• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.721-733
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• 2018

BACKGROUND: Because three-dimensional (3D) models more closely mimic native tissues, one of the goals of 3D in vitro tissue models is to aid in the development and toxicity screening of new drug therapies. In this study, a 3D skin wound healing model comprising of a collagen type I construct with fibrin-filled defects was developed. METHODS: Optical imaging was used to measure keratinocyte migration in the presence of fibroblasts over 7 days onto the fibrin-filled defects. Additionally, cell viability and growth of fibroblasts and keratinocytes was measured using the $alamarBlue^{(R)}$ assay and changes in the mechanical stiffness of the 3D construct was monitored using compressive indentation testing. RESULTS: Keratinocyte migration rate was significantly increased in the presence of fibroblasts with the cells reaching the center of the defect as early as day 3 in the co-culture constructs compared to day 7 for the control keratinocyte monoculture constructs. Additionally, constructs with the greatest rate of keratinocyte migration had reduced cell growth. When fibroblasts were cultured alone in the wound healing construct, there was a 1.3 to 3.4-fold increase in cell growth and a 1.2 to 1.4-fold increase in cell growth for keratinocyte monocultures. However, co-culture constructs exhibited no significant growth over 7 days. Finally, mechanical testing showed that fibroblasts and keratinocytes had varying effects on matrix stiffness with fibroblasts degrading the constructs while keratinocytes increased the construct's stiffness. CONCLUSION: This 3D in vitro wound healing model is a step towards developing a mimetic construct that recapitulates the complex microenvironment of healing wounds and could aid in the early studies of novel therapeutics that promote migration and proliferation of epithelial cells.

• BMB Reports
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• v.45 no.3
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• pp.189-193
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• 2012

Cyst formation is a major characteristic of ADPKD and is caused by the abnormal proliferation of epithelial cells. Renal cyst formation disrupts renal function and induces diverse complications. The mechanism of cyst formation is unclear. mIMCD-3 cells were established to develop simple epithelial cell cysts in 3-D culture. We confirmed previously that Mxi1 plays a role in cyst formation in Mxi1-deficient mice. Cysts in Mxi1 transfectanted cells were showed by collagen or mebiol gels in 3-D cell culture system. Causative genes of ADPKD were measured by q RT-PCR. Herein, Mxi1 transfectants rarely formed a simple epithelial cyst and induced cell death. Overexpression of Mxi1 resulted in a decrease in the PKD1, PKD2 and c-myc mRNA relating to the pathway of cyst formation. These data indicate that Mxi1 influences cyst formation of mIMCD-3 cells in 3-D culture and that Mxi1 may control the mechanism of renal cyst formation.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.4
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• pp.259-269
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• 2022

Objective: Animal-free scaffolds have emerged as a potential foundation for consistent, chemically defined, and low-cost materials. Because of its good potential for high biocompatibility with reproductive tissues and well-characterized scaffold design, we investigated whether polyglycolic acid (PGA) could be used as an animal-free scaffold instead of natural fibrin-agarose, which has been used successfully for three-dimensional human endometrial cell culture. Methods: Isolated primary endometrial cells was cultured on fibrin-agarose and PGA polymers and evaluated various design parameters, such as scaffold porosity and mean fiber diameter. Cytotoxicity, scanning electron microscopy (SEM), and immunostaining experiments were conducted to examine cell activity on fabricated scaffolds. Results: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and SEM results showed that endometrial cells grew and proliferated on both scaffolds. Immunostaining showed cytokeratin and vimentin expression in seeded cells after 7 days of culture. On both scaffolds, an epithelial arrangement of cultured cells was found on the top layer and stromal arrangement matrix on the bottom layer of the scaffolds. Therefore, fibrin-agarose and PGA scaffolds successfully mimicked the human endometrium in a way suitable for in vitro analysis. Conclusion: Both fibrin-agarose and PGA scaffolds could be used to simulate endometrial structures. However, because of environmental and ethical concerns and the low cost of synthetic polymers, we recommend using PGA as a synthetic polymer for scaffolding in research instead of natural biomaterials.

• Bulletin of the Korean Chemical Society
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• v.32 no.2
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• pp.431-433
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• 2011

Alginate microfibers were fabricated by self assembly of alginate monomers exuded from alginate beads (~2 mm in diameter) containing calcium phosphate. Upon incubation of the beads in cell culture medium at $37^{\circ}C$ for a few days, fibers with a diameter of about $7{\mu}m$ started to sprout from the bead surface, and these grew up to about 10 mm in length, resulting in the beads being covered with fiber forests similar to chestnut bur. The combined system of the alginatebased microfiber forest and bead is considered to be useful as a novel 3-dimensional scaffold for cell culture and tisssue growth.

• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.238-247
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• 2022

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

• Journal of the Korean Society for Precision Engineering
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• v.26 no.3
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• pp.137-143
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• 2009

It has been reported that mechanical stimulation takes a role in improving eel/ growth in skeletal system. Various research groups have been showed their own bioreactors which stimulate cell-seed three-dimensional scaffold. In this study, we hypothesized that the various conditions of mechanical stimulation would affect cell growth and proliferation. To prove our hypothesis, we designed a custom-made bioreactor capable of applying controlled compression to cell-encapsulated scaffolds. This device consisted of a circulation system and a compression system. Each parts of the bioreactor was fabricated using the rapid prototyping technology By using the rapid prototyping technology, we can modify and improve the bioreactor very rapidly For dynamic cell-culture, cell-encapsulated agarose gel was fabricated in 2% concentration. We performed dynamic cell-culture using this agarose gel and developed bioreactor in 3 days.

• Biomolecules & Therapeutics
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• v.24 no.3
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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.

• Korean Journal of Microbiology
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• v.22 no.1
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• pp.19-28
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• 1984

The nematode-trapping process by Arthrobotrys conoides was investigated with the aid of scanning and transmission electron microscopy. 1. A. conoides captures nematode by means of three-dimensional network. 2. The wall of trap cell was thicker than that of vegetative hypha and the trap cell was more rich in cell organelles such as endoplasmic reticulum, mitochondria and electrondense granule. 3. The electron-dense granule, which could be found only in trap organs, gradually disappeared during its penetration into nematode cuticle. 4. The osmiophilic area was found at adhering site between the trap organ and nematode cuticle. 5. In some cases, any appressorium was not found at the site of penetration. 6. When the fungal-nematode culture was conserved for 2~3 weeks, numerous young nematodes were found to be adhered to spores, resulting in death.

• Journal of Life Science
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• v.33 no.8
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• pp.612-622
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• 2023

T-cell deficiency may occur in various clinical conditions including congenital defects, cell/organ transplantation, HIV infection and aging. In this regard, the development of artificial thymus has recently been attracting much attention. To achieve this aim, the development of techniques for 3D culture of thymic stromal cells is necessary because thymocytes grown only in a 3D thymic microenvironment can be differentiated fully to become mature, immunocompetent T cells; the same cannot be achieved for thymocytes grown in 2D. This study aimed to develop a nanotechnology-based 3D culture technique using polymeric scaffolds for thymic epithelial cells (TECs), the main component of thymic stromal cells. Scanning electron microscopic observation revealed that the pores of both PCL and PCL/PLGA scaffolds were filled with TECs. Interestingly, TECs grown in 3D on polydopamine-coated scaffolds exhibited enhanced cell attachment and proliferation compared to those grown on non-coated scaffolds. In addition, the gene expression of thymopoietic factors was upregulated in TECs cultured in 3D on polydopamine-coated scaffolds compared to those cultured in 2D. Taken together, the results of the present study demonstrate an efficient 3D culture model for TECs using polymeric scaffolds and provide new insights into a novel platform technology that can be applied to develop functional, biocompatible scaffolds for the 3D culture of thymocytes. This will eventually shed light on techniques for the in vitro development of T cells as well as the synthesis of artificial thymus.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.49-60
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• 2008

Objective: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-${\beta}1$ on the development of 2-cell mouse embryos. Methods: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-${\beta}1$, its receptor-1, -2, integrin-${\beta}3$ and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. Results: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-${\beta}1$, its receptor-1, integrin-${\beta}3$ and prolactin except TGF-${\beta}1$-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). Conclusion: The administration of progesterone, estrogen and TGF-$\beta$ could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.