• Molecules and Cells
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• 제24권1호
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• pp.9-15
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• 2007

To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase $C{\alpha}$ ($PKC{\alpha}$) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12-myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated $PKC{\alpha}$. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that $PKC{\alpha}$ and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system.

• Toxicological Research
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• 제8권2호
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• pp.285-302
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• 1992

Human and rat hepatocytes were isolated by nonperfusion method and cultured for longer than 5 days. Human liver biopsy sample and rat liver were used as hepatocyte source. Several physical and chemical factors which were influencing on hepatocyte isolation procedure were examined and a batch isolation procedure was established for small size sample of rat liver. Isolated hepatocytes showed normal morphlologica characteristics in microscopy and electron microscopical examinations and a morphologica response to phalloidin. Isolated cells were cultured as a monolayer and proven to have intact morphological characteristics for longer than 15 days. Because human liver sample is harder and tighter compared with rat liver, a standard procedure for rat hepatocytes was slightly modified to reduce mechanical damage. Similarly with rat hepatocytes, isolated human hepatocytes showed a normal morphological characteristics and could be cultured for longer than 15days. Human and rat hepatocytes were examined on their functional integrities including cytochrome-P450 related enzyme activity and it's inducibility, hormonal inducibility of AIB uptake and TAT activity, albumin synthesis, DNA synthesis, cellular protein maintenance. In all parameters used in the present study, human and rat hepatocytes showed normal functional characteristics.

• International Journal of Stem Cells
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• 제17권1호
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• pp.91-98
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• 2024

The development of in vitro models is essential in modern science due to the need for experiments using human material and the reduction in the number of laboratory animals. The complexity of the interactions that occur in living organisms requires improvements in the monolayer cultures. In the work presented here, neuroepithelial stem (NES) cells were differentiated into peripheral-like neurons (PLN) and the phenotype of the cells was confirmed at the genetic and protein levels. Then RNA-seq method was used to investigate how stimulation with pro-inflammatory factors such as LPS and IFN𝛾 affects the expression of genes involved in the immune response in human fibroblast-like synoviocytes (HFLS). HFLS were then cultured on semi-permeable membrane inserts, and after 24 hours of pro-inflammatory stimulation, the levels of cytokines secretion into the medium were checked. Inserts with stimulated HFLS were introduced into the PLN culture, and by measuring secreted ATP, an increase in cell activity was found in the system. The method used mimics the condition that occurs in the joint during inflammation, as observed in the development of diseases such as rheumatoid arthritis (RA) or osteoarthritis (OA). In addition, the system used can be easily modified to simulate the interaction of peripheral neurons with other cell types.

• Journal of Korean Neurosurgical Society
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• 제43권3호
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• pp.149-154
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• 2008

Objective: The aim of this study is to elucidate the effects of transforming growth factor-${\beta}$ (TGF-${\beta}$)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-${\beta}1$ in intervertebral disc cells. Methods: Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in $37^{\circ}C$, 5% $CO_2$ incubator. When intervertebral disc cells were cultured with TGF-${\beta}1$ or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-${\beta}1$ were detected by RT-PCR and Western blot analysis in each. Results: The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-${\beta}1$ 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-${\beta}1$ and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-${\beta}1$, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group. Conclusion: This study suggests that TGF-${\beta}1$ would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.

• Parasites, Hosts and Diseases
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• 제46권1호
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• pp.37-40
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• 2008

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 ${\mu}M$ did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron ($24.6{\pm}2.1%$) compared with $62.7{\pm}2.8\;and\;63.1{\pm}1.4%$ of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed $69.1{\pm}4.3%\;and\;72.6{\pm}5.7%$ of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells ($2.8{\pm}0.2%$). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.

• Journal of Periodontal and Implant Science
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• 제32권1호
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• pp.129-142
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• 2002

Epithelial-mesenchymal interaction plays a important role in cell growth and differentiation. This interaction is already well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular microenvironment which provide a epithelial-mesenchymal interaction. Because conventional monolayer culture lacks epithelial-mensenchymal interaction, cultivated cells have an morphologic, biochemical, and functional characteristics differ from in vivo tissue. Moreover, it's condition is not able to induce cellular differention due to submerged culture condition. Therefore, the aims of this study were to develop and evaualte the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted oral keratinocytes by histological and immunohistochemical analysis. The results were as follow; 1. Gingival keratinocytes reconstituted by three-dimensional organotypic culture revealed similar morphologic characteristics to biopsied patient specimen showing stratification, hyperkeratinosis, matutation of epithelial architecture. 2. Connective tissue structure was matured, and there is no difference during stratification period of epithelial 3-dimensional culture. 3. The longer of air-exposure culture on three-dimensionally reconstituted cells, the more epithelial maturation, increased epithelial thickness and surface keratinization 4. In reconstitued mucosa, the whole epidermis was positively stained by anti-involucrin antibody, and there is no difference according to air-exposured culture period. 5. The Hsp was expressed in the epithelial layer of three-dimensionally cultured cells, especially basal layer of epidermis. The change of Hsp expression was not significant by culture stratification. 6. Connexin 43, marker of cell-cell communication was revealed mild immunodeposition in reconstitued epithelium, and there is no significant expression change during stratification. These results suggest that three-dimensional oragnotypic co-culture of normal gingival keratinocytes with dermal equivalent consisting type I collagen and gingival fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. And this culture system seems to provide adequate micro-environment for in vitro tissue reconstitution. Therefore, further study will be focused to study of in vitro gingivitis model, development of novel perioodntal disease therapeutics and epithelial-mensenchymal interaction.

• Biomolecules & Therapeutics
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• 제24권3호
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• pp.260-267
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• 2016

Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes increased in a time-dependent manner. After re-attachment of MSC spheroids to the plastic dish, their adhesion capacity and morphology were not altered. MSC spheroids showed enhanced production of hypoxia-induced angiogenic cytokines such as vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF), and hepatocyte growth factor (HGF). In addition, spheroid culture promoted the preservation of extracellular matrix (ECM) components, such as laminin and fibronectin, in a culture time- and spheroid size-dependent manner. Furthermore, phosphorylation of AKT, a cell survival signal, was significantly higher and the expression of pro-apoptotic molecules, poly (ADP ribose) polymerase-1 (PARP-1) and cleaved caspase-3, was markedly lower in the spheroids than in MSCs in monolayers. In the murine hindlimb ischemia model, transplanted MSC spheroids showed better proliferation than MSCs in monolayer. These findings suggest that MSC spheroids promote MSC bioactivities via secretion of angiogenic cytokines, preservation of ECM components, and regulation of apoptotic signals. Therefore, MSC spheroid-based cell therapy may serve as a simple and effective strategy for regenerative medicine.

• Journal of Animal Science and Technology
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• 제57권8호
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• pp.27.1-27.6
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• 2015

This study was designed to investigate the effects of ${\alpha}$-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of ${\alpha}$-tocopherol (0, 5, 10, $15{\mu}g/ml$), oocytes were incubated at $39^{\circ}C$ with 5 % $CO_2$ for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that ${\alpha}$-tocopherol had no effect on GVBD and MII as compared to control group, but when ${\alpha}$-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2002년도 제42차 춘계학술대회
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• pp.93-94
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• 2002

• Radiation Oncology Journal
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• 제7권2호
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• pp.149-156
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• 1989

HeLa 세포의 spheroid를 배 양시켜 cis-platinum과 carboplatin으로 처리한 후 그 반응을 세포의 생존분획으로 분석하였다. 체외실험 model인 spheroid를 사용하여 platinum유사체의 약효와 방사선 감수성을 평가하고 약제에 대한 단층 세포와 spheroid의 감수성 차이를 세포-생존곡선에서 규명하기 위하여 본 실험을 시행하였다. Cis-platinum 농도-곡선에서 spheroid의 $Cq=3.4{\mu}M$이고 $Co=1.2{\mu}M$이었다. 이 것은 단층세포에 비하여 Co는 큰 변화가 없으나 Cq가 증가되어 cis-platinum이 저산소층 세포보다 활동적으로 분화하는 표면세포에 주로 작용하였으며, 반대로 carboplatin의 효과는 spheroid에 대한 $(Co=15.0{\mu}M)$ 감수성이 단층세포$(Co=32.5{\mu}M)$에 비하여 크게 증가되어, spheroid의 심층 세포에 주로 작용하였다. 방사선과 carboplation의 병용효과를 세포 생존분획이 0.01 수준에서 isobologram으로 분석한 결과 상호작용으로 supra-additive 효과를 나타내었다. 따라서, carboplatin은 cis-platinum에 비하여 신장과 위장에 대한 독성작용이 적고, 방사선과 병용함으로써 향후 더욱 효과적 인 종양 치료에 중요한 역할을 할 것으로 기대한다.