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ABSTRACT: To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines, FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the three clones was decreased by 21-39%, which was because of a decreased specific glucose uptake rate and yield of lactate from glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO cell culture can be efficiently reduced.
Applied Microbiology and Biotechnology 10/2007; 76(3):659-65. · 3.42 Impact Factor
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ABSTRACT: Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75-89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54-87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45-79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.
Applied Microbiology and Biotechnology 03/2007; 74(1):152-9. · 3.42 Impact Factor
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ABSTRACT: To characterize the changes in cell growth rate and adenovirus vector (AdV) production capability of 293 cells during culture passages, 293 cells obtained at the 31st culture passage from ATCC (293M #31) were maintained as a monolayer culture and 293 cells obtained at an unknown culture passage from Invitrogen (293S) were maintained as suspension culture. In monolayer culture, the specific growth rate (micro) of 293M cells increased rapidly with culture passage up to passage 65 and thereafter became saturated. The micro of 293M passage 43 (#43) was 0.29 day(-1), while the average micro of 293M from #66 to #86 was 0.74+/-0.01 day(-1) (average +/- standard deviation). It was also noted that the cells became smaller in size during early culture passages. AdV production was also influenced by the number of culture passages. The AdV titer in the culture of 293M #66 was ca. tenfold higher than that of 293M #44, resulting from both a higher cell concentration and a higher AdV titer per cell at #66. In contrast, the micro, cell size, and AdV production of 293S cells in suspension culture did not change significantly as the culture passage number increased up to #40. Taken together, the culture passage influenced cell growth and AdV production of 293M cells in monolayer culture, but not those of 293S cells in suspension culture.
Applied Microbiology and Biotechnology 11/2004; 65(5):553-8. · 3.42 Impact Factor
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Chung-Hyun Cho,
Richard A Kammerer,
Hyuek Jong Lee,
Kunio Yasunaga,
Kyung-Tae Kim,
Han-Ho Choi,
Won Kim, Sung Hyun Kim,
Sung Kwang Park,
Gyun Min Lee,
Gou Young Koh
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ABSTRACT: Radiation therapy is a widely used cancer treatment, but it causes side effects even when localized radiotherapy is used. Extensive apoptosis of microvascular endothelial cells of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Many in vitro studies suggest that angiopoietin-1 (Ang1) has potential therapeutic applications in enhancing endothelial cell survival. For in vivo use, we developed a soluble, stable, and potent Ang1 variant, COMP-Ang1. COMP-Ang1 is more potent than native Ang1 in phosphorylating the Tie2 receptor in lung endothelial cells in vivo. Interestingly, COMP-Ang1 administered i.v. was mainly localized to microvascular endothelial cells of the intestinal villi and lung but not to microvascular endothelial cells of the liver. In irradiated mice, i.v. COMP-Ang1 protected against radiation-induced apoptosis in microcapillary endothelial cells of the intestinal villi and prolonged survival. Thus, COMP-Ang1 could be used as a therapeutic protein for specific protection against endothelial cell injury.
Proceedings of the National Academy of Sciences 05/2004; 101(15):5553-8. · 9.68 Impact Factor
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Chung-Hyun Cho,
Richard A Kammerer,
Hyuek Jong Lee,
Michel O Steinmetz,
Young Shin Ryu,
Sung Ho Lee,
Kunio Yasunaga,
Kyung-Tae Kim,
Injune Kim,
Han-Ho Choi,
Won Kim, Sung Hyun Kim,
Sung Kwang Park,
Gyun Min Lee,
Gou Young Koh
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ABSTRACT: Angiopoietin-1 (Ang1) has potential therapeutic applications in inducing angiogenesis, enhancing endothelial cell survival, and preventing vascular leakage. However, production of Ang1 is hindered by aggregation and insolubility resulting from disulfide-linked higher-order structures. Here, by replacing the N-terminal portion of Ang1 with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP), we have generated a soluble, stable, and potent Ang1 variant, COMP-Ang1. This variant is more potent than native Ang1 in phosphorylating the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor and Akt in primary cultured endothelial cells, enhancing angiogenesis in vitro and increasing adult angiogenesis in vivo. Thus, COMP-Ang1 is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo.
Proceedings of the National Academy of Sciences 05/2004; 101(15):5547-52. · 9.68 Impact Factor
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ABSTRACT: Lowering the culture temperature has been suggested as a useful tool for improving the production of recombinant proteins in Chinese hamster ovary (CHO) cells. In an effort to improve anti-4-1BB antibody production in recombinant CHO (rCHO) cells, rCHO cells producing anti-4-1BB antibody (LGA31-56) were cultivated at three different temperatures, 30, 33, and 37 degrees C. Lowering the culture temperature led to suppressed cell growth, cell cycle arrest in G(0)/G(1) phase, and improved cell viability for a longer period. However, antibody production and q(Ab) were not increased at low culture temperature. The maximum antibody concentration and q(Ab) at 37 degrees C were 110.6 +/- 2.6 microg mL(-)(1) and 0.43 +/- 0.03 microg (10(6) cells h)(-)(1), respectively, whereas those at 30 degrees C were 28.3 +/- 3.8 microg mL(-)(1) and 0.44 +/- 0.07 (10(6) cells h)(-)(1), respectively. Northern blot analysis revealed that lowering the culture temperature did not increase the transcription level of heavy and light chains. These results were quite in contrast with the improved production of erythropoietin, which is expressed in the same CHO host and driven by the same CMV promoters, by lowering the temperature. Taken together, the results obtained imply that the beneficial effect of low culture temperature on recombinant protein production in rCHO cells is cell-line-specific.
Biotechnology Progress 19(4):1383-6. · 2.34 Impact Factor
