• KSBB Journal
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• v.13 no.4
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• pp.411-417
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• 1998

The possibility of application of membrane type immobilized adsorbent to the fed-batch or perfusion culture system with anchorage-independent cells as well as batch system was investigated. The improvement in cell density and cell viability due to the combination of immobilized adsorbent with each culture system was evaluated for the investigation, and the optimum culture system employing immobilized adsorbent system was suggested based on the results. It was observed that the system with immobilized adsorbent showed better cell growth and cell viability than that without immobilized adsorbent in every operation system of batch, fed-batch, and perfusion. In case of batch system, 200% improvement of maximum cell density was observed in the system where ammonium chloride was added on purpose. And 50% improvement of maximum cell density was observed in the fed-batch system where ammonium ion accumulates significantly, while small increase in maximum cell density was observed in the perfusion system where dilution of waste byproducts exists. Especially, the fed-batch system showed the most significant improvement on cell growth because both compensation of nutrient and removal of ammonium ion occurred simultaneously in the system. Therefore a combined system of immobilized adsorbent and fed-batch operation could be suggested as an optimum system with in situ removal of ammonium ion.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.168-173
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• 2004

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -20$-20^{\circ}C$, $-30^{\circ}C$ or $-80^{\circ}C$ in the presence of 5% DMSO used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstracted from the somatic cells, 62.3%, 76.6% to 65% showed cleavage 70.5%, 81.9% to 78.5% reached the stage of morula formation and 39.7%, 43.2% or 47.6% reached the blastocyst stage. There was no significant difference in development when the NT embryos were compared with those reconstracted from fresh somatic cell derieved skin tissues (72%, 75.3%, and 45.2%, for cleavage, and development to morula and blastocyst stage, respectively). NT embryos were then placed in a portable $CO_2$ incubator and carried to China from Japan by air. After reaching to farm, two NT embryos were transferred to each of 5 recipients. We obtained 2 NT calves which birth weights is 30kg and 36kg female, and gestation periods is 281 and 284 days, respectively. There were no observation any abnormality from those calves. The results indicated that cell lines derieved from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer using the portable $CO_2$ incubator.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.122-126
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• 2004

A digestion trial in vitro was conducted to study effects of supplementation of NSP (non-starch polysaccharides) degrading enzyme (feed grade) on cell wall degradation and digestibility of nutrients in barley. The slices of barley were soaked in distilled water with or without 0.15% non-starch polysaccharides degrading enzyme. Microscopic examination of the slices showed that the endosperm cell wall of barley was completely degraded by the non-starch polysaccharides degrading enzyme. The residues and supernatant of digesta in vitro were separated by filtration with 0.1 mm nylon fabric. The residues were used for measurement of crude protein, crude fat, crude fiber, and moisture. The supernatant was used for determination of viscosity, as well as amino-nitrogen and glucose content. The results showed that compared with the control, the amino-nitrogen and glucose content of the supernatant increased by 17.58% (p<0.05) and 10.26% (p<0.05), respectively, while viscosity did not change. Enzyme supplementation increased the digestibilities of dry matter, crude protein, nitrogen-free extract, crude fat and crude fiber of barley by 18.1% (p<0.05), 20.3% (p<0.05), 16.4% (p<0.05), 26.9% (p<0.05) and 30.0% (p<0.05), respectively. The present study suggests that cell wall hydrolysis may contribute to improved nutrient digestion in vivo when non-starch polysaccharides degrading enzymes are fed to swine.

• Asian-Australasian Journal of Animal Sciences
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• v.4 no.2
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• pp.151-156
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• 1991

Bovine in vitro fertilization experiment was carried out using ovary-derived follicular oocytes and frozen-thawed spermatozoa to determine the optimal preincubation time of spermatozoa and the insemination time for successful in vitro fertilization rate. The possibility of parthenogenetic cell division of unfertilized oocytes during culture without spermatozoa was also examined. There was no significant (p>0.05) difference in percent ratio of embryos developed to blastocyst stage between 0 and 3 h preincubation times of spermatozoa, showing a tendency to have higher percentage for 0 h of preincubation time. The 6 h insemination time seemed to be better for producing higher percentage of ova cleavage compared with those of 1 and 3 h treatments. Approximately 10% of unfertilized oocytes divided into 2 to 4-cell stage, and some of them cleaved to 5 up to 8-cells. The results obtained from this study suggested that 0 h of sperm preincubation time and 6 h of insemination time would be suitable for producing better in vitro fertilization rate of bovine oocytes. It is also likely that unfertilized bovine oocytes probably cleave to some cell stages with irregular divisions of the cells. On the one hand, considerable variation was also found in spermatozoa function among individual bulls.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.20-20
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• 2001

It has been reported that cloning cattle is inefficient. One of the problems was placental abnormality, finally resulting in fetal mortality after transfer of nuclear transfer (NT) bovine embryos. This study was focused on the allocations of embryonic cells to the inner cell mass (ICM) or to the trophectoderm(TE) in NT bovine blastocysts. Somatic cells were derived from a Day 45 fetus of gestation, individually transferred into enucleated oocytes and developed to the blastocyst stage in vitro. Differential staining was used to assess the qualify of blastocysts derived from NT, IVF and in vivo. Development rate of NT embryos to blastocysts (25.0%, 41/164) was similar to that of IVF embryos (28.7%, 49/171). The total cell number of NT blastocysts (101.3$\pm$45.9) was not different compared with that of IVF embryos (107.9$\pm$34.2, P>0.05), but was lower than in vivo embryos (122.5$\pm$21.6, P<0.05). Ratio of ICM/total cells was higher in NT embryos (51.6$\pm$ 18.6%) than in IVF and in vivo embryos (42.3$\pm$ 15.3% and 34.9$\pm$8.9%, respectively) (P<0.05). Most IVF (56.8%, 25/44) and in vivo blastocysts(80.8%, 21/26) was distributed in the proportion of ICM/total cells ranging from 20 to 40% group. However, most NT blastocysts was biased in the 40-60%(34.1%, 15/44) and >60% (31.8%, 14/44) groups. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocation of NT embryos to the ICM cells.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.109-120
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• 2023

Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.19-19
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• 2002

This study was to examine whether the in vitro differentiated neural cells derived from human embryonic stem (hES, MB03) cells can be survived and expressed tyrosin hydroxylase(TH) in grafted normal or PD rat brain. To differentiate in vitro into neural cells, embryoid bodies (EB: for 5 days, without mitogen) were formed from hES cells, neural progenitor cells(neurosphere, for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) were produced from EB, and then finally neurospheres were differentiated into mature neuron cells in N2 medium(without bFGF) for 2 weeks. In normal rat brain, neural progenitor cells or mature neuron cells (1×10/sup 7/ cells/㎖) were grafted to the striatum of normal rats. After 2 weeks, when the survival of grafted hES cells was examined by immunohistochemical analysis, the neural progenitor cell group indicated higher BrdU, NeuN＋, MAP2＋ and GFAP＋ than mature neuron cell group in grafted sites of normal rats. This result demonstrated that the in vivo differentiation of grafted hES cells be increased simultaneously in both of neuronal and glial cell type. Also, neural progenitor cell grafted normal rats expressed more TH pattern than mature neuron cells. Based on this data, as a preliminary test, when the neural progenitor cells were grafted into the striatum of 6-hydroxydopamine lesioned PD rats, we confirmed the cell survival (by double staining of Nissl and NeuN) and TH expression. This result suggested that in vitro differentiated neural progenitor cells derived from hES cells are more usable than mature neuron cells for the neural cell grafting in animal model and those grafted cells were survived and expressed TH in normal or PD rat brain.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.501-510
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• 2007

The present study was conducted to establish primary bovine muscle satellite cell (MSC) culture conditions and to investigate the effects of various steroid hormones on transcription of the genes involved in muscle cell proliferation and differentiation. Of three different types of proteases (type II collagenase, pronase and trypsin-EDTA) used to hydrolyze the myogenic satellite cells from muscle tissues, trypsin-EDTA treatment yielded the highest number of cells. The cells separated by hydrolysis with type II collagenase and incubated on gelatin-coated plates showed an enhanced cell attachment onto the culture plate and cell proliferation at an initial stage of cell growth. In this study, the bovine MSCs were maintained in vitro up to passage 16 without revealing any significant morphological change, and even to when the cells died at passage 21 with decreased or almost no cell growth or deformities. When the cells were incubated in a steroid-depleted environment (DMEM(-)/10% CDFBS (charcoal-dextran stripped FBS)), they grew slowly initially, and were widened and deformed. In addition, when the cells were transferred to an incubation medium containing steroid (DMEM(+)/10% FBS), the deformed cells resumed their growth and returned to a normal morphology, suggesting that steroid hormones are crucial in maintaining normal MSC morphology and growth. The results demonstrated that treatments with 19-nortestosterone and testosterone significantly increased AR gene expression (p<0.05), implying that both testosterone and 19-nortestosterone bind with AR and that the hormone bound-AR complex up-regulates the genes of its own receptor (AR) plus other genes involved in satellite cell growth and differentiation in bovine muscle.

• Food Science of Animal Resources
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• v.43 no.6
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• pp.1017-1030
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• 2023

Fetal bovine serum (FBS), which contains various nutrients, comprises 20% of the growth medium for cell-cultivated meat. However, ethical, cost, and scientific issues, necesitates identification of alternatives. In this study, we investigated commercially manufactured serum-free media capable of culturing Hanwoo satellite cells (HWSCs) to identify constituent proliferation enhancing factors. Six different serum-free media were selected, and the HWSC proliferation rates in these serum-free media were compared with that of control medium supplemented with 20% FBS. Among the six media, cell proliferation rates were higher only in StemFlex^TM Medium (SF) and Mesenchymal Stem Cell Growth Medium DXF (MS) than in the control medium. SF and MS contain high fibroblast growth factor 2 (FGF2) concentrations, and we found upregulated FGF2 protein expression in cells cultured in SF or MS. Activation of the fibroblast growth factor receptor 1 (FGFR1)-mediated signaling pathway and stimulation of muscle satellite cell proliferation-related factors were confirmed by the presence of related biomarkers (FGFR1, FRS2, Raf1, ERK, p38, Pax7, and MyoD) as indicated by quantitative polymerase chain reaction, western blotting, and immunocytochemistry. Moreover, PD173074, an FGFR1 inhibitor suppressed cell proliferation in SF and MS and downregulated related biomarkers (FGFR1, FRS2, Raf1, and ERK). The promotion of cell proliferation in SF and MS was therefore attributed to FGF2, which indicates that FGFR1 activation in muscle satellite cells may be a target for improving the efficiency of cell-cultivated meat production.