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Strategies for ex vivo Expansion and Cytotoxicity Enhancement of NK Cells Using Mechanical Stimuli
Myeongkwan SONG,Soonjo KWON 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
Adoptive cell therapy, also known as cellular immunotherapy, has recently attracted attention as a new cancer treatment method. Natural killer (NK) cells with the advantage of allogenic transfer are used as a cell therapy strategy along with Tumor-Infiltrating Lymphocyte (TIL), Engineered T Cell Receptor (TCR), and Chimeric Antigen Receptor (CAR) T Cell. However, for clinical applications, NK cells have problems with low ex vivo expansion and NK cell mediated cytotoxicity. To enhance NK cell expansion and cell-mediated cytotoxicity, we exposed NK cells to two types of mechanical stimuli: vibration and hyper-gravity. Vibration stimulus was calculated by sinusoidal acceleration, and hyper-gravity stimulus was calculated as g-force. CCK-8 assay was performed to determine whether vibration and hyper-gravity stimulus affect cell viability. There was no statistically significant decrease in cell viability at all intensities. We analyzed changes in the expression of cell-mediated cytotoxicity-related genes (granzyme B, perforin, TNF-α, and IFN-γ) and apoptosis related genes (BCL-2, BAX) following exposure of NK cells to two types of mechanical stimuli. In both mechanical stimuli, cytotoxicity enhancement and apoptosis inhibitory effects were observed. In addition, we are observing the changes in cell-mediated cytotoxicity compared to the control group by LDH assay in cancer cells in co-cultures. These findings provide useful insight for improving ex vivo expansion of NK cells and enhancing cell-mediated cytotoxicity.
Biodentine<sup>TM</sup>의 경화시간에 따른 미세누출과 전단결합강도
송용호,이난영,이상호,지명관,Song, Yong Ho,Lee, Nanyoung,Lee, Sangho,Jih, Myeongkwan 대한소아치과학회 2018 大韓小兒齒科學會誌 Vol.45 No.3
이 연구의 목적은 mineral tiroxide aggregate의 긴 경화시간과 치아변색 등의 단점으로 인해 이를 대체하고자 최근 주목받고 있는 재료인 tricalcium silicate를 기반으로 한 치수복조제 중 Biodentine의 미세누출과 최종수복제로 가장 많이 쓰이는 composite resin간의 전단결합강도에 대하여 경화시간에 따른 차이를 비교 평가하는 것이다. 미세누출 평가를 위해 소 치아 70개를 이용하여 순면에 와동을 형성한 후 Biodentine을 충전하고 무작위로 10개씩 하위 군으로 나누어 12분, 45분, 24시간, 48시간, 1주일, 2주일, 1개월로 경화시간을 달리한 후 상방에 composite resin을 적용하여 시편을 제작하였다. 미세누출 평가를 위해 표본을 24시간 동안 0.5% fuchsin 용액에 침적한 뒤, 수세하고 건조하여 각 표본을 수주 하 디스크를 이용해 절반으로 나누어 20배의 비율로 실체현미경(Olympus SZ61, Olympus, Tokyo, Japan)을 사용하여 관찰하였다. 전단결합강도 평가를 위해 중심구를 가진 아크릴 레진 블록을 210개를 제작하고 중심구에 Biodentine을 채운 후 각각 30개씩 7개 군으로 나누어 12분, 45분, 24시간, 48시간, 1주일, 2주일, 1개월 경화시간 경과 후 상방에 composite resin을 적용하여 시편을 제작하였다. Universal testing machine을 이용하여 경화시간에 따른 전단결합강도를 측정하였다. 연구결과, 미세누출에 있어서 24시간 이상 Biodentine 경화 후 상방에 composite resin 수복을 시행한 경우 미세누출을 최소화 하는 것으로 나타났다. 또한 전단결합강도에 있어서도 Biodentine의 경화시간이 24시간 이상일 경우 composite resin의 중합수축을 상쇄할 수 있는 전단결합강도가 측정되었다. composite resin 수복 전 Biodentine을 24시간 이상 경화시키는 것이 임상적으로 더 나은 결과를 나타낼 것이다. The purposes of this study were to evaluate microleakage of Biodentine, one of the tricalcium silicate based pulp-capping materials, and to compare the shear bond strength between composite resin and Biodentine with different setting times. For microleakage evaluation, 70 bovine teeth were used. Cavities were formed on the labial surfaces and filled with Biodentine. The teeth were divided into seven groups, each consisting of 10 teeth. The specimens were prepared by applying the composite resin on the upper side after different setting times. To evaluate shear bond strength, 210 acrylic resin blocks with central grooves were prepared, and the grooves were filled with Biodentine. The acrylic resin blocks were divided into seven groups of 30 specimens each, and the specimens were prepared by applying the composite resin on the upper side after different setting times. In samples with setting time of 24 hours or longer period, the microleakage between composite resin and Biodentine was reduced significantly while the shear bond strength increased to offset the polymerization shrinkage of the composite resin. Setting Biodentine for more than 24 hours before composite resin restoration would lead to more favorable clinical result.
Ha Yeon PARK,Sehwan JEONG,Myeongkwan SONG,Kyobum KIM,Hyun Jin KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
In vitro transcribed (IVT) mRNA is considered as a new gene regulation tool for therapeutic application with its rapid expression and low immunogenicity. Natural killer (NK) cells have attracted attention due to proinflammatory and cytotoxic function toward solid tumors. Genetically-modified NK cells show high viability and tumor targeting ability but there are few reports on optimal IVT mRNA transfection conditions for NK cells. We formulated polymeric nanoparticles between cationic polymer, PAsp(DET/CHE) and firefly-luciferase (F-Luc) mRNA. The polymer contains two moieties; DET triggers endosomal escape and CHE stabilizes particles. We introduced the mRNA nanoparticles into KHYG-1, human NK cell line at various transfection conditions of dose (50-500ng), N/P ratio (N/P 2-15), and incubation time (4-72hr). Through luminescence measurements, the most effective condition showed 1000-fold higher intensities than negative controls. This study provides optimal ex vivo transfection conditions for NK cells, which intrinsically exhibit low translation efficacy of mRNA. Ultimately, we will advance the next generation cancer therapy using NK cells overexpressing therapeutic receptor mRNA.