• Journal of Periodontal and Implant Science
• /
• v.32 no.1
• /
• pp.235-247
• /
• 2002

The aim of this study was to evaluate the effects of chitosan coating on the attachment, proliferation, functional and morphological change of human gingival fibroblasts. Primary culture of human gingival fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. In experimental group, cells were inoculated in the multiwell plates coated with chitosan in concentration of 0.02, 0.2, and 2 mg/ml. Cell counting and MTT assay were done after 0.5, 1.5, 3, 6 and 24 hours of incubation to evaluate the cell attachment, and then after 2 and 7 days of culture to evaluate the cell proliferation. The alkaline phosphatase activity was measured after 4 and 7 days of culture and the ability to produce mineralized nodules was evaluated after 21 days of culture. The results were as follows : The morphology of cells on the chitosan-coated well was round or spheric. Round cells were aggregated since 6 hours of culture and showed nodule-like appearance after 24 hours of culture and did not achieved confluency at 7 days. The attachment of gingival fibroblasts was inhibited by chitosan coating with a tendency of dose dependent pattern. But, cellular activity of unit cell was higher than control. The proliferation of gingival fibroblasts was inhibited by chitosan coating at 2 mg/ml(P<0.01), while the cell proliferation at 0.02, 0.2 $mg/m{\ell}$ was comparable to the control well. Total alkaline phosphatase activity was inhibited by chitosan coating and decreased in the course of time. While ALP activity of unit cell was the highest at 2mg/ml after 4 days of culture. Finally, gingival fibroblasts produced the mineralized nodule at 2 mg/ml. In summary, the attachment, proliferation, and alkaline phosphatase activity of gingival fibroblasts were influenced differently by the concentration of coated chitosan. From this study, it could be used as the matrix of tissue engineering for gingiva without inhibition on proliferation of gingival fibroblasts using chitosan at the optimal concentration (0.02mg/ml).

• Journal of the korean academy of Pediatric Dentistry
• /
• v.41 no.1
• /
• pp.1-7
• /
• 2014

If it is possible to preserve and culture cells from exfoliated deciduous teeth in a readily available storage medium within each family, more stem cells would be obtained. This research is about the effect of storage media and time on pulpal cell viability of exfoliated deciduous teeth. 330 exfoliated deciduous teeth were randomly divided into 11 groups; fresh group, dry group, groups stored in cell culture medium (2, 4, 7 days each), in milk (2, 4, 7 days each), and in saline (2, 4, 7 days each). Primary culture of pulpal cells was conducted in each group and the success rates were compared by calculating the number of teeth with viable cells. The result of primary culture shows that the success rate decreases as the time of storage gets longer. There was no statistical difference between groups stored in the cell culture medium, milk, and saline for 2 and 4 days. However, the groups stored in milk and saline for 7 days showed dramatic decrease in success rate compared to the group stored in the cell culture medium. In conclusion, exfoliated or extracted deciduous teeth can be used to culture pulpal cells when they are stored in milk and saline for a certain period of time; however obtaining viable pulpal cells becomes harder as the storage time gets longer.

• Journal of The Korean Society of Integrative Medicine
• /
• v.8 no.3
• /
• pp.53-62
• /
• 2020

Purpose :Periodontal ligament stem cells maintain tissue homeostasis in periodontal ligament. The purpose of this study was to determine the characteristics of periodontal ligament stem cells isolated from premolar teeth and observe protective effects against oxidative damage caused by Triethylene glycol dimethacrylate (TEGDMA) following treatment with N-acetylsysteine amide (NACA) drug known as enzymatic antioxidants. Methods : Primary periodontal ligament stem cell (PDSC) culture was performed from simply extracted human premolar of orthodontic patients. The characteristics of the primary cultured PDSCs was analyzed using the FACS system. PDSCs was incubated with TEGDMA and NACA. The cell proliferation and survival was determined using WST-1 assay. Collected data were analyzed using SPSS Window 20. Results : Primary cultured PDSCs grow on the floor and develop rapidly in a cluster form from up to 14 days. The morphology of PDSCs showed the spindle-shaped cells and grew directionally. FACS analysis, In addition, positive expression of visible cells were observed in mesenchymal stem cell biomarkers. PDLSCs cell viability was significantly decreased at high concentration in both 3 and 6 hours after TEGDMA treatment. We observed a decrease in the number of cells as well as a morphological change of PDLSCs. Antioxidative effect was notable since the death of PDLSC death was significantly inhibited compared to the control group at 24 and 48 hours after NACA treatment. Conclusion : Therefore, based on the results of this study, further research should be encouraged considering the development of clinical treatment methods using various antioxidants as well as regenerative engineering techniques utilizing periodontal ligament stem cells.

• 한국생물공학회:학술대회논문집
• /
• 2003.04a
• /
• pp.302-305
• /
• 2003

It is difficult to obtain sufficient healthy skin for coverage of a wide area of skin wound. In the skin, an additional population of living epithelial cells is located in the outer root sheath (ORS) of hair $follicles.^{1),2)}$ ORS cells should be a good source of epithelium because they are easily obtainable and patients do not have to suffer from scar formation at donor sites. We modified ordinary primary culture technique for the purpose of solving such problem that epithelial cells have a low propagation and easy aging during culture periods. First of all, we improved primary cultivation methods. In the ordinary primary culture, average yield of human ORS cells was $2\;{\times}\;10^3$ cells/follicle by direct incubation with trypsin (0.1%)/EDTA (0.02%) solution for 15 min at $37^{\circ}C$ but we could obtain about $6.5\;{\times}\;10^3$ cells/follicle by two step enzyme digestion method with dispase (1.2 U/ml) and trypsin (0.1%)/EDTA (0.02%) solution. So we could achieve three times higher primary cultured ORS cell yield. Secondly, we could obtain total $2\;{\times}\;10^7$ cells in serum free medium and even more total $6\;{\times}\;10^7$ cells in modified E-medium with mitomycin C-treated feeder cells during 17 days. Using the cultured ORS cells, and we could make bioartificial skin equivalent in vitro and concluded that ORS cells were progenitor cells for skin epithelial cell.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.6 no.4
• /
• pp.264-268
• /
• 2001

Aggregates of specific cells are often regarded as better from in artificial organs and mammalian cell bioreactors in terms of cell-specific functionality. In this study, the morphology and liver specific functions of freshly harvested primary rat hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide(98 mole % in feed) and acrylic acid (poly (NiPAAm-co-AAC)), a thermo- reversible copolymer gel ma- trix, was used to entrap hepatocytes either in spheroids or single cells. During a 7-day culture pe-riod, the spheroids maintained higher viability and produced albumin and urea at a relatively con-stant rate, while, the single cell culture showed a slight increase in cell numbers and a reduction in albumin secretion Hepatocytes cultrured as spheroids present a potentially useful three-dimensional cell culture system for application in bioartificial liver device.

• Korean Journal of Veterinary Research
• /
• v.36 no.3
• /
• pp.551-563
• /
• 1996

This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium. The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum- supplemented medium(p < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${\gamma}$-GTP) were increased(p < 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p < 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p < 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum-supplemented medium. The activities of $Na^+$glucose cotransporter($^{14}C$-a-MG uptake), $Na^+$phosphate cotransportere($^{32}P$ uptake) and $Na^+$ transporter($^{22}Na^+$ uptake) in the brush border membrane, and of $Na^+/K^+$-ATPase($^{86}Rb$ uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p < 0.05). In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.2
• /
• pp.274-278
• /
• 2002

A culture system for lactating rat mammary acini was evaluated, where the primary indicator of performance was lactose secretion, measured by a sensitive bioluminescence assay. Lactose secretion was reduced by half (p<0.01) over the first 6 h of culture by overnight feed withdrawal (FW) from tissue donors but was sensitive to increased glucose concentration in the culture media (p<0.001) up to 30 mM. Lactose production of cells from fed donors over the first 6 h in culture in 30 mM glucose was 8.9 fmol/cell/h - a rate calculated to be about half that in vivo. No significant difference was shown in lactose secretion by cells from fed or FW rats over 6-24 h. Lactose secretion was 3.6 fmol/cell/h by cells from fed animals in 40 mM glucose concentration media over the 6-24 h culture period. Addition of insulin to the culture media had no effect on rates of lactose secretion while addition of prolactin and hydrocortisone, with or without insulin, significantly (p<0.001) decreased lactose production over both 0-6 h and 6-24 h culture periods. Lactose synthesis in vitro was significantly enhanced by aeration of the media during collagenase digestion of mammary tissue (p<0.05). No improvement in lactose secretion was effected by shaking of cells during culture, Matrigel coating of culture dishes or change in cell density over a range up to 2.5 million cells per ml.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.18 no.10
• /
• pp.551-558
• /
• 2017

To study and validate tissue-specific promoters and vectors, it is important to develop cell culture systems that retain the tissue and species specificity. Such systems are attractive alternatives to transgenic animal models. This study established a line of porcine mammary gland epithelial cells (PMECs) from a primary culture based on the cellular morphology and mRNA levels of porcine beta-casein (CSN2). The selected PMECs were stained with the cytokeratin antibody, and were shown to express milk protein genes (CSN2, lactoferrin, and whey acidic protein). In addition, to confirm the acini structure of PMEC932-7 in 3D culture, live cells were stained with SYTO-13 dye, which binds to nucleic acid. The acini of these PMECs on matrigel were formed by the aggregation of peripheral cells and featured a hollow lumens. The system was demonstrated by testing the effects of the culture conditions to cell culture including cell density and matrigel methods of the PMECs. These results suggest that PMECs possess the genetic and structural features of mammary epithelial cells.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.7
• /
• pp.980-987
• /
• 2008

The objective of this study was to investigate the effects of cell status of BOEC on development of bovine IVM/IVF embryos and gene expression in BOEC before or after culturing of embryos. The developmental rates beyond morula stage in the BOEC co-culture group was significantly higher than in the control group (p<0.05). In particular, blastocyst production in the BOEC co-culture group (28.3%) was dramatically increased compared with the control group (7.2%). In the in vitro development of bovine IVM/IVF embryos according to cell status, the developmental rates beyond morula stage in the primary culture cell (PCC) co-culture group were the highest of all experimental groups. Expression of genes related to growth (TGF-${\beta}$ EGF and IGFBP), apoptosis (Bax, Caspase-3 and p53) and antioxidation (CuZnSOD, MnSOD, Catalase and GPx) in different status cells of BOEC for embryo culture was detected by RT-PCR. While EGF gene was detected in isolated fresh cells (IFC) and PCC, TGF-${\beta}$ and IGFBP were found in IFC or PCC after use in the embryo culture, respectively. Caspase-3 and Bax genes were detected in all experimental groups regardless of whether the BOEC was used or not used in the embryo culture. However, p53 gene was found in IFC of both conditions for embryo culture and in frozen/thawed culture cells (FPCC) after use in the embryo culture. Although antioxidant genes examined were detected in all experimental groups before using for the embryo culture, these genes were not detected after use. This study indicated that the BOEC co-culture system used for in vitro culture of bovine IVF embryos can increase the developmental rates, and cell generations and status of BOEC might affect the in vitro development of bovine embryos. The BOEC monolayer used in the embryo culture did not express the growth factors (TGF-${\beta}$ and EGF) and enzymatic antioxidant genes, thereby improving embryo development in vitro.

• The Journal of Korean Society of Virology
• /
• v.28 no.4
• /
• pp.317-325
• /
• 1998

The primary culture of human nasal epithelial cells was performed using the inferior nasal turbinate tissues, and infected with Hantaan virus to examine the hypothesis of airborne transmission of Hantaan virus in humans. The primary culture cells were identified as epithelial cells by morphologic and immunologic analyses. The viral antigens were detected in the primary human nasal epithelial cells infected with Hantaan virus by immunofluorescence staining. The ICAM-1 induction by Hantaan virus or $IFN-{\gamma}$ was examined in the primary human nasal epithelial cells and human lung fibroblasts (WI-38). Hantaan virus induced the surface ICAM-1 in WI-38 cells in a time-dependent manner, and $IFN-{\gamma}$ induced the surface ICAM-1 in a dose-dependent manner in HNEC and WI-38 cells. These results revealed that the human nasal epithelial cells are susceptible to Hantaan viral infection supporting the hypothesis of airborne transmission of Hantaan virus in humans. The human lung fibroblasts also might have an important role in the pathogenesis of Hantaan virus through the induction of ICAM-1.