Cytotechnology 7: 165-172, 1991. 9 1991 KluwerAcademic Publishers. Printed in the Netherlands.
A novel Vero cell line for use as a mammalian host-vector system in serum-free medium Tadao Ohno, Xinhai Wang, Junko Kurashima, Kaoru Saijo-Kurita and Masafumi Hirono RIKEN Cell Bank, RIKEN (The Institute of Physical and Chemical Research), Koyadai, Tsukuba Science City, 305, Japan Received 9 July 1991; acceptedin revised form 17 October 1991
Abstract
We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots $3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33~ to 39.5~
Introduction For efficient biotechnological production of compounds in mammalian cells, selection of a suitable host-cell line is important from the view point of productivity, the scale of the production system, subsequent purification, and the physiological activity and safety of the final products. The serum requirement of the cell line is therefore a key factor in host-cell selection. In mammalian host-vector systems, after transfection of the required exogenous gene, the host cells are usually cloned to obtain a line in which the exogenous gene is integrated and which expresses the gene product constitutively. Then the culture conditions can be changed and the cells can be adapted to serum-free medium. COS-1 and COS-7 ceils have been widely used as host cells for transient expression of exogenous genes (reviewed in Gluzman, 1982). These cells express the large T antigen of SV40 virus
constitutively, and transfected plasmids containing the replication origin of SV40 DNA are episomally amplified. Rio et al. (1985), Portela et al. (1986), Kern and Basilico (1986), and Sidvey (1988) improved this type of mammalian hostvector systems to make it conditionally regulatable by introducing the temperature-sensitive large T antigen gene into simian kidney cells. However, all these newly developed cell lines must be cultured in serum-containing medium: we know of no report of host-cell lines that can be cultured in protein-free medium. In 1978, Yasumura et al. isolated a subline of simian kidney Vero cells, Veto-317, that can grow in protein-free, biotin-containing MEM without any other supplements (Yasumura, 1978). Here we report the establishment of a novel cell line derived from Vero-317 that constitutively expresses temperature-sensitive SV40 large T antigen, therefore allowing regulation of its expression of exogenous genes in replication origin-
166 containing plasmids in serum-, protein- and lipidfree conditions.
Materials and methods
,y,
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Cells
Vero-317 (RCB272), COS-1 (RCB143), and COS-7 (RCB539) cells were obtained from RIKEN Cell Bank, Tsukuba Science City, Japan. Vero-317 cells were maintained at 37~ in autoclavable and protein-free Eagle's MEM (MEM 'Nissui' (3)) supplemented with phenol red, glutamine, and sodium bicarbonate at concentrations of 6 mg/l, 292 mg/1, and 2 g/l, respectively (hereafter referred to as MEM). This MEM contains biotin at 0.02 mg/1, which is essential for maintenance of Vero-317 cells and their daughter cells in protein-free conditions. COS cells were maintained in MEM containing 5% newborn bovine serum (NBS). P~smids
A plasmid DNA which was ligated at the BamHIsites of both pBR322 and the temperature-sensitive A58 mutant SV40 DNA was a gift from Dr. M. Obinata (Institute for Anti-tuberculosis and Cancer, Tohoku University, Sendal, Japan). This plasmid was digested with the restriction enzyme SfiI, and with Mung Bean nuclease to make blunt ends, and then ligated with T4 DNA ligase. A DNA clone of the resulting circular plasmid in which the BglI-sensitive SV40 DNA replication origin was replaced by the SacII-sensitive site was selected and named pSVtsA58ori(-)-2. Its restriction map is shown in Fig. 1. pSV2neo, a gift from Dr. Y. Sugimoto (RIKEN Life Science Tsukuba Research Center, Tsukuba Science City, Japan), was digested with Hind III and Pvu II, and the resulting 330 bp DNA fragment containing the SV40 DNA replication origin was separated by agarose gel electrophoresis. To the Pvu II terminal of this fragment, a synthetic DNA hexamer (double strand, 5'CTCGAG-3' complemented with 3'-GAGCTC-
/ Bg,,- \
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Fig. 1. Restrictionmap of pSVtsA58ori(-)-2.
5') was ligated, and then the fragment was digested with XhoI. The resulting fragment was inserted into the HindlII-SalI site of the plasmid pXGH5 and named pXGH5ori, pXGH5 containing the human growth hormone (hGH) gene (Seldin et al., 1986) was purchased from Japan Mediphysics Co. Ltd. (Takarazuka-shi, Hyogo, Japan) as part of a Human Growth Hormone Radioimmunoassay Kit (Allegro Human Growth Hormone, Nichols Institute Diagnostics). Transfection
For establishment of Verots cell lines, 105 Vero317 cells were suspended in 0.5 ml of mannitol medium (mannitol 250 mM, NaC1 10 mM, and HEPES 10 mM, adjusted to pH 7.2 with HC1) and placed in a well of a 24-well culture plate (Falcon Co. Ltd.). Then 10 gg of pSVtsA58ori(-)-2, which had been digested with BamHI and BglI, and 2 gg of pSV2neo, which had been digested with BamHI and PvuII, were dissolved in 20 gl of water and added to the well. The resulting suspension in mannitol medium was subjected to electroporation in the well at 480 V direct voltage for 1 msec on a Pro-Genetor (Hoefer Scientific Instruments Co, CA). The application of a direct voltage was repeated 5 times at intervals of about
167 1 mira. Then the cells were washed with MEM5% NBS, seeded into a 60 mm culture dish with 5 ml of MEM-5% NBS, and cultured for 5 days. The medium was replaced by MEM-5% NBS containing 400 ktg/ml of G418 (Sigma, Co. Ltd.). After 25 days of culture, the clones that developed were screened by indirect immunofluorescence staining with anti-SV40 large T monoclonal antibody (a gift from Dr. N. Yamaguchi, Institute of Medical Science, University of Tokyo). The resulting cloned cells were named Verots S 1, Verots $2, and Verots $3. For production of hGH, the plasmid pXGH5 or pXGH5ori was transfected into the cells. The cells to be transfected were seeded at 5 • 105 cells in 5 ml of MEM-5% NBS and cultured at 37~ overnight, and then for one day at 33~ They were then washed 3 times with MEM, and incubated with 50 Bg of DOTMA (Boehringer Mannheim Co. Ltd.) and 20 ~tg of plasmid DNA in 3 ml of MEM at 33~ for 6 h. They were again washed 3 times with MEM, and incubated in 4 ml of MEM-5% NBS at the temperature indicated. The final culture medium was renewed every 24 h.
Southern dot-blot analysis 32p-Labeled pXGH5ori, used as a probe, was prepared using 32p-CTP and a random primer labeling kit (PRIME-IT TM, STRATAGENE, La Jolla). Plasmid DNAs, together with a carrier salmon sperm DNA, were extracted from pXGH5orior pXGH5-transfected cells in 35-ram dishes by the alkaline extraction method (Sambrook, et al., 1989). A sample of 200 gl of the final plasmid DNA fraction was dot-blotted onto a 86.5 mm 2 area of a membrane filter using HYBRI-SLOT TM (Bethesda Research Laboratories, Bethesda). Prehybridization and hybridization were carried out under the conditions described by Shilo and Weinberg (1981), and the filter was washed at 65~ The filter (4 x 17 cm) was then hybridized with 1 x 106 cpm of the 32pqabeled probe. 32p Radioactivity bound to the membrane filter was determined with a FUJIX Bio-Image Analyzer
BAS2000 (FUJI Film Co., Tokyo) and expressed as the relative radioactive intensity, PSL, on the imaging plate after its exposure to the filter for 60 min.
Assay of human growth hormone A Human Growth Hormone Radioimmunoassay Kit (Allegro Human Growth Hormone, Nichols Institute Diagnostics) was used for measuring the hGH contents of culture media.
Results
Transformation of Vero-317 cells We constructed a plasmid containing the large T antigen gene derived from the temperature-sensitive (ts) A58 mutant of SV40 virus and lacking the replication origin of the SV40 DNA. The tsA58 SV40 DNA was inserted into the BamI-II site of pBR322, cut with SfiI, which cleaved the origin of SV40 DNA replication, blunt ended by digestion with Mung Bean nuclease and then ligated with T4 DNA ligase to create a BglIresistant but Sac II-sensitive ligated site. This plasmid, pSVtsA58ori(-)-2 (Fig. 1), was digested with BamHI and BglI. The resulting DNA fragments were transfected into Vero-317 cells together with the plasmid DNA, pSV2neo, that had been linearized and shortened by digestion with BamHI and PvuII. After culture of approximately 106 cells in G-418 containing medium, 23 colonies were obtained. Three of these clones, named Verots S1, Verots $2, and Verots $3, expressed large T antigen constitutively as shown by indirect immunofluorescent staining as described in the Materials and methods. During expansion and maintenance culture of these 3 cell lines in MEM containing 5% NBS at 33~ no cytopathic effect was observed for more than a month, suggesting that SV40 virions were not produced. The growths of the Verots $3 cells and the mother cell line, Vero-317, in proteinfree MEM are shown in Fig. 2.
168
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5
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Fig. 2. Growth of Verots $3 and Vero-317 ceils in protein-free MEM. Cells were grown in 35 mm dishes containing 2 ml of autoclavable and protein-free Eagle's MEM (MEM 'Nissui'(3)) supplemented with phenol red, glutamine, and sodium bicarbonate at concentrations of 6 mg/l, 292 mg/1, and 2 g/l, respectively. The dishes were cultured under 5% CO 2 in air at 37~ or 33~ Values are means for duplicate dishes.
Transient expression of human growth hormone gene To confirm the ability of the cloned cells to act as a host cell line in a transient expression system, we used a commercially available human growth
Effect of ori on hGH Production 400
300
E 200
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Fig. 4. Restriction map of pXGH5ori.
hormone (hGH) gene. The replication origin of SV40 DNA was inserted into the HindIII-SalI site of the plasmid pXGH5 (Fig. 3). Then 106 Verots $3 cells were transfected with 20 ~tg of this plasmid, pXGH5ori, and cultured for 5 days in 4 ml of MEM containing 5% NBS, with daily change of the medium. The cells produced hGH at the high level of 364 ng/ml (i.e., 1456 ng per day), while cells transfected with pXGH5 as a control produced less than 40 ng per day (Fig. 4). Measurement of the contents of transfected plasmid DNA in the Verots $3 cell layer by Southern blotting showed that the transfected pXGH5 DNA decreased rapidly and continuously, whereas the content of pXGH5ori DNA decreased for 1 day after transfection and then remained at a steady level for 4 days during culture in serum-containing medium (Fig. 5). Even after the culture medium was changed to protein-free MEM on day 5, the plasmid DNA content of the cell layer did not decrease until at least day 11.
pXGH5
1
2
3
4
5
Comparison of Verots cells with COS cells
Days after transfeclion
Fig. 3. Human growth hormone production by Verots $3 cells transfected with pXGFl5ori or pXGH5. The cells were cultured in MEM supplemented with 5% NBS. Transfection was carried out as described in Materials and methods. Values are means for duplicate dishes.
The abilities of the 3 clones, Verots $1, $2, and $3, to produce hGH were compared with those of the widely used COS-1 cells. For this, samples of 106 cells were transfected with 20 ~tg of pXGH5ori and cultured in 4 ml of MEM contain-
169 The production rates of hGH of all the cell lines except Verots $3 decreased after the peak. Verots $3 cells maintained a high production rate of hGH for at least 17 days after transfection. The total amount of hGH produced in this particular experiment by Verots $3 reached 24.3 ~tg per dish, which was 6.4 times higher than that produced by COS-1 cells.
15000 MEM+5%NBS
MEM
10000
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Effect of temperature 0
! 4
i
2
i
i
i
6
8
10
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Days after transfection
Fig. 5. Quantification of transfected plasmid DNAs by Southern blot analysis. Transfection and Southern blot analysis were carded out as described in Materials and methods. Cells were cultured for 5 days after transfection in MEM supplemented with 5% NBS, and then in protein-free MEM.
ing 5% NBS at 33~ As shown in Fig. 6, all these cells produced considerable amounts of hGH, but their highest production rates per day were different. The highest production rates by Verots S1 and $2 were 341 and 106 ng/ml, respectively, on day 7, while those of Verots $3 and COS-1 were 448 and 134 ng/ml, respectively, on day 5. The parent cell line, Veto-317, produced less than 18 ng/ml throughout the experiment.
Verots $3 cells showed higher daily rates of production o f h G H at above 33~ than at 33~ in the first 3 or 5 days after transfection (Fig. 7: 37~ vs. 33~ Fig. 8: 39.5~ vs. 33~ However, at both 37~ and 39.5~ the production rate subsequently decreased to a lower level than that at 33~ The production rate of hGH by Verots $3 was consistently higher than that by COS-7 cells at 33~ and 37~ (Fig. 7). To determine whether the ceils continued to produce hGH after their subcultivation, we trypsinized the transfected Verots $3 cells and subcultivated them at 1:4 dilution on day 5 and, again, on day 9. After subculture, the cells continued to produce hGH at roughly 25% of the rate before subcultivation (Fig. 8).
1200 100O 46O
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Days after transfection
8
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Days after transfection
Fig. 7. Comparison of production of human growth hormone by Fig. 6. Production of human growth hormone by Verots, COS-l, and Vero-317 cells at 33~ Transfection of pXGH5ori was carried out as described in the Materials and methods. Cells were cultured in MEM supplemented with 5% NBS. Values are means for duplicate dishes.
Verots $3 and COS-7 cells at 33~ and 37~ Transfection of pXGH5ori was carried out as described in Materials and methods. Cells were cultured in MEM supplemented with 5% NBS. Values are means for duplicate dishes.
170
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Days after transfection
DME/Fll*Ins&Tf
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Days after medium change
Fig. 9. Human growth hormone production by Verots $3 cells in Fig. 8. Production of human growth hormone by Verots $3 cells at different temperatures. Transfection of pXGH5ori was cartied out as described in Materials and methods. Cells were cultured in MEM supplemented with 5% NBS. On days 5 and 9, the ceils at 33~ were subcultured at a split ratio of 1:4 (arrows). Values are means for duplicate dishes.
Effect of media on hGH production To confirm that Verots $3 cells could continue not only to proliferate (Fig. 2) but also to produce hGH in serum-free or protein-free medium, we changed the culture medium from MEM-5% NBS to the media shown in Fig. 9 on day 5 after transfection. In this particular experiment, the production rate of hGH reached 1000 ng/ml on day 5. Although the production rate decreased for two days in cultures in protein-free MEM or protein-free DME/F12 (1:1 volume mixture), the rate no longer decreased but instead increased by day 11 after changing the medium to serum-free DME/F12 supplemented with 1 ~tg/ml insulin and 10 ~tg/ml transferrin. Moreover when the proteinfree MEM was changed to serum-free DME/F12 supplemented with insulin and transferrin on day 11, the production rate increased rapidly from 435 ng/ml to 1286 ng/ml on day 13.
Discussion Vero cells, the parent cell line of Veto-317, were derived from kidney of an African green monkey. They are widely used for vaccine production and
a serum-free hormone-supplemented medium or in protein-free media. Transfection of pXGH5ori was carried out as described in Materials and methods. Cells were cultured at 33~ in 4 ml of MEM supplemented with 5% NBS for 5 days after transfection, and then in the indicated media. DME/F12 consists of equal volumes of Dulbecco's modified MEM and Ham's F12. Ins & Tf indicates supplementation with insulin (1 ~tg/ml) and transferrin (10 ~tg/ml). Protein-free MEM was replaced by DME/F12 supplemented with insulin and transferrin 6 days after transfer to the variOus media, i.e., 11 days after transfection. The media, 4 ml/dish,, was renewed every day. Values are means for duplicate dishes.
detection of virus contamination, because they show clear cytopathic effects on infection and are highly susceptible to infection by many kinds of viruses, such as human and simian adenoviruses, herpes, myxovirus, papova, picorna, pox, reo, arbo, rubella (Rhim et al., 1969), Korean haemorrhagic fever (McCormick et al., 1982), Lassa fever (Buckley and Casals, 1970), and others (Simizu and Terasima, 1988). One reason for this high susceptibility is that the cell line is defective in interferon synthesis (Mosca and Pitha, 1986). Vero-317 cells also show high susceptibility to viruses (Yasumura, personal communication), so Verots cells are probably also highly susceptible. Thus if the cell line is accidentally contaminated with one of these viruses, the culture will die. This seems an advantageous characteristic for a cell line used for producing medicinal products, since obviously contamination of the products with viruses is undesirable. A transformant of Veto-317, Verots $3, showed a high production rate of hGH after its transfec-
171 tion with the plasmid p X G H 5 o r i containing the SV40 replication origin (Fig. 4). This reflects the maintenance of a high level of pXGH5or~ in the cells (Fig. 5), presumably because of amplification o f p X G H 5 o r i DNA, as amplification of replication-origin containing plasmids occurs in COS7 cells (Rio et al., 1985). Verots $3 ceils have advantages over COS-1 and COS-7 cells for use as a host-vector system as shown in Figs. 6 and 7. COS-7 cells have usually been found to produce more proteins expressed from transfected genes in SV40 replication-origin containing plasmids than COS-1 cells at 37~ (Todokoro, K., RIKEN, personal commtmication). However, although Verots $3 cells produced a relatively large amount of h G H at the permissive temperature (33~ for the tsA58 large T antigen (Fig. 6) after transfection with pXGH5ori, they did not show temperature-sensitive shut-down of h G H production at the nonpermissive temperature (39.5~ during the first 3 days after transfection (Fig. 8). This is in contrast to a report that no factor VIII m R N A or protein expression by the factor VIII gene in BSC-40tsA58 was detectable at 39.5~ (Sidvey, 1988). At present, we have no explanation to this unexpected behaviour o f the Verots $3 cells. However, in Verots $3 cells, temperature-sensitive shut-down o f h G H production was observed 4 days after transfection. Unlike previously reported temperature-sensitive host cell lines, which all require serum, Verots $3 cells can grow in protein-free M E M at 37~ or even, at a lower rate, at 33~ (Fig. 2). This characteristic m a y contribute to the maintenance o f the high h G H production in protein-free M E M or protein-free DME/F12 shown in Fig. 9, and would be advantageous for industrial use of these cells, since it would simplify purification steps of the products and reduce the production cost. However, in protein-free m e d i u m without any growth factors, the production rate of h G H by transfected Verots $3 cells was less than that in serum-containing m e d i u m in the first 5 days (Fig. 9), although the plasmid p X G H 5 o r i content of the cell layer did not decrease in protein-free M E M
(Fig. 5). This low production of h G H in proteinfree M E M is presumably because some serum factors contributed to the h G H production. These factors could be insulin and/or transferrin because cells cultured in M E M produced more h G H on transfer to serum-free DME/F12 containing insulin and transferrin than the cells cultured in protein-free DME/F12, and the production rate in the former was restored to that o f cells in M E M supplemented with serum (Fig. 9). Verots $3 cells seem better than the rodent derived cell lines such as CHO-K1 and B H K as a host cell line for production o f medicinal products derived from exogenous genes because monkeys are closer to humans than rodents, and so, products o f simian-derived Verots $3 cells should have less likelihood of causing immunological problems in humans. Moreover, Verots $3 cells can be used as a host cell line not only for transient expression o f exogenous genes but also, like COS-1 cells, for stable integrated gene expression.
Acknowledgements W e are grateful to Dr. I. Ike, RIKEN, and Dr. H. Matsui, R I K E N and Tsukuba University for critical discussion and technical advice.
References Buckley SM & Casals J (1970) Lassa fever, a new virus disease of man from West Africa. III. Isolation and characterization of the virus. Am. J. Trop. Med. Hyg. 19: 680-691. Gluzman Y (ed.) (1982) Eukaryotic viral vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Kern FG & Basilico C (1986) An inducible eukaryotic hostvector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a therrnolabilelarge T antigen. Gene 43: 237-245. McCormick JB, Sasso DR, Palmer EL & Kiley HP (1982) Morphological identificationof the agent of Korean haemorrhagic fever (Hantaan virus) as a member of the bunyaviridae. Lancet April 3: 765-768. Mosca JD & Pitha PM (1986) Transcriptional and post-transcriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Mol. Cell. Biol. 6: 2279-2283.
172 Portela A, Melero JA, de la Luna S & Ortin J (1986) Construction of cell lines and expression of influenza virus nonstructural protein genes. EMBO J. 5: 2387-2392. Rhirn JS, Schell K, Creasy B & Case W (1969) Biological characteristics and viral susceptibility of an African green monkey kidney cell line (Vero) (34285). Proc. Soc. Exp. Biol. IVied. 132: 670-678. Rio DC, Clark SG & Tjian R (1985) A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction. Science 227: 23-28. Sambrook J, Fritsch EF & Maniates T (eds.) (1989) Molecular Cloning. A Laboratory Manual. Second Edition, pp. 1.251.28. Seldin R, Howie KB, Rowe ME, Goodman HM & Moore D (1986) Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol. 6: 3173-3179.
Shilo B & Weinberg RA (1981) DNA sequences homologous to vertebrate oncogenes are conserved in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 78: 6789-6792. Sidvey JM (1988) New genetic methods for mammalian cells. Bin/Technology 6:1192-1196. Simizu B & Terasima T (eds.) (1988) Vero cells - Origin, properties and biomedical applications. Department of Microbiology, School of Medicine Chiba University, Chiba University, Chiba. Yasumura Y, Niwa A & Yamamoto K (1978) Phenotypic requirement for glutamine of kidney cells and for glutamine and arginine of liver cells in culture. In: Katsuta H (ed.) Nutritional Requirements of Cultured Cells. Japan Scientific Societies Press, Tokyo, and University Press, Baltimore, pp. 223-255.
Address for offprints: T. Ohno, RIKEN Cell Bank, RIKEN (The Institute of Physical and Chemical Research), Koyadai, Tsukuba Science City, 305, Japan
A novel Vero cell line for use as a mammalian host-vector system in serum-free medium Tadao Ohno, Xinhai Wang, Junko Kurashima, Kaoru Saijo-Kurita and Masafumi Hirono RIKEN Cell Bank, RIKEN (The Institute of Physical and Chemical Research), Koyadai, Tsukuba Science City, 305, Japan Received 9 July 1991; acceptedin revised form 17 October 1991
Abstract
We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots $3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33~ to 39.5~
Introduction For efficient biotechnological production of compounds in mammalian cells, selection of a suitable host-cell line is important from the view point of productivity, the scale of the production system, subsequent purification, and the physiological activity and safety of the final products. The serum requirement of the cell line is therefore a key factor in host-cell selection. In mammalian host-vector systems, after transfection of the required exogenous gene, the host cells are usually cloned to obtain a line in which the exogenous gene is integrated and which expresses the gene product constitutively. Then the culture conditions can be changed and the cells can be adapted to serum-free medium. COS-1 and COS-7 ceils have been widely used as host cells for transient expression of exogenous genes (reviewed in Gluzman, 1982). These cells express the large T antigen of SV40 virus
constitutively, and transfected plasmids containing the replication origin of SV40 DNA are episomally amplified. Rio et al. (1985), Portela et al. (1986), Kern and Basilico (1986), and Sidvey (1988) improved this type of mammalian hostvector systems to make it conditionally regulatable by introducing the temperature-sensitive large T antigen gene into simian kidney cells. However, all these newly developed cell lines must be cultured in serum-containing medium: we know of no report of host-cell lines that can be cultured in protein-free medium. In 1978, Yasumura et al. isolated a subline of simian kidney Vero cells, Veto-317, that can grow in protein-free, biotin-containing MEM without any other supplements (Yasumura, 1978). Here we report the establishment of a novel cell line derived from Vero-317 that constitutively expresses temperature-sensitive SV40 large T antigen, therefore allowing regulation of its expression of exogenous genes in replication origin-
166 containing plasmids in serum-, protein- and lipidfree conditions.
Materials and methods
,y,
.........
\ \
Cells
Vero-317 (RCB272), COS-1 (RCB143), and COS-7 (RCB539) cells were obtained from RIKEN Cell Bank, Tsukuba Science City, Japan. Vero-317 cells were maintained at 37~ in autoclavable and protein-free Eagle's MEM (MEM 'Nissui' (3)) supplemented with phenol red, glutamine, and sodium bicarbonate at concentrations of 6 mg/l, 292 mg/1, and 2 g/l, respectively (hereafter referred to as MEM). This MEM contains biotin at 0.02 mg/1, which is essential for maintenance of Vero-317 cells and their daughter cells in protein-free conditions. COS cells were maintained in MEM containing 5% newborn bovine serum (NBS). P~smids
A plasmid DNA which was ligated at the BamHIsites of both pBR322 and the temperature-sensitive A58 mutant SV40 DNA was a gift from Dr. M. Obinata (Institute for Anti-tuberculosis and Cancer, Tohoku University, Sendal, Japan). This plasmid was digested with the restriction enzyme SfiI, and with Mung Bean nuclease to make blunt ends, and then ligated with T4 DNA ligase. A DNA clone of the resulting circular plasmid in which the BglI-sensitive SV40 DNA replication origin was replaced by the SacII-sensitive site was selected and named pSVtsA58ori(-)-2. Its restriction map is shown in Fig. 1. pSV2neo, a gift from Dr. Y. Sugimoto (RIKEN Life Science Tsukuba Research Center, Tsukuba Science City, Japan), was digested with Hind III and Pvu II, and the resulting 330 bp DNA fragment containing the SV40 DNA replication origin was separated by agarose gel electrophoresis. To the Pvu II terminal of this fragment, a synthetic DNA hexamer (double strand, 5'CTCGAG-3' complemented with 3'-GAGCTC-
/ Bg,,- \
,,,322
.X
/
Fig. 1. Restrictionmap of pSVtsA58ori(-)-2.
5') was ligated, and then the fragment was digested with XhoI. The resulting fragment was inserted into the HindlII-SalI site of the plasmid pXGH5 and named pXGH5ori, pXGH5 containing the human growth hormone (hGH) gene (Seldin et al., 1986) was purchased from Japan Mediphysics Co. Ltd. (Takarazuka-shi, Hyogo, Japan) as part of a Human Growth Hormone Radioimmunoassay Kit (Allegro Human Growth Hormone, Nichols Institute Diagnostics). Transfection
For establishment of Verots cell lines, 105 Vero317 cells were suspended in 0.5 ml of mannitol medium (mannitol 250 mM, NaC1 10 mM, and HEPES 10 mM, adjusted to pH 7.2 with HC1) and placed in a well of a 24-well culture plate (Falcon Co. Ltd.). Then 10 gg of pSVtsA58ori(-)-2, which had been digested with BamHI and BglI, and 2 gg of pSV2neo, which had been digested with BamHI and PvuII, were dissolved in 20 gl of water and added to the well. The resulting suspension in mannitol medium was subjected to electroporation in the well at 480 V direct voltage for 1 msec on a Pro-Genetor (Hoefer Scientific Instruments Co, CA). The application of a direct voltage was repeated 5 times at intervals of about
167 1 mira. Then the cells were washed with MEM5% NBS, seeded into a 60 mm culture dish with 5 ml of MEM-5% NBS, and cultured for 5 days. The medium was replaced by MEM-5% NBS containing 400 ktg/ml of G418 (Sigma, Co. Ltd.). After 25 days of culture, the clones that developed were screened by indirect immunofluorescence staining with anti-SV40 large T monoclonal antibody (a gift from Dr. N. Yamaguchi, Institute of Medical Science, University of Tokyo). The resulting cloned cells were named Verots S 1, Verots $2, and Verots $3. For production of hGH, the plasmid pXGH5 or pXGH5ori was transfected into the cells. The cells to be transfected were seeded at 5 • 105 cells in 5 ml of MEM-5% NBS and cultured at 37~ overnight, and then for one day at 33~ They were then washed 3 times with MEM, and incubated with 50 Bg of DOTMA (Boehringer Mannheim Co. Ltd.) and 20 ~tg of plasmid DNA in 3 ml of MEM at 33~ for 6 h. They were again washed 3 times with MEM, and incubated in 4 ml of MEM-5% NBS at the temperature indicated. The final culture medium was renewed every 24 h.
Southern dot-blot analysis 32p-Labeled pXGH5ori, used as a probe, was prepared using 32p-CTP and a random primer labeling kit (PRIME-IT TM, STRATAGENE, La Jolla). Plasmid DNAs, together with a carrier salmon sperm DNA, were extracted from pXGH5orior pXGH5-transfected cells in 35-ram dishes by the alkaline extraction method (Sambrook, et al., 1989). A sample of 200 gl of the final plasmid DNA fraction was dot-blotted onto a 86.5 mm 2 area of a membrane filter using HYBRI-SLOT TM (Bethesda Research Laboratories, Bethesda). Prehybridization and hybridization were carried out under the conditions described by Shilo and Weinberg (1981), and the filter was washed at 65~ The filter (4 x 17 cm) was then hybridized with 1 x 106 cpm of the 32pqabeled probe. 32p Radioactivity bound to the membrane filter was determined with a FUJIX Bio-Image Analyzer
BAS2000 (FUJI Film Co., Tokyo) and expressed as the relative radioactive intensity, PSL, on the imaging plate after its exposure to the filter for 60 min.
Assay of human growth hormone A Human Growth Hormone Radioimmunoassay Kit (Allegro Human Growth Hormone, Nichols Institute Diagnostics) was used for measuring the hGH contents of culture media.
Results
Transformation of Vero-317 cells We constructed a plasmid containing the large T antigen gene derived from the temperature-sensitive (ts) A58 mutant of SV40 virus and lacking the replication origin of the SV40 DNA. The tsA58 SV40 DNA was inserted into the BamI-II site of pBR322, cut with SfiI, which cleaved the origin of SV40 DNA replication, blunt ended by digestion with Mung Bean nuclease and then ligated with T4 DNA ligase to create a BglIresistant but Sac II-sensitive ligated site. This plasmid, pSVtsA58ori(-)-2 (Fig. 1), was digested with BamHI and BglI. The resulting DNA fragments were transfected into Vero-317 cells together with the plasmid DNA, pSV2neo, that had been linearized and shortened by digestion with BamHI and PvuII. After culture of approximately 106 cells in G-418 containing medium, 23 colonies were obtained. Three of these clones, named Verots S1, Verots $2, and Verots $3, expressed large T antigen constitutively as shown by indirect immunofluorescent staining as described in the Materials and methods. During expansion and maintenance culture of these 3 cell lines in MEM containing 5% NBS at 33~ no cytopathic effect was observed for more than a month, suggesting that SV40 virions were not produced. The growths of the Verots $3 cells and the mother cell line, Vero-317, in proteinfree MEM are shown in Fig. 2.
168
25 Verots
20
$3,
37"C
Bglll
EcoRI\ SacI~ \ j~
370C
"~ lO
B~HI,,\\m pXGHSori
Sail
ori $
Vero~ $3, 33~
o
I
I
I
I
I
1
2
3
4
5
i
1
I
6
7
7.0 k b p
HindIII
"--EcoRI
Days
Fig. 2. Growth of Verots $3 and Vero-317 ceils in protein-free MEM. Cells were grown in 35 mm dishes containing 2 ml of autoclavable and protein-free Eagle's MEM (MEM 'Nissui'(3)) supplemented with phenol red, glutamine, and sodium bicarbonate at concentrations of 6 mg/l, 292 mg/1, and 2 g/l, respectively. The dishes were cultured under 5% CO 2 in air at 37~ or 33~ Values are means for duplicate dishes.
Transient expression of human growth hormone gene To confirm the ability of the cloned cells to act as a host cell line in a transient expression system, we used a commercially available human growth
Effect of ori on hGH Production 400
300
E 200
== 100 /
Fig. 4. Restriction map of pXGH5ori.
hormone (hGH) gene. The replication origin of SV40 DNA was inserted into the HindIII-SalI site of the plasmid pXGH5 (Fig. 3). Then 106 Verots $3 cells were transfected with 20 ~tg of this plasmid, pXGH5ori, and cultured for 5 days in 4 ml of MEM containing 5% NBS, with daily change of the medium. The cells produced hGH at the high level of 364 ng/ml (i.e., 1456 ng per day), while cells transfected with pXGH5 as a control produced less than 40 ng per day (Fig. 4). Measurement of the contents of transfected plasmid DNA in the Verots $3 cell layer by Southern blotting showed that the transfected pXGH5 DNA decreased rapidly and continuously, whereas the content of pXGH5ori DNA decreased for 1 day after transfection and then remained at a steady level for 4 days during culture in serum-containing medium (Fig. 5). Even after the culture medium was changed to protein-free MEM on day 5, the plasmid DNA content of the cell layer did not decrease until at least day 11.
pXGH5
1
2
3
4
5
Comparison of Verots cells with COS cells
Days after transfeclion
Fig. 3. Human growth hormone production by Verots $3 cells transfected with pXGFl5ori or pXGH5. The cells were cultured in MEM supplemented with 5% NBS. Transfection was carried out as described in Materials and methods. Values are means for duplicate dishes.
The abilities of the 3 clones, Verots $1, $2, and $3, to produce hGH were compared with those of the widely used COS-1 cells. For this, samples of 106 cells were transfected with 20 ~tg of pXGH5ori and cultured in 4 ml of MEM contain-
169 The production rates of hGH of all the cell lines except Verots $3 decreased after the peak. Verots $3 cells maintained a high production rate of hGH for at least 17 days after transfection. The total amount of hGH produced in this particular experiment by Verots $3 reached 24.3 ~tg per dish, which was 6.4 times higher than that produced by COS-1 cells.
15000 MEM+5%NBS
MEM
10000
50oo
Effect of temperature 0
! 4
i
2
i
i
i
6
8
10
12
Days after transfection
Fig. 5. Quantification of transfected plasmid DNAs by Southern blot analysis. Transfection and Southern blot analysis were carded out as described in Materials and methods. Cells were cultured for 5 days after transfection in MEM supplemented with 5% NBS, and then in protein-free MEM.
ing 5% NBS at 33~ As shown in Fig. 6, all these cells produced considerable amounts of hGH, but their highest production rates per day were different. The highest production rates by Verots S1 and $2 were 341 and 106 ng/ml, respectively, on day 7, while those of Verots $3 and COS-1 were 448 and 134 ng/ml, respectively, on day 5. The parent cell line, Veto-317, produced less than 18 ng/ml throughout the experiment.
Verots $3 cells showed higher daily rates of production o f h G H at above 33~ than at 33~ in the first 3 or 5 days after transfection (Fig. 7: 37~ vs. 33~ Fig. 8: 39.5~ vs. 33~ However, at both 37~ and 39.5~ the production rate subsequently decreased to a lower level than that at 33~ The production rate of hGH by Verots $3 was consistently higher than that by COS-7 cells at 33~ and 37~ (Fig. 7). To determine whether the ceils continued to produce hGH after their subcultivation, we trypsinized the transfected Verots $3 cells and subcultivated them at 1:4 dilution on day 5 and, again, on day 9. After subculture, the cells continued to produce hGH at roughly 25% of the rate before subcultivation (Fig. 8).
1200 100O 46O
$3,
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330C
400
20o
20o 10O 2 $
10
15
4
6
Days after transfection
8
10
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20
Days after transfection
Fig. 7. Comparison of production of human growth hormone by Fig. 6. Production of human growth hormone by Verots, COS-l, and Vero-317 cells at 33~ Transfection of pXGH5ori was carried out as described in the Materials and methods. Cells were cultured in MEM supplemented with 5% NBS. Values are means for duplicate dishes.
Verots $3 and COS-7 cells at 33~ and 37~ Transfection of pXGH5ori was carried out as described in Materials and methods. Cells were cultured in MEM supplemented with 5% NBS. Values are means for duplicate dishes.
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80
~u 1000
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40
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MEN 0
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Days after transfection
DME/Fll*Ins&Tf
r
i
i
i
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8
Days after medium change
Fig. 9. Human growth hormone production by Verots $3 cells in Fig. 8. Production of human growth hormone by Verots $3 cells at different temperatures. Transfection of pXGH5ori was cartied out as described in Materials and methods. Cells were cultured in MEM supplemented with 5% NBS. On days 5 and 9, the ceils at 33~ were subcultured at a split ratio of 1:4 (arrows). Values are means for duplicate dishes.
Effect of media on hGH production To confirm that Verots $3 cells could continue not only to proliferate (Fig. 2) but also to produce hGH in serum-free or protein-free medium, we changed the culture medium from MEM-5% NBS to the media shown in Fig. 9 on day 5 after transfection. In this particular experiment, the production rate of hGH reached 1000 ng/ml on day 5. Although the production rate decreased for two days in cultures in protein-free MEM or protein-free DME/F12 (1:1 volume mixture), the rate no longer decreased but instead increased by day 11 after changing the medium to serum-free DME/F12 supplemented with 1 ~tg/ml insulin and 10 ~tg/ml transferrin. Moreover when the proteinfree MEM was changed to serum-free DME/F12 supplemented with insulin and transferrin on day 11, the production rate increased rapidly from 435 ng/ml to 1286 ng/ml on day 13.
Discussion Vero cells, the parent cell line of Veto-317, were derived from kidney of an African green monkey. They are widely used for vaccine production and
a serum-free hormone-supplemented medium or in protein-free media. Transfection of pXGH5ori was carried out as described in Materials and methods. Cells were cultured at 33~ in 4 ml of MEM supplemented with 5% NBS for 5 days after transfection, and then in the indicated media. DME/F12 consists of equal volumes of Dulbecco's modified MEM and Ham's F12. Ins & Tf indicates supplementation with insulin (1 ~tg/ml) and transferrin (10 ~tg/ml). Protein-free MEM was replaced by DME/F12 supplemented with insulin and transferrin 6 days after transfer to the variOus media, i.e., 11 days after transfection. The media, 4 ml/dish,, was renewed every day. Values are means for duplicate dishes.
detection of virus contamination, because they show clear cytopathic effects on infection and are highly susceptible to infection by many kinds of viruses, such as human and simian adenoviruses, herpes, myxovirus, papova, picorna, pox, reo, arbo, rubella (Rhim et al., 1969), Korean haemorrhagic fever (McCormick et al., 1982), Lassa fever (Buckley and Casals, 1970), and others (Simizu and Terasima, 1988). One reason for this high susceptibility is that the cell line is defective in interferon synthesis (Mosca and Pitha, 1986). Vero-317 cells also show high susceptibility to viruses (Yasumura, personal communication), so Verots cells are probably also highly susceptible. Thus if the cell line is accidentally contaminated with one of these viruses, the culture will die. This seems an advantageous characteristic for a cell line used for producing medicinal products, since obviously contamination of the products with viruses is undesirable. A transformant of Veto-317, Verots $3, showed a high production rate of hGH after its transfec-
171 tion with the plasmid p X G H 5 o r i containing the SV40 replication origin (Fig. 4). This reflects the maintenance of a high level of pXGH5or~ in the cells (Fig. 5), presumably because of amplification o f p X G H 5 o r i DNA, as amplification of replication-origin containing plasmids occurs in COS7 cells (Rio et al., 1985). Verots $3 ceils have advantages over COS-1 and COS-7 cells for use as a host-vector system as shown in Figs. 6 and 7. COS-7 cells have usually been found to produce more proteins expressed from transfected genes in SV40 replication-origin containing plasmids than COS-1 cells at 37~ (Todokoro, K., RIKEN, personal commtmication). However, although Verots $3 cells produced a relatively large amount of h G H at the permissive temperature (33~ for the tsA58 large T antigen (Fig. 6) after transfection with pXGH5ori, they did not show temperature-sensitive shut-down of h G H production at the nonpermissive temperature (39.5~ during the first 3 days after transfection (Fig. 8). This is in contrast to a report that no factor VIII m R N A or protein expression by the factor VIII gene in BSC-40tsA58 was detectable at 39.5~ (Sidvey, 1988). At present, we have no explanation to this unexpected behaviour o f the Verots $3 cells. However, in Verots $3 cells, temperature-sensitive shut-down o f h G H production was observed 4 days after transfection. Unlike previously reported temperature-sensitive host cell lines, which all require serum, Verots $3 cells can grow in protein-free M E M at 37~ or even, at a lower rate, at 33~ (Fig. 2). This characteristic m a y contribute to the maintenance o f the high h G H production in protein-free M E M or protein-free DME/F12 shown in Fig. 9, and would be advantageous for industrial use of these cells, since it would simplify purification steps of the products and reduce the production cost. However, in protein-free m e d i u m without any growth factors, the production rate of h G H by transfected Verots $3 cells was less than that in serum-containing m e d i u m in the first 5 days (Fig. 9), although the plasmid p X G H 5 o r i content of the cell layer did not decrease in protein-free M E M
(Fig. 5). This low production of h G H in proteinfree M E M is presumably because some serum factors contributed to the h G H production. These factors could be insulin and/or transferrin because cells cultured in M E M produced more h G H on transfer to serum-free DME/F12 containing insulin and transferrin than the cells cultured in protein-free DME/F12, and the production rate in the former was restored to that o f cells in M E M supplemented with serum (Fig. 9). Verots $3 cells seem better than the rodent derived cell lines such as CHO-K1 and B H K as a host cell line for production o f medicinal products derived from exogenous genes because monkeys are closer to humans than rodents, and so, products o f simian-derived Verots $3 cells should have less likelihood of causing immunological problems in humans. Moreover, Verots $3 cells can be used as a host cell line not only for transient expression o f exogenous genes but also, like COS-1 cells, for stable integrated gene expression.
Acknowledgements W e are grateful to Dr. I. Ike, RIKEN, and Dr. H. Matsui, R I K E N and Tsukuba University for critical discussion and technical advice.
References Buckley SM & Casals J (1970) Lassa fever, a new virus disease of man from West Africa. III. Isolation and characterization of the virus. Am. J. Trop. Med. Hyg. 19: 680-691. Gluzman Y (ed.) (1982) Eukaryotic viral vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Kern FG & Basilico C (1986) An inducible eukaryotic hostvector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a therrnolabilelarge T antigen. Gene 43: 237-245. McCormick JB, Sasso DR, Palmer EL & Kiley HP (1982) Morphological identificationof the agent of Korean haemorrhagic fever (Hantaan virus) as a member of the bunyaviridae. Lancet April 3: 765-768. Mosca JD & Pitha PM (1986) Transcriptional and post-transcriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Mol. Cell. Biol. 6: 2279-2283.
172 Portela A, Melero JA, de la Luna S & Ortin J (1986) Construction of cell lines and expression of influenza virus nonstructural protein genes. EMBO J. 5: 2387-2392. Rhirn JS, Schell K, Creasy B & Case W (1969) Biological characteristics and viral susceptibility of an African green monkey kidney cell line (Vero) (34285). Proc. Soc. Exp. Biol. IVied. 132: 670-678. Rio DC, Clark SG & Tjian R (1985) A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction. Science 227: 23-28. Sambrook J, Fritsch EF & Maniates T (eds.) (1989) Molecular Cloning. A Laboratory Manual. Second Edition, pp. 1.251.28. Seldin R, Howie KB, Rowe ME, Goodman HM & Moore D (1986) Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol. 6: 3173-3179.
Shilo B & Weinberg RA (1981) DNA sequences homologous to vertebrate oncogenes are conserved in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 78: 6789-6792. Sidvey JM (1988) New genetic methods for mammalian cells. Bin/Technology 6:1192-1196. Simizu B & Terasima T (eds.) (1988) Vero cells - Origin, properties and biomedical applications. Department of Microbiology, School of Medicine Chiba University, Chiba University, Chiba. Yasumura Y, Niwa A & Yamamoto K (1978) Phenotypic requirement for glutamine of kidney cells and for glutamine and arginine of liver cells in culture. In: Katsuta H (ed.) Nutritional Requirements of Cultured Cells. Japan Scientific Societies Press, Tokyo, and University Press, Baltimore, pp. 223-255.
Address for offprints: T. Ohno, RIKEN Cell Bank, RIKEN (The Institute of Physical and Chemical Research), Koyadai, Tsukuba Science City, 305, Japan
