Effect of static pressure on human granulocyte-macrophage stimulating factor (hGM-CSF) production by Chinese hamster ovary cells

JOURNALOFBIOSCIENCE ANDBIOENGINEERING Vol. 94, No. 3,271-274. 2002

Effect of Static Pressure on Human Granulocyte-Macrophage Stimulating Factor (hGM-CSF) Production by Chinese Hamster Ovary Cells HENG GONG,’ MUTSUMI TAKAGI,‘* TETSUHIRO MORIYAMA,’ TSUYOSHI OHNO,’ AND TOSHIOMI YOSHIDA’ International Centerfor Biotechnology, Osaka University, 2-l Yamada-oka, Suita, Osaka 565-0871, Japan’ Received 9 May 2002/Accepted 5 July 2002

The effect of static pressure on the production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by the Chinese hamster ovary (CHO) cell line DRlOOOL4N was investigated. Although constant static pressures between 0.15 and 0.9 MPa had little influence on specific growth rate, the specific production rate of hGM-CSF increased from 1.23 to 1.62~10~‘~ mg/cell/h in proportion to static pressure. The specific rate of tPA production by the human embryo lung cell line MRC-5 also increased from 2.92 to 6.38x10-‘* mg/cell/h as static pressure increased. The expression level of hGM-CSF mRNA increased with the pressurization even for 4 h. [Key words: static pressure, CHO

cells, GM-CSF, protein production,mRNA]

Static pressure is one of the operational parameters that are effective in increasing the supply rate of dissolved oxygen in mammalian cell cultures. Although the effects of pH, temperature, dissolved oxygen concentration and agitation rate on mammalian cell cultures have been well documented, there are only few reports describing the effect of static pressure on mammalian cells. Weak pressurization of up to 15 cm H,O ( 1.5 x 10m3MPa) was reported to induce the release of basic fibroblast growth factors and elongation of endothelial cells (1). Intermittent pressurization of up to 6.87 MPa stimulated the production of an extracellular matrix by chondrocytes (2). Pressurization at 0.2 MPa of a culture of murine marrow cells caused a decrease in the expression level of mRNA coding for the membrane-bound form of macrophage colony stimulating factors and led to the formation of few osteoclast-like cells (3). However, there are few engineering studies describing the effect of static pressure on protein production by mammalian cells. We have previously demonstrated that the conversion rate of glucose to lactate in human embryo lung (HEL) cells at 0.25 MPa was less than that at 0.12 MPa and the production rate of tPA increased by 16% as pressure increased (4). In the cultures of mouse hybridoma cells (AFP-27) grown at various constant static pressures ranging between 0.1 and 0.9 MPa, the specific production rate of a monoclonal antibody increased from 4.5 to 5.6x IO-‘O mg/cell/h in proportion to the pressure increase (5). In these reports, the partial pressures of oxygen and carbon dioxide were controlled to be constant independent of the pressure in order to avoid the difference in pH and dissolved oxygen concentration at different pressures. Recently, the Chinese hamster ovary (CHO) cell line has * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-7456 fax: +86-(0)6-6879-7454 271

been one of the most useful mammalian cell lines as a host for gene engineering and has been employed extensively in industrial cultivation processes for production of pharmaceutical substances using mammalian cells. Thus, it may be important to determine the effect of static pressure on protein production by CHO cells. For example, human granulocyte-macrophage stimulating factor (hGM-CSF) is an important hematopoietic growth factor (6), which induces a large variety of biological effects, such as proliferation induction in early progenitors, stimulation of differentiation of various myeloid lineages (7), cooperative function with erythroid cytokines (8), as well as regulation of mature cell function (6). In clinical applications, GM-CSF has been shown to dramatically elevate neutrophil levels in primates in vivo (9), and thus, can be used to adjust neutrophil levels induced by cytotoxic chemotherapy ( 10). In this study, the effect of static pressure on the production of human granulocyte-macrophage colony-stimulating factors (hGM-CSFs) by the CHO cell line DRlOOOL4N was investigated. MATERIALS

AND METHODS

Cells and medium CHO DRlOOOL4N cells producing hGMCSF (11) were cultivated employing Iscove’s Modified Dulbecco’s Medium (catalog no. 17633; Sigma, Louis, MO, USA) supplemented with 10% dialyzed fetal calf serum (lot no. 1025398; Gibco, Grand Island, NY, USA), 0.1 mgll streptomycin (Gibco), 100 U/I penicillin (Gibco) and 1 PM methotrexate (catalog no. M8407; Sigma). Dullbecco’s Modified Eagle’s Medium (catalog no. 1033120; ICN, OH, USA) supplemented with 10% new born bovine serum (lot no. 1000620; Gibco), streptomycin, and penicillin was used for the culturing human embryo lung cells (MRC-5; IF0 50073) that produce tissue plasminogen activator (tPA). Cell culture T-flasks for cell culture (25 cm2; Coming, NY, USA) containing 5 ml of the medium were inoculated with respec-

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FIG. 1. Schematic diagram of the pressurized incubator system. tive cells (DRlOOOL4N, 2.4 x IO5cells; MRC-5, 1.1 x lo5 cells), incubated in a conventional CO, incubator (37’C, 5% CO,) for 24 h to induce cell adhesion and then, further incubated in a pressurized incubator as described below. The pressurized incubator system (Fig. 1) consisted of a stainless steel vessel (length, 120 mm; inner diameter, 95 mm), closed using flanges fitted with valves, a pressure gauge, and a PVA filter. After the T-flasks were put into the stainless steel vessel and the gas in the vessel was replaced with a gas mixture of 5% CO,+95% air, they were pressurized up to desired pressures between 0.1 (1 atm) and 0.9 MPa using nitrogen gas. The culture temperature was maintained at 37°C using a water bath. The partial pressures of 0, and CO, in the T-flasks were, respectively, 0.021 and 0.005 MPa independent of static pressure in order to avoid the difference in pH and dissolved oxygen concentration at different pressures. The vessel was not opened until the end of cultivation. The pH of the medium in the centrifuge tube placed in the pressurized incuba-

tor was measured immediately after opening the incubator in order to confirm that there is no difference in CO, gas partial pressure between the pressurized incubators (data not shown). All culture experiments were performed more than twice and the reproduction of the tendency of results were confirmed. The cell concentration in the cultures was General analysis determined by nuclei staining, in which the adhering cells were incubated in a solution of 21 g/Z citrate and 1 8/Z crystal violet, and nuclei were counted under a microscope. hGM-CSF and tPA concentrations in the culture supematant were determined triplicate using enzyme-linked immunosorbent assay kits, respectively (Endogen, Woburn, MA, USA and Biopool, Umea, Sweden) and the average value was shown in Fig. 2. Determination of hGM-CSF mFWA Total RNA from CHO DRlOOOL4N cells (5 x 1OScells) was eluted using 80 pl of RNase free water from the RNeasy mini kit (Qiagen, Hilden, Germany). The eluate (2 ~1) was used for the reverse transcription reaction employing Omniscript Reverse Transcriptase (Qiagen) and an Oligo d(T) primer. This reverse transcription product was used for PCR employing HotStarTaq Master Mix (Qiagen) and biotinylated primers for hGM-CSF mRNA. The amplified product was determined using a probe-coated plate (hGM-CSF mRNA XpressPack kit; Chemicon, Temecula, CA, USA) and a calorimetric OligoDetect detection system (Chemicon). Results are expressed as meansfS.D. Statistical analysis Comparisons were performed using a t-test. Differences were considered significant at PcO.05.

RESULT Effect of static pressure on hGM-CSF production by After inducing adhesion of CHO cells in the CHO cells

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FIG. 2. Effects of static pressure on growth and hGM-CSF production by CHO DRlOOOL4N cells. Growth cultures of CHO cells were performed at various static pressures. (A) The specific cell growth rate; (B) the specific hGM-CSF production rate. Each assay done in triplicate. Average of duplicate culture was shown.

T-flask at 0.1 MF’a for 24 h, the cells were simultaneously cultured at 0.1 MPa and in the pressurized incubator at con-

stant static pressures of 0.15, 0.4, 0.75 and 0.9 MPa. Monitoring of the cell concentration during the conventional CO, incubator cultivation showed logarithmical cell growth, even at 120 h when pressurization was terminated (data not shown). The specific growth rates of the CHO cells decreased slightly as static pressure increased resulting in 92% rates at 0.9 MPa compared with that at 0.15 MPa (Fig. 2A). The specific production rate of hGM-CSF increased in proportion to the pressure increase and the increment at 0.9MPa was 32% of that at 0.15 MPa (Fig. 2B). This dependence on static pressure agreed with our previous results for HEL and hybridoma AFP-27 cells (4,5). In order to further confirm the above-mentioned dependence, tPA-producing MRC-5 cells as another example of protein producer cells were cultured in the same manner as above. As shown in Fig. 3A, the specific cell growth rate of MRC-5 cells did not differ even with increasing static pressure but the specific production rate of tPA increased with increasing static pressure up to 0.75 MPa. (Fig. 3B)

EFFECT OF STATIC PRESSURE ON GM-CSF PRODUCTION

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FIG. 3. Effects of static pressure on growth and tPA production by MRC-5 cells. Growth cultures of MRC-5 cells were performed at various static pressures. (A) The specific cell growth rate; (B) the specific tPA production rate. Each assay done in triplicate. Average of duplicate culture was shown.

Effect of static pressure on LGM-CSF mRNA expression level in CHO cells In order to determine the cause of the increase in hGM-CSF production rate at high static pressure, the effect of static pressure on hGM-CSF mRNA expression level was investigated. After cell adhesion at 0.1 MPa for 24 h, CHO cells were simultaneously cultured for 72 h in the pressurized incubator at constant static pressures of 0.1 and 0.9 MPa. The relative hGM-CSF mRNA expression levels (absorbance at 450 mn) in cells grown at 0.1 and 0.9MPa were 0.33kO.05 (n=Q 0.40+0.07 (n=8), respectively (Fig. 4A). On the other hand, the absorbance of pactin mRNA did not differ with increasing static pressure (PcO.05) (Fig. 4B). Consequently, the ratios of hGM-CSF mRNA expression level to p-actin mRNA expression level in the cells grown at 0.1 and 0.9MPa were 0.56kO.07 (n=3) and 0.75+0.08 (n=3), respectively (Fig. 4C). These values were significantly different (PcO.05). It was concluded that the hGM-CSF mRNA expression level in the cells grown at 0.9 MPa was higher than that at 0.1 MPa. To further study the effect of static pressure on the hGMCSF mRNA expression level in CHO cells, cells grown to near confluence at 0.1 MPa in a conventional CO, incubator were further incubated for 4 h at constant static pressures of

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FIG. 4. Effect of static pressure on mRNA expression level (*: WO.05). Each mRNA level was determined at the end of growth culture of CHO cells at 0.1 and 0.9 MPa. (A) hGM-CSF mRNA level; (B) 5-actin mRNA level; (C) the ratio of hGM-CSFQ-actin mRNA level. The bar showed the SD of measurements (n23).

0.1 and 0.9 MPa. The absorbances of hGM-CSF mRNA in the CHO cells after the incubation at 0.1 and 0.9 MPa were 0.13+0.01 (n=3) and 0.21kO.02 (n=3), respectively (Fig. 5). These significantly different (PcO.05) values indicated that high static pressure rapidly enhanced the expression of hGM-CSF mRNA even without cell growth. DISCUSSION We have previously demonstrated (4) that the conversion rate from glucose to lactate in HEL cells cultured at 0.25 MPa was less than that at 0.12 MPa and the production rate of tPA increased by 16% as pressure increased. The specific production rate of a monoclonal antibody by hybridoma

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REFERENCES

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expression level. Cells grown near confluence at 0.1 MPa were further incubated for 4 h at constant static pressuresof 0.1 and 0.9 MPa (*: PiO.05).

cells (AFP-27) grown at various constant static pressures between 0.1 and 0.9 MPa increased from 4.5 to 5.6x lo-lo mg/cell/h in proportion to the pressure increase (5). Here, we showed that the specific production rate of hGM-CSF by CHO DRlOOOL4N cells increased markedly in proportion to the pressure increase and the increment at 0.9 MPa was 32% of that at with 0.15 MPa. On the other hand, the specific growth rate of CHO DRlOOOL4N cells decreased slightly as static pressure increased and resulted in a 92% increase at 0.9 MPa relative to that at 0.15 MPa (Fig. 2). The production of tPA by MRC-5 cells was also stimulated at high static pressure (Fig. 3). All these results implies that static pressure, as well as pH, temperature, dissolved oxygen concentration and agitation rate, plays an important role in protein production by mammalian cells. Moreover, it is interesting that the production of proteins increased at higher static pressure independent of the kinds of cells used and proteins determined. It is suggested that increasing static pressure is a strategy to increase protein production in mammalian cells. The hGM-CSF mRNA expression level in the CHO cell line DRlOOOL4N cells grown at 0.9 MPa was higher than that at 0.1 MPa. This effect was observed even after a short incubation period without cell growth. It is strongly suggested that the expression of the hGM-CSF gene must be regulated by pressure, which might be the major reason for the increase in hGM-CSF productivity at high static pressure, while there remains the possibility that the membrane permeability for the secretion of these proteins was influ-

1. Acevedo, A. D., Bowser, S. S., Gerritsen, M. E., and Bizios, R.: Morphological and proliferative responses of endothelial cells to hydrostatic pressure: role of fibroblast growth factor. J. Cell. Physiol., 157,603-614 (1993). 2. Carver, S. E. and Heath, C.A.: Increasing extracellular matrix production in regenerating cartilage with intermittent physiological pressure. Biotechnol. Bioeng., 62, 166-I 74 (1999). 3. Rubin, J., Blskobing, D., Fan, X., Rubin, C., Mcleod, K., and Taylor, W. R.: Pressure regulates osteoclast formation and MCSF expression in marrow culture. J. Cell. Physiol., 170, 81-87 (1997). 4. Takagi, M., Okumura, H., Okada, T., Kobayashi, N., Kiyota, T., and Ueda, K.: An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture. J. Ferment. Bioeng., 77, 301-306 (1994). 5. Takagi, M., Ohara, K., and Yosbida, T.: Effect of hydrostatic pressure on hybridoma cell metabolism. J. Ferment. Bioeng., 80,619-621 (1995). 6. Metcalf, D.: Clonal analysis of proliferation and differentiation of paired daughter cells: action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors. Proc. Natl. Acad. Sci. USA, 77,5327-5330 (1980). I. Metcalf, D.: The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature, 339,27-30 (1989). 8. Nisbijlma, I., Nakabata, T., Hirabayashi, Y., Inoue, T., Kurata, H., Miyajima, A., Hayashi, N., Iwakura, Y., Arai, K., and Yokota, T.: A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF. Mol. Biol. Cell, 6,497-508 (1995). 9. Donahue, R E., Wang, E. A., Stone, D. K., Kamen, R, Wong, G. G., Sebgal, P. K., Nathan, D. G., and Clark, S. C.: Stimulation of haematopoiesis in primates by continuous infusion of recombinant human GM-CSF. Nature, 321, 872-875 (1986). 10. Baldwin, G. C., Gasson, J. C., Quan, S. G., Fleischmann, J., Weisbart, R, Oette, D., Mitsuyasu, RT., and Golde, D. W.: Granulocyte-macrophage colony-stimulating factor enhances neutrophil function in acquired immunodeficiency syndrome patients. Proc. Natl. Acad. Sci. USA, 85, 27632766 (1988). 11. Omasa, T., Itami, S., Kameoka, D., Katakura, Y., and Suga, K.: Selection and stability for a recombinant CHO cell line expressing human GM-CSF in gene amplification, p. 2933. In Ikura, K., Nagao, M., Masuda, S., and Sasaki, R. (ed.), Animal cell technology: basic and applied aspects, vol. 9. Kluwer Academic Publishers, London (1998).