• Journal of Pharmaceutical Investigation
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• v.36 no.5
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• pp.309-314
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• 2006

The multicellular layers(MCL) of human cancer cells is a three dimensional(3D) in vitro model for human solid tumors which has been used primarily for the assessment of avascular penetration of anti-cancer drugs. For anti-cancer drugs with penetration problem, MCL represents a good experimental model that can provide clinically relevant data. Calcein-AM is a fluorescent dye that demonstrates the cellular vitality in a graded manner in cancer cell culture system. In the present study, we evaluated the use of calcein-AM for determination of anti-proliferative activity of anti-cancer agents in MCL model of DLD-1 human colorectal cancer cells. Optical sectioning of confocal imaging was compromised with photonic attenuation and penetration barrier in the deep layers of MCL. By contrast, fluorescent measurement on the cryo-sections provided a feasible alternative. Cold pre-incubation did not enhance the calcein-AM distribution to a significant degree in MCL of DLD-1 cells. However, the simultaneous determination of drug penetration and cellular vitality appeared to be possible in drug treated MCL. In conclusion, these data suggest that calcein-AM can be used for the simultaneous determination of drug-induced anti-proliferative effect and drug penetration in MCL model.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.366.1-366.1
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• 2002

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defective site to initiate de novo tissue regeneration. In this respect. the availability of an appropriate biomaterial scaffold is crucial to allow chondrocyte growth and cartilaginous matrix deposition in a three-dimensional geometry. Hyaluronic acid (HA) molecules are anchored to the chondrocyte membrane via receptors, such as CD44. (omitted)

• ETRI Journal
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• v.42 no.6
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• pp.837-845
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• 2020

The objective of this study was to implement an in vitro exposure system for 28 GHz to investigate the biological effects of fifth-generation (5G) communication. A signal source of 28 GHz for 5G millimeter-wave (MMW) deployment was developed, followed by a variable attenuator for antenna input power control. A power amplifier was also customized to ensure a maximum output power of 10 W for high-power 28-GHz exposure. A 3-dB uniformity over the 80 mm × 80 mm area that corresponds to four Petri dishes of three-dimensional cell cultures can be obtained using a customized choke-ring-type antenna. An infrared camera is employed for temperature regulation during exposure by adjusting the airflow cooling rate via real-time feedback to the incubator. The reported measurement results confirm that the input power control, uniformity, and temperature regulation for 28-GHz exposure were successfully accomplished, indicating the possibility of a wide application of the implemented in vitro exposure system in the fields of various MMW dose-response studies.

• BMB Reports
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• v.57 no.7
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• pp.311-317
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• 2024

Brain organoid is a three-dimensional (3D) tissue derived from stem cells such as induced pluripotent stem cells (iPSCs) embryonic stem cells (ESCs) that reflect real human brain structure. It replicates the complexity and development of the human brain, enabling studies of the human brain in vitro. With emerging technologies, its application is various, including disease modeling and drug screening. A variety of experimental methods have been used to study structural and molecular characteristics of brain organoids. However, electrophysiological analysis is necessary to understand their functional characteristics and complexity. Although electrophysiological approaches have rapidly advanced for monolayered cells, there are some limitations in studying electrophysiological and neural network characteristics due to the lack of 3D characteristics. Herein, electrophysiological measurement and analytical methods related to neural complexity and 3D characteristics of brain organoids are reviewed. Overall, electrophysiological understanding of brain organoids allows us to overcome limitations of monolayer in vitro cell culture models, providing deep insights into the neural network complex of the real human brain and new ways of disease modeling.

• Applied Microscopy
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• v.41 no.1
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• pp.69-73
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• 2011

High-voltage electron microscope (HVEM) has higher resolution and penetration power than conventional transmission electron microscope that could be load thick specimen. Some researchers have taken this advantage of HVEM to explore 3-dimensional configuration of the biological structures including tissue and cells. Whole mount preparations has been employed to study some cell lines and primary culture cells. In this study, we would like to introduce useful whole mount preparation method for neuronal studies. The plastic coverslips were punched, covered by formvar membrane and coated with carbon. The neurons obtained embryonic 18 rat hippocampus were seeded on the prepared cover slip. The coverslips were fixed, dried in freeze drier and kept in a descicator until HVEM observation. We could observe detailed neuronal structures such as soma, dendrite and spine under HVEM without conventional thin section and heavy metal stain. The anaglyphic image based on stereo paired image ($-8^{\circ},+8^{\circ}$) provides three dimensional perception of the neuronal dendrites and their spines. This method could be applied to sophisticated analysis of dendritic spine under the various experimental conditions.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.321-329
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• 2019

Hair loss, also known as alopecia, is a common dermatological condition of psychosocial significance; development of therapeutic candidates for the treatment of this condition is, hence, important. Silibinin, a secondary metabolite from Silybum marianum, is an effective antioxidant that also prevents various cutaneous problems. In this study, we have investigated the effect of silibinin on hair induction using three-dimensional (3D) cultured, human dermal papilla (DP) spheroids. Silibinin was found to significantly increase viability through AKT serine/threonine kinase (AKT) activation in 3D DP spheroids. This was correlated with an increase in the diameter of the 3D DP spheroids. The activation of the wingless and INT-1 (Wnt)/${\beta}$-catenin signaling pathway, which is associated with hair growth induction in the DP, was evaluated using the T cell-specific transcription factor and lymphoid enhancer-binding factor (TCF/LEF) transcription factor reporter assay; results indicated significantly increased luciferase activity. In addition, we were able to demonstrate increased expression of the target genes, WNT5a and LEF1, using quantitative real-time PCR assay. Lastly, significantly elevated expression of signature genes associated with hair induction was demonstrated in the 3D DP spheroids treated with silibinin. These results suggest that silibinin promotes proliferation and hair induction through the AKT and Wnt/${\beta}$-catenin signaling pathways in 3D DP spheroids. Silibinin can be a potential candidate to promote hair proliferation.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.3
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• pp.175-184
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• 2022

Objective: The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization. Methods: In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction. Results: In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups. Conclusion: Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

• Tuberculosis and Respiratory Diseases
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• v.87 no.1
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• pp.52-64
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• 2024

Chronic respiratory diseases such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and respiratory infections injure the alveoli; the damage evoked is mostly irreversible and occasionally leads to death. Achieving a detailed understanding of the pathogenesis of these fatal respiratory diseases has been hampered by limited access to human alveolar tissue and the differences between mice and humans. Thus, the development of human alveolar organoid (AO) models that mimic in vivo physiology and pathophysiology has gained tremendous attention over the last decade. In recent years, human pluripotent stem cells (hPSCs) have been successfully employed to generate several types of organoids representing different respiratory compartments, including alveolar regions. However, despite continued advances in three-dimensional culture techniques and single-cell genomics, there is still a profound need to improve the cellular heterogeneity and maturity of AOs to recapitulate the key histological and functional features of in vivo alveolar tissue. In particular, the incorporation of immune cells such as macrophages into hPSC-AO systems is crucial for disease modeling and subsequent drug screening. In this review, we summarize current methods for differentiating alveolar epithelial cells from hPSCs followed by AO generation and their applications in disease modeling, drug testing, and toxicity evaluation. In addition, we review how current hPSC-AOs closely resemble in vivo alveoli in terms of phenotype, cellular heterogeneity, and maturity.

• Journal of Life Science
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• v.28 no.6
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• pp.681-687
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• 2018

Recently, several researchers have been developing cosmetics from natural ingredients for skin whitening and anti-aging products. The red sea cucumber (RSC), Apostichopus japonicas, is a species of sea cucumber in the family stichopodiae, which is widely distributed in China, Japan, and Korea. To use Red Sea Cucumber as a cosmetic ingredient, its inhibitory effects on melanogenesis and the anti-aging effects of RSC extracts were investigated. First, a tyrosinase activity assay was performed, which showed that RSC inhibited tyrosinase activity at a concentration of $200{\mu}g/ml$. An MTT assay was carried out to evaluate cell toxicity, and the results showed that RSC extract has no cytotoxicity in HaCaT cells. Furthermore, the mRNA expression levels of tyrosinase, tyrosinase related protein 1 (TRP-1), tyrosinase related protein 2 (TRP-2), microphthalmia-associated transcription factor (MITF), and matrix metalloproteinase (MMPs) genes treated with RSC extract in B16F10 and HaCaT cells decreased. Moreover, a wound-healing assay was performed to identify the cell regeneration effect of RSC extracts. Also, a skin turnover effect was confirmed by creating a three-dimensional cell culture with HaCaT and human fibroblasts. Altogether, the results suggested that Red Sea Cucumber may possess a high ability to induce whitening and anti-wrinkle effects as a cosmeceutical ingredient.

• Journal of Life Science
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• v.34 no.7
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• pp.476-484
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• 2024

Although two-dimensional (2D) monolayer cell culture models are still widely used as the optimal models for anticancer activity research, three-dimensional (3D) multicellular tumor spheroid (3D MTS) models that can better approximate the tumor environment can offer an alternative to bridge the gap between in vitro and animal model studies. Isoalantolactone is among the sesquiterpene lactones found in medicinal plants, including the roots of Elecampane (Inula helenium L.), and is known to have various pharmacological activities, including anticancer activity. In this study, we investigated whether the anticancer activity of isoalantolactone observed in 2D models could be reproduced in a 3D MTS model derived from human hepatocellular carcinoma (HCC) Hep3B cells. According to our results, isoalantolactone inhibited the formation of MTSs in a manner dependent on the treatment concentration, which was accompanied by an increase in reactive oxygen species (ROS) generation. In particular, as isoalantolactone treatment and the culture time increased, the area of proliferating cells was replaced by cells in which apoptosis was induced. Additionally, in MTSs, isoalantolactone increased the expression of death-receptor-related proteins and the activity of caspase-3, and it decreased the expression of the Bax/Bcl-2 expression ratio and total poly(ADP-ribose) polymerase. However, when the production of ROS was artificially blocked, all these changes caused by isoalantolactone were attenuated and the cell survival rate of MTS cells was restored. Therefore, the results of this study suggest that the induction of apoptosis in Hep3B cell-derived MTSs by isoalantolactone is achieved through the activation of extrinsic and intrinsic pathways and is ROS-dependent.