• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.246-254
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• 2022

Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and anti-inflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient's synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RA-hMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.

• BMB Reports
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• v.46 no.5
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• pp.276-281
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• 2013

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures.

• Animal cells and systems
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• v.13 no.2
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• pp.235-245
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• 2009

Embryonic stem (ES) cells have a capability to generate all types of cells. However, the mechanism by which ES cells differentiate into specific cell is still unclear. Using microarray technology, the differentiation process in mouse embryonic stem cells was characterized by temporal gene expression changes of mouse ES cells during differentiation in a monolayer culture. A large number of genes were differentially regulated from 1 day to 14 days, and less number of genes were differentially expressed from 14 days to 28 days. The number of up-regulated genes was linearly increased throughout the 28 days of in vitro differentiation, while the number of down-regulated genes reached the plateau from 14 days to 28 days. Most differentially expressed genes were functionally classified into transcriptional regulation, development, extra cellular matrix (ECM),cytoskeleton organization, cytokines, receptors, RNA processing, DNA replication, chromatin assembly, proliferation and apoptosis related genes. While genes encoding ECM proteins were up-regulated, most of the genes related to proliferation, chromatin assembly, DNA replication, RNA processing, and cytoskeleton organization were down-regulated at 14 days. Genes known to be associated with embryo development or transcriptional regulation were differentially expressed mostly after 14 days of differentiation. These results indicate that the altered expression of ECM genes constitute an early event during the spontaneous differentiation, followed by the inhibition of proliferation and lineage specification. Our study might identify useful time-points for applying selective treatments for directed differentiation of mouse ES cells.

• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.781-791
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• 2018

BACKGROUND: Glucosamine hydrochloride (GlcN HCl) has been shown to inhibit cell growth and matrix synthesis, but not with N-acetyl-glucosamine (GlcNAc) supplementation. This effect might be related to an inhibition of critical growth factors (GF), or to a different metabolization of the two glucosamine derivatives. The aim of the present study was to evaluate the synergy between GlcN HCl, GlcNAc, and GF on proliferation and cartilage matrix synthesis. METHOD: Bovine chondrocytes were cultivated in monolayers for 48 h and in three-dimensional (3D) chitosan scaffolds for 30 days in perfusion bioreactors. Serum-free (SF) medium was supplemented with either growth factors (GF) $TGF-{\beta}$ ($5ng\;mL^{-1}$) and IGF-I ($10ng\;mL^{-1}$), GlcN HCl or GlcNAc at 1mM each or both. Six groups were compared according to medium supplementation: (a) SF control; (b) SF + GlcN HCl; (c) SF + GlcNAc; (d) SF + GF; (e) SF + GF + GlcN HCl; and (f) SF + GF + GlcNAc. Cell proliferation, proteoglycan, collagen I (COL1), and collagen II (COL2) synthesis were evaluated. RESULTS: The two glucosamines showed opposite effects in monolayer culture: GlcN HCl significantly reduced proliferation and GlcNAc significantly augmented cellular metabolism. In the 30 days 3D culture, the GlcN HCl added to GF stimulated cell proliferation more than when compared to GF only, but the proteoglycan synthesis was smaller than GF. However, GlcNAc added to GF improved the cell proliferation and proteoglycan synthesis more than when compared to GF and GF/GlcN HCl. The synthesis of COL1 and COL2 was observed in all groups containing GF. CONCLUSION: GlcN HCl and GlcNAc increased cell growth and stimulated COL2 synthesis in long-time 3D culture. However, only GlcNAc added to GF improved proteoglycan synthesis.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.113-120
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• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.

• Toxicological Research
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• v.5 no.1
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• pp.9-15
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• 1989

The effect of butylated hydroxytoluene (BHT) and its major metabolite, 3, 5-di-tert-butyl-4-hydroxybenzoic acid (BHT-acid) on the uptake of taurocholate into hepatocytes was studied using the primary culture of rat hepatocytes. Hepatocyte were isolated by an in situ collagenase perfusion technique and maintained as a monolayer in serum-free meadia for 24 hours before use. The uptake of taurocholate was saturable with an apparent Km of 12.8+2.8 MuM and Vmax of 0.18+0.01 nmol/mg/min. Both BHT and BHT-acid inhibited the hepatocellular uptake of taurocholate when they were added to the culture.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.73-83
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• 1990

These studies were carried out to find the proper conditions for in vitro maturation and fertilization of bovine follicular oocytes and culture methods capable of further developing early embryos. For these objectives, the cleavage rate of oocytes matured and fertilized in vitro was investigated under medium supplemented with hormones and estrous cow serum and season of oocytes collection as well as different cumulus cell stage before insemination. Finally, 2~8 cell embryos were cultured in in vitro and in vitro culture system to investigate developmental capacity into morula. 1. Cleavage rate of oocytes matured in vitro was 27%(20/73) for A(LH＋FSH＋estradil-17$\beta$＋10% FBS), 38%(27/71) for B(LH＋10% ECS) and 27%(15/56) for C(10% ECS), respectively. Supplement B showed more higher rate and 4~8 cell embryos were also obtained much more in this group(67%, 18/27). In vitro maturation rate of follicular oocytes cultured in TCM 199 supplemented withLH and 10% ECS was 88%(75/85). 2. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes with partially removed cumulus cell before insemination was more higher than that(44%, 27/62) of oocytes with intact cumulus cell. 4. The frequency of development from early cleaved embryos into morula was 6%(4/65), 12%(4/33) for co-culture of cumulus cell monolayer and bovine oviduct epithelial cell monolayer, respectively and 25%(6/25) in ligated rabbit oviduct.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.173-178
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• 1998

The objective of this study was to evaluate the embryonic development ability and the appearance of blastocysts of bovine in vitro fertilized oocytes cultured in different culture media, and also to evaluate survival rate after thawing of frozen embryos by using 1.5 or 1.8M ethylene glycol(EG) with sucrose or trehalose. Fertilized oocytes were divided into three groups; i ) monolayer of cumulus /granulosa cell prepared by TGM 199+5% calf serum(TGM199), ii)GRlaa+5% CS, iii)SOF+5% CS, and they were cultured after insemination for 9 days, at 39˚C, under 5% $CO_2$ in air, but SOF+5% CS was cultured at 39˚C, under 5% 02, 5% GO2, 99% N2. Blastocysts derived from GRlaa + 5% CS on day 7~8 after insemination were frozen by using 1.5M EG or 1.8M EG with/without 0.2M sucrose or O.1M trehalose. The development rate of blastocysts on day 7 after insemination in SOF+5% CS was significant higher than in TCM199 or CR1aa(P<0.05). The appearance rate of blastocysts on day 7-8 after insemination was higher than in TCM199, when fertilized oocytes were cultured in GRlas or SOF. The survival rate of frozen blastocysts after thawing tended to increase, when blastocysts were frozen by using 1.8M EG with 0.2M sucrose or O.1M trehalose. These results indicated that SOF or CRlaa media with amino acids was superior to TCM199 with monolayer in terms of blastocyst development in culturing of in vitro fertilized bovine nocytes, and sucrose or trehalose was supposed to prevent embryos from the freezing shock.

• Archives of Plastic Surgery
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• v.33 no.5
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• pp.616-620
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• 2006

Purpose: The isolated human chondrocytes for cartilage reconstruction and transplantation presents a major problem as these cells would change biologically in vitro. For more effective applications of these cells in the clinical field, it is necessary to get a large amount of cells in a short period without affecting their function and phenotype. Methods: This study reports the effects of placenta extract on chondrocytes in vitro. We initiated this study on the basis of the hypothesis that placenta extract can influence both the proliferation of chondrocytes and their biologic functions(for example, to express cell specific gene or to produce their own extracellular matrix). Chondrocytes in monolayer culture with or without placenta extract were collected and analyzed by MTT assay, ECM assay, and RT-PCR. Results: Placenta extract stimulated the proliferation of chondrocytes in monolayer culture. The phenotype of chondrocytes was well maintained during the expansion in monolayers. Chondrocytes expanded in the presence of placenta extract produced ECM, glycosaminoglycan, abundantly. Compared to chondrocyte expanded in culture medium only, chondrocytes expanded with placenta extract demonstrated higher COL2A1 expression that was biochemically comparable to primary chondrocytes. This study provides an evidence that placenta extract is helpful to expand chondrocytes during tissue cultivation, to maintain their differentiated phenotype and to promote their function. Conclusion: These results suggest that placenta extract during cultivation play an important role in controlling cell behaviors. Furthermore, these results provide a biologic basis for cartilage tissue engineering.

• Tuberculosis and Respiratory Diseases
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• v.50 no.2
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• pp.205-212
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• 2001

Background : The information on nasal transport and the metabolism of peptides have been obtained from pharmacokinetic investigations in experimental animals. However, there are no transport and metabolic studies of human nasal epithelial cells. In this study, the permeability characteristics and the metabolic properties of in vitro human nasal cell monolayers were investigated. Material and Methods : Normal human inferior nasal conchal tissue samples were obtained from patients undergoing endoscopic nasal cavitary surgery. The specimens were cultured in a transwell using an air-liquid Interface (ALI) culture, and the transepithelial electrical resistance (TEER) value of the blank filter and confluent cell monolayers were measured. To determine the % leakage of mannitol, $4{\mu}mol%$ $^{14}C$-labelled mannitol was added and the % leakage was measured every 10 minute for 1 hour. Result : Human nasal epithelial cells in the primary culture grew to a confluent monolayer within 7 days and expressed microvilli. The tight junction between the cells was confirmed by transmission electron microscopy. The TEER value of the blank filter, fifth day and seventh day reached $108.5\;ohm.cm^2$, $141\;ohm.cm^2$ and $177.5\;ohm.cm^2$, respectively. Transcellular % leakage of the $^{14}$-mannitol at 10, 20, 30, 40, 50 and 60 minutes was $35.67{\pm}5.43$, $34.42{\pm}5.60$, $32.75{\pm}5.71$, $31.76{\pm}4.22$, $30.96{\pm}3.49$ and $29.60{\pm}3.68\;%$, respectively. Conclusion : The human nasal epithelial monolayer using ALI culture techniques is suitable for a transcellular permeability study. The data suggests that human nasal epithelial cells In an ALI culture technique shows some promise for a nasal transport and metabolism study.