• Korean Journal of Ichthyology
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• v.20 no.1
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• pp.1-6
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• 2008

Three types of clonal cell lines were isolated according to their size and phenotype from the adherent cell populations in long-term liquid cultures from the embryonic fibroblast cells of Zebrafish, Danio rerio. All kind of cell lines were well proliferated. The size and number of clonal cell lines derived colonies from stable embryonic cells were significantly increased in the presence of NAC and A2P conditioned medium from the cell lines. The stable cell lines and clonal cell lines were cap-able of well proliferation in vitro. These cell lines have been maintained in continuous culture without change in characteristics. A majority of the clonal cells (80%) was shown a normal chromosomal complement (50 chromosomes, 2N) in according with FACs analysis. Majority of cells were positive to vimentin staining and none of them were positive for nestin and Oct -4 by immunocytochemistry. These results indicate that the clonal cell lines obtained from cultured cells are fibroblasts and may be extremely useful in genetic manipulation for further nuclear transfer and fish cloning.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.7-12
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• 2004

This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or $\beta$-mercaptoethanol ($\beta$ME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P＜0.05). The establishment efficiency of $\beta$ME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p＜0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p＜0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and $\beta$ME.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.201-208
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• 2004

This study was performed to examine the effects of catalase and $\beta$-mercaptoethanol ($\beta$ME) on the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were passaged two times before use. A single cell was seeded in 96-well plates and cultured in medium supplemented with or without catalase or $\beta$ ME. Cell colonies were passaged two times into 4-well dish. Cell lines with proliferating potential were classified as an established clonal cell line. In experience 1, the establishment efficiencies were examined by addition of catalase (100ng/$m\ell$) or $\beta$ME (100 uM) in culture medium. The establishment efficiency of $\beta$ME-added group (8.3％) was significantly higher than that of control group (3.2％, P<0.05). However, catalase did not have a positive efffct on the establishment efficiency. In experience 2, the establishment efficiencies were examined by addition of different concentrations of catalase (0-1,000 ng/$m\ell$) in culture medium. However, establishment efficiencies were not different among the different concentrations of catalase (0-2.6％). In experience 3. the establishment efficiencies were examined by addition of different concentrations of $\beta$ME(0-1,000 uM) in culture medium. The establishment efficiency was significantly higher in 100 uM $\beta$ME-added group (9.4％) compare to others (0-1.6％). The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 100uM $\beta$ME. However, catalase did not have a positive effect on the establishment efficiency.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.20-25
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• 2009

Xenotransplantation of pig organs into humans is a potential solution for the shortage of donor organs for transplantation. However, multiple immune barriers preclude its clinical application. In particular, the initial type of rejection in xenotransplantation is an acute cellular rejection by host $CD8^+$ cytotoxic T lymphocyte (CTL) cells that react to donor major histocompatibility complex (MHC) class I. The human cytomegalovirus (HCMV) glycoprotein Unique Short (US) 2 specifically targets MHC class I heavy chains to relocate them from the endoplasmic reticulum (ER) membrane to the cytosol, where they are degraded by the proteasome. In this study we transfected the US2 gene into minipig fetal fibroblasts and established four US2 clonal cell lines. The integration of US2 into transgenic fetal cells was confirmed using PCR and Southern blot assay. The reduction of Swine Leukocyte Antigen (SLA)-I by US2 was also detected using Flow cytometry assay (FACS). The FACS analysis of the US2 clonal cell lines demonstrated a substantial reduction in SLA-I surface expression. The level (44% to 76%) of SLA-I expression in US2 clonal cell lines was decreased relative to the control. In cytotoxicity assay the rate of $CD8^+$ T cell-mediated cytotoxicity was significantly reduced to 23.8${\pm}$15.1% compared to the control (59.8${\pm}$8.4%, p<0.05). In conclusion, US2 can directly protect against $CD8^+$-mediated cell lysis. These results indicate that the expression of US2 in pig cells may provide a new approach to overcome the CTL-mediated immune rejection in xenotransplantation.

• Toxicological Research
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• v.12 no.2
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• pp.271-275
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• 1996

Carcinogenic potential of HPV-16 DNA and NNK in a human keratinocyte cell line was assessed to study effects of viral-chemical interaction. Human cells were transfected with HPV-16 DNA and 6 clonal cell lines were subsequently obtained. Clonal line-3 and 6 at passage 7 showed characteristics of tumor cells such as increases of saturation density, soft-agar colony formation, cell aggregation and foci appearance. Among cells treated with 1$\mu M$, 10$\mu M$, 100$\mu M$ or 1 mM of NNK for 4 weeks, 100$\mu M$ treatment showed most tumorigenic characteristics at passage 7. These results indicate that either HPV-16 or NNK alone is tumorigenic in this in human in vitro model. When cells transfected with HPV-16 were subsequently exposed by 100 uM NNK for 4 weeks, all the clonal cells except clone-1 showed higher levels of tumor cell characteristics than HPV-16 DNA or NNK exposure alone. Clonal line-6, the most tumorigenic cells, showed higher transcriptional level of fibronectin and lower level of TGF-$\beta_1$, as compared to control cells, suggesting that alteration of growth factor or extracellular matrix may play a role in carcinogenesis process induced by HPV-16 and NNK. Taken together, the present study indicates that viral-chemical interactions between HPV-16 DNA and NNK enhance carcinogenic potentials of human cells and implies that smoking among people infected with human papillomavirus may pose an additional risk of causing cancer.

• Fisheries and Aquatic Sciences
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• v.16 no.3
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• pp.177-185
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• 2013

The development of species-specific fish cell lines has become a valuable tool for biological research. In recent years, marine medaka Oryzias dancena has been recognized as a good experimental model fish but there are no reports of establishment of cell lines from this fish. In this study, two cell lines from O. dancena blastula embryos were established from 41 total trials (4.9%). The two cell lines displayed typical in vitro morphology and have been cultured for >121 passages, which corresponds to 293 days. The doubling times of the cell lines were 29.84 and 28.59 h, respectively, and both possessed the potential to expand in a clonal manner, albeit with significant differences between the two cell lines. The absence of any of the four main medium supplements; i.e., fish serum, fetal bovine serum, basic fibroblast growth factor, and medaka embryo extract, significantly inhibited growth. The proportion of cells possessing normal chromosome number was 45% and 46.7% of the cell lines, respectively. Taken together, two cell lines that proliferate continuously were established from marine medaka and these cell lines may provide a basic tool for characterizing the unique features of this fish species.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.73-73
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• 2003

본 연구는 돼지 체세포의 clonal cell lines을 효율적으로 확립할 수 있는 배양방법을 제시하기 위하여 실시하였다. 50일령의 돼지태아로부터 섬유아세포를 회수하여 2회 계대배양 후 단일 세포를 96-well plate와 4-well dish에서 배양하여 배양액내의 FBS농도(10, 30, 50%), 첨가제(catalase, $\beta$-mercaptoethanol; BME), 세포의 크기(<16, 16~20, 20<) 및 형태(smooth, rough)에 따른 cell line 확립 효율을 검토하였다. FBS 농도에 따른 clonal line 확립 효율과 PD를 검토한 결과, 효율에 있어서 30% FBS 처리구(5.1%)가 10%(0.3%) 및 50%처리구(2.1%)보다 비교적 높게 나타났으나 유의적 차이는 없었으며, PD의 경우는 10, 30, 50%처리구에서 각각 36.7, 29.4, 26.3 시간으로 FBS 농도가 증가할수록 PD 시간이 유의적으로 짧아졌다(p<0.05). 배양액 첨가제에 있어서 BME 가시 무처리구나 catalase 첨가시보다 유의적으로 높은 효율을 보였으며(11.4%, 3.2%, 3.2%; p<0.05), PD 시간은 짧게 나타났다(23.6, 25.5, 28.1; p<0.05). 그러나 catalase는 cell line 확립 효율에 영향을 미치지 않는 것으로 나타났다. 세포의 크기에 따른 cell line 확립효율은 5.2~8.2%로 크기에 따른 차이는 보이지 않았으며, PD 또한 23.7~27.9 시간으로 세포 크기에 따른 차이는 없었다. 세포의 형태에 따른 세포의 부착율은 smooth(55.8%)구가 rough(73.0%)구보다 유의적으로 낮게 나타났으나(p<0.05), clonal line 확립 효율(7.7%n vs. 6.6%) 및 PD(23.5h vs. 24.0h)에서는 차이가 없었다. 본 연구의 결과, 세포의 형태에 따른 cell line 확립 효율은 큰 차이가 없었으나, 세포 배양액내에 30% FBS와 BME의 첨가로 clonal cell line 확립 효율을 향상시킬 수 있음을 확인할 수 있었다. 또한 cell line 확립 효율이 높아질수록 PD 시간이 짧아지는 경향을 볼 수 있었다.

• BMB Reports
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• v.29 no.5
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• pp.417-422
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• 1996

In clonal sublines with different metastatic ability derived from Dunning rat prostate tumor, phosphoamino acid levels of cellular proteins were determined. Cell lines with high metastatic ability exhibited 5-fold higher phosphotyrosine level than did cell lines with low metastatic ability, while the contents of phosphoserine and phosphothreonine were similar among cell lines examined, All cell lines showed similar activities of protein tyrosine kinases as well as overall protein kinases. Phosphotyrosine protein phosphatase (PTPP) activities of the cells with high metastatic ability were very low, compared to those of the cells with low metastatic ability, suggesting that the different phosphotyrosine levels among the cell lines were due to the difference in PTPP activities rather than protein tyrosine kinase activities. Cellular activities of prostatic acid phosphatase (PAcP), which has been reported to possess phosphotyrosine protein phosphatase activity, were shown to be inversely related to the phosphotyrosine levels and metastatic abilities of the prostate tumor cells, These results suggest that cellular PAcP activity, regulating phosphotyrosine levels of cellular proteins, is closely connected with the metastatic process in prostate tumor cells and can be utilized as a good biochemical marker for the diagnosis of metastasis of prostate tumor.

• IMMUNE NETWORK
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• v.13 no.4
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• pp.133-140
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• 2013

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and $IFN-{\gamma}$ production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.209-209
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• 2004

This cDNAs were cloned with the method of the polymerase chain reaction (PCR) by sequences based on cloned rat LH/CG and FSH receptor cDNAs. A cDNAs of LHR and FSHR were transfected into the CHO cells. Several clonal cell lines were obtained expressing different numbers of cell surface receptors. (omitted)