• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.44-47
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• 2000

Cell growth and DNA synthesis were studied from a cultured early- and late- pas- sage mouse aorta smooth muscle cell (MASMC) because the proliferation of vascular smooth muscle cell (VSMC) is a key factor in development of atherosclerosis. In this study, the cells were cultured in fetal bovine serum (FBS) and stimulated by growth factors such as thrombin and platelet-derived growth factor-BB (PDGF-BB). Compared to the number of early-passage MASMC (passage 3 to 9) the number of late-passage MASMC (passage 30 to 40) in a normal serum state was increased 2 fold at Day 1, 3 and 6 in culture, respectively. Incorporation of $[^3H]$ thymidine into DNA induced by serum, PDGF and thrombin in late-passage MASMC was greater than those in early-passage MASMC. We also examined whether intracellular zinc levels would be an aging factor or not. The intracellular zinc level in early- and late-passage MASMC was monitored by using the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide. It is interested that late-passage MASMC increased the intracellular fluorescence level of zinc, more than the early passage MASMC did. The alterations of intracellular zinc level occur concurrently with changes in MASMC proliferation rate during aging. This data suggest that the age-associated changes in zinc concentrations may provide a new in vitro model for the study of smooth muscle cell differentiation.

• Journal of the Korean Society of Marine Environment & Safety
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• v.23 no.3
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• pp.273-278
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• 2017

This paper is to study a technique to detect the real-time route aberrance on the passage route using bumper area of the ship domain theory. In order to evaluate the risk of route aberrance, a quarter line was created between the center line and the outer line, and a passage route with the image line outside the outer line was designed. It calculated the real-time route aberrance with the vessel bumper area to measure the risk level on the passage route. The route aberrance using overlap bumper area was simulated through three kinds of scenario vessel at the designed passage route. In this paper, we proposed Ratio to Aberrance Risk as one of the evaluation parameter to detect the route aberrance risk at each sector in the passage route and to give the evaluation criteria of 5 levels for seafarer's navigation safety. The purpose of this work is to provide the information of the route aberrance to seafarer automatically, to make it possible to prevent the human errors of seafarer on the high risk aberrance route. As the real-time risk of route aberrance on the passage route is automatically evaluated, it was well thought that seafarer can have only a little workload in order to know the risk of route aberrance at early-time. Following the further development of this work, the techniques for detecting the real-time route aberrance will be able to use the unmanned vessel.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.144-153
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• 2021

Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Culture-conditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1800-1807
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• 2016

To understand how human cytomegalovirus (HCMV) might change and evolve after reactivation, it is very important to understand how the nucleotide sequence of cultured HCMV changes after in vitro passaging in cell culture, and how these changes affect the genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV was propagated in vitro for more than 40 passages and its biological and genetic changes were monitored. For each passage, real-time PCR was performed in order to determine the genome copy number, and a plaque assay was employed to get virus infection titers. The infectious virus titers gradually increased with passaging in cell culture, whereas the number of virus genome copies remained relatively unchanged. A linear correlation was observed between the passage number and the log₁₀ infectious virus titer per virus genome copy number. To understand the genetic basis underlying the increase in HCMV infectivity with increasing passage, the whole-genome DNA sequence of the high-passage strain was determined and compared with the genome sequence of the low-passage strain. Out of 100 mutations found in the high-passage strain, only two were located in an open reading frame. A G-T substitution in the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and UL122 genes might be related to the increase in virus infectivity, although the role of the mutations found in noncoding regions could not be excluded.

• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.771-779
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• 2018

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent stem cells that can differentiate into several cell types. In addition, many studies have shown that MSCs modulate the immune response. However, little information is currently available regarding the maintenance of immunomodulatory characteristics of MSCs through passages. Therefore, we investigated and compared cytokine and gene expression levels from adipose (AD) and bone marrow (BM)-derived MSCs relevant to immune modulation from early to late passages. METHODS: MSC immunophenotype, growth characteristics, cytokine expressions, and gene expressions were analyzed. RESULTS: AD-MSCs and BM-MSCs had similar cell morphologies and surface marker expressions from passage 4 to passage 10. Cytokines secreted by AD-MSCs and BM-MSCs were similar from early to late passages. AD-MSCs and BM-MSCs showed similar immunomodulatory properties in terms of cytokine secretion levels. However, the gene expressions of tumor necrosis factor-stimulated gene (TSG)-6 and human leukocyte antigen (HLA)-G were decreased and gene expressions of galectin-1 and -3 were increased in both AD- and BM-MSCs with repeated passages. CONCLUSION: Our study showed that the immunophenotype and expression of immunomodulation-related cytokines of AD-MSCs and BM-MSCs immunomodulation through the passages were not significantly different, even though the gene expressions of both MSCs were different.

• Journal of Life Science
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• v.18 no.3
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• pp.344-349
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• 2008

Normal somatic cells proliferate for a limited number of doublings in culture and then enter an irreversible growth-arrest state called replicative senescence. Replicative senescence has been believed a reason for the limited cellular turnover and deterioration of tissue function in aged animals. However, there is no experimental evidence supporting this assumption. Furthermore, cells from aged person have been poorly characterized with an exception of the cases of T cells. In this study, we examined cell biological changes occurring in replicative senescence of fibroblast strains originated from a new-born (NHF-NB) and a 87 year old man (NHF-87). NHF-87 (and the cells from a 75-year old) proliferated to smaller population doublings and with longer doubling times than NHF-NB did. At early passages, NHF-87 exhibited a low senescence-associated ${\beta}-Gal$ (SA ${\beta}-Gal$) activity and lipofuscin level, typical markers for cellular senescence. Furthermore, they maintained low levels of lysosome and reactive oxygen species (ROS). All of these levels increased dramatically in the late passage NHF-87 quite similarly as those in the late passaged NHF-NB did. These results indicate that most cells originated from the aged maintain a phenotype of the cells originated from new-born donors and undergo replicative senescence with the same kinetics as that of the cells from new-born. It is also indicated that not SA ${\beta}-gal$ activity but cell proliferation rate may be qualified as a biomarker for cells aged in vivo.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.64-64
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• 2001

To facilitate the widespread application of somatic cell cloning, improvements in blastocyst production efficiency and subsequent fetal viability are required. Area where technical improvements are needed include donor cell treatments, starvation and passage numbers. This study was carried out to investigate the effect of serum-starvation and passage on the development of ear skin fibroblast cells cloned embryos. A skin biopsy was obtained from the ear of a 2-year-old Korean Hanwoo female. The cells were cultured in 10% FBS＋DMEM up to 2-3 months(up to 10 passages) and then used. In Experiment 1, the Korean bovine Ear Skin Fibroblast cells (KbESF) were either serum starved (culture in 0.05% FBS＋DMEM) or serum fed (10% FBS＋DMEM) for 4-7 days Prior to NT In Experiment 2, the KbESF cells used for nuclear transfer in these experiments were from passages 2 to 10. The development of 208 nuclear transfer (NT) embryos reconstructed from either serum starved or serum fed ear skin fibroblast was assessed. NT embryos reconstructed from serum starved and serum fed cells showed the same developmental rate (cleavage 80.16 vs. 85.37%; blastocyst 20.63 vs. 19,51%). The development of 590 nuclear transfer (NT) embryos reconstructed from passage 2 to 10 was assessed. We observed the same developmental rates for embryos derived from later Passages as compared with those embryos from early passages(blastocyst from 16.69 to 27.91%, average 20.17%). There was no significant difference between serum-fed and serum-starved donor cells. We observed no difference in developmental rates for embryos derived from 2 to 10 passages. These data show that prolonged culture and serum starvation does not affects the cloning competence of adult somatic cells.

• Phonetics and Speech Sciences
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• v.6 no.1
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• pp.31-38
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• 2014

The current study presents an instrumental phonetic analysis of Korean monophthong vowels in the early twentieth century Seoul Korean, based on audio recordings of elementary school textbooks Botonghakgyo Joseoneodokbon (Korean Reading Textbook for Elementary School). The data examined in this study were a list of the Korean mono syllables (Banjeol), and a short passage, recorded by one 41-year-old male speaker in 1935, as well as a short passage recorded by one 11-year-old male speaker in 1935. The Korean monophthongs were examined in terms of acoustic analysis of the vowel formants (F1, F2) and compared to those recorded by 18 male speakers of Seoul Korean in 2013. The results show that in 1935, 1) /e/ and /ɛ/ were clearly separated in the vowel space; 2) /o/ and /u/ were also clearly separated without any overlapping values; 3) some tokens of /y/ and /ø/ were produced as monophthongs, not as diphthongs. Based on the results, we can observe the historical change of the Korean vowels over 80-90 years such as 1) /e/ and /ɛ/ have been merged; and 2) /o/ has been raised and overlapped with /u/.

• Journal of the korean academy of Pediatric Dentistry
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• v.48 no.3
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• pp.291-301
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• 2021

The aim of this study is to compare the properties of odontoblast gene of early passage cells and late passage cells derived from impacted maxillary supernumerary teeth. Impacted supernumerary teeth with maxilla were extracted from 12 patients (8 males, 4 females) between 6 - 9 years old without medical history. Real-time polymerase chain reaction (PCR) was conducted to compare characterization of odontoblast cell in the 3rd and 10th passage, and between with bone inducing additive group and without additive group. Genes for odontoblasts characteristics are osteonectin (ONT), alkaline phosphatase (ALP), osteocalcin (OCN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP). The level of gene expression was in a decreasing order of ONT, ALP, OCN, DMP-1 and DSPP in the 3rd passage, and in decreasing order of ONT, DMP-1, OCN, ALP, and DSPP in the 10th passage in the undifferentiation and differentiation group. The order of ONT, DMP-1, and OCN did not changed. ALP and DMP-1 were switched in order. ALP and DMP-1 may be used as important markers for differentiating between the 3rd passage and 10th passage cells. Considering that supernumerary tooth was extracted young age and the time required to cultured 10th passage was short, supernumerary tooth can be considered a useful donor site of dental pulp stem cells.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.171-174
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• 2012

Various small molecules can be used to control major signaling pathways to enhance stemness and inhibit differentiation in murine embryonic stem cell (mESC) culture. Small molecules inhibiting the fibroblast growth factor (FGF)/ERK pathway can preserve pluripotent cells from stimulation of differentiation. In this study, we aimed to evaluate the effect of pluripotin (SC-1), an inhibitor of the FGF/ERK pathway, on the colony formation of outgrowing presumptive mESCs. After plating the zona pellucida-free blastocyst on the feeder layer, attached cell clumps was cultured with SC-1 until the endpoint of the experiment at passage 10. In this experiment, when the number of colonies was counted at passage 3, SC-1-treated group showed 3.4 fold more mESC colonies when compared with control group. However, after passage 4, there was no stimulating effect of SC-1 on the colony formation. In conclusion, SC-1 treatment can be used to promote mESC generation by increasing the number of early mESC colonies.