• Biomolecules & Therapeutics
• /
• v.24 no.3
• /
• pp.260-267
• /
• 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.

• Archives of Pharmacal Research
• /
• v.21 no.4
• /
• pp.398-405
• /
• 1998

We investigated the effects of triterpene acids (TAs), ursolic acid (UA) and oleanolic acid (OA), on the induction of proliferation and differentiation of normal rat mammary epithelial cells (RMEC) or organoids cultured in Matrigel or primary culture system. To elucidate the effects, we tested their differentiation inducing activities with intercellular communication ability, cell cycle patterns, induction of apoptosis, and morphological differentiation in the three dimensional extracellular culture system. To study the changes of RMEC subpopulation in culture, the cultured cells were isolated, immunostained with peanut lectin (PNA) and anti-Thy-1.1 antibody and then analyzed with flow cytometry. Four different subpopulations, such as PNA and Thy-1.1 negative cells (B-), PNA positive cells (PNA+), Thy-1.1 positive cells (Thy-1.1+), PNA and Thy-1.1 positive cells (B+), were obtained and the size of each subpopulation was changed in culture with time in the presence of TAs. Intercellular communication was observed in culture for 7 days in TAs-treated cells, but not in culture for 4 days with scrape-loading dye transfer technique. $G_2$/M phase cells and the number of apoptotic population were increased in TAs-treated groups in cell cycle analyses. S phase fractions were reduced and the change of $G_1$ phase cells was not observed. The colonies with distinct multicelfular structures, such as stellate, ductal, webbed, squamous, lobulo-ductal colonies, were observed in Matrigel culture and the frequencies of each colony were changed in the presence of TAs. These results suggest that UA and OA have differentiation inducing effects on rat mammary epithelial cells in primary or in Matrigel culture.

• Journal of Mechanical Science and Technology
• /
• v.14 no.8
• /
• pp.861-866
• /
• 2000

Flow induced shape change is important for spatial interpretation of vascular response and for understanding of mechanotransduction in a single cell. We investigated the possible shapes of endothelial cell (EC) in a mathematical model and compared these with experimental results. The linearized analytic solution from the sinusoidal wavy wall and Stokes flow was applied with the constraint of EC volume. The three dimensional structure of the human umbilical vein endothelial cell was visualized in static culture or after various durations of shear stress (20 $dyne/cm^2$ for 5, 10, 20, 40, 60, 120min). The shape ratio (width: length: height) of model agreed with that of the experimental result, which represented the drag force minimizing shape of stream-lining. EC would be streamlined in order to accommodate to the shear flow environmented by active reconstruction of cytoskeletons and membranes through a drag force the sensing mechanism.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.31 no.6
• /
• pp.478-484
• /
• 2009

Three-dimensional porous scaffolds play an important role in tissue engineering strategies. They provide a void volume in which vascularization, new tissue formation, and remodeling can occur. Like any grafted materials, the ideal scaffold for bone tissue engineering should be biocompatible without causing an inflammatory response. It should also possess biodegradability, which provides a suitable three-dimensional environment for the cell function together with the capacity for gradual resorption and replacement by host bone tissue. Various scaffolds have already been developed for bone tissue engineering applications, including naturally derived materials, bioceramics, and synthetic polymers. The advantages of biodegradable synthetic polymers include the ability to tailor specific functions. The purpose of this study was to examine the osteogenic activity of periosteal-derived cells in a polydioxanone/pluronic F127 scaffold. Periosteal-derived cells were successfully differentiated into osteoblasts in the polydioxanone/pluronic F127 scaffold. ALP activity showed its peak level at 2 weeks of culture, followed by decreased activity during the culture period. Similar to biochemical data, the level of ALP mRNA in the periosteal-derived cells was also largely elevated at 2 weeks of culture. The level of osteocalcin mRNA was gradually increased during entire culture period. Calcium content was detactable at 1 week and increased in a time-dependent manner up to the entire duration of culture. Our results suggest that polydioxanone/pluronic F127 could be a suitable scaffold of periosteal-derived cells for bone tissue engineering.

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

• Journal of Life Science
• /
• v.26 no.11
• /
• pp.1313-1319
• /
• 2016

Cancer is a complex disease heterogeneously composed of various types of cells including cancer stem-like cells responsible for relapse and chemoresistance in the tumor microenvironment. The conventional two-dimensional cell culture-based platform has critical limitations for representing the heterogeneity of cancer cells in the three-dimensional tumor niche in vivo. To overcome this insufficiency, three-dimensional cell culture methods in a scaffold-dependent or -free physical environment have been developed. In this study, we improved and simplified the HCT-8 colon cancer cell-based spheroid culture protocol and evaluated the relationship between cancer stemness and responses of chemosensitivity to 5- Fluorouracil (5-FU), a representative anticancer agent against colon cancer. Supplementation with defined growth factors in the medium and the culture dish of the regular surface with low attachment were required for the formation of constant-sized spheroids containing $CD44^+$ and $CD133^+$ colon cancer stem cells. The chemo-sensitivities of $CD44^+$ cancer stem cells in the spheroids were much lower than those of $CD44^-$ non-stem-like cancer cells, indicating that the chemoresistance to 5-FU is due to the stemness of colon cancer cells. Taken together, the inflammation and oncogenic gut environment-sensitive HCT-8 cell-based colon cancer spheroid culture and comparative evaluation using the simplified model would be an efficient and applicable way to estimate colon cancer stemness and pharmaceutical response to anticancer drugs in the realistic tumor niche.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.10
• /
• pp.1577-1582
• /
• 2006

Cartilage tissue engineering has emerged as an alternative approach for reconstruction or repair of injured cartilage tissues. In this study, rabbit chondrocytes were cultured in a three-dimensional environment to fabricate a new cartilaginous tissue with the application of tissue engineering strategies based on biodegradable PLGA microspheres. Chondrocytes were seeded on PLGA microspheres and cultured on a rocking platform for 5 weeks. The PLGA microspheres provided more surface area to adhere chondrocytes compared with PLGA sponge scaffolds. The novel system facilitated uniform distribution of the cells on the whole of the PLGA microspheres, thus forming a new cartilaginous construct at 4 weeks of culture. The histological and immunohistochemical analyses verified that the number of chondrocytes and the amount of extracellular matrix components such as proteoglycans and type II collagen were significantly greater on the PLGA microspheres constructs as compared with those on the PLGA sponge scaffolds. Therefore, PLGA microspheres enhanced the function of chondrocytes compared with PLGA sponge scaffolds, and thus might be useful for formation of cartilage tissue in vitro.

• Journal of Animal Science and Technology
• /
• v.66 no.1
• /
• pp.1-30
• /
• 2024

Interest and investment in cultivated meat are increasing because of the realization that it can effectively supply sufficient food resources and reduce the use of livestock. Nevertheless, accurate information on the specific technologies used for cultivated meat production and the characteristics of cultivated meat is lacking. Authorization for the use of cultivated meat is already underway in the United States, Singapore, and Israel, and other major countries are also expected to approve cultivated meat as food once the details of the intricate process of producing cultivated meat, which encompasses stages such as cell proliferation, differentiation, maturation, and assembly, is thoroughly established. The development and standardization of mass production processes and safety evaluations must precede the industrialization and use of cultivated meat as food. However, the technology for the industrialization of cultivated meat is still in its nascent stage, and the mass production process has not yet been established. The mass production process of cultivated meat may not be easy to disclose because it is related to the interests of several companies or research teams. However, the overall research flow shows that equipment development for mass production and cell acquisition, proliferation, and differentiation, as well as for three-dimensional production supports and bioreactors have not yet been completed. Therefore, additional research on the mass production process and safety of cultivated meat is essential. The consumer's trust in the cultivated meat products and production technologies recently disclosed by some companies should also be analyzed and considered for guiding future developments in this industry. Furthermore, close monitoring by academia and the government will be necessary to identify fraud in the cultivated meat industry.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.305-306
• /
• 2003

To treat fulminant hepatic failure (FHF) patients, various extracorporeal bioartificial liver (BAL) systems have been developed. Several requirements should be met for the development of BAL systems: hepatocytes should be cultured in a sufficiently high density; their metabolic functions should be of a sufficiently high level and duration; and the BAL systems module should permit scaling-up and aseptic handling. Several investigators have found that freshly isolated primary hepatocytes can be cultured into three dimensional, tightly packed, freely suspended, multicellular aggregates, or spheroids. These specialized cell structures exhibited enhanced liver specific functions and a prolonged differentiated state compared to cells maintained in a monolayer culture. Cells in spheroids appear to mimic the morphology and ultrastructure of the in vivo liver lobule. The ability of hepatocytes to organize into three-dimensional structures was hypothesized to contribute to their enhanced liver-specific activities. In this study, the ammonia removal rate and urea secretion rate of pig hepatocytes spheroids encapsulated in Ca-alginate bead were determined. A packed-bed bioreactor with encapsulated pig hepatocytes was devised as BAL support system. The efficacy of the system was evaluated in vitro.

• 한국생물공학회:학술대회논문집
• /
• 2002.04a
• /
• pp.289-292
• /
• 2002

The corneal tissue consists of three layers : epithelium, stroma, and endothelium. Central cornea is a highly differentiated tissue whereas the limbus contains the epithelial stem cell. In the present study. we report the engineering of the three-dimensional reconstructed cornea derived from rabbit limbal epithelial and stromal cells. The differentiation degree of corneal stem cells were assessed in serum concentration and inoculation density of stromal cells. Optimal condition differentiation of corneal stem cells is achieved when 5% FBS was supplemented to culture medium and $1-2{\times}10^5$ cells/ml inoculation density of stromal cells.