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연골세포와 중간엽줄기세포의 3차원 Co-culture를 통한 연골화 향상
황슬기(Seul-Gee Hwang),차현명(Hyun-Myoung Cha),임진혁(Jin-Hyuk Lim),이지희(Ji-Hee Lee),심혜은(Hye-Eun Shim),김동일(Dong-Il Kim) 한국생물공학회 2016 KSBB Journal Vol.31 No.2
Two-dimensional cultivation is typically used for cell growth, but the method reduces the characteristics of chondrocytes and stem cells, and limits culture area. Therefore, development of three-dimensional culture method is needed to mimic in vivo environment, improve quality of cells and scale-up efficiently. Improving proliferation and chondrogenesis is available by co-culture of chondrocytes and mesenchymal stem cells (MSCs) that leads to interaction between two kinds of cells. However, the co-culture has problems that permeability of sphere diminishes as aggregate size increased and ratio of two kinds of cells composing each spheres is different. In this work, co-cultivation method using controlled sphere composed of chondrocytes and MSCs was established and enhanced chondrogenesis. Periosteum-derived progenitor cells (PDPCs) that are appropriate for cell therapy source of articular cartilage were used as MSCs. Controlled spheres were formed in the hanging-drop plates and shifted for being induced chondrogenesis in 35-mm non-adhesive culture dishes at a rotation rate of 60 rpm. After inducing chondrogenesis, gene expressions related with chondrogenesis were found to be improved and it was apparent that the utilization of controlled spheres promoted chondrogenesis. As a result, available numbers of cells per unit area were increased and chondrogenic differentiation ability was improved compared to typical two-dimensional culture. This approach shows the potential in cartilage regeneration as it can provide sufficient numbers of chondrocytes.
Guk, Kyeonghye,Hwang, Seul Gee,Lim, Jaewoo,Son, Hye-young,Choi, Yuna,Huh, Yong-Min,Kang, Taejoon,Jung, Juyeon,Lim, Eun-Kyung The Royal Society of Chemistry 2019 Chemical communications Vol.55 No.24
<P>We have proposed a novel strategy for miRNA detection through enzyme-free signal amplification by self-circulation of the hybridization between the miRNAs and molecular beacon (MB) circuits. Unlike general MB-based miRNA detection based on the one-to-one (1 : 1) hybridization between MBs and miRNA, our system consists of four species of MBs (MBs A, B, C and D) (MB circuits) and is activated by a hybridization chain reaction. MBs stably coexist as hairpin structures that hardly show fluorescence signals in the absence of target miRNA. After miRNA detection, this MB circuit is able to generate fluorescence signals and amplify the fluorescence signal, contributing to improvement in detection sensitivity under iso-thermal conditions without an enzyme. Furthermore, <I>in vitro</I> and <I>in vivo</I> studies have proven that MB circuits can detect low levels of miRNA with high sensitivity, compared to when only one MB alone is used. Therefore, the MB circuits can provide a useful platform for target miRNA detection.</P>
Naked Eye Detection of Salmonella typhimurium Using Scanometric Antibody Probe
Yi, So Yeon,Hwang, Seul-Gee,Moon, Jeong,Eom, Gayoung,Hwang, Ahreum,Sim, Jieun,Lim, Eun-Kyung,Jeong, Jinyoung,Kim, Bongsoo,Kang, Taejoon American Scientific Publishers 2017 Journal of Nanoscience and Nanotechnology Vol.17 No.7
<P>Salmonella is one of the most common foodborne pathogens, and Salmonella outbreaks are mostly associated with the intake of contaminated food or drink. Therefore, the rapid and sensitive on-site detection of Salmonella is very important. We report a naked eye detection method for Salmonella typhimurium using scanometric antibody probe. The antibody-attached glass substrate was treated with Salmonella typhimurium and the scanometric antibody probe was applied. After Ag enhancement of the probe, Salmonella typhimurium could be detected with the naked eye. The scanometric antibody probe was prepared by simply mixing Au nanoparticles, gold binding peptide-protein G, and antibody against Salmonella typhimurium. This probe can act as a signal enhancer and thus allows for an extremely simple, rapid, and efficient analysis of Salmonella typhimurium by the naked eye. We detected Salmonella typhimurium at a low concentration of 10(3) CFU/ml and clearly distinguished this bacterium from other foodborne pathogens. Furthermore, we successfully detected Salmonella typhimurium in milk, suggesting that this method can be useful in real-life samples. Because the scanometric antibody probe can be expanded to various types of antibodies, this naked eye detection method could be employed for the detection of various types of pathogens.</P>
Guk, Kyeonghye,Keem, Joo Oak,Hwang, Seul Gee,Kim, Hyeran,Kang, Taejoon,Lim, Eun-Kyung,Jung, Juyeon Elsevier 2017 Biosensors & bioelectronics Vol.95 No.-
<P><B>Abstract</B></P> <P>Rapid and reliable diagnosis of methicillin-resistant <I>Staphylococcus aureus</I> (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe. A dCas9/sgRNA-SG I based detection approach has advantages over monoclonal antibody in conventional immunoassay systems due to its ability to interact with the target gene in a sequence-specific manner. The detection limit of MRSA was as low as 10 cfu/ml and was found to be sufficient to effectively detect MRSA. Unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, the target gene can be detected within 30min with high sensitivity without performing a gene separation step by using cell lysates. We showed that the fluorescence signal of the MRSA cell lysate was more than 10-fold higher than that of methicillin-susceptible S. aureus (MSSA). Importantly, the present approach can be applied to any target other than MRSA by simply changing the single-guide RNA (sgRNA) sequence. Because dCas9/sgRNA-SG I based detection approach has proved to be easy, fast, sensitive, and cost-efficient, it can be applied directly at the point of care to detect various pathogens as well as MRSA in this study.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CRISPR-mediated DNA FISH system for the simple and sensitive detection of methicillin-resistant staphylococcus aureus (MRSA). </LI> <LI> The target gene can be detected within 30min without PCR amplification or a gene purification steps. </LI> <LI> Applicable to targets other than MRSA by simply altering the single-guide RNA (sgRNA) sequence. </LI> </UL> </P>