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Changes in aggregation and differentiation of cartilage cells grown in monolayer cultures
Y. Kuroda
446
Summary.-Growth and differentiating patterns of normal and malignant stratified epithelia of human cervix were compared with nucleic acid and protein measurement of 235 normal cells and 244 malignant cells with Caspersson’s photoelectric microspectrophotometer. In normal stratified epithelia, as the cells are going from growth to differentiation, an inhibitory factor to nucleic acid and protein concentrations were observed. In malignant stratified epithelia, this inhibitory factor was found missing. REFERENCES 1. CASPERSSON,
T., JACOBSSON,
F., LOMAKKA,
G., SVENSSON,
G. and
SKFSTRGM,
R.,
Exptl.
Cell
Res. 5, 560 (1953). CASPERSSON, T. and SANTESSON, L., Acta Radiol. Suppl. XLVI, 37 (1942). DE, P. and CHATTERJEE, R., Rrif. J. Cancer XVI, 141 (1962). DE, P., CHATTERJEE, R., BHATTACHARYA, K. and MITRA, S., Cancer 14,502 (1961). GRIFFIN, A. C., Metabolism of cancer cell in fundamental aspects of normal and malignant growth, p. 877. W. W. NOWINSKI (ed.). Elsevier Pub. Co., Amsterdam, 1960. 6. MITRA, S. and DE, P. K., &it. J. Cancer 8, 107 (1954). 7. MOBERGER, G., LINDSTRGM, B. and ANDERSSON, L., Expfl. Cell Res. 6, 228 (1954). 2. 3. 4. 5.
CHANGES
IN AGGREGATION
CARTILAGE
CELLS
AND
GROWN
DIFFERENTIATION
IN MONOLAYER
OF
CULTURES’
Y. KURODAa Department
of Zoology, University Received
of Chicago, Chicago, Ill., U.S.A.
January
14, 1963
IT
is well known that freshly dissociated embryonic cells, when maintained in vitro under conditions favorable for aggregation, tend to form multicellular structures in which they proceed to differentiate histotypically [3, 41. On the other hand, cells cultured dispersed in monolayers undergo various changes and tend to lose their original structural and functional traits [l, 21. A detailed survey of events involved in such loss is of obvious importance to the understanding of factors that control and stabilize differentiation of cells. This report deals briefly with changes in the aggregability of cartilage cells grown in monolayer, and with the differentiation of aggregates formed by monolayer-grown cartilage cells. Material and Methods.-Suspensions of cartilage cells were obtained by dissociation of the articular and epiphyseal cartilages from femora of 13-day chick embryos using a slight modification of Moscona’s method [3]. Full details of procedure will be described elsewhere. The cells were dispersed in standard culture medium (Eagle’s 1 Research supported by grant C-4274, National Cancer Institute, U.S. Public Health 2 International Postdoctoral Fellow of the National Institutes of Health, U.S. Public Service; Grant PHS-FFG304. Present address: Department of Biology, Faculty of Osaka University, Osaka, Japan.
Experimental
Cell Research 30
Service. Health Science,
Changes in cartilage
cells
basal medium with 1 m&f L-glutamine, 10 per cent horse serum, 2 per cent chick embryo extract, and penicillin-streptomycin at a concentration of 50 units each per ml. For cultivation in monolayers the cells in suspension were distributed into T-60 culture flasks which were gassed with 5 per cent CO,-air mixture and incubated at 37.5%. After various periods of cultivation in monolayer the ability of the cells to form aggregates was tested by the rotation procedure [3]. The monolayer sheets were dispersed into single cells by brief treatment with 0.5 per cent trypsin solution and resuspended in standard medium. 3 ml aliquots of cell suspension containing 3.0 x lo6 cells were distributed into 25 ml Erlenmeyer flasks which were rotated on a gyratory shaker with a constant speed of 70 rpm, at 38°C. Aggregates harvested after 24 to 48 hr were counted, measured, sampled for histology and transferred for organ culture to nutrient agar (1 per cent agar in glucosol with 10 per cent horse serum and 2 per cent embryo extract) for further differentiation. Results.-Freshly dissociated cartilage cells, tested by rotation for aggregability, aggregated readily and in 24 hr cohered into one or two large aggregates. Precultivation in monolayer caused a progressive decrease in the mutual adhesiveness of the cells resulting in increasingly smaller and more numerous aggregates and a larger number of cells that remained free. Tested after one day in monolayer, the cells formed several smaller aggregates and loose clusters of cells, with an average diameter less than one half of that of aggregates of freshly dissociated cells. After two days in monolayer there was a further decline in the size of aggregates and increase in their number. After three days in monolayer the cells formed aggregates with an average diameter one-tenth of the original. The pattern of decline in the size of aggregates formed by monolayer-precultured cartilage cells is seen in Text-Fig. 1. It will be noted that the decrease in the average diameter of aggregates was sharpest in the first 2-3 days; from then on the decline was slight and gradual and a practically uniform size distribution was established. Examined histologically, 24-hr aggregates of freshly dissociated cartilage cells were found to consist of procartilage, with the cells embedded in a continuous matrix. Maintained on nutrient agar for 2-5 days, such aggregates differentiated into typical, hypertrophic cartilage. Similarly, 24-hr aggregates of cells precultured for 1 day in monolayer also formed procartilage capable of further typical differentiation. Cells precultured for two days in monolayer formed aggregates in which there was considerably less matrix. This decline in matrix formation progressed with longer precultivation and, after six days of growth in monolayer the aggregated cells showed no histological features characteristic of cartilage; on nutrient agar such aggregates attained only prechondral stage. In the clusters formed by cells precultured in monolayer for more than nine days, there was no noticeable capacity for chondrogenic activity even after prolonged maintenance on nutrient agar. Remarks.--Cultivation in monolayer rapidly reduced the aggregability of cartilage cells. Similar findings were reported for cells from other tissues [2]. In the aggregation procedure used in this sudy, decrease in size of aggregates indicates a decline in the mutual adhesiveness of cells in the population [3]; since the only experimental variable examined here was length of cultivation of the cells in monolayer, it appears (a) that maintenance of the cells in a dispersed state results in a change in their attachment properties; (b) that this change progresses with increased duration of Experimental
Cell Research
30
448
Y. Kuroda
growth in dispersion; (c) that it is accompanied by changes in the developmental properties of the cells. As reported for vertebral cartilage cells [l], femoral cartilage cells grown in monoIayer progressively lose their tendency to form cartilage so that eventually, even though recombined in aggregates, they do not synthesize matrix.
0’
’ I ’ ’ ’
0123456
:Ii ’ ’
9
DAYS IN MONOLAYER
In
both
is of the
cases,
the
order
of
time nine
Text-Fig.
12
I5
I.-Effect of precultivation in monolayer of dissociated cartilage cells on the size of aggregates formed. Cells derived from the femora of 13-day chick embryos. Dots represent average diameter of aggregates; line indicates diameter distribution. 24 hr rotation cultures; cell concentration and other conditions equal.
CULTURE
in
monolayer
days.
However,
required the
for present
loss study
of
detectable of aggregates
matrix has
formation shown
that
a marked decline in the original histotypic functions of the cells is noticeable already after three days in monolayer. It seems particularly interesting that this decline starts after the sharp drop in the aggregability of the cells. This suggests, perhaps, that modifications in the mechanisms involved in cell attachment either precede or represent an early symptom of the changes that result in loss of histotypic traits in monolayer-grown cells. Whether these modifications are due to metabolic “adaptation” of cells to the in vitro conditions, or represent effects of selection or mutation is a matter of further study. The author wishesto express his gratitude to Professor A. A. Moscona for advice and criticism throughout the course of this work. REFERENCES J. and HOLTZER, S., Proc. Natl. Acad. Sci. 46, 1533 Systems and their Control, 18th Growth Symposium, Ronald Press Co., New York, 1960. Exptl. Cell Reb. 22, 455 (1961). J. Cellular Comp. Physiol. 6, Suppl. 1, 65 (1962).
1. HOLTZER, H., 2. MOSCONA; A.; D. Rudnick
3. __ 4. __ Experimental
ABBOT, J., in Developing (ed.) The
Cell Research
LASH,
Cell
30
(1960). 1959.
446
Summary.-Growth and differentiating patterns of normal and malignant stratified epithelia of human cervix were compared with nucleic acid and protein measurement of 235 normal cells and 244 malignant cells with Caspersson’s photoelectric microspectrophotometer. In normal stratified epithelia, as the cells are going from growth to differentiation, an inhibitory factor to nucleic acid and protein concentrations were observed. In malignant stratified epithelia, this inhibitory factor was found missing. REFERENCES 1. CASPERSSON,
T., JACOBSSON,
F., LOMAKKA,
G., SVENSSON,
G. and
SKFSTRGM,
R.,
Exptl.
Cell
Res. 5, 560 (1953). CASPERSSON, T. and SANTESSON, L., Acta Radiol. Suppl. XLVI, 37 (1942). DE, P. and CHATTERJEE, R., Rrif. J. Cancer XVI, 141 (1962). DE, P., CHATTERJEE, R., BHATTACHARYA, K. and MITRA, S., Cancer 14,502 (1961). GRIFFIN, A. C., Metabolism of cancer cell in fundamental aspects of normal and malignant growth, p. 877. W. W. NOWINSKI (ed.). Elsevier Pub. Co., Amsterdam, 1960. 6. MITRA, S. and DE, P. K., &it. J. Cancer 8, 107 (1954). 7. MOBERGER, G., LINDSTRGM, B. and ANDERSSON, L., Expfl. Cell Res. 6, 228 (1954). 2. 3. 4. 5.
CHANGES
IN AGGREGATION
CARTILAGE
CELLS
AND
GROWN
DIFFERENTIATION
IN MONOLAYER
OF
CULTURES’
Y. KURODAa Department
of Zoology, University Received
of Chicago, Chicago, Ill., U.S.A.
January
14, 1963
IT
is well known that freshly dissociated embryonic cells, when maintained in vitro under conditions favorable for aggregation, tend to form multicellular structures in which they proceed to differentiate histotypically [3, 41. On the other hand, cells cultured dispersed in monolayers undergo various changes and tend to lose their original structural and functional traits [l, 21. A detailed survey of events involved in such loss is of obvious importance to the understanding of factors that control and stabilize differentiation of cells. This report deals briefly with changes in the aggregability of cartilage cells grown in monolayer, and with the differentiation of aggregates formed by monolayer-grown cartilage cells. Material and Methods.-Suspensions of cartilage cells were obtained by dissociation of the articular and epiphyseal cartilages from femora of 13-day chick embryos using a slight modification of Moscona’s method [3]. Full details of procedure will be described elsewhere. The cells were dispersed in standard culture medium (Eagle’s 1 Research supported by grant C-4274, National Cancer Institute, U.S. Public Health 2 International Postdoctoral Fellow of the National Institutes of Health, U.S. Public Service; Grant PHS-FFG304. Present address: Department of Biology, Faculty of Osaka University, Osaka, Japan.
Experimental
Cell Research 30
Service. Health Science,
Changes in cartilage
cells
basal medium with 1 m&f L-glutamine, 10 per cent horse serum, 2 per cent chick embryo extract, and penicillin-streptomycin at a concentration of 50 units each per ml. For cultivation in monolayers the cells in suspension were distributed into T-60 culture flasks which were gassed with 5 per cent CO,-air mixture and incubated at 37.5%. After various periods of cultivation in monolayer the ability of the cells to form aggregates was tested by the rotation procedure [3]. The monolayer sheets were dispersed into single cells by brief treatment with 0.5 per cent trypsin solution and resuspended in standard medium. 3 ml aliquots of cell suspension containing 3.0 x lo6 cells were distributed into 25 ml Erlenmeyer flasks which were rotated on a gyratory shaker with a constant speed of 70 rpm, at 38°C. Aggregates harvested after 24 to 48 hr were counted, measured, sampled for histology and transferred for organ culture to nutrient agar (1 per cent agar in glucosol with 10 per cent horse serum and 2 per cent embryo extract) for further differentiation. Results.-Freshly dissociated cartilage cells, tested by rotation for aggregability, aggregated readily and in 24 hr cohered into one or two large aggregates. Precultivation in monolayer caused a progressive decrease in the mutual adhesiveness of the cells resulting in increasingly smaller and more numerous aggregates and a larger number of cells that remained free. Tested after one day in monolayer, the cells formed several smaller aggregates and loose clusters of cells, with an average diameter less than one half of that of aggregates of freshly dissociated cells. After two days in monolayer there was a further decline in the size of aggregates and increase in their number. After three days in monolayer the cells formed aggregates with an average diameter one-tenth of the original. The pattern of decline in the size of aggregates formed by monolayer-precultured cartilage cells is seen in Text-Fig. 1. It will be noted that the decrease in the average diameter of aggregates was sharpest in the first 2-3 days; from then on the decline was slight and gradual and a practically uniform size distribution was established. Examined histologically, 24-hr aggregates of freshly dissociated cartilage cells were found to consist of procartilage, with the cells embedded in a continuous matrix. Maintained on nutrient agar for 2-5 days, such aggregates differentiated into typical, hypertrophic cartilage. Similarly, 24-hr aggregates of cells precultured for 1 day in monolayer also formed procartilage capable of further typical differentiation. Cells precultured for two days in monolayer formed aggregates in which there was considerably less matrix. This decline in matrix formation progressed with longer precultivation and, after six days of growth in monolayer the aggregated cells showed no histological features characteristic of cartilage; on nutrient agar such aggregates attained only prechondral stage. In the clusters formed by cells precultured in monolayer for more than nine days, there was no noticeable capacity for chondrogenic activity even after prolonged maintenance on nutrient agar. Remarks.--Cultivation in monolayer rapidly reduced the aggregability of cartilage cells. Similar findings were reported for cells from other tissues [2]. In the aggregation procedure used in this sudy, decrease in size of aggregates indicates a decline in the mutual adhesiveness of cells in the population [3]; since the only experimental variable examined here was length of cultivation of the cells in monolayer, it appears (a) that maintenance of the cells in a dispersed state results in a change in their attachment properties; (b) that this change progresses with increased duration of Experimental
Cell Research
30
448
Y. Kuroda
growth in dispersion; (c) that it is accompanied by changes in the developmental properties of the cells. As reported for vertebral cartilage cells [l], femoral cartilage cells grown in monoIayer progressively lose their tendency to form cartilage so that eventually, even though recombined in aggregates, they do not synthesize matrix.
0’
’ I ’ ’ ’
0123456
:Ii ’ ’
9
DAYS IN MONOLAYER
In
both
is of the
cases,
the
order
of
time nine
Text-Fig.
12
I5
I.-Effect of precultivation in monolayer of dissociated cartilage cells on the size of aggregates formed. Cells derived from the femora of 13-day chick embryos. Dots represent average diameter of aggregates; line indicates diameter distribution. 24 hr rotation cultures; cell concentration and other conditions equal.
CULTURE
in
monolayer
days.
However,
required the
for present
loss study
of
detectable of aggregates
matrix has
formation shown
that
a marked decline in the original histotypic functions of the cells is noticeable already after three days in monolayer. It seems particularly interesting that this decline starts after the sharp drop in the aggregability of the cells. This suggests, perhaps, that modifications in the mechanisms involved in cell attachment either precede or represent an early symptom of the changes that result in loss of histotypic traits in monolayer-grown cells. Whether these modifications are due to metabolic “adaptation” of cells to the in vitro conditions, or represent effects of selection or mutation is a matter of further study. The author wishesto express his gratitude to Professor A. A. Moscona for advice and criticism throughout the course of this work. REFERENCES J. and HOLTZER, S., Proc. Natl. Acad. Sci. 46, 1533 Systems and their Control, 18th Growth Symposium, Ronald Press Co., New York, 1960. Exptl. Cell Reb. 22, 455 (1961). J. Cellular Comp. Physiol. 6, Suppl. 1, 65 (1962).
1. HOLTZER, H., 2. MOSCONA; A.; D. Rudnick
3. __ 4. __ Experimental
ABBOT, J., in Developing (ed.) The
Cell Research
LASH,
Cell
30
(1960). 1959.