- Email: [email protected]
Primary culture of human diploid cells and its long-term transfer in a serum-free medium
470
Preliminary notes
cells and could implicate them as myoepithelial. The authors would like to thank Mrs P. Purkis for her excellent assistance in collecting the milk samples.
References I. Buehring, G C, J natl cancer inst 49 (1972) 1433. 2. Holquist, D G & Papanicolau, G N, Ann NY acad sci 63 (1956) 1422. 3. Taylor-Papadimitriou, J, Shearer, M & Tilly, R J, J natl cancer inst 58 (1977) 1563. 4. Ceriani, R L, Taylor-Papadimitriou, J, Peterson, J A &Brown, P, In vitro 15 (1979) 356. 5 Fentiman, I S, Taylor-Papadimitriou, J & Stoker, M, Nature 264 (1976) 760. 6. Fentiman, I S & Taylor-Papadimitriou, J, Nature 269 (1977) 156. 7. Fentiman, I S, Hurst, J, Ceriani, R L & TaylorPapadimitriou, J, Cancer res 39 (1979) 4739. 8. Taylor-Papadimitriou, J, Purkis, P & Fentiman, I S, J cell physiol 102(1980) 321. 9. Taylor-Papadimitriou, J, Shearer, M & Stoker, M, Int j cancer 20 (1977) 903. 10. Hallowes, R C, Bone, E J & Jones, W, Tissue culture in medical research (ed R J Richards & K T Rajan) vol. 2, p. 213. Pergamon Press, Oxford (1980). 11. Pitts, J D & Simms, J W, Exp cell res 104 (1977) 153. 12. Lane, E B, J cell biol (1981). In press. 13. Taylor-Papadimitriou, J, Peterson, J A, Arklie, J, Burchell, J, Ceriani, R L & Bodmer, W F, Int j cancer (1981). In press. 14. Hackett, A J, Smith, H S, Springer, E L, Owens, R B, Nelson Rees, W A, Riggs, J L & Gardner, M B: J natl cancer inst 58 (1977) 1795. 15. Bennett, D C, Peachey, L A, Durbin, H & Rudland, P S, Cell I5 (1978) 283. Received January 12, 1981 Revised version received April 10, 1981 Accepted April 15, 1981
Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 00 14.4827B l/080470-05$02.0010
term serial culture. The basal medium consists of the components of modified Eagle’s minimum essential medium (MEM) and non-essential amino acids, various growth factors and trace metals. Human fibronection (FN) and bovine serum albumin (BSA) were added. BSA was found to be essential for long-term serial culture in the presence of FN. Incubation in a gaseous environment of low oxygen (7 % 0,) and lowtemperature trypsinization at the time of transfer were also found to be important for growth in serum-free medium.
To date, various serum-free culture media for mammalian cells have been reported by many workers [l-4]. Recently serum-free media containing various growth factors for clonal growth were also reported by Bottenstein et al. [5]. However, most of these media were developed for transformed or neoplastic cells and not for untraxformed or normal cells. Early this year, Phillips & Cristofalo [6] and Walthall & Ham [7] simultaneously reported serum-free media which supported the growth of human embryo diploid fibroblasts, but could not be used for primary cultivation or for longterm serial culture. Previously we described a serum-free medium for human fibroblasts [8], but it too did not support long-term serial culture. We have now improved the medium and succeeded in both primary culture of human diploid cells and its long-term serial transfer, as well as using serum-containing conventional medium, although our medium is not wholly defined, chemically. Materials and Methods The cells were obtained from lung tissue of 16-week-old human embryos, by dispersing the tissue with trypsin (Difco Labs, 1 : 250). For experiments on growth kinetics, the cells were cultured in Eagle’s Basal Medium (BME) supplemented with 10% fetal bovine serum (FBS) and subcultured at 1 : 2 dilution for 20 cell generations. They were then cultivated further in serum-free medium. For long-term cultivation, cells just isolated from human embryo lung were obtained as follows: Lung tissue was minced with knife and scissors. This mince was sieved through a stainless steel 30 mesh net and then treated with a solution of 300 pg/ml trypsin (x2 crystalline: Miles Co.) at room temperature for
Cells and stock cultures.
Primary culture of human diploid cells and its long-term transfer in a serum-free medium ISA0 YAMANE, MIKIO KAN, HIROYOSHI HOSHI and YOSHIKI MINAMOTO, Demzrtmenf of Ceil Biology, Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980, Japan
Using a serum-free culture medium, primary human embryo tibroblasts can be grown in long-
Summary.
Exp Cdl Rrs 134 (1981)
Preliminary notes
471
Table 1. Composition of medium RITC 80-7
Modified MEM (ref. [9]) Amino acids L-Aspartic acid L-Glutamine Glycine L-Glutamic acid L-Proline L-Serine Vitamin and hormones Folinic acid 3,3’,5-Triiodo-L-thyronine m-EGF Transferrin (bovine) Insulin Vitamin Bi2 Biotin Other organic compounds Putrescine 2HCI Sodium pyruvate Choline chloride Thymidine Hypoxanthine Trace elements CuSO, 5H,O FeSO, 7H;O MnSO, 7H,O WHMbOz4 Hz0 NiCI, 6H,O NH,i’O, H*SeO, Buffer Hepes NaOH NaHCO,
and is omitted of glutamine, sodium bicarbonate. Insulin was added to the medium after dissolution in small amounts of 1 N HCI and hvooxanthine was mdl added after dissolution in small amounts of 1 N NaOH. Zinc was not added to the medium, but it was 9 400 detected there by means of the atomic absorption method in a concentration of 0.2 mg/l, possibly-as a 13.3 contaminant. Medium pH was adjusted to 7.3 with Hepes and NaOH in air (NaHCO,-free). It was usually 292 enriched with FN and BSA. The control medium was 7.5 BME supplemented with 10% FBS. 0.15 Cell trypsinization and plating. Cell trypsinization 3.5 was carried out with crystalline trypsin (x2 crys10.5 tallized; Miles Co.) at a low temperature (4-5°C) using 300 pg/ml of the trypsin for cultures grown in o.oOOo5 serum-containing medium, and 30 pg/ml for cultures 0.0002 grown in serum-free medium [lo]. The trypsinized 0.01 cells were washed three times with Ca”+-, Me’+-free 10 PBS and inoculated into plastic Petri dishes
Results Growth kinetics and serial cultures of human diploid cells in RITC 80-7 medium. Powdered MEM was purchased from Nissui Seiyaku We developed a serum-free medium, RITC Co., Ltd, Tokyo, Japan. Insulin (24.4 unit/mg) was 80-7, by estimating the growth-promoting purchased from Shimizu Seiyaku Co., Ltd (Tokyo), effect of each medium component (by mouse-epidermal growth factor (m-EGF) from Collaborative Research Inc. (Waltham, Mass.), bovine adopting those described and omitting such transferrin from US Biochemical Corp. (Cleveland, ineffective components as fibroblast- and Ohio), 3,3’5-triiodo-L-thyronine (T3) from Fluka AG, Chemische Fabrik, Switzerland. platelet-derived growth factors). The initial inoculum was 20000 cells/l.5 ml medium and incubation was for 5-7 days at 37°C. IO min. The treated cells and cell clusters were washed As shown in table 1, this medium contains three times with PBS and inoculated to 3 ml of RITC 80-7 supplemented with human fibronectin (FN) and the ingredients of modified MEM and is bovine serum albumin (BSA) in the cell number of 4x supplemented with non-essential amino lO*/dish. The primary culture reached confluence in 5 hormones and trace days and was then transferred serially with the use of acids, vitamins, low temperature trypsinization in the cell number of amounts of rare metals, as well as growth 4x 104/dish. Culture media. Table 1 shows the ingredients of factors such as insulin, epidermal growth the new serum-free medium RITC 80-j. Modified factor (EGF) and transferrin. Fig. 1 shows MEM is different from original MEM, in that it contains succinate (75 mg/l), sodium succinate (100 mg/l) growth curves of human diploid fibroblasts 3 300 300 1400
Exp Cd
Rrs 134 (198/J
412
Preliminary notes
: z
IO
0 FN BSA-
Fi,e. 1. Time course of the growth of human dinloid fibroblasts (PDL-22). Stock-cultures fully grown in BME plus 10% FBS medium were shifted to various media including serum-free ones. Cells were grown in BME olus 10% FBS (0). RITC 80-7 DIUSFN (12 me/l). BSA (5 g/l) (0), RITC 80-7 (A) medium respectivily: BSA was added into the medium after 1 h incubation. The average values of duplicate experiments, which varied less than lo%, are presented.
in serum-free RITC 80-7 and in BME containing 10% FBS. The initial lag time in RITC 80-7 was longer than in serum-supplemented BME. When serial cultivation was attempted using RITC 80-7, only a few transfers (about 15 cell generations) could be carried out (fig. 3). However, cell dispersion treatment with cold trypsin was found to be quite helpful in the transfer of cultures grown in serum-free medium. The effect of human fibronection (FN) and bovine serum albumin (BSA). BSA was shown by us to be markedly growthenhancing for various cells with suspension growth [ 171,but in its concentration of more than 200 pg/ml, BSA was shown to interfere with the cellular attachment to the culture vessel. However, BSA was growth-promoting when added only after the cells tightly attached to the substrate (24 h incubation at 37°C after cell inoculation). This was Erp Cdl Res 134 (198/J
-
I
5
3 -
Ii 3 5
12 -
12 I
I2 5
rn
g/l g/l
Fig. 2. Effect of FN and BSA on cell growth. The cells were cultured in BME plus 10% FBS medium until 24-PDL, and shifted to various media. White column, growth in RITC 80-7 medium for 5 days culture; dotted column, growth in BME plus 10% FBS medium. BSA was added into the medium after I h
interpreted to mean that during 24 h of incubation, the fibroblasts produced the FN necessary for cellular attachment [13]. Accordingly, FN (10 mg/l) was added to the medium together with BSA (5 g/l) at the time when cells were inoculated. This permitted satisfactory attachment and the same cellular yield as BME supplemented with 10% FBS (figs 1, 2). Primary culture of the diploid fibroblasts and its long-term, serial transfer. Fig. 3 shows cumulative growth curves of both serial cultures in RITC 80-7 and in serumcontaining BME. As shown in fig. 3, cumulative growth so far increased straight on with use of RITC up to 76 DPL (population doubling level), whereas the culture with serum-containing medium reached up to 73 PDL and did not grow any further. The discrepancy in lifespan between these two cultures is not certain to be significant, but is anyhow shown in the same or similar lines of the experimental cultures.
Preliminary notes
473
We employed trypsinization at cold temperature to diminish the cellular damage following McKeehan [IO], because serum-free culture did not contain any protease inhibitor present in serum. The gaseous environment (7% 0,) was chosen after preliminary experiments under various conditions. Balin et al. [15] reported that the most favorable partial tension of oxygen was about 3% or less for the culture of human diploid fibroblasts, but they employed agitated cultures, where0 0 60 20 40 80 100 as we used stationary cultures. Therefore, Culture days the difference in optimal oxygen tension Fig. 3. Long-termed serial culture of human diploid may be due to the effect of agitation [16]. fibroblasts derived from embryonic lung tissue. The culture was made in BME plus 10% FBS medium The growth-enhancing effect of low oxygen (0) and RITC SO-7plus FN (12 mg/l), BSA (5 g/l), tension was observed, whether serum was medium (O), RITC SO-7medium (A). The subculture was made every 5 days. BSA was added into the me- present or not, but became more promidium after I h incubation. nent as the amount of BSA was increased. The mechanism by which BSA promotes cellular growth has been exhaustively studDiscussion ied using cultured Yoshida sarcoma cells: Employing serum-free RITC 80-7 medium Unsaturated fatty acids bound to BSA ensupplemented with FN and BSA, we suc- hance the cellular growth and membrane ceeded in culturing primary human embryo fluidity of cultured cells [17, 181which was fibroblasts and transferring them through preliminarily proved with human fibrolong-term serial passages. An earlier me- blasts. This should also function in the case dium deficient in thymidine, hypoxanthine, of fibroblast culture because when employand vitamin Blz (RITC 78-6 medium) did ing defatted BSA, there was no growthnot support more than three serial transfers promoting activity (unpublished data). [14]. Inclusion of FN and BSA in RITC Also, insulin was not detected in the BSA SO-7medium improved both cellular growth sample by means of radioimmuno assay, and the recovery from the cellular damage although the BSA sample used for this due to trypsinization. When BSA was added study was not pure (95-99 % purity). On the to the medium beginning 24 h later after other hand, transferrin and c-u,-antitrypsin inoculation of the cells, FN supplementa- was detected by means of immunoelectrotion could be omitted perhaps because of phoresis, but the content of these proteins FN synthesis during the first 24 h, but was too low to promote cellular growth cultivation was not successful without FN (data not shown). when BSA was added at the very beginThe serum-free medium described here ning (unpublished data). We attempted to can be used to grow all kinds of human reduce the amount of BSA added to the established epithelial cell lines including medium, but so far we could not reduce it HeLa, Hep 2, FL, as well as human diploid below 1 g/l medium. fibroblasts (unpublished data). Also the
474
Preliminary notes
slight modification of this medium enables the abundant growth of various human lymphoblasts in the absence of FN and in the presence of BSA. This fact may contradict Ham’s opinion that each cell requires an individual separate culture medium [ 193, but our serum-free medium contains BSA, whereas the medium of Ham does not. This is the first report which deals with both primary culture of human diploid fibroblasts in a serum-free medium and its long-term serial transfer. Such long-term serial cultures have so far only been achieved when excellent quality FBS was added to the culture medium, as the lot quality of each serum batch distinctly controls the results of the serial culture. Therefore the use of serum for such cultures is unfavorable and inconvenient from the standpoint of experimental reproducibility, taking into account the popular and widespread employment of long-term cultures of human diploid cells for various gerontological studies in vitro. This serum-free medium should serve as an efficient tool for research when employing human cultured cells, as this medium is not wholly chemically defined, though more defined than whole serum-supplemented medium. This work was partially supported by Grant-inAid (Cancer Special Research) of the Ministry of Education, Science and Culture, Japan.
References I. Sanford. K K. Dunree. L T & Covaleskv. A B, Exp cell res 31 (1983) 345. 2. Ham, R G, Proc natl acad sci US 53 (1965) 288. 3. Higuchi, K, J cell physiol 75 (1970) 65. 4. Takaoka, T & Katsuta, H, Exp cell res 67 (1971) 295. 5. Bottenstein, J, Hayashi, I, Hutchings, S, Masui, H, Mather, J, McClure, D B, Ohasa, S, Rizzino, A, Sato, G, Serrero, G, Wolfe, R & Wu, R, Method in enzymology, vol. 58. Academic Press, New York (1979). 6. Phillips, P D & Cristofalo, V J, In vitro 16 (1980) 250. 7. Walthall, B J & Ham, R G, In vitro I6 (1980) 250. 8. Yamane, I. Kan, M & Hoshi. H. Growth and Exp Cell RES 134 (198/J
growth factors. University of Tokyo Press, Tokyo (1980). 9. Yamane, I, Matsuya, Y & Jimbo, K, Proc sot exp biol med 127(1968) 335. IO. McKeehan, W L, Cell biol int rep I (1977) 335. I I. Taylor, W G, Camalier, R F & Sanford, K K, J cell ohvsiol 95 (1978) 33. 12. biento, M‘& Vaheri, A, Biochem I83 (1979) 33 I. 13. Grinnell. F & Feld. M K. Cell I7 (1979) 117. 14. Kan, M ‘& Hoshi. H. Soshikibaiyb (in japanese) 5 (1979) 22 I. 15. Balin, A K, Goodman, D B P, Rasmussen. H & Cristofalo, V J, J cell physiol89 (1976) 235. 16. Werrlein, R J & Glinos, A D, Nature 251 (1974) 317. 17. Yamane, I, Murakami, 0 & Kato, M, Proc sot exp biol med 149 (1975) 439. IS. Yamane, I & Tomioka, F, Cell biol int rep 3 (1979) 515. 19. Ham, R G, The handbook of experimental pharmacology. Springer Verlag, Berlin (1980). Received January 16, 1981 Revised version received April 6, 1981 Accepted April 15, I981
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/81/080474-04~2.00/0
Binding of thrombin to normal and transformed fibroblasts depends on cell density PIRKKO
POHJANPELTO
and ANTTI
Department of Virology, University 00290 Helsinki 29, Finland
VAHERI,
of Helsinki,
SF-
Summary. Binding of biologically active [‘251]thrombin
to several normal and transformed human and chicken cell lines was found to depend on cell density; more [‘ZSl]thrombin per cell was bound to sparse than to dense cultures. When normal and transformed cells were compared at equal densities the previously reported difference in [‘251]thrombin binding was not evident.
Thrombin is a potent growth factor for normal chicken fibroblasts [l], human fibroblasts [2] and Chinese hamster lung cells [3], but some transformed cells, e.g. SV40transformed human fibroblasts, HeLa cells, and the continuous monkey kidney cell lines BS-C-I and Vero can also be stimulated by thrombin [4]. The difficulty with
Preliminary notes
cells and could implicate them as myoepithelial. The authors would like to thank Mrs P. Purkis for her excellent assistance in collecting the milk samples.
References I. Buehring, G C, J natl cancer inst 49 (1972) 1433. 2. Holquist, D G & Papanicolau, G N, Ann NY acad sci 63 (1956) 1422. 3. Taylor-Papadimitriou, J, Shearer, M & Tilly, R J, J natl cancer inst 58 (1977) 1563. 4. Ceriani, R L, Taylor-Papadimitriou, J, Peterson, J A &Brown, P, In vitro 15 (1979) 356. 5 Fentiman, I S, Taylor-Papadimitriou, J & Stoker, M, Nature 264 (1976) 760. 6. Fentiman, I S & Taylor-Papadimitriou, J, Nature 269 (1977) 156. 7. Fentiman, I S, Hurst, J, Ceriani, R L & TaylorPapadimitriou, J, Cancer res 39 (1979) 4739. 8. Taylor-Papadimitriou, J, Purkis, P & Fentiman, I S, J cell physiol 102(1980) 321. 9. Taylor-Papadimitriou, J, Shearer, M & Stoker, M, Int j cancer 20 (1977) 903. 10. Hallowes, R C, Bone, E J & Jones, W, Tissue culture in medical research (ed R J Richards & K T Rajan) vol. 2, p. 213. Pergamon Press, Oxford (1980). 11. Pitts, J D & Simms, J W, Exp cell res 104 (1977) 153. 12. Lane, E B, J cell biol (1981). In press. 13. Taylor-Papadimitriou, J, Peterson, J A, Arklie, J, Burchell, J, Ceriani, R L & Bodmer, W F, Int j cancer (1981). In press. 14. Hackett, A J, Smith, H S, Springer, E L, Owens, R B, Nelson Rees, W A, Riggs, J L & Gardner, M B: J natl cancer inst 58 (1977) 1795. 15. Bennett, D C, Peachey, L A, Durbin, H & Rudland, P S, Cell I5 (1978) 283. Received January 12, 1981 Revised version received April 10, 1981 Accepted April 15, 1981
Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 00 14.4827B l/080470-05$02.0010
term serial culture. The basal medium consists of the components of modified Eagle’s minimum essential medium (MEM) and non-essential amino acids, various growth factors and trace metals. Human fibronection (FN) and bovine serum albumin (BSA) were added. BSA was found to be essential for long-term serial culture in the presence of FN. Incubation in a gaseous environment of low oxygen (7 % 0,) and lowtemperature trypsinization at the time of transfer were also found to be important for growth in serum-free medium.
To date, various serum-free culture media for mammalian cells have been reported by many workers [l-4]. Recently serum-free media containing various growth factors for clonal growth were also reported by Bottenstein et al. [5]. However, most of these media were developed for transformed or neoplastic cells and not for untraxformed or normal cells. Early this year, Phillips & Cristofalo [6] and Walthall & Ham [7] simultaneously reported serum-free media which supported the growth of human embryo diploid fibroblasts, but could not be used for primary cultivation or for longterm serial culture. Previously we described a serum-free medium for human fibroblasts [8], but it too did not support long-term serial culture. We have now improved the medium and succeeded in both primary culture of human diploid cells and its long-term serial transfer, as well as using serum-containing conventional medium, although our medium is not wholly defined, chemically. Materials and Methods The cells were obtained from lung tissue of 16-week-old human embryos, by dispersing the tissue with trypsin (Difco Labs, 1 : 250). For experiments on growth kinetics, the cells were cultured in Eagle’s Basal Medium (BME) supplemented with 10% fetal bovine serum (FBS) and subcultured at 1 : 2 dilution for 20 cell generations. They were then cultivated further in serum-free medium. For long-term cultivation, cells just isolated from human embryo lung were obtained as follows: Lung tissue was minced with knife and scissors. This mince was sieved through a stainless steel 30 mesh net and then treated with a solution of 300 pg/ml trypsin (x2 crystalline: Miles Co.) at room temperature for
Cells and stock cultures.
Primary culture of human diploid cells and its long-term transfer in a serum-free medium ISA0 YAMANE, MIKIO KAN, HIROYOSHI HOSHI and YOSHIKI MINAMOTO, Demzrtmenf of Ceil Biology, Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980, Japan
Using a serum-free culture medium, primary human embryo tibroblasts can be grown in long-
Summary.
Exp Cdl Rrs 134 (1981)
Preliminary notes
471
Table 1. Composition of medium RITC 80-7
Modified MEM (ref. [9]) Amino acids L-Aspartic acid L-Glutamine Glycine L-Glutamic acid L-Proline L-Serine Vitamin and hormones Folinic acid 3,3’,5-Triiodo-L-thyronine m-EGF Transferrin (bovine) Insulin Vitamin Bi2 Biotin Other organic compounds Putrescine 2HCI Sodium pyruvate Choline chloride Thymidine Hypoxanthine Trace elements CuSO, 5H,O FeSO, 7H;O MnSO, 7H,O WHMbOz4 Hz0 NiCI, 6H,O NH,i’O, H*SeO, Buffer Hepes NaOH NaHCO,
and is omitted of glutamine, sodium bicarbonate. Insulin was added to the medium after dissolution in small amounts of 1 N HCI and hvooxanthine was mdl added after dissolution in small amounts of 1 N NaOH. Zinc was not added to the medium, but it was 9 400 detected there by means of the atomic absorption method in a concentration of 0.2 mg/l, possibly-as a 13.3 contaminant. Medium pH was adjusted to 7.3 with Hepes and NaOH in air (NaHCO,-free). It was usually 292 enriched with FN and BSA. The control medium was 7.5 BME supplemented with 10% FBS. 0.15 Cell trypsinization and plating. Cell trypsinization 3.5 was carried out with crystalline trypsin (x2 crys10.5 tallized; Miles Co.) at a low temperature (4-5°C) using 300 pg/ml of the trypsin for cultures grown in o.oOOo5 serum-containing medium, and 30 pg/ml for cultures 0.0002 grown in serum-free medium [lo]. The trypsinized 0.01 cells were washed three times with Ca”+-, Me’+-free 10 PBS and inoculated into plastic Petri dishes
Results Growth kinetics and serial cultures of human diploid cells in RITC 80-7 medium. Powdered MEM was purchased from Nissui Seiyaku We developed a serum-free medium, RITC Co., Ltd, Tokyo, Japan. Insulin (24.4 unit/mg) was 80-7, by estimating the growth-promoting purchased from Shimizu Seiyaku Co., Ltd (Tokyo), effect of each medium component (by mouse-epidermal growth factor (m-EGF) from Collaborative Research Inc. (Waltham, Mass.), bovine adopting those described and omitting such transferrin from US Biochemical Corp. (Cleveland, ineffective components as fibroblast- and Ohio), 3,3’5-triiodo-L-thyronine (T3) from Fluka AG, Chemische Fabrik, Switzerland. platelet-derived growth factors). The initial inoculum was 20000 cells/l.5 ml medium and incubation was for 5-7 days at 37°C. IO min. The treated cells and cell clusters were washed As shown in table 1, this medium contains three times with PBS and inoculated to 3 ml of RITC 80-7 supplemented with human fibronectin (FN) and the ingredients of modified MEM and is bovine serum albumin (BSA) in the cell number of 4x supplemented with non-essential amino lO*/dish. The primary culture reached confluence in 5 hormones and trace days and was then transferred serially with the use of acids, vitamins, low temperature trypsinization in the cell number of amounts of rare metals, as well as growth 4x 104/dish. Culture media. Table 1 shows the ingredients of factors such as insulin, epidermal growth the new serum-free medium RITC 80-j. Modified factor (EGF) and transferrin. Fig. 1 shows MEM is different from original MEM, in that it contains succinate (75 mg/l), sodium succinate (100 mg/l) growth curves of human diploid fibroblasts 3 300 300 1400
Exp Cd
Rrs 134 (198/J
412
Preliminary notes
: z
IO
0 FN BSA-
Fi,e. 1. Time course of the growth of human dinloid fibroblasts (PDL-22). Stock-cultures fully grown in BME plus 10% FBS medium were shifted to various media including serum-free ones. Cells were grown in BME olus 10% FBS (0). RITC 80-7 DIUSFN (12 me/l). BSA (5 g/l) (0), RITC 80-7 (A) medium respectivily: BSA was added into the medium after 1 h incubation. The average values of duplicate experiments, which varied less than lo%, are presented.
in serum-free RITC 80-7 and in BME containing 10% FBS. The initial lag time in RITC 80-7 was longer than in serum-supplemented BME. When serial cultivation was attempted using RITC 80-7, only a few transfers (about 15 cell generations) could be carried out (fig. 3). However, cell dispersion treatment with cold trypsin was found to be quite helpful in the transfer of cultures grown in serum-free medium. The effect of human fibronection (FN) and bovine serum albumin (BSA). BSA was shown by us to be markedly growthenhancing for various cells with suspension growth [ 171,but in its concentration of more than 200 pg/ml, BSA was shown to interfere with the cellular attachment to the culture vessel. However, BSA was growth-promoting when added only after the cells tightly attached to the substrate (24 h incubation at 37°C after cell inoculation). This was Erp Cdl Res 134 (198/J
-
I
5
3 -
Ii 3 5
12 -
12 I
I2 5
rn
g/l g/l
Fig. 2. Effect of FN and BSA on cell growth. The cells were cultured in BME plus 10% FBS medium until 24-PDL, and shifted to various media. White column, growth in RITC 80-7 medium for 5 days culture; dotted column, growth in BME plus 10% FBS medium. BSA was added into the medium after I h
interpreted to mean that during 24 h of incubation, the fibroblasts produced the FN necessary for cellular attachment [13]. Accordingly, FN (10 mg/l) was added to the medium together with BSA (5 g/l) at the time when cells were inoculated. This permitted satisfactory attachment and the same cellular yield as BME supplemented with 10% FBS (figs 1, 2). Primary culture of the diploid fibroblasts and its long-term, serial transfer. Fig. 3 shows cumulative growth curves of both serial cultures in RITC 80-7 and in serumcontaining BME. As shown in fig. 3, cumulative growth so far increased straight on with use of RITC up to 76 DPL (population doubling level), whereas the culture with serum-containing medium reached up to 73 PDL and did not grow any further. The discrepancy in lifespan between these two cultures is not certain to be significant, but is anyhow shown in the same or similar lines of the experimental cultures.
Preliminary notes
473
We employed trypsinization at cold temperature to diminish the cellular damage following McKeehan [IO], because serum-free culture did not contain any protease inhibitor present in serum. The gaseous environment (7% 0,) was chosen after preliminary experiments under various conditions. Balin et al. [15] reported that the most favorable partial tension of oxygen was about 3% or less for the culture of human diploid fibroblasts, but they employed agitated cultures, where0 0 60 20 40 80 100 as we used stationary cultures. Therefore, Culture days the difference in optimal oxygen tension Fig. 3. Long-termed serial culture of human diploid may be due to the effect of agitation [16]. fibroblasts derived from embryonic lung tissue. The culture was made in BME plus 10% FBS medium The growth-enhancing effect of low oxygen (0) and RITC SO-7plus FN (12 mg/l), BSA (5 g/l), tension was observed, whether serum was medium (O), RITC SO-7medium (A). The subculture was made every 5 days. BSA was added into the me- present or not, but became more promidium after I h incubation. nent as the amount of BSA was increased. The mechanism by which BSA promotes cellular growth has been exhaustively studDiscussion ied using cultured Yoshida sarcoma cells: Employing serum-free RITC 80-7 medium Unsaturated fatty acids bound to BSA ensupplemented with FN and BSA, we suc- hance the cellular growth and membrane ceeded in culturing primary human embryo fluidity of cultured cells [17, 181which was fibroblasts and transferring them through preliminarily proved with human fibrolong-term serial passages. An earlier me- blasts. This should also function in the case dium deficient in thymidine, hypoxanthine, of fibroblast culture because when employand vitamin Blz (RITC 78-6 medium) did ing defatted BSA, there was no growthnot support more than three serial transfers promoting activity (unpublished data). [14]. Inclusion of FN and BSA in RITC Also, insulin was not detected in the BSA SO-7medium improved both cellular growth sample by means of radioimmuno assay, and the recovery from the cellular damage although the BSA sample used for this due to trypsinization. When BSA was added study was not pure (95-99 % purity). On the to the medium beginning 24 h later after other hand, transferrin and c-u,-antitrypsin inoculation of the cells, FN supplementa- was detected by means of immunoelectrotion could be omitted perhaps because of phoresis, but the content of these proteins FN synthesis during the first 24 h, but was too low to promote cellular growth cultivation was not successful without FN (data not shown). when BSA was added at the very beginThe serum-free medium described here ning (unpublished data). We attempted to can be used to grow all kinds of human reduce the amount of BSA added to the established epithelial cell lines including medium, but so far we could not reduce it HeLa, Hep 2, FL, as well as human diploid below 1 g/l medium. fibroblasts (unpublished data). Also the
474
Preliminary notes
slight modification of this medium enables the abundant growth of various human lymphoblasts in the absence of FN and in the presence of BSA. This fact may contradict Ham’s opinion that each cell requires an individual separate culture medium [ 193, but our serum-free medium contains BSA, whereas the medium of Ham does not. This is the first report which deals with both primary culture of human diploid fibroblasts in a serum-free medium and its long-term serial transfer. Such long-term serial cultures have so far only been achieved when excellent quality FBS was added to the culture medium, as the lot quality of each serum batch distinctly controls the results of the serial culture. Therefore the use of serum for such cultures is unfavorable and inconvenient from the standpoint of experimental reproducibility, taking into account the popular and widespread employment of long-term cultures of human diploid cells for various gerontological studies in vitro. This serum-free medium should serve as an efficient tool for research when employing human cultured cells, as this medium is not wholly chemically defined, though more defined than whole serum-supplemented medium. This work was partially supported by Grant-inAid (Cancer Special Research) of the Ministry of Education, Science and Culture, Japan.
References I. Sanford. K K. Dunree. L T & Covaleskv. A B, Exp cell res 31 (1983) 345. 2. Ham, R G, Proc natl acad sci US 53 (1965) 288. 3. Higuchi, K, J cell physiol 75 (1970) 65. 4. Takaoka, T & Katsuta, H, Exp cell res 67 (1971) 295. 5. Bottenstein, J, Hayashi, I, Hutchings, S, Masui, H, Mather, J, McClure, D B, Ohasa, S, Rizzino, A, Sato, G, Serrero, G, Wolfe, R & Wu, R, Method in enzymology, vol. 58. Academic Press, New York (1979). 6. Phillips, P D & Cristofalo, V J, In vitro 16 (1980) 250. 7. Walthall, B J & Ham, R G, In vitro I6 (1980) 250. 8. Yamane, I. Kan, M & Hoshi. H. Growth and Exp Cell RES 134 (198/J
growth factors. University of Tokyo Press, Tokyo (1980). 9. Yamane, I, Matsuya, Y & Jimbo, K, Proc sot exp biol med 127(1968) 335. IO. McKeehan, W L, Cell biol int rep I (1977) 335. I I. Taylor, W G, Camalier, R F & Sanford, K K, J cell ohvsiol 95 (1978) 33. 12. biento, M‘& Vaheri, A, Biochem I83 (1979) 33 I. 13. Grinnell. F & Feld. M K. Cell I7 (1979) 117. 14. Kan, M ‘& Hoshi. H. Soshikibaiyb (in japanese) 5 (1979) 22 I. 15. Balin, A K, Goodman, D B P, Rasmussen. H & Cristofalo, V J, J cell physiol89 (1976) 235. 16. Werrlein, R J & Glinos, A D, Nature 251 (1974) 317. 17. Yamane, I, Murakami, 0 & Kato, M, Proc sot exp biol med 149 (1975) 439. IS. Yamane, I & Tomioka, F, Cell biol int rep 3 (1979) 515. 19. Ham, R G, The handbook of experimental pharmacology. Springer Verlag, Berlin (1980). Received January 16, 1981 Revised version received April 6, 1981 Accepted April 15, I981
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/81/080474-04~2.00/0
Binding of thrombin to normal and transformed fibroblasts depends on cell density PIRKKO
POHJANPELTO
and ANTTI
Department of Virology, University 00290 Helsinki 29, Finland
VAHERI,
of Helsinki,
SF-
Summary. Binding of biologically active [‘251]thrombin
to several normal and transformed human and chicken cell lines was found to depend on cell density; more [‘ZSl]thrombin per cell was bound to sparse than to dense cultures. When normal and transformed cells were compared at equal densities the previously reported difference in [‘251]thrombin binding was not evident.
Thrombin is a potent growth factor for normal chicken fibroblasts [l], human fibroblasts [2] and Chinese hamster lung cells [3], but some transformed cells, e.g. SV40transformed human fibroblasts, HeLa cells, and the continuous monkey kidney cell lines BS-C-I and Vero can also be stimulated by thrombin [4]. The difficulty with