. Aggregates formed by freshly obtained mouse cartilage-forming cells alone had a considerably wider size distribution and an average diameter of 0.19 mm. In flasks with two parts of monolayer-grown cells and one part freshly obtained cells, large aggregates with a diameter of up to 0.79 mm formed. This maximum size of aggregates did not noticeably increase in the presence of higher proportions of freshly dissociated cells, but the average diameter of aggregates increased with the rise in proportion of mouse cells. Effect of cartilage-forming cells on differentiation in aggregates of monolayer modified chondrocytes.-As described above, aggregates formed by chondrocytes maintained continuously in monolayer for 15 days showed no histologically detectable evidence of chondrification; even when organ-cultured for several days on nutrient agar, they failed to differentiate into cartilage. It seemed of interest to examine the effect of cartilage-forming cells on those monolayer-modified cells. It was first found that the two kinds of cells could be associated: 24hr aggregates that formed in suspensions comprising monolayer modified chondrocytes and freshly obtained mouse cartilageforming cells (2 : 1) had a chimeric composition and consisted of interspersed Experimental
Cell Research
35
346
Y. Kuroda
chick and mouse cells easily distinguishable by their size and staining differences (Fig. 13). The larger aggregates contained also masses of mouse procartilage cells surrounded by interspersed chick and mouse cells. After cultivation on nutrient agar, these aggregates contained scattered chondrified nodules which consisted of interspersed chick and mouse cells (Fig. 14) in addition to mouse cartilage. 24-hr aggregates formed in suspensions comprising equal proportions of both cell kinds also were of chimeric composition. In organ cultures, many of these aggregates differentiated into cartilage, and many of the nodules consisted of both kinds of cells closely associated in a common cartilaginous matrix. M’hile there were always areas of mouse cartilage only there were no regions that consisted of chondrified chick cells. Organ cultures of aggregates formed in suspensions of one part chick with tlvo parts mouse cells differentiated into cartilage consisting predominantly of mouse cells with scattered chimeric areas. The significance of the effects of freshly dissociated cells on monolayermodified chondrocytes, \vas tested by intermingling the latter with cells from other than cartilage-forming tissues. Dissociated liver and kidney cells from newborn mice were commingled in equal proportions with the dispersed, monolayer-modified chondrocytes. In aggregates formed by rotation of such cultures, the cells of each kind tended to group separately and showed no such close intermingling as in the co-aggregates with cartilage-forming cells. There was no noticeable chondrogenesis in any of these cultures. It appears thus, that the ability to enter into close architectural association \vith the monolayer-grown chondrocytes and to elicit chondrification was restricted, in these series of tests, to the freshly dissociated cartilage-forming cells.
DISCUSSION
It was shown that chondrocytes maintained in monolayer cultures decline in their tendency to form aggregates and in their ability to differentiate into cartilage. The second of these findings confirms the results of Holtzer et al. [ 1 ] obtained with cartilage cells from another tissue. It is of interest that the decline in aggregability of the monolayer-grown cells was steepest during the first days of cultivation and that it preceded the decline in chondrogenic activity of the cells. Both changes coincided with the progressive reduction in the number of round cells and their transformation into fibroblastic forms [2]. Another facet in this course of events was the progressively increasing Experimental
Cell Research
35
Aggregation
of
CaFtdage
CdS
tendency of the monolayer-grown chondrocytes to attach to glass, rather than to each other or to remain in suspension. The progressive decline in aggregability of monolayer-cultured chondrocytes agrees with similar findings on other types of cells [J]. It was postulated that it reflects changes in the adhesive properties of cells consequent upon metabolic adaptation to in vitro conditions [‘L]. It is possible that, since mutual attachment of cells may be mediated by specific cell products functioning at the cell surface, the biogenesis of these products may be affected by the metabolic adjustments related to the persistence of cells in a dispersed state 161. The decline in chondrogenic activity in monolayer-grown cells may represent another aspect of the syndrome of functioning in a dispersed state. It may represent a diversion to other tasks of the biosynthetic machinery of the cells primarily towards proliferation but perhaps also towards elaboration of other, as yet unidentified differentiation products. Other interpretations are, of course, conceivable at present. Of suggestive interest is the finding that monolayer-modified chondrocytes, though by themselves unable to elaborate chondrihed matrix, appear to form cartilage when co-aggregated with freshly dissociated cartilage-forming cells. These findings call for certain obvious reservations. First, in the present experiments, cells maintained for 15 days in monolayer were used; whether cells from older monolayer cultures would react similarly is unknown since the relatively low frequency of detected “reversions” to chondrogenic activity suggests that the competence to react to the chondrogenic stimulus may decline further with longer cultivation in monolayer. Second, the close proximity in composite aggregates between the monolayer-derived cells and the cartilage-forming cells leaves open the possibility that the first were passively entrapped in the matrix elaborated by the latter. However, the presence of separate cartilage capsules around many of the monolayer-derived cells suggests formation of matrix by these cells and supports the possibility that “reversion” to chondrogenic activity may have indeed occurred. If so, then the question of mechanism arises, with reference to the nature of the decline and gain of chondrogenic competence, and of the causal conditions. It may be speculated that the freshly obtained, cartilage-forming cells provided the monolayer-modified cells with metabolites not present in the culture medium but essential for chondrogenesis; or that a transfer is involved of a more specific “primer” or “template” conducive to biosynthesis of chondrihable matrix but modified, dilated, or lost in the course of monolayer cultivation of the cells. The testing of such assumptions is feasible within the framework of available methodologies. 23-641804
Experimental
Cell Research
35
Y. Kuroda SUMMARY
1. Articular and epiphyseal cartilage cells, dissociated from femora of 13-day chick embryos were cultured in monolayer for various lengths of time. The cells were redispersed, tested for aggregability by rotation in flasks, and the aggregates examined for presence of cartilage. 2. Freshly dissociated chondrocytes always formed one or two giant aggregates. Cultivation in monolayer reduced progressively the tendency of cells to aggregate, resulting in smaller and less compact clusters. Decrease in size of aggregates was steep following the first days of cultivation, then levelled off. 3. Aggregates of freshly dissociated chondrocytes differentiated readily into cartilage. Cultivation in monolayer resulted in a decline in the chondrogenic activity of the cells. The steep decrease in aggregability of the monolayergrown cells preceded the marked decline in cartilage forming activity. After 9 days of growth in monolayer, the cell, when aggregated, displayed no cartilage forming activity. 4. Chondrocytes maintained in monolayer for 15 days and devoid of chondrogenic activity were coaggregated with freshly dissociated cartilageforming cells from mouse embryos. Chimeric aggregates, were formed and after further cultivation showed lumps of cartilage constructed by both kinds of cells. The significance of these results is briefly discussed. This work was carried out during the tenure by the author of an International Postdoctoral Research Fellowship of the National Institutes of Health. It was aided by Grant No. C-4272(to A. A. Moscona)from the National Cancer Institute, U.S.P.H.S. The author wishes to express his deepest appreciation to Professor A. A. Moscona, for his constant interest and advice throughout the course of this investigation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
HOLTZER, H., ABBOTT, J., LASH, J. and HOLTZER, S., Proc. Natl. KURODA, Y., Exptl Cell Res. 35, 326 (1964). MOSCONA, A., Proc. Nat!. Acad. Sei. 43, 184 (1957). in Developing Cell Systems and their Control. The Ronald __ Exvtl Cell Res. 22, 455 (1961). __ J. kellular Comp. Physibl. 60, Suppl. 1, 65 (1962). 86, 287 (1952). MOSCONA, A. and MOSCONA, H., J. Anat. TRINKHAUS, J. P. and GROVES, P. W., Proc. Natl Acad. Sci. 41, WEISS, P. and MOSCONA, A., J. Embryol. Exptl Morphol. 6, 238
Experimenfal
Cell
Research
35
Acad.
Sci.
46,
Press
Co.,
New
787 (1955). (1958).
1533
York,
(1960).
1960.