Acta histoch em. Bd. 56, S . 115-119 (1976)
Institut fur Bi ophysik und Strahlenbiologie der Universitat Hamburg (Goschaftsfuhrender Direktor: Prof. Dr. H. JUNO)
A comparative study of four sublines of mouse fibroblast cells") By
SURAM
B.
REDDY, WAL]'RIED
A.
LINDEN
and
KLAUS LUBBERS
With 2 figures (Received September 4, 1975)
Summary Cell doubling time, DNA distribution pattern, frequen cy distribution of chromosome numbers per cell and degree of synchrony obtained after mitotic sel ection were studied in 4 different sublines of mouse fibroblast cell s, N CTC L.929, in cult u re . The 4 su bline s did not differ with respect to their cell kinetic data but showed changes of the ch rom osome pattern compared to the stemline and compared to ea ch other sugges t ing that ch romosome analysis provides a very sensitive means t o detect ev olu t ia na ry cha nges of a permanent cell line.
Introduction It is of obvious interest to note that permanent cell lines are in a steady evolution with increasing number of subcultivations. This evolution of a cell population may involve eno rmous differences in karyology, growth potentialities and other characteristic features in comparison with the original cell line. Also for mouse fibroblasts, NCTC line 929, cyclic and directional changes of population structure were reported (Hsu 1960). In our present study attempts were made to characterize 4 sublines of L-strain mouse fibroblasts (NCTC 929) by measuring chromosome number, cellular growth, degree of synchronization, and cellular DNA distribution patterns. The results will show differences between the sublines demonstrating the importance of selecting the appropriate subline in each experimental design.
Materials and Methods Cell system Four su blines of L-929 m ou se f ibroblast ce lls HH, D, F and E wer e employed in our investigation . The mon olayer cultu res were initiated in 180 ml flask s in a modified EAGLE'S medium (No . 47 340 A, S erva, H e idelb erg) supplemented with 5 % fetal ca lf se rum , peni cillin and stre p t om yci n . I) Supported by the D eutsche Forschungsgemeinschaft, B onn-Bad God esb crg.
116
S. B. REDDY, W. A. LINDEN and K. LUBBERS
Growth assays Approximately 1()6 cells were planted in 180 ml glass flasks in 15 ml medium. The growth curves of the cells were dertermined by counting the cells for every 12 h over a period of 5 days. For this purpose the cells were trypsinized with 0.05 % trypsin (Difeo, 1: 250), released from monolayer and counted in a haemocytometer,
Chromosome preparation The cells were grown on 2 glass slides placed in a glass flask. 48 h later, the cultures obtained a hypotonic shock by exposure to 0.075 M KCI at 37°C for 12 min. To accumulate metaphases the cultures were treated with 0.02 % colcemid 1 h before harvesting.
Synchronization The cells were propagated in 1200 ml Raux flasks with an input of 7.5 X 106 in 80 ml medium. After 48 h of incubation ,when the cells had reached a confluent layer, mitotic cells were released by manual shaking. The details of the synchronization procedure were the same as described earlier (LINDEN et al. 1974). For each subline the synchronization procedure was repeated 5 times over a period of 10 h at 2 h intervals. For determination of mitotic index at least 500 cells were scored from each slide.
Pulse cytophotometry DNA distribution patterns of all the sublines were measured using a pulse cytophotometer (ICP 11, Phywe, GOttingen). For this purpose cells in exponential growth i. e. 48 h after subcultivation were fixed and stained with ethidium bromide. The details of the pulse cytophotometric technique were described elsewhere (GOHDE and DITTRICH 1971, LINDEN et al. 1973). A computer evaluation of the DNA histograms obtained yielded the percentage of cells in the different phases of the cell cycle (BAISCH et al. 1975).
Results and Discussion The karyology studies revealed that the 4 sublines have different distributions of chromosome numbers per cell (Fig. 1). Although subline HH shows a similar frequency distribution around the maximum as was found for subline F, the latter contains a substantial portion of cells having chromosome numbers higher than 80. The same holds when we compare sublines D and E. The most frequent chromosome numbers, the average chromosome numbers and the percentages of "polyploid" cells having chromosome numbers greater than 80 are plotted in Table 1. The fact that the lines D and F show nearly equal figures of the mean chromosome numbers whereas their frequency distributions are obviously quite different derives from subline D having 4.9 % polyploid cells compared to 12.6 % in subline F. This finding indicates once more that the mean chromosome number does not contain sufficient information to enable a reasonable comparison between 2 sublines of mammalian cells. It has been described by SHANNON (1972) that the modal chromosome number of L-strain mouse fibroblast cells derived from the parental strain from normal subcutaneous areolar and adipose tissue of a 100 day-old male C3 H/A mouse was 66. After a couple of years the same strain has undergone remarkable changes in chromosome constitution. Hsu (1960) cited that in subline L-P55 after 2 years of routine
117
A comparative study of four sublines 30
HH 20 10 0
D 20 VI
...J ...J
ILl U u,
0
10 0
0= ILl III
~
::J Z
F
20 10 0 20 10
oL..-_-'---_......................LLL._.....J._----'_---J 40 o 20 100 120 60 80 CHROMOSOME NUMBER PER CELL Fig.!. Frequency distributions of chromosome numbers per cell of Hamburg (HH), Diisseldorf (D), Freiburg (F) and Eppendorf (E) sublines of L-929 mouse fibroblasts. Table 1. Chromosome analysis of 4 different sublines of L-929 mouse fibroblasts. Subline
Most frequent chromosome number
Mean chromosome Percentage of "polyploid" cells number
HH D F E
60 64 59 64
61.2 64.4 64.7 61.7
1.0 4.9 12.6 0
subculture system the original stem line's chromosome number dropped from 68 to 64. From foregoing arguments it appears that this spectrum of population changes in vitro is a common observation. Also changes of the chromosome type can be observed. While in the cells of the original stem line 77 cells out of 100 cells had' 1 metacentric chromosome (SHANNON 1972) the sublines investigated had 6 to 12 metacentric chromosomes per cell. The data concerning the doubling time of the sublines are presented in Table 2. The results are based on a regression analysis of the growth curves and show that all the 4 lines did not vary significantly from each other in their doubling time.
llS
S. B. REDDY, W. A, LINDEN and K. LUBBERS
4_103
r-- - - - - .,.,. .- - - - - - - - - - - - - --,
3 ~
-... "ii 0 0
2
GI .0
E ::::J
Z
/\,
~
00
35
70
Relative DNA content Fig. 2. A typical DNA distribution pattern of L-strain mouse fibroblast cells, subline F, obtained with the pulse cytophotometer. The first peak at 35 represents G1 DNA content and the second peak at 70 (G 2 + M) DNA content. Table 2. Cell doubling times obtained with 4 different sublines of L-929 mouse fibroblasts. Subline
Doubling time [h]
95 % Confidence interval [h]
L-929 HH L-929 F L-929 D L-929 E
23.4 24.9 20.8 22.9
20.5 27.4 23.4 26,5 18.1. .. 24.4 21.1. .. 25.0
Table 3. Phase distribution patterns of 4 exponentially growing sublinss of mouse fibroblasts as revealed by pulse cytophotometry. The percentage of cells in each phase was calculated by a computer program. Subline
L-929 L-929 L-929 L-929
HH F D E
Percentage of cells in the different phases of cell cycle
G1
S
G2
62 59 53 63
29 25 32 24
9 16 15 13
+M
A typical DNA histogram of L-cells in culture is presented in Fig. 2. From the DNA distributions of the individual sublines the percentages of cells in the different phases of the cell cycle were derived (Table 3). The values are means from 5 repetitive experiments. No statistically significant differences could be detected.
S. B. REDDY u. a., A comparative study of four sublines,
119
Table 4. The mitotic indices [%] of 4 sublines of mouse fibroblasts after mechanical synchronization. Subline
L-929 L-929 L-929 L·929
HH F D E
Mitotic selection No. 1
2
3
4
5
23 72 24 38
43 76 28 47
44 79 22 54
42 83 29 56
40 84 26 58
The synchronization data presented in Table 4 show that the mitotic index achieved with the 4 sublines ranged from 29% to 84 %. BREMERSKOV and LINNEMANN (1969) indicate that the sublines of L-929 mouse fibroblasts used in their experiments yielded different degrees of synchronization ranging from 66.8 % to 79.8 %. The different yield of synchronized cells obtained with the 4 sublines investigated could be due to the different morphology of the sublines, the HH-cells being more spindle shaped and the F-cells having a more polyangular appearance while the E- and Dcells showed up like typical fibroblasts. Though the 4 sublines did not differ significantly in their growth characteristics (doubling time, phase distribution) considerable changes in karyology compared to the stem line could be observed. Also compared to each other the 4 sublines showed differences in karyology, morphology and degree of synchrony obtained after mitotic selection. The results show that karyology is a sensitive marker showing changes of a cell line earlier than the analysis of growth characteristics. Thus, rather frequent chromosome analysis of tissue culture cells can be recommended as a prerequisite for reproducible results. Literature BAISCH, H., GOHDE, W., and LINDEN, W. A., Analysis of PCP-Data to Determine the Fraction of Cells in the Various Phases of Cell Cycle. Rad. Environ. Biophys. 12,31-39 (1975). BREMERSKOV, V., and LINNEMANN, R., Some Effects of Daunomycin on the Nucleic Acid Synthesis in Synchronized L-cells. Europ. J. Cancer 5,317-330 (1969). GOHDE, W., and DITTRICH,W., Impulsfluorometrie - Ein neuartiges DurchfluJ3verfahren zur ultraschnellen Mengenbestimrnung von Zellinhaltsstoffen. Acta histochem. (Jena) 10 (Suppl.), 429 bis 437 (1971). Hsu, T. C., Mammalian Chromosomes in Vitro. XIII. Cyclic and Directional Changes of Population Structure. J. Nat. Cancer Inst. 25, 1339-1350 (1960). LINDEN, W. A., BAISCH, R., v. CANSTEIN, L., KONIG, K., and v. CANSTEIN, M., Impulsecytophotometric Studies on the Effects of Daunomycin on Synchronised L-cells. Europ. J. Cancer 10, 647 to 651 (1974). ZYWIETZ, F., LANDEN, R., and WENDT, C., Synchronisation des Teilungszyklus von L-Zellen in der G 2-Phase durch fraktionierte Rontgenbestrahlung und Daunomycin. Strahlentherapie 146, 216-225 (1973). SHANNON, J. E. (Ed.), American Type Culture Collection. Registry of Animal Cell Lines. CCL 1.1 ATCC. Rockville 1972. Address: Doz. Dr. Dr. W. LINDEN, Dr. S. B. REDDY und K. LUBBERS, Institut ftir Biophysik, 2000 Hamburg 20, Martinistral3e 52.
Institut fur Bi ophysik und Strahlenbiologie der Universitat Hamburg (Goschaftsfuhrender Direktor: Prof. Dr. H. JUNO)
A comparative study of four sublines of mouse fibroblast cells") By
SURAM
B.
REDDY, WAL]'RIED
A.
LINDEN
and
KLAUS LUBBERS
With 2 figures (Received September 4, 1975)
Summary Cell doubling time, DNA distribution pattern, frequen cy distribution of chromosome numbers per cell and degree of synchrony obtained after mitotic sel ection were studied in 4 different sublines of mouse fibroblast cell s, N CTC L.929, in cult u re . The 4 su bline s did not differ with respect to their cell kinetic data but showed changes of the ch rom osome pattern compared to the stemline and compared to ea ch other sugges t ing that ch romosome analysis provides a very sensitive means t o detect ev olu t ia na ry cha nges of a permanent cell line.
Introduction It is of obvious interest to note that permanent cell lines are in a steady evolution with increasing number of subcultivations. This evolution of a cell population may involve eno rmous differences in karyology, growth potentialities and other characteristic features in comparison with the original cell line. Also for mouse fibroblasts, NCTC line 929, cyclic and directional changes of population structure were reported (Hsu 1960). In our present study attempts were made to characterize 4 sublines of L-strain mouse fibroblasts (NCTC 929) by measuring chromosome number, cellular growth, degree of synchronization, and cellular DNA distribution patterns. The results will show differences between the sublines demonstrating the importance of selecting the appropriate subline in each experimental design.
Materials and Methods Cell system Four su blines of L-929 m ou se f ibroblast ce lls HH, D, F and E wer e employed in our investigation . The mon olayer cultu res were initiated in 180 ml flask s in a modified EAGLE'S medium (No . 47 340 A, S erva, H e idelb erg) supplemented with 5 % fetal ca lf se rum , peni cillin and stre p t om yci n . I) Supported by the D eutsche Forschungsgemeinschaft, B onn-Bad God esb crg.
116
S. B. REDDY, W. A. LINDEN and K. LUBBERS
Growth assays Approximately 1()6 cells were planted in 180 ml glass flasks in 15 ml medium. The growth curves of the cells were dertermined by counting the cells for every 12 h over a period of 5 days. For this purpose the cells were trypsinized with 0.05 % trypsin (Difeo, 1: 250), released from monolayer and counted in a haemocytometer,
Chromosome preparation The cells were grown on 2 glass slides placed in a glass flask. 48 h later, the cultures obtained a hypotonic shock by exposure to 0.075 M KCI at 37°C for 12 min. To accumulate metaphases the cultures were treated with 0.02 % colcemid 1 h before harvesting.
Synchronization The cells were propagated in 1200 ml Raux flasks with an input of 7.5 X 106 in 80 ml medium. After 48 h of incubation ,when the cells had reached a confluent layer, mitotic cells were released by manual shaking. The details of the synchronization procedure were the same as described earlier (LINDEN et al. 1974). For each subline the synchronization procedure was repeated 5 times over a period of 10 h at 2 h intervals. For determination of mitotic index at least 500 cells were scored from each slide.
Pulse cytophotometry DNA distribution patterns of all the sublines were measured using a pulse cytophotometer (ICP 11, Phywe, GOttingen). For this purpose cells in exponential growth i. e. 48 h after subcultivation were fixed and stained with ethidium bromide. The details of the pulse cytophotometric technique were described elsewhere (GOHDE and DITTRICH 1971, LINDEN et al. 1973). A computer evaluation of the DNA histograms obtained yielded the percentage of cells in the different phases of the cell cycle (BAISCH et al. 1975).
Results and Discussion The karyology studies revealed that the 4 sublines have different distributions of chromosome numbers per cell (Fig. 1). Although subline HH shows a similar frequency distribution around the maximum as was found for subline F, the latter contains a substantial portion of cells having chromosome numbers higher than 80. The same holds when we compare sublines D and E. The most frequent chromosome numbers, the average chromosome numbers and the percentages of "polyploid" cells having chromosome numbers greater than 80 are plotted in Table 1. The fact that the lines D and F show nearly equal figures of the mean chromosome numbers whereas their frequency distributions are obviously quite different derives from subline D having 4.9 % polyploid cells compared to 12.6 % in subline F. This finding indicates once more that the mean chromosome number does not contain sufficient information to enable a reasonable comparison between 2 sublines of mammalian cells. It has been described by SHANNON (1972) that the modal chromosome number of L-strain mouse fibroblast cells derived from the parental strain from normal subcutaneous areolar and adipose tissue of a 100 day-old male C3 H/A mouse was 66. After a couple of years the same strain has undergone remarkable changes in chromosome constitution. Hsu (1960) cited that in subline L-P55 after 2 years of routine
117
A comparative study of four sublines 30
HH 20 10 0
D 20 VI
...J ...J
ILl U u,
0
10 0
0= ILl III
~
::J Z
F
20 10 0 20 10
oL..-_-'---_......................LLL._.....J._----'_---J 40 o 20 100 120 60 80 CHROMOSOME NUMBER PER CELL Fig.!. Frequency distributions of chromosome numbers per cell of Hamburg (HH), Diisseldorf (D), Freiburg (F) and Eppendorf (E) sublines of L-929 mouse fibroblasts. Table 1. Chromosome analysis of 4 different sublines of L-929 mouse fibroblasts. Subline
Most frequent chromosome number
Mean chromosome Percentage of "polyploid" cells number
HH D F E
60 64 59 64
61.2 64.4 64.7 61.7
1.0 4.9 12.6 0
subculture system the original stem line's chromosome number dropped from 68 to 64. From foregoing arguments it appears that this spectrum of population changes in vitro is a common observation. Also changes of the chromosome type can be observed. While in the cells of the original stem line 77 cells out of 100 cells had' 1 metacentric chromosome (SHANNON 1972) the sublines investigated had 6 to 12 metacentric chromosomes per cell. The data concerning the doubling time of the sublines are presented in Table 2. The results are based on a regression analysis of the growth curves and show that all the 4 lines did not vary significantly from each other in their doubling time.
llS
S. B. REDDY, W. A, LINDEN and K. LUBBERS
4_103
r-- - - - - .,.,. .- - - - - - - - - - - - - --,
3 ~
-... "ii 0 0
2
GI .0
E ::::J
Z
/\,
~
00
35
70
Relative DNA content Fig. 2. A typical DNA distribution pattern of L-strain mouse fibroblast cells, subline F, obtained with the pulse cytophotometer. The first peak at 35 represents G1 DNA content and the second peak at 70 (G 2 + M) DNA content. Table 2. Cell doubling times obtained with 4 different sublines of L-929 mouse fibroblasts. Subline
Doubling time [h]
95 % Confidence interval [h]
L-929 HH L-929 F L-929 D L-929 E
23.4 24.9 20.8 22.9
20.5 27.4 23.4 26,5 18.1. .. 24.4 21.1. .. 25.0
Table 3. Phase distribution patterns of 4 exponentially growing sublinss of mouse fibroblasts as revealed by pulse cytophotometry. The percentage of cells in each phase was calculated by a computer program. Subline
L-929 L-929 L-929 L-929
HH F D E
Percentage of cells in the different phases of cell cycle
G1
S
G2
62 59 53 63
29 25 32 24
9 16 15 13
+M
A typical DNA histogram of L-cells in culture is presented in Fig. 2. From the DNA distributions of the individual sublines the percentages of cells in the different phases of the cell cycle were derived (Table 3). The values are means from 5 repetitive experiments. No statistically significant differences could be detected.
S. B. REDDY u. a., A comparative study of four sublines,
119
Table 4. The mitotic indices [%] of 4 sublines of mouse fibroblasts after mechanical synchronization. Subline
L-929 L-929 L-929 L·929
HH F D E
Mitotic selection No. 1
2
3
4
5
23 72 24 38
43 76 28 47
44 79 22 54
42 83 29 56
40 84 26 58
The synchronization data presented in Table 4 show that the mitotic index achieved with the 4 sublines ranged from 29% to 84 %. BREMERSKOV and LINNEMANN (1969) indicate that the sublines of L-929 mouse fibroblasts used in their experiments yielded different degrees of synchronization ranging from 66.8 % to 79.8 %. The different yield of synchronized cells obtained with the 4 sublines investigated could be due to the different morphology of the sublines, the HH-cells being more spindle shaped and the F-cells having a more polyangular appearance while the E- and Dcells showed up like typical fibroblasts. Though the 4 sublines did not differ significantly in their growth characteristics (doubling time, phase distribution) considerable changes in karyology compared to the stem line could be observed. Also compared to each other the 4 sublines showed differences in karyology, morphology and degree of synchrony obtained after mitotic selection. The results show that karyology is a sensitive marker showing changes of a cell line earlier than the analysis of growth characteristics. Thus, rather frequent chromosome analysis of tissue culture cells can be recommended as a prerequisite for reproducible results. Literature BAISCH, H., GOHDE, W., and LINDEN, W. A., Analysis of PCP-Data to Determine the Fraction of Cells in the Various Phases of Cell Cycle. Rad. Environ. Biophys. 12,31-39 (1975). BREMERSKOV, V., and LINNEMANN, R., Some Effects of Daunomycin on the Nucleic Acid Synthesis in Synchronized L-cells. Europ. J. Cancer 5,317-330 (1969). GOHDE, W., and DITTRICH,W., Impulsfluorometrie - Ein neuartiges DurchfluJ3verfahren zur ultraschnellen Mengenbestimrnung von Zellinhaltsstoffen. Acta histochem. (Jena) 10 (Suppl.), 429 bis 437 (1971). Hsu, T. C., Mammalian Chromosomes in Vitro. XIII. Cyclic and Directional Changes of Population Structure. J. Nat. Cancer Inst. 25, 1339-1350 (1960). LINDEN, W. A., BAISCH, R., v. CANSTEIN, L., KONIG, K., and v. CANSTEIN, M., Impulsecytophotometric Studies on the Effects of Daunomycin on Synchronised L-cells. Europ. J. Cancer 10, 647 to 651 (1974). ZYWIETZ, F., LANDEN, R., and WENDT, C., Synchronisation des Teilungszyklus von L-Zellen in der G 2-Phase durch fraktionierte Rontgenbestrahlung und Daunomycin. Strahlentherapie 146, 216-225 (1973). SHANNON, J. E. (Ed.), American Type Culture Collection. Registry of Animal Cell Lines. CCL 1.1 ATCC. Rockville 1972. Address: Doz. Dr. Dr. W. LINDEN, Dr. S. B. REDDY und K. LUBBERS, Institut ftir Biophysik, 2000 Hamburg 20, Martinistral3e 52.
