• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.77-77
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• 2003

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. $CD34^+/ $ cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 $\times$ $10^5$ cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 $\times$ $10^5$ cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was $26.6 \pm 8.4.$ In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 $\pm$ 0.5 and that of HSCs cultured onto an insert was $46.9 \pm 8.4.$ The percentage of BM-MSCs cells remained being fluorescent was $97.9 \pm 0.3%$ after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.

• 한국수정란이식학회지
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• 제10권3호
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• pp.251-256
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• 1995

This study was experimented that developmental effects of bovine in vitro fertilized embryos by coculture system and supplementation of energy materials into simple media. With the ovaries from slaughter house in vitro maturation by 24h, in vitro fertilization was performed with sperms collected by Percoll gradient method. Fertilized embryos were cocultured in 15% FCS+CZB medium with BOEC(bovine oviductal epithelial cell), GCM (granulosa cell monolayer) and MEFC(mouse embryonic fihrohlast cell). And also in this study, there was trying to improve the early developmental rate of embryos by addition of concentration-controlled Na-pyruvate, D-glucose which were used as energy sources into CZB medium. In vitro developmental rate was confirmed by the cleavage rate of 48h post-IVF and the embryo development rate at 240h culture. In the coculture system BOEC had 20.0% of blastocysts rate, which was higher than that of other coculture systems. To determine the optimum concentration for early embryo developmental rate rapidly, through the gradient of concentrations of Na-pyruvate and D-glucose, we focused on the cleavage rate at 48h and blastocysts rate at 240h. In case of Na-pyruvate, cleavage rate and developmental rate over 3-cell were lower at the concentration of 1.OOrnM than the other treatment concentrations, otherwise the blastocysts rate was higher as 23.2% than the others. That result showed that as like reported group which had higher develop-mental rate over 3-cell was also higher to the blastocysts rate. In case of D-glucose, there was no effects through the concentration changes. It was the result of this study for which the use of BOEC coculture system and 1.OOmM Na-pyruvate as an energy source had an effect upon embryo development.

• Experimental and Molecular Medicine
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• 제50권11호
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• pp.1.1-1.10
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• 2018

Bone marrow-derived mesenchymal stem cells (BMMSCs) are used extensively for cardiac repair and interact with immune cells in the damaged heart. Macrophages are known to be modulated by stem cells, and we hypothesized that priming macrophages with BMMSCs would enhance their therapeutic efficacy. Rat bone marrow-derived macrophages (BMDMs) were stimulated by lipopolysaccharide (LPS) with or without coculture with rat BMCs. In the LPS-stimulated BMDMs, induction of the inflammatory marker iNOS was attenuated, and the anti-inflammatory marker Arg1 was markedly upregulated by coculture with BMMSCs. Myocardial infarction (MI) was induced in rats. One group was injected with BMMSCs, and a second group was injected with MIX (a mixture of BMMSCs and BMDMs after coculture). The reduction in cardiac fibrosis was greater in the MIX group than in the BMC group. Cardiac function was improved in the BMMSC group and was substantially improved in the MIX group. Angiogenesis was better in the MIX group, and anti-inflammatory macrophages were more abundant in the MIX group than in the BMMSC group. In the BMMSCs, interferon regulatory factor 5 (IRF5) was exclusively induced by coculture with macrophages. IRF5 knockdown in BMMSCs failed to suppress inflammatory marker induction in the macrophages. In this study, we demonstrated the successful application of BMDMs primed with BMMSCs as an adjuvant to cell therapy for cardiac repair.

• 한국임상수의학회지
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• 제27권2호
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• pp.147-153
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• 2010

연골밑뼈와 연골이식편 사이의 생유합성은 임상적으로 중요한 과제이다. 현재까지 탈회골기질의 이용은 생체내 뼈세포 증식에 있어서 가장 효과적인 방법이다. 본 연구에서는 연골세포와 탈회골기질의 공배양을 통해 뼈와 연골의 유도 목적에 부합되는 모든 필수적인 요소를 갖는 재료로 이용가능 여부를 확인하고자 실시되었다. 본 연구의 목적은 두 종류의 배양법 즉, 펠렛 배양과 알긴산 배양에서 탈회골기질과 공배양된 소 연골세포의 증식과 표현형을 비교, 평가하는 것이다. 알긴산 배양에서는 세포 군집의 형성 및 연골세포의 수적 증가가 관찰되었다. 전형적인 연골세포의 표현형이 시험기간인 8주에 걸쳐 유지되었으며, 조직학적인 검사에서 연골세포는 일반적인 원형의 형태를 유지하였고, 연골세포방과 연골세포가 점진적으로 증가하였다. 대조군(연골세포 단독배양)에 비해 탈회골기질과 공배양한 두군 모두에서 많은 세포증식이 관찰되었으며, 글리코사미노글리칸의 생성 또한 증가되었다.

• 한국가축번식학회지
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• 제20권3호
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• pp.279-288
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• 1996

본 연구는 EGF가 체외성숙과 수정에 의해 생산된 소 수정란의 발달과 inner cell mass (ICM)와 trophectoderm (TE) 세포수에 미치는 영향 및 공동배양시의 첨가효가를 조사하고 그와 더불어 간적면역 형광법을 이용하여 EGF-R 단백의 발현 유무를 조사하기 위해 실시하였다. 그 결과를 요약하면 다음과 같다. EGF 1, 10, 100 ng/ml의 농도로 처리되었던 4-세포기와 8-세포기 배는 대조군에 비하여 유의차는 인정되지 않았으나 양호한 배반포기 배 발달과 ICM과 TE 세포수 증가 양상을 나타내었다. 특히, 발달단계에 따른 EGF (10ng/ml) 효과를 조사하였던 바, 8-세포기 이후 배에서 대조군에 비하여 배반포기까지 유의한 배 발달을 유도하는 것을 확인할 수 있었지만 (p<0.05), ICM과 TE 세포수 증가에는 유의한 영향을 미치지 못하는 것을 알 수 있었다. 또한, 간접 면역 형광에 의한 EGF-R의 발현 유무를 조사한 결과, EGF-R는 4-세포기 이후에 발현되며 그 강도는 발달단계가 진행되면서 다양하게 나타난다는 것을 알 수 있었다. 한편, 수정란과 난구세포 공동배양 군은 EGF의 첨가 유무에 상관없이 대조군에 비하여 유의한 배 발달과 총세포수의 증가를 나타내며, 공동배양군에 대한 EGF 첨가는 수정란과 난구세포와의 공동배양 효과를 증진시키는 것으로 나타났다. 따라서, EGF는 착상전 소 수정란의 4-세포기 이후에 발현디는 EGF-R에 반응하여 배 발달을 유기하고, 공동배양시의 배 발달에 유용한 물질형성을 촉진시키지만, 배반포기 배의 ICM과 TE 세포수 증가에는 유의한 영향을 나타내지 못한다는 것을 알 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Rapid Communication in Photoscience
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• 제3권3호
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• pp.48-49
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• 2014

For in vitro myelination system, Schwann cells and neuronal cells of rat were cocultured. Schwann cells and neuronal cells, respectively, were obtained from dorsal root ganglion of rat embryos (E15). This method includes four steps: first step of suspension of the embryonic dorsal root ganglion cells, second step of addition of anti-mitotic cocktail, third step of purification of dorsal root cells, and fourth step of addition of Schwann cells to dorsal root ganglion cells. We made a highly purified population of myelination in a short period through this procedure and identified myelination basic protein using antibody of myelination basic protein.

• 한국수정란이식학회지
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• 제20권1호
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• pp.35-41
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• 2005

본 연구는 가축유전자원의 효율적 보존을 위해 정조세포를 줄기세포 형태로 장기보관하면서 필요에 따라 증식, 분화를 통해 가축의 복원에 활용하기 위한 연구의 일부로 진행되었다. 정조세포를 분리하여 배양한 결과 배양온도는 다른 세포들과는 달리 $32^{\circ}C$에 세포분열이 활발하였으며, TCM199에 $10\%$ FCS를 첨가한 배양액과 세르톨리세포 공배양으로 정조세포의 배양을 지지하였다. 40일령이 지나면서 정조세포 콜로니 즉 germline stem cells를 형성하였으며, 일부에서는 외형상 ES-like cells를 형성하거나, 세정관 형태로 정조세포들이 재구성되었다. 40일령까지 배양한 상태에서는 정조세포의 정모세포나 정자세포로 분화하는 징후를 관찰할 수 없었으며, 추후 이들 세포로 분화를 유기하는 실험이 진행되어야 할 것이다.

• Journal of Microbiology and Biotechnology
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• 제32권2호
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• pp.176-186
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• 2022

Continued fenvalerate use has caused serious environmental pollution and requires large-scale remediation. Dibutyl phthalate (DBP) was discovered in fenvalerate metabolites degraded by Citrobacter freundii CD-9. Coculturing is an effective method for bioremediation, but few studies have analyzed the degradation pathways and potential mechanisms of cocultures. Here, a DBP-degrading strain (BDBP 071) was isolated from soil contaminated with pyrethroid pesticides (PPs) and identified as Stenotrophomonas acidaminiphila. The optimum conditions for DBP degradation were determined by response surface methodology (RSM) analysis to be 30.9 mg/l DBP concentration, pH 7.5, at a culture temperature of 37.2℃. Under the optimized conditions, approximately 88% of DBP was degraded within 48 h and five metabolites were detected. Coculturing C. freundii CD-9 and S. acidaminiphila BDBP 071 promoted fenvalerate degradation. When CD-9 was cultured for 16 h before adding BDBP 071, the strain inoculation ratio was 5:5 (v/v), fenvalerate concentration was 75.0 mg/l, fenvalerate was degraded to 84.37 ± 1.25%, and DBP level was reduced by 5.21 mg/l. In addition, 12 fenvalerate metabolites were identified and a pathway for fenvalerate degradation by the cocultured strains was proposed. These results provide theoretical data for further exploration of the mechanisms used by this coculture system to degrade fenvalerate and DBP, and also offer a promising method for effective bioremediation of PPs and their related metabolites in polluted environments.

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.733-744
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• 2002

The fibroblasts are the principal cells in the periodontal ligament of peridontium. As the periodontal ligament fibroblasts (PDLF) show similar phenotype with osteoblasts, the PDLF are thought to play an important role in alveolar bone remodeling. Cell-to-cell contacted signaling is crucial for osteoclast formation. Recently it has been reported that PDLJ enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to $clarify\;^{1)}$ the mechanism of PDLF-induced osteoclastogenesis $and\;^{2)}$ whether we can use preosteoclast cell line instead of primary hematopoietic preosteoclast cells for studying the mechanism of PDLF-induced osteoclastogenesis. Osteoclastic differentiation of mouse macrophage cell line RAW264.7 was compared with that of mouse bone marrow-derived M-CSF dependent cell (MDBM), a well-known hematopoietic preosteoclast model, by examining, 1) osteoclast-specific gene expression such as calcitonin receptor, M-CSF receptor (c-fms), cathepsin K, receptoractivator nuclear factor kappa B (RANK) ,2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the $OAAS^{TM}$ plate. RAW264.7 cultured in the medium containing of soluble osteoclast differentiation Factor (sODF) showed similar phenotype with MDBM-derived osteoclasts, those are mRNA expression pattern of osteoclast-specific genes, TRAP(+) MNCs generation, and bone resorbing abivity. Formation of resorption pits by osteoclastic MNCs differentiated from sODF-treated RAW264.7, was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for ODF, to the sODF-containing culture me야um. The effects of PDLF on differentiation of RAW264.7 into　the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fxed with paraformaldehyde, followed by coculture with RAW264.7, which induced formation of TRAP(+) MNCs in the absence of additional treatment of sODF. When compared with untreated and fixed PDLF (fPDLF), IL-1 ${\beta}$-treated, or lipopolysaccha-ride-treated and then fixed PDLF showed two-folld increase in the supporting activity of osteoclastogenesis from RAW264.7 coculture system. There were no TRAP(+) MNCs formation in coculture system of RAW264.7 with PDLF of no fixation. These findigs suggested that we can replace the primary hematopoietic preosteoclasts for RAW264. 7 cell line for studying the mechanism of PDLF-induced osteoclastogenesis, and we hypothesize that PDLF control osteoclastogenesis through ODF expression which might be enhanced by inflammatory signals.