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Cell culture technology has evolved into three-dimensional (3D) artificial tissue models for better reproduction of human native tissues. However, there are some unresolved limitations that arise due to the adhesive properties of cells. In this study, we developed a hexanoyl glycol chitosan (HGC) as a non-cell adhesive polymer for scaffold-based and -free 3D culture. The uniform cell distribution in a porous scaffold was well maintained during the long culutre period on the HGC-coated substrate by preventing ectopic adhesion and migration of cells on the substrate. In addition, when culturing many spheroids in one dish, supplementation of the culture medium with HGC prevented the aggregation of spheroids and maintained the shape and size of spheroids for a long culture duration. Collectively, the use of HGC in 3D culture systems is expected to contribute greatly to creating excellent regenerative therapeutics and screening models of bioproducts.

3D spheroids, which have the potential to bridge the gap between 2D cell culture and native tissue, are used as tissue models in many applications, particularly in cancer, stem cell, and pharmaceutical research. A considerable amount of effort has focused on the development of more relevant physiological models. However, spheroids still have limitations in that they cannot replicate the components and structure of the ECM in the natural environment. In this study, we proposed new concept of scaffold-based techniques for the generation of spheroids. Spheroids were successfully generated by single cell or small number of aggregated cells between HA particles. The size of each spheroid was uniform, a necrotic core didn't form, and the system showed high viability. The expression levels of the proteins and genes required to maintain cell-specific functions increased. Thus, our system provides more physiologically relevant models and could be applied to regenerative medicine or drug screening.

Background We investigated whether electrical stimulation via indium tin oxide (ITO) could enhance the in vitro culture of neonatal rat ventricular myocytes (NRVMs), which are important in vitro models for studying the mechanisms underlying many aspects of cardiology. Methods Cardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. To evaluate function of NRVMs cultured on ITO with electrical stimulation, the cell viability, change of cell morphology, immunochemistry using cardiac-specific antibodies, and gene expression were tested. Results Defined sarcomeric structure, cell enlargement, and increased distribution of NRVMs appeared in the presence of electrical stimulation. These characteristics were absent in NRVMs cultured under standard culture conditions. In addition, the expression levels of cardiomyocyte-specific and ion channel markers were higher in NRVMs seeded on ITO-coated dishes than in the control group at 14 days after seeding. ITO-coated dishes could effectively provide electrical cues to support the in vitro culture of NRVMs. Conclusions These results provide supporting evidence that electrical stimulation via ITO can be effectively used to maintain culture and enhance function of cardiomyocytes in vitro.

Additional file 1: Supplementary 1. Table S1. List of primers used in qRT-PCR

The use of injectable hydrogel formulations have been suggested as a promising strategy for the treatment of degenerative disc disease to both restore the biomechanical function and reduce low back pain. In this work, a new thermo-sensitive injectable hydrogels with tunable thermo-sensitivity and enhanced stability were developed with N-hexanoylation of glycol chitosan (GC) for treatment of degenerative disc disease, and their physico-chemical and biological properties were evaluated. The sol-gel transition temperature of the hydrogels was controlled in a range of 23–56 °С, depending on the degree of hexanoylation and the polymer concentration. In vitro and in vivo tests showed no cytotoxicity and no adverse effects in a rat model. The hydrogel filling of the defective IVD site in an ex vivo porcine model maintained its stability for longer than 28 days. These results suggest that the hydrogel can be used as an alternative material for treatment of disc herniation.

We report a biocompatible and in situ cross-linkable hydrogel derived from hyaluronic acid via a bioorthogonal reaction and confirm the clinical potential of our hydrogel through in vivo cartilage regeneration. Gelation is attributed to copper-free click reactions between an azide and dibenzyl cyclooctyne. HA-PEG4-DBCO was synthesized and cross-linked via 4-arm PEG azide. The effects of the ratio of HA-PEG4-DBCO to 4-arm PEG azide on the gelation time, microstructure, surface morphology, equilibrium swelling, and compressive modulus were examined. The potential of a hydrogel as an injectable scaffold was demonstrated by the encapsulation of chondrocytes within the hydrogel matrix in vitro and in vivo. The results demonstrated that the hydrogel supported cell survival, and the cells regenerated cartilaginous tissue. In addition, these characteristics provide potential opportunities for the use of injectable hydrogels in tissue engineering applications.

Metabolic labeling is one of the most powerful methods to label the live cell for in vitro and in vivo tracking. However, the cellular mechanisms by modified glycosylation due to metabolic agents are not fully understood. Therefore, metabolic labeling has not yet been widely used in EPC tracking and labeling. In this study, cell functional properties such as proliferation, migration and permeability and gene expression patterns of metabolic labeling agent-treated hUCB-EPCs were analyzed to demonstrate cellular effects of metabolic labeling agents. As the results, 10 μM Ac4ManNAz treatment had no effects on cellular function or gene regulations, however, higher concentration of Ac4ManNAz (>20 μM) led to the inhibition of functional properties (proliferation rate, viability and rate of endocytosis) and down-regulation of genes related to cell adhesion, PI3K/AKT, FGF and EGFR signaling pathways. Interestingly, the new blood vessel formation and angiogenic potential of hUCB-EPCs were not affected by Ac4ManNAz concentration. Based on our results, we suggest 10 μM as the optimal concentration of Ac4ManNAz for in vivo hUCB-EPC labeling and tracking. Additionally, we expect that our approach can be used for understanding the efficacy and safety of stem cell-based therapy in vivo.

The major limitations of nanocarriers for drug delivery are the poor biocompatibility and low stability that may induce the unintended burst release of loaded drugs during blood circulation. To overcome these limitations, a photocrosslinkable amphiphilic block copolymer consisting of hydrophobic poly(e-caprolactone) (PCL) block and a hydrophilic hyperbranched polyglycerol (hbPG) block with improved biocompatibility and stability for drug delivery is developed. They are readily prepared via UV-triggered chemical crosslinking with 4-hydroxycinnamic acid (CA) modification in the hbPG block. The photocrosslinked hbPG-b-PCL-CA nanoparticles are spherical and the size of nanoparticles is increased to 60 ± 30 nm. Photocrosslinked hbPG-b-PCL-CA nanoparticles exhibit significantly high stability in a physiological buffer and the loaded drug in sustained manner. In addition, photocrosslinked hbPG-b-PCL-CA nanoparticles show good biocompatibility in vitro and in vivo. These data imply the promising potential of photocrosslinked hbPG-b-PCL-CA nanoparticles as nanocarriers for drug delivery.

Metabolic labeling techniques are powerful tools for cell labeling, tracking and proteomic analysis. However, at present, the effects of the metabolic labeling agents on cell metabolism and physiology are not known. To address this question, in this study, we analyzed the effects of cells treated with Ac4ManNAz through microarray analysis and analyses of membrane channel activity, individual bio-physiological properties, and glycolytic flux. According to the results, treatment with 50 mu M Ac4ManNAz led to the reduction of major cellular functions, including energy generation capacity, cellular infiltration ability and channel activity. Interestingly, 10 mu M Ac4ManNAz showed the least effect on cellular systems and had a sufficient labeling efficiency for cell labeling, tracking and proteomic analysis. Based on our results, we suggest 10 mu M as the optimum concentration of Ac4ManNAz for in vivo cell labeling and tracking. Additionally, we expect that our approach could be used for cell-based therapy for monitoring the efficacy of molecule delivery and the fate of recipient cells.

Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy.

Wilms’ tumor 1-associating protein (WTAP) is a nuclear protein that has been associated with the regulation of proliferation and apoptosis. Although its dynamic expression and physiological functions in vascular cells have been reported, its expression and roles in cholangiocarcinoma cells are poorly characterized. To examine the expression of WTAP in patient tissues, we performed immunohistochemistry. To examine motility of cholangiocarcinoma cells, we employed Boyden chamber, wound healing and Matrigel invasion assays, and a liver xenograft model. Immunohistochemistry in patient tissues showed WTAP overexpression in cholangiocarcinoma tissues and correlation of WTAP expression with metastasis of cholangiocarcinoma cells. Overexpression or knockdown of WTAP significantly increased or decreased the motility of cholangiocarcinoma cells. Moreover, WTAP overexpression or knockdown significantly increased or decreased tumorigenicity of cholangiocarcinoma cells in an orthotopic xenograft model. Furthermore, microarray study showed that WTAP induce the expressions of MMP7, MMP28, cathepsin H and Muc1. WTAP is overexpressed in cholangiocarcinoma and regulates motility of cholangiocarcinoma cells

Ego integrity, the last developmental task in Erikson's psychological theory, develops naturally among older people. However, the presence of loss–like physical disability–can considerably affect the quality of life, interactions, and well being of older adults. Hence, older people with physical disabilities need more assistance in accomplishing tasks of ego integrity. This study aimed to utilize traditional Filipino art to capture the essence of the lived experiences of a select group of older people with physical disabilities in relation to their ego integrity. Four Filipino elderly with physical disabilities were purposely selected for this study. Picture prompting and doodling sessions were held as pretest and posttest to ascertain the effectiveness of puni-making in facilitating ego integrity of participants. A series of interviews were pursued to elicit experiences and feelings of participants regarding their disability and old age. From the analysis of the triangulated set of data, two findings eme...

Epigallocatechin-3-gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast differentiation. However, the precise molecular mechanisms underlying the inhibitory action of EGCG in osteoclastogenesis and the effect of EGCG on inflammation-mediated bone destruction remain unclear. In this study, we found that EGCG inhibited osteoclast formation induced by osteoclastogenic factors in bone marrow cell-osteoblast cocultures but did not affect the ratio of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to osteoprotegerin induced by osteoclastogenic factors in osteoblasts. We also found that EGCG inhibited osteoclast formation from bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor plus RANKL in a dose-dependent manner without cytotoxicity. Pretreatment with EGCG significantly inhibited RANKL-induced the gene expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. EGCG suppressed RANKL-induced activation of c-Jun N-terminal protein kinase (JNK) pathway, among the three well known mitogen-activated protein kinases and also inhibited RANKL-induced phosphorylation of the NF-kappaB p65 subunit at Ser276 and NF-kappaB transcriptional activity without affecting the degradation of IkappaBalpha and NF-kappaB DNA-binding in BMMs. The inhibitory effect of EGCG on osteoclast formation was somewhat reversed by retroviral c-Fos overexpression, suggesting that c-Fos is a downstream target for antiosteoclastogenic action of EGCG. In addition, EGCG treatment reduced interleukin-1-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo. Taken together, our data suggest that EGCG has an antiosteoclastogenic effect by inhibiting RANKL-induced the activation of JNK/c-Jun and NF-kappaB pathways, thereby suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

Three-dimensional (3D) cell culture systems have promising applications compared with conventional two-dimensional cell culture systems. Herein, we report a facile method for the formation of 3D spheroids using novel thermo-reversible polysaccharide-based hydrogels. A series of thermo-reversible hydrogels consisting of N-acyl glycol chitosans (NAGCs) are synthesized through a simple N-acylation reaction, and the degree of acylation is finely tuned to obtain adequate thermo-reversible properties and gel stability. Among the NAGCs, N-hexanoyl glycol chitosan is the most thermo-sensitive and is highly effective for forming multi-cellular spheroids when used to coat the surfaces of cell culture dishes. Cell spheroids are effectively formed at various cell concentrations, and their spheroid shape and cellular functions are well maintained for longer times. The hydrogel culture system is also useful for co-cultures that mimic a biological microenvironment. Our thermo-reversible hydrogels may offer a convenient method for the development of in vitro 3D cell culture systems to provide enhanced performance in tissue regeneration, organ-on-chips, drug screening research and other biomedical applications. Modifying the crustacean-derived molecule chitosan produces thermosensitive hydrogels for realistic, spherical cell growth studies. Three-dimensional (3D) cell environments are better platforms for exploring cell structure and function than traditional Petri dishes, but typically require complex ‘hanging drop’ substrates or micromoulds. Researchers from the Korea Institute of Toxicology and Chungnam National University have developed a simpler 3D cell culture using a water-filled polysaccharide that can transform into a gel on demand thanks to the thermo-responsive property. In experiments, cells such as cardiomyocytes spontaneously aggregated to form spheroids after only 24 hours immersion in the chitosan-based hydrogel. Harvesting was performed using temperature-reduction techniques suitable for large-scale processes. Cytotoxicity tests revealed that these cultures were safe and could be used to co-culture different cells to realize better biomimicking. We developed glycol chitosan-based thermo-reversible hydrogels that can provide a facile, convenient and reproducible method for the formation of 3D cell spheroids. The spontaneous formation of spheroids on HGC-coated plates was completed within 1 day. Furthermore, this system can be useful for co-culturing heterotypic cells to form spheroids that mimic biological systems.

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM(+)/CD44(+) population accounts for 4.5% of tumor cells. EpCAM(+)/CD44(+) gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) and EpCAM(-)/CD44(+) cells failed to do so. Xenografts of EpCAM(+)/CD44(+) gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM(+)/CD44(+), but not EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) or EpCAM(-)/CD44(+) cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM(+)/CD44(+) cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM(+)/CD44(+) subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

A low serum level of vitamin D has been associated with an increased incidence of gastrointestinal tract cancers. However, the effects of vitamin D3 have not been investigated in gastric cancer and cholangiocarcinoma. In the present study, we found that vitamin D3 treatment significantly suppressed the viability of gastric cancer and cholangiocarcinoma cells. Moreover, vitamin D3 had a synergistic effect with other anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine, for suppressing cell viability. To determine the underlying mechanism involved in the regulation of viability by vitamin D3, we examined the effects of vitamin D3 on expression of hedgehog signaling target genes, which has been associated with gastric cancer and cholangiocarcinoma. Vitamin D3 treatment decreased the level of mRNA expression of patched1, Gli1, cyclin D1, and Bcl2, suggesting the possibility that vitamin D3 may act through regulation of hedgehog signaling. From the above results, we conclude that vitamin D3 regulates cell viability in gastric cancer and cholangiocarcinoma.