• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.470-475
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• 2000

Genetic chracterstics of the structural gene of guamerin (a novel elastase inhibitor from Korean leech), integrated into the HIS4 locus of chromosomal DNA of Pichia pastoris along with the $\alpha$-factor leader sequence, were investigated. In the selected clone from candidates, two copies of the integration cassette including the structural gene copies of the integration cassette including the structural gene of guamerin were found in the integration site of the chromosomal DNA of P.pastoris. It was demonstrated that the integrated structural gene of guamerin was stable up to about 70 generations in the relay flask culture. Then, a high-cell-density culture could be fulfilled easily by DO-stat fed-batch culture, in which the cell growth and the recombinant guamerin production reached about 250 of OD600nm and 260 mg/l, respectively. Finally, it was revealed that the DNA sequence of the integrated structural gene of guamerin in P. pastoris was maintained correctly in the end of production cells of relay flask culture and high-cell-density culture.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.2
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• pp.110-117
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• 2000

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production rate increased with increasing specifis growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomana, MAb mPNA experession and cell cycle distribution were investigated in bacth cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromodeoxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb nRNA expression until dead phase, which was longer than in suspended cell. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated Mab productivity of T0405 cells.

• Microbiology and Biotechnology Letters
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• v.24 no.6
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• pp.717-725
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• 1996

We have developed an efficient immobilized cell separator for continuous operation of immobilized fungal cell cultures, and applied this separator to actual fermentation process for the production of cyclosporin A (CyA), a powerful immunosuppressant. In the experiments employing highly viscous polymer (carboxymethyl cellulose) solution, the decantor showed good separating performances at high solution viscosites and fast dilution rates. Air duct and cylindrical separator installed inside the decantor turned out to play key roles for the efficient separation of the immobilized cells. By installing the decantor in an immobilized perfusion reactor system (IPRS), continuous immobilized culture was stably carried out even at high dilution rate for a long period, leading to high productivities of free cells and CyA. Almost no immobilized biomass existed in effuluent stream of the IPRS, demonstrating the effectiveness of the decan- tor system for a long-term continuous fermentation. It was noteworthy that we could obtain these results despite of the unfavorable fermentation conditions, i.e., reduced density of the biosupports caused by overgrowth of cells inside the bead particles and existence of high density of suspended fungal cells (10g/l) in the fermentation broth.

• KSBB Journal
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• v.8 no.5
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• pp.478-482
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• 1993

Perfusion culture was conducted in Celligen perfusion culture system using a self-constructed hybridoma cell and low serum medium. The culture system employed hollow fiber to separate cells from the culture broth. Maximum cell density of $2.1\times10^7$ ce11s/m1, 10 times higher than in batch culture, could be achieved. Concentration of monoclonal antibody (MAb) was 4 times higher and production rate at maximum feed rate was 9 times higher than in batch culture. Glucose concentration was very important for the cell growth and MAb production. When glucose concentration was below 1g/l, i. e. 0.5~0.9g/l, specific MAb production rate decreased but cell concentration still increased. As the glucose concentration goes above 1g/l, specific MAb production rate increased and remained at maximum value at more than 1.5g glucose/l. The maximum value of the specific Mab production rate was similar to that of batch culture.

• Journal of Microbiology and Biotechnology
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• v.6 no.3
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• pp.221-224
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• 1996

Pseudomonas putida BM01 was grown efficiently on glucose as the sole carbon source with a supply of a nitrogen source in pH-stat mode using a low setpoint limit. A final cell concentration of 100 g/l was obtained in 30 h of fed-batch cultivation by controlling glucose concentration within the range of 5-20 g/l and maintaining dissolved oxygen tension above 10$%$ saturation using pure oxygen. This high cell density culture technique is believed highly useful for the production of poly(3-hydroxyalkanoates) by this strain.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.1
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• pp.41-46
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• 2003

Difficulties associated with bioartificial liver (BAL) preservation limit not only the commercialization of BAL, but also its clinical trials. In this study, the possibility of cold preservation of BAL cartridges containing porcine hepatocytes was examined at 4$^{\circ}C$. In an in vitro perfusion culture System, BAL cartridges maintained cytochrome P450 metabolic function for at least 50 days. However, all BAL cartridges completely lost their ammonia eliminating ability when stored at 4$^{\circ}C$. We a1so studied the effect of cell density on the maintenance of BAL liver function In a highly differentiated and healthy state. As expected, BALs containing a larger number of hepatocytes demonstrated higher metabolic functions. When metabolic functions were compared per gram of hepatotytes, no large differences were observed between devices containing different densities of hepatocytes. Decreased cell density did not Successfully prolong BAL function. The viability and function of isolated hepatotytes highly depend on the culture conditions, such as cell density, substrata, culture media, and additives to the culture media. Perfusion culture of BAL cartridges at 4$^{\circ}C$ gave a promosing result with respect to the maintenance of P450 activity. However, as indicated by the rapid loss of ammonia metabolic activity, many factors still remain to be optimized for preservation of BAL keeping high metabolic functions for a longer time.

• Journal of Embryo Transfer
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• v.15 no.2
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• pp.129-136
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• 2000

This study was carried out to investigate the effect of biological factors on the in vitro production(IVP) of bovine oocytes for development of simple culture methods and medium. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol and this study was examined if there were necessary to co-culture, media change, media type and embryo density. This results were as follows: 1. The development rate according to co-culture with cumulus cells and non co-culture as drop culture was not significantly different in cleavage (68.9 vs 71.7%), 8-cell stage (41.2 vs 44.1%) and blastocyst stage (12.2 vs 13.8%), respectively (p<0.05) 2. The blastocyst development rates in YS and CRIaa were higher than that in TCM199 (12.4, 10.4$ vs 3.7%), but the cleavage (69.0, 77.8 and 61.0%) and 8-cell stage (31.7, 37.0 and 35.7%) development accoring to YS, TCM199 and CRIaa ws not significantly different, respectively (p<0.05). 3. There was no significantly different in cleavage (62.6, 59.5 and 61.2%), 8-cell(34.7, 37.9 and 34.0%) and blastocyst (9.5, 11.6 and 12.8%) development among medium change time as control, Group I and Group II, respectively (p<0.05). 4. Blastocyst formation of 8-cell stage according to embryo density was not significantly different in 1, 10 and 25 embryos (27.3, 22.5 and 34.0%), respectively (p<0.05). These results indicated that simple culture system could reduce bovine IVP embryos as drop culture as non co-culture system, high density embryo (25 embryos/50 $\mu$1 drop). YS defined medium and no medium change for whole culture period, although other biological factors need to examine in order to produce efficient IVP bovine embryos.

• KSBB Journal
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• v.13 no.3
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• pp.272-278
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• 1998

Cell recovery from high cell density broths of Alcaligenes eutrophus by pretreatment with aluminum-based coagulants such as aluminum sulfate, polyaluminum hydrooxide chloride silicate (PACS), and polyaluminum hydrooxide chloride (Hi-PAX) was carried out. Cells coagulated with coagulants could be successfully recovered above 95-99% by centrifugation or filtration. The optimum initial pH of fermentation broths for cell recovery was in the range of 10 to 12. Optimum coagulants dosage for cell recovery increased with increasing of cell concentrations (21-160 g/L). The optimum coagulant dosages to recover cells with more than 95% cell recovery by centrifugation for the cell concentrations ranged 21-160 g/L were as follows: aluminum sulfate, 416-1708 mg Al/L; PACS, 211-826 mg Al/L; Hi-PAX, 320-960 mg Al/L. At optimum conditions for the coagulation of cells, centrifugal forces for 95% of cell recovery were dependent on the cell concentration. The centrifugal forces at 82 g/L and 160 g/L of cell concentration were only 45${\times}$g and 1600${\times}$g, respectively.

• KSBB Journal
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• v.13 no.2
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• pp.181-186
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• 1998

In order to obtain high-level production of recombinant human thrombopoietin (rhTPO) in insect cell line, HTI-TN-5B1-4 (TN5), conditions for optimal rhTPO expression such as multiplicity of infection (MOI), the cell density at infection, harvesting time and type of culture method as well as growth media were determined. When TN5 cells were cultured as anchorage-dependent state in 60-mm dish, cell density $2\times^6$ cells,MOI of 10 and Garvesting the culture media at 72 hr post-infection wrere the cinditions for highest rh TPO production. High production of rhTPO was also achieved by using EXPRESS FIVE serum free media rather than SF900II serum free media-1. Anchorage-dependent TN5 cells were adapted as a suspension culture when they were grown in the presence of heparin. TN5 cells were successfully cultured at 0.2 L scale in suspension culture without having aggregation. When TN5 cells were cultured as suspension state, cell density of $0.6\times10^6$ cells/mL, MOI of 1 and harvesting the culture media at 72 hr post-infection, gave the highest yield of rhTPO.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.600-603
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• 2000

DNA chip containing 207 E. coli genes related to important metabolisms such as (TCA cycle, glycolysis, fermentation and etc) were used to carry out a comprehensive investigation of the change in metabolism and physiology during high cell density culture of E. coli by fed-batch cultivation.