• Journal of Microbiology and Biotechnology
• /
• v.23 no.12
• /
• pp.1747-1756
• /
• 2013

The aim of this paper was to study the effect of spent medium recycle on Spodoptera exigua Se301 cell line proliferation, metabolism, and baculovirus production when grown in batch suspension cultures in Ex-Cell 420 serum-free medium. The results showed that the recycle of 20% of spent medium from a culture in mid-exponential growth phase improved growth relative to a control culture grown in fresh medium. Although both glucose and glutamine were still present at the end of the growth phase, glutamate was always completely exhausted. The pattern of the specific glucose and lactate consumption and production rates, as well as the specific glutamine and glutamate consumption rates, suggests a metabolic shift at spent medium recycle values of over 60%, with a decrease in the efficiency of glucose utilization and an increase in glutamate consumption to fuel energy metabolism. Baculovirus infection provoked a change in the metabolic pattern of Se301 cells, although a beneficial effect of spent medium recycle was also observed. Both growth rate and maximum viable cell density decreased relative to uninfected cultures. The efficiency of glucose utilization was dramatically reduced in those cultures containing the lowest percentages of spent medium, whereas glutamine and glutamate consumption was modulated, thereby suggesting that infected cells were devoted to virus replication, retaining their ability to incorporate the nutrients required to support viral replication. Recycle of 20% of spent medium increased baculovirus production by around 90%, thus showing the link between cell growth and baculovirus production.

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.1
• /
• pp.9-14
• /
• 1999

Factors affecting gene gun-mediated expression of the human erythropoietin gene were investigated in primary cultured oviduct cells from laying hens. The human erythropoietin gene was transfected by a gene gun method at $1.25{\mu}g$ per dish, and cultured in a synthetic serum-free medium for 72 hrs. The concentration of human erythropoietin mRNA was determined by RNA : RNA solution hybridization. In experiment 1, the effect of changing the shooting pressure of DNA-coated microparticles with nitrogen gas was tested at 20 and $60kgf/cm^2$. The results showed that the erythropoietin mRNA concentration was significantly higher at 60 than $20kgf/cm^2$. In experiment 2, the effects of supplementing the medium with fetal calf serum at 10%, and raising the shooting pressure from 60 to $80kgf/cm^2$ on the cell number and erythropoietin gene expression were examined. Although supplementation with fetal calf serum significantly increased the cell numbes compared with no supplemented controls (p < 0.05), erythropoietin mRNA concentration per $10^3$ cells was not affected. Raising the shooting pressure from 60 to $80kgf/cm^2$ did not affect either of the parameters, In experiment 3, the effect of supplementing ascorbate 2-phosphate at 0.5 mM was tested. The results indicated that the ascorbate supplementation significantly increased the cell number (p < 0.05), and tended to increase erythropoietin mRNA concentration (p < 0.1). Thus, for human erythropoietin gene expression by using the gene gun method, shooting pressure with nitrogen gas should be sufficient at $60kgf/cm^2$ and supplementation with ascorbate phosphate would be useful to enhance not only the cell proliferation but also erythropoietin gene expression.

• Korean Chemical Engineering Research
• /
• v.43 no.1
• /
• pp.103-109
• /
• 2005

Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. $1.92{\pm}0.59{\times}10^6$ viable cells were isolated by trypsin enzymatic digestion method from $0.25cm^2$ biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 0.15 mM $Ca^{2+}$ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn't have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 2.0 mM $Ca^{2+}$, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.

• Journal of Nutrition and Health
• /
• v.29 no.2
• /
• pp.159-165
• /
• 1996

This study was performed to investigate the effects of concentration and degree of unsaturation of fatty acids on cellular proliferation and lipid peroxide production, using primary skin fibroblasts from neonate rats Fibroblasts (CPD : 2.8-5.4). Cells were cultured either in control medium (Dulbecco's modified Eagle's medium supplement with 10% fetal bovine serum) or in media supplemented with various kinds (stearic, oleic, linoleic, arachidonic, linolenic, eicosapentaenoic acid) and amounts (5, 10, 25, 50, 100, 150uM)of fatty acids. Cellular proliferation ratio and lipid peroxice production were measured and morphological changes were observed. Cellular proliferation was inhibited and morphological changes were observed. Cellular proliferation was inhibited and morphological changes were observed in cells grown in stearic containing media. Oleic, arachidonic, and eicosapentaenoic aicd tend to stimulate cellualar proliferation, and linolenic acid had no effects. Lipid peroxide concentrations in fibroblasts increased in proportion to the contents and unsaturation of fatty acids in media. Especially supplementation of arachidonic acid accelerated cellualr lipid peroxidation. Free radicals may cause severs damage to biological molecules, so lipid peroxidation probably contributes cellular membrane damages. However there were little relationship between lipid peroxide production and cellular proliferation in this study. (Korean J Nutrition 29(2) : 159~165, 1996)

• Biomedical Science Letters
• /
• v.5 no.1
• /
• pp.85-93
• /
• 1999

Nitric oxide (NO) plays an important role in immunologic defense, and influences upon the functioning of secretory tissues and cells. It also exhibits cytotoxic/cytostatic activity as one of major operating effectors of the cellular immunity system. We investigated the effects of serum on the cell damages and NO production in the mouse liver cell line BNL CL.2 to establish the role of NO. We observed that, when BNL CL.2 cells were cultured in serum-free medium, they were induced to cell damage by the stimulation of IFN-$\gamma$ alone or IFN-$\gamma$ plus LPS. Serum-starved cells showed large amount of nitrite accumulation and NO synthase (NOS) expression in response to IFN-$\gamma$ alone in dose- and time- dependent manners, but serum-supplied cells did not The production of NO was blocked by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin. These results suggest that the deprivation of serum in the BNL CL.2 cell culture medium might primed with the cells to produce NO when the cells are triggered by IFN-$\gamma$ and the involvement of PTK signal transduction pathway in the expression of NOS gene in murine hepatocytes.

• Journal of Sasang Constitutional Medicine
• /
• v.13 no.1
• /
• pp.51-60
• /
• 2001

Effects of Taeumjowetang on Lipid Peroxidation by Free Radicals and Oxidative Damage of Hepatocytes by tert-Butyl Hydroperoxide. 1. Purpose The present study was carried out to evaluate the antioxidant effects of Taeumjowetang in vitro. 2. Methods In this study, antioxidant effects of TJT on lipid peroxidation were determined according to the method of TBA. (Abbreviation) TJT : Taeumjowetang, TBA : 2-thiobarbituric acid. 3. Results : 1) TJT inhibited markedly peroxidation of linoleic acid during the autoxidation. 2) TJT inhibited lipid peroxidation induced by hydroxyl radical derived from H2O2-Fe2+ in rat liver homogenate. 3) TJT showed 66% scavenging effect on DPPH radical. 4) TJT exhibited a 25% inhibitory effect on superoxide generation from xanthine-xan thine oxidase system. 5) To investigate the antioxidative effects of TJT on the hepatocytes, cultured normal rat liver cells(Ac2F) were prepared and incubated with or without TJT. After 16~18hr, cells placed in DMEM medium without serum, and then incubated with 1mM t-BHP for 2hr. Viable cells were detected by MTT assay. In this test, TJT protected the cell death induced by t-BHP and significantly increased cell viability in the normal rat liver cell. (Abbreviation) DPPH : ${\alpha},{\alpha}$-diphenyl-${\beta}$-picryl hydrazyl, DMEM : Dulbecco's Modified Eagle Medium, t-BHP : terr-butyl hydroperoxide, 4. Conclusion These results suggested that TJT might play a protective role in lipid peroxidation by free radicals.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.6
• /
• pp.842-849
• /
• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.

• Development and Reproduction
• /
• v.20 no.1
• /
• pp.63-71
• /
• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

• 한국생물공학회:학술대회논문집
• /
• 2005.04a
• /
• pp.214-217
• /
• 2005

Mammalian cell culture in the presence of sodium butyrate has been shown to enhance protein biosynthesis. In the present study, the effect of sodium butyrate on growth of recombinant Chinese Hamster Ovary cells and on the production of Iduronate 2-sulphatase were investigated in serum-containing and serum-free media. The culture with addition of 0-5mM sodium butyrate showed enhancement of both intracellular and extracellular IDS production. But, Cell death was observed in a dose-dependent manner. The optimal sodium butyrate concentration was observed to be 5mM. Also, The relative productivity of IDS was significantly increased when sodium butyrate was added to medium at 48 hour, the rapid growth phase. These results suggest that sodium butyrate are efficient agent for increasing the productivity of IDS with recombinant CHO cells.

• Journal of Nutrition and Health
• /
• v.30 no.7
• /
• pp.770-780
• /
• 1997

This study was performed to investigate the effect of chitosan and NOCC from different sources and of different molecular weights on lipid metabolism. Sprague-Dawley rats were blocked into 26 groups according to body weight, and were raised for 4 weeks. Cadmium chloride was given at the level of 0 or 400 ppm in diet. Various chitosan and NOCC sources were given at the level of 0%(w/w) of diet. Total serum cholesterol and serum triglyceride concentrations were little affected by chitosan and NOCC supplements. However, HDL-cholesterol concentration and HDL : total cholesterol ratio were increased , and liver lipid, cholesterol , and triglyceride concentration were decreased by chitosan and NOCC supplements. The cholesterol and lipid lowering activity depends on fiber sources(crab and shrimp); type (chitosan and NOCC); and molecular weight(low , medium , and high). Among cadmium-free groups, chitosan-fed groups showed greater activity than NOCC -fed groups in lowering cholesterol and lipid levels, and greater fecal excretion of lipids and bile acids. Crab chitosans were more effective in cholesterol and lipid lowering activity than shrimp chitosans. The group fed high molecular weight crab chitosan showed the highest fecal excretion of lipids and bile acids. This indicated that high molecular weight crab chitosan was most effective in interfering with cholesterol and lipid absorption.