Cytotechnology 10: 9-14, 1992. 9 1992KluwerAcademicPublishers.Printedin the Netherlands.
Chemically defined medium for the production of biologically active substances of CHO cells Jun-ichiro Hata 1, Takeyoshi Tamura 2, Soji Yokoshima 2, Shinya Yamashita 1, Shoko Kabeno 1, Ken Matsumoto 2 and Kazukiyo Onodera 3
1Central Research Laboratory, Nippon Suisan Kaisha, Ltd., Hachioji-shi, Tokyo, 190; 2Research Laboratory, Nissui Pharmaceutical Co. Ltd., Yuuki-shi, Ibaraki, 307; and 3Department of Agricultural Chemistry, The University of Tokyo, Bunkyou-ku, Tokyo, 113, Japan Received 13 January 1992; acceptedin revisedform 14 July 1992
Key words: recombinant CHO, hGH, dhfr, MTX, chemically defined medium Abstract
A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.
Abbreviations: CHO: Chinese hamster ovary; hGH: human growth hormone; dFCS: dialyzed fetal calf serum; dhfr: dihydroforate reductase; MTX: methotrexate.
Introduction
Chinese hamster ovary (CHO) cells established by Kao and Puck (1968) have often been used as a host cell in the production of recombinant proteins of mammalian origin, because genes transfected into the cells are stably expressed (Sano et al., 1988) and their transformants easily obtained due to the formation of a large number of colonies. Furthermore, CHO cells which were defective in dihydroforate reductase (dhfr) were isolated and had been used as host cells into which the foreign gene was transfected. The cells become resistant to high concentrations of methotrexate (MTX) by gene amplification (Urlaub and Chasin, 1980;
Kaufman and Sharp, 1982). With this property of the cells, dhfr-deficient CHO cell lines have been exploited in order to enhance the productivity of proteins encoded by co-introduced genes. To obtain high levels of the gene product by the cells, transcription units in expression plasmids have been also studied with inducible promoters (Friedman et al., 1989; Israel and Kaufman, 1989). The parental CHO cells grow relatively easily in serum-free medium (Hamilton and Ham, 1977; Gasser et al., 1985), but detailed studies of media to enhance the productivity of recombinant proteins in CHO cells have not been conducted. From this point of view, we constructed a expression plasmid containing the human growth hormone
10 (hGH) genomic gene (Selden et al., 1986) under SV40 transcriptional control, and constructed a CHO cell subline, designated GT19, carrying this plasmid. The GT19 cells continuously secreted a high level of hGH. We report here that basal medium components affected the productivity of exogenous gene-encoding proteins in CHO cells using GT19 cells. The exploitation of chemically defined medium allowed us to identify the chemical components which were suitable not only for the enhancement of the productivity of gene products by CHO cells, but also to get information on the regulation of gene expression and secretion of the product.
Hindlll dhfr
Pstl SV40 early promoter
pSV2-hGH / dhfr (7.2 Kb)
SV40 splice + poly (A}
SV40 early promoter
\ B,glll hGH
Materials and methods Fig. 1. Construction of the hGH expression plasmid pSV2-hGH/
Construction of an expression plasmid for hGH The plasmid p0GH containing the genomic sequence encoding hGH was purchased from Nichols Institute (Gasser et al., 1985). An expression plasmid pSV2-dhfr (ATCC 37146) containing the cDNA sequence of mouse dihydroforate reductase (dhfr) was digested with HindIII and BglII to remove the dhfr sequence. The 2.1 Kbp BamHIEcoRI hGH gene fragment containing a poly(A) addition site was then ligated to the HindIII-BglII site of pSV2 after treating both DNA insert and vector with DNA polymerase Klenow fragment. The EcoRI site of this plasmid, and the PvuIIBglII fragment isolated from pSV2-dhfr containing both SV40 promoter and dhfr sequences were blunt-ended using DNA polymerase Klenow fragment and were ligated. The clone thus obtained was designated pSV2-hGH/dhfr, and is shown in Fig. 1.
Culture and transfection of CHO cells CHO (dhfr-) cells, DXB 11 line (Urlaub and Chasin, 1980), were grown in alpha MEM medium with nucleosides (GIBCO) supplemented with 5% fetal calf serum (FCS). The expression plasmid pSV2-hGH/dhfr was linearized by EcoRI digestion, then transfected into CHO cells using
dhfr. The plasmid contains a 2.1 Kbp fragment of the hGH gene placed under control of the SV40 early promoter as described in Materials and methods. Thin single line represents plasmid pSV2 containing ampicillin-resistance gene.
electroporation (3.0 KV/cm, 50 Its, twice) (Potter et al., 1984). The transfected cells were selected by growing them in alpha MEM medium without nucleosides (GIBCO) supplemented with 5% dialyzed FCS (dFCS). Sublines which expressed a high level of hGH were isolated from a dhfr+ transformants expressing hGH by growing them in media containing indicated concentrations of methotrexate (MTX) (see text).
Assay of hGH productivities of CHO cells Concentrations of hGH which was expressed and secreted into medium by transformants were determined using RIA kit (Pharmacia). Culture supernatants were diluted with 0.5% BSA/PBS into the measurement range, and their final hGH concentration determined.
Results and discussion
The expression plasmid pSV2-hGH/dhfr containing both the hGH and the mouse dhfr sequences
11
under SV40 transcriptional control (Fig. 1), was transfected into CHO (dhfr-) cells using an electroporation technique. A cell line GF32 constitutively secreted the highest level of hGH into the medium among 48 dhfr+ transformants. The quantity of hGH secreted was determined using radioimmunoassay as described in Materials and methods. We could not detect any hGH in the culture supematant of the parental CHO cells by this method. In order to increase hGH productivity of the cells, GF32 cells were selected with MTX, because the introduced hGH gene would be amplified with the dhfr gene by MTX selection (Urlaub and Chasin, 1980; Kaufman and Sharp, 1982). The hGH expression of a subline GT19 which became resistant against 500 nM MTX reached approximately 20 times that of the original transformant GF32, and the expression has been stable for up to 50 passages (data not shown). Thus we obtained a cell line GT19 producing stably a high level of hGH and performed further studies using this cell line. We first examined basal medium which was suitable for growth and hGH productivity of GT19 cells. With 5% dFCS, growth of GT19 cells in alpha MEM (Stanners et al., 1971) was better than that in various medium such as MEM, DME, ES (Ayusawa et al., 1981) and RPMI 1640 (data not shown). Parental CHO cells grew similarly well in alpha MEM medium. With 5% dFCS, alpha MEM, DME and ES were suitable for hGH production by GT19 cells. Although Ham F-12 medium was also appropriate, alpha MEM was better suited to our research because of its simple formulation. Thus we chose alpha MEM as basal medium for GT19 cells. The alpha MEM supported the growth of GT 19 cells with 5% dFCS, but not in the presence of 1% dFCS (Fig. 2). Thus we searched chemical components which supported growth and hGH productivity of GT19 ceils by addition to alpha MEM under 1% dFCS. As shown in Fig. 3, ferrous ion showed an especially remarkable effect on cell growth. Although this effect had been recognized in other studies in serum-free growth medium for CHO cells (Hamilton and Ham, 1977; Gasser et al., 1985), a similar effect was confirmed for
alpha M E M
UC-202
108
1o 6
~: 105
~: 10 5
dFCS
1"/. 0.5%
.$
0.1"/.
~
'~
10 4
i
i
i
i
1
4
7
10
104
i i 0 1
i 4
day
i 7
i 10
day
Fig. 2. Comparison of growth promoting activity of UC202 medium to alpha MEM in presence of various concentrations of dFCS. GT19 cells were inoculated at 1 • 104 per well. Each of the wells contained 0.5 ml of medium with an indicated concentration of dFCS.
GT19 line derived from CHO cells. This growth stimulation might have resulted from the deficiency of transferrin due to the low concentration of dFCS. The most effective dose of ferrous ion sulfate was 0.8 mg/l. Ferrous ion was also effective in increasing the yield of gene product hGH (data not shown). Additions of choline and i-inositol, which had already been present as components of the original medium, were effective on the cell growth and the hGH productivity when ferrous iron was present 3.0
8
2.0
~t o
,5
1.0
u
Z
o
o'-'
0 . 0 2 5 0 . 0'5 0
O. ', 0
0 . 2' 0
0 . 4. 0
.
0 . 8.0
.
1.6
3.2
FeSO 4"7H 2 0 conc. (#g / ml)
Fig. 3. Effect of FeSO4 to the growth of the GT19 cells. GT19 cells were inoculated at 5 x 103/well of 24-well plate, in which 0.5 ml of 1% dFCS/alpha MEM medium was added with indicated concentrations of FeSO 4. Five days after inoculation, cells in each well were counted.
12
_E 5.0 modified
medium
oo
|
4.0
12
E
3.0
~t o
2.0
o
x v
'-'r"
Chemically defined medium for the production of biologically active substances of CHO cells Jun-ichiro Hata 1, Takeyoshi Tamura 2, Soji Yokoshima 2, Shinya Yamashita 1, Shoko Kabeno 1, Ken Matsumoto 2 and Kazukiyo Onodera 3
1Central Research Laboratory, Nippon Suisan Kaisha, Ltd., Hachioji-shi, Tokyo, 190; 2Research Laboratory, Nissui Pharmaceutical Co. Ltd., Yuuki-shi, Ibaraki, 307; and 3Department of Agricultural Chemistry, The University of Tokyo, Bunkyou-ku, Tokyo, 113, Japan Received 13 January 1992; acceptedin revisedform 14 July 1992
Key words: recombinant CHO, hGH, dhfr, MTX, chemically defined medium Abstract
A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.
Abbreviations: CHO: Chinese hamster ovary; hGH: human growth hormone; dFCS: dialyzed fetal calf serum; dhfr: dihydroforate reductase; MTX: methotrexate.
Introduction
Chinese hamster ovary (CHO) cells established by Kao and Puck (1968) have often been used as a host cell in the production of recombinant proteins of mammalian origin, because genes transfected into the cells are stably expressed (Sano et al., 1988) and their transformants easily obtained due to the formation of a large number of colonies. Furthermore, CHO cells which were defective in dihydroforate reductase (dhfr) were isolated and had been used as host cells into which the foreign gene was transfected. The cells become resistant to high concentrations of methotrexate (MTX) by gene amplification (Urlaub and Chasin, 1980;
Kaufman and Sharp, 1982). With this property of the cells, dhfr-deficient CHO cell lines have been exploited in order to enhance the productivity of proteins encoded by co-introduced genes. To obtain high levels of the gene product by the cells, transcription units in expression plasmids have been also studied with inducible promoters (Friedman et al., 1989; Israel and Kaufman, 1989). The parental CHO cells grow relatively easily in serum-free medium (Hamilton and Ham, 1977; Gasser et al., 1985), but detailed studies of media to enhance the productivity of recombinant proteins in CHO cells have not been conducted. From this point of view, we constructed a expression plasmid containing the human growth hormone
10 (hGH) genomic gene (Selden et al., 1986) under SV40 transcriptional control, and constructed a CHO cell subline, designated GT19, carrying this plasmid. The GT19 cells continuously secreted a high level of hGH. We report here that basal medium components affected the productivity of exogenous gene-encoding proteins in CHO cells using GT19 cells. The exploitation of chemically defined medium allowed us to identify the chemical components which were suitable not only for the enhancement of the productivity of gene products by CHO cells, but also to get information on the regulation of gene expression and secretion of the product.
Hindlll dhfr
Pstl SV40 early promoter
pSV2-hGH / dhfr (7.2 Kb)
SV40 splice + poly (A}
SV40 early promoter
\ B,glll hGH
Materials and methods Fig. 1. Construction of the hGH expression plasmid pSV2-hGH/
Construction of an expression plasmid for hGH The plasmid p0GH containing the genomic sequence encoding hGH was purchased from Nichols Institute (Gasser et al., 1985). An expression plasmid pSV2-dhfr (ATCC 37146) containing the cDNA sequence of mouse dihydroforate reductase (dhfr) was digested with HindIII and BglII to remove the dhfr sequence. The 2.1 Kbp BamHIEcoRI hGH gene fragment containing a poly(A) addition site was then ligated to the HindIII-BglII site of pSV2 after treating both DNA insert and vector with DNA polymerase Klenow fragment. The EcoRI site of this plasmid, and the PvuIIBglII fragment isolated from pSV2-dhfr containing both SV40 promoter and dhfr sequences were blunt-ended using DNA polymerase Klenow fragment and were ligated. The clone thus obtained was designated pSV2-hGH/dhfr, and is shown in Fig. 1.
Culture and transfection of CHO cells CHO (dhfr-) cells, DXB 11 line (Urlaub and Chasin, 1980), were grown in alpha MEM medium with nucleosides (GIBCO) supplemented with 5% fetal calf serum (FCS). The expression plasmid pSV2-hGH/dhfr was linearized by EcoRI digestion, then transfected into CHO cells using
dhfr. The plasmid contains a 2.1 Kbp fragment of the hGH gene placed under control of the SV40 early promoter as described in Materials and methods. Thin single line represents plasmid pSV2 containing ampicillin-resistance gene.
electroporation (3.0 KV/cm, 50 Its, twice) (Potter et al., 1984). The transfected cells were selected by growing them in alpha MEM medium without nucleosides (GIBCO) supplemented with 5% dialyzed FCS (dFCS). Sublines which expressed a high level of hGH were isolated from a dhfr+ transformants expressing hGH by growing them in media containing indicated concentrations of methotrexate (MTX) (see text).
Assay of hGH productivities of CHO cells Concentrations of hGH which was expressed and secreted into medium by transformants were determined using RIA kit (Pharmacia). Culture supernatants were diluted with 0.5% BSA/PBS into the measurement range, and their final hGH concentration determined.
Results and discussion
The expression plasmid pSV2-hGH/dhfr containing both the hGH and the mouse dhfr sequences
11
under SV40 transcriptional control (Fig. 1), was transfected into CHO (dhfr-) cells using an electroporation technique. A cell line GF32 constitutively secreted the highest level of hGH into the medium among 48 dhfr+ transformants. The quantity of hGH secreted was determined using radioimmunoassay as described in Materials and methods. We could not detect any hGH in the culture supematant of the parental CHO cells by this method. In order to increase hGH productivity of the cells, GF32 cells were selected with MTX, because the introduced hGH gene would be amplified with the dhfr gene by MTX selection (Urlaub and Chasin, 1980; Kaufman and Sharp, 1982). The hGH expression of a subline GT19 which became resistant against 500 nM MTX reached approximately 20 times that of the original transformant GF32, and the expression has been stable for up to 50 passages (data not shown). Thus we obtained a cell line GT19 producing stably a high level of hGH and performed further studies using this cell line. We first examined basal medium which was suitable for growth and hGH productivity of GT19 cells. With 5% dFCS, growth of GT19 cells in alpha MEM (Stanners et al., 1971) was better than that in various medium such as MEM, DME, ES (Ayusawa et al., 1981) and RPMI 1640 (data not shown). Parental CHO cells grew similarly well in alpha MEM medium. With 5% dFCS, alpha MEM, DME and ES were suitable for hGH production by GT19 cells. Although Ham F-12 medium was also appropriate, alpha MEM was better suited to our research because of its simple formulation. Thus we chose alpha MEM as basal medium for GT19 cells. The alpha MEM supported the growth of GT 19 cells with 5% dFCS, but not in the presence of 1% dFCS (Fig. 2). Thus we searched chemical components which supported growth and hGH productivity of GT19 ceils by addition to alpha MEM under 1% dFCS. As shown in Fig. 3, ferrous ion showed an especially remarkable effect on cell growth. Although this effect had been recognized in other studies in serum-free growth medium for CHO cells (Hamilton and Ham, 1977; Gasser et al., 1985), a similar effect was confirmed for
alpha M E M
UC-202
108
1o 6
~: 105
~: 10 5
dFCS
1"/. 0.5%
.$
0.1"/.
~
'~
10 4
i
i
i
i
1
4
7
10
104
i i 0 1
i 4
day
i 7
i 10
day
Fig. 2. Comparison of growth promoting activity of UC202 medium to alpha MEM in presence of various concentrations of dFCS. GT19 cells were inoculated at 1 • 104 per well. Each of the wells contained 0.5 ml of medium with an indicated concentration of dFCS.
GT19 line derived from CHO cells. This growth stimulation might have resulted from the deficiency of transferrin due to the low concentration of dFCS. The most effective dose of ferrous ion sulfate was 0.8 mg/l. Ferrous ion was also effective in increasing the yield of gene product hGH (data not shown). Additions of choline and i-inositol, which had already been present as components of the original medium, were effective on the cell growth and the hGH productivity when ferrous iron was present 3.0
8
2.0
~t o
,5
1.0
u
Z
o
o'-'
0 . 0 2 5 0 . 0'5 0
O. ', 0
0 . 2' 0
0 . 4. 0
.
0 . 8.0
.
1.6
3.2
FeSO 4"7H 2 0 conc. (#g / ml)
Fig. 3. Effect of FeSO4 to the growth of the GT19 cells. GT19 cells were inoculated at 5 x 103/well of 24-well plate, in which 0.5 ml of 1% dFCS/alpha MEM medium was added with indicated concentrations of FeSO 4. Five days after inoculation, cells in each well were counted.
12
_E 5.0 modified
medium
oo
|
4.0
12
E
3.0
~t o
2.0
o
x v
'-'r"
