Fully automated high-throughput cardiac toxicity screening platform using interlocking-structured 192 SU-8 cantilever arrays

Kim, Jong Yun,Oyunbaatar, Nomin-E.,Lee, Dong-Weon Elsevier 2019 Sensors and actuators. B Chemical Vol.285 No.-

<P><B>Abstract</B></P> <P>This paper proposes a high-throughput drug-screening platform integrated with 48 well plates containing 192 SU-8 cantilevers that assess cardiac toxicity levels by measuring changes in the contractile force of cardiomyocytes in vitro environment. To improve the reliability and reproducibility of the measured data, four interlocking-structured cantilevers were employed in each well and the displacement changes were measured at the nanometer scale using a laser-based sensor. The use of the mean value of the cantilever displacement in each well greatly improved the accuracy of the drug toxicity results. In addition, microgrooves patterned onto the SU-8 cantilevers greatly enhanced the alignment of cardiomyocytes, resulting in an increase in the contraction force by approximately 2.4 times. After the preliminary experiment, the contractile behaviors of cardiomyocytes on the surface-patterned SU-8 cantilevers were measured under various drugs, namely, Bay K8644, Verapamil, Isoproterenol, Quinidine, Lidocaine, E-4031, and Astemizole. In the case of Bay K8644, Verapamil, and Isoproterenol, the contractile force of cardiomyocytes was increased or decreased by 30%, 50%, and 20%, respectively, at the value of IC<SUB>50</SUB>. However, Quinidine only increased the half-value duration (HaVD) by approximately 22%, even though it did not affect the contractile force at IC<SUB>50</SUB>. Potassium-channel inhibitors E-4031 and Astemizole also showed no change in contractile force at low concentrations. However, the HaVD increased by approximately 3.4 times and abnormal peaks were observed at higher drug concentrations. The fully automated high-throughput screening capability of the proposed platform has immense potential as a preliminary drug-screening system in the next generation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 192 SU-8 cantilever arrays integrated with a 48-well plate is proposed for high-throughput screening in drug-induced cardiac toxicity. </LI> <LI> Changes in cantilever displacement and beating frequency of cardiomyocytes treated with various drugs were systematically analyzed. </LI> <LI> The fully automated high-throughput screening approach has great potential to further improve the accuracy of drug discovery studies. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>The manuscript introduced a novel method of fully automated high-throughput cardiac toxicity screening system platform. This system can precisely and continuously measure the displacement of 192 cantilever arrays using a laser vibrometer and a motorized XY-stage for the purposed of high-throughput drug screening.</P> <P>[DISPLAY OMISSION]</P>

Tissue-Engineered 3D In Vitro Disease Models for High-Throughput Drug Screening

Huskin Gillian,Chen Jun,Davis Trenton,Jun Ho-Wook 한국조직공학과 재생의학회 2023 조직공학과 재생의학 Vol.20 No.4

During high-throughput drug screening, in vitro models are fabricated and the effects of therapeutics on the models evaluated in high throughput—for example, with automated liquid handling systems and microplate reader-based high-throughput screening (HTS) assays. The most frequently-used model systems for HTS, 2D models, do not adequately model the in vivo 3D microenvironment—an important aspect of which is the extracellular matrix—and therefore, 2D models may not be appropriate for drug screening. Instead, tissue-engineered 3D models with extracellular matrix-mimicking components are destined to become the preferred in vitro systems for HTS. However, for 3D models, such as 3D cell-laden hydrogels and scaffolds, cell sheets, and spheroids as well as 3D microfluidic and organ-on-a-chip systems, to replace 2D models in HTS, they must be compatible with high-throughput fabrication schemes and evaluation methods. In this review, we summarize HTS in 2D models and discuss recent studies that have successfully demonstrated HTS-compatible 3D models of high-impact diseases, such as cancers or cardiovascular diseases.

Simple and efficient model systems of screening anti-<i>Toxoplasma</i> drugs <i>in vitro</i>

Jin, Chunmei,Jung, Suk-Yul,Kim, Sung Yeon,Song, Hyun-Ok,Park, Hyun Informa UK, Ltd. 2012 Expert opinion on drug discovery Vol.7 No.3

<P><B><I>Introduction:</I></B> A lot of <I>in vitro</I> technologies have been developed to screen drugs for toxoplasmosis, which is caused by <I>Toxoplasma gondii</I> and is one of the most serious infectious diseases in the world. However, developed screening methods still have limitation such as inaccuracy, labor-intensive and time-consuming procedure. Therefore, the development of simpler, more efficient and accurate high-throughput screening assay is needed.</P><P><B><I>Areas covered:</I></B> The present review gives the overview of <I>in vitro</I> screening technologies described in literatures so far including morphological assay, incorporation of [<SUP>3</SUP>H]uracil assay, enzyme-linked immunosorbent assay (ELISA), colorimetric microtiter assay (β-galactosidase assay), flow cytometric quantification assay, yellow fluorescent protein assay and cell viability assay. The authors discuss how these methods are efficient and/or limited for screening anti-<I>T. gondii</I> drugs. The authors further suggest brand-new technologies which are faster, simpler, more effective and available for high-throughput screening.</P><P><B><I>Expert opinion:</I></B> Options for clinical treatment of toxoplasmosis are currently very limited. Thus, more accurate <I>in vitro</I> screening methods must be established to identify the most effective anti-<I>T. gondii</I> drugs from random screening of compounds. At the same time, based on genome information, combination of an appropriate screening technology, combinatorial chemistry and computational biology may increase the efficiency of target-based drug discovery against <I>T. gondii.</I></P>

약물처리된 심장세포의 세포 수축력 측정을 위한 병렬 폴리머 캔틸레버 제작

김동수 ( Dong-su Kim ),이동원 ( Dong-weon Lee ) 한국센서학회 2020 센서학회지 Vol.29 No.2

Thus far, several in vivo biosensing platforms have been proposed to measure the mechanical contractility of cultured cardiomyocytes. However, the low sensitivity and screening rate of the developed sensors severely limit their practical applications. In addition, intensive research and development in cardiovascular disease demand a high-throughput drug-screening platform based on biomimetic engineering. To overcome the drawbacks of the current state-of-the-art methods, we propose a high-throughput drug-screening platform based on 16 functional high-sensitivity well plates. The proposed system simulates the physiological accuracy of the heart function in an in vitro environment. We fabricated 64 cantilevers using highly flexible and optically transparent silicone rubber and placed in 16 independent wells. Nanogrooves were imprinted on the surface of the cantilever to promote cell alignment and maturation. The adverse effects of the cardiovascular drugs on the cultured cardiomyocytes were systematically investigated. The 64 cantilevers demonstrated a highly reliable and reproducible mechanical contractility of the drug-treated cardiomyocytes. Real-time high-throughput screening and simultaneous evaluation of the cardiomyocyte mechanical contractility under multiple drugs verified that the proposed system could be used as an efficient drug-toxicity test platform.

High-throughput genetic screens using CRISPR–Cas9 system

권지연,김용섭 대한약학회 2018 Archives of Pharmacal Research Vol.41 No.9

The CRISPR–Cas9 system is a powerful tool for genome engineering, and its programmability and simplicity have enabled various types of gene manipulation such as gene disruption and transcriptional and epigenetic perturbation. Particularly, CRISPR-based pooled libraries facilitate high-throughput screening for functional regulatory elements in the human genome. In this review, we describe recent advances in CRISPR–Cas9 technology and its use in high-throughput genetic screening. We also discuss its potential for drug target discovery and current challenges of this technique in biomedical research.

High-throughput Cell Culture Platform

Hye-Young LEE,Wonmo KANG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

When producing protein medicines from the production cell line, the composition of the growth of cells and protein production as well as changes in quality factors occur, and optimization of cell culture medium is a necessary process step to ensure targeted protein quality and high productivity. The cell culture media screening platform’s cell culture medium screening is performed in the steps of basic media screening, feed media screening, and flask confirm. The Cell culture media screening platform is a high-throughput cell culture system (HTS) that can search hundreds of different types of badges in a small space, and based on Design of Experiments (DoE), the optimal culture rpm and culture volume of HTS was established. This cell culture medium selection is selected by applying 100 basic culture mediums and 30 types of feed mediums through a business agreement with multinational culture medium manufacturers. Cell line & Media Development Team expects to raise the level of bio-medicine development in Korea by addressing the request of the client agency to develop culture medium optimized for cell owners at a rapid and efficient cost.

High-throughput screening strategy for efficient photosynthesis using phototaxis of Chlamydomonas reinhardtii in microfluidic system

최진원,곽호석,심상준 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report high-throughput screening system using phototaxis of Chlamydomonas reinhardtii, for rapid isolation of strains with enhanced photosynthetic efficiencies in microfluidic system. We proved the correlation between phototaxis and PSII efficiency by quantitative analysis of phototaxis. We enriched the strains with improved PSII efficiency by isolating cells showing fast phototactic response from 10,000 mutants. Among 147 strains isolated after screening, 94.6% showed enhanced growth over the parental strain. Two mutants showed much improved performances with up to 1.9- and 8.1-fold increases in growth and lipid production, respectively. Our approach provides a powerful screening tool for rapid improvement of microalgae to enhance photosynthetic efficiency.

High-throughput Screening Strategy Used for Enhanced Production of Pigment by Monascus purpureus D39-4

Jun Tan,Ju Chu,Wenjuan Shi,Cheng Lin,Yuanxin Guo,Ying-ping Zhuang,Siliang Zhang,Tadayuki Imanaka 한국식품과학회 2012 Food Science and Biotechnology Vol.21 No.6

Most of the fermentation experiment designs were limited by the low-throughput of shake flask, especially for the medium optimization. A simple high-throughput screening system was developed for the determination of pigment in Monascus purpureus fermentation samples. This downscaled system was designed to optimize medium composition combined with statistical methods. The total 29 experiments designed by the Box–Behnken were used to study the 4 most important operating variables on pigment production. The analysis revealed that the optimum concentrations of glucose, peptone, NaNO3, and KH2PO4were 51.42, 4.91, 1.00, and 1.00 g/L, respectively. A production of 69.5 U/mL was achieved in agreement with the prediction (68.9 U/mL) fermented in 24-deep-well microtiterplates. Furthermore, the fermentation medium optimized in the high-throughput system was verified in shake flasks, and the pigment production could be enhanced from 206.5 U/mL in un-optimized medium to 265.8 U/mL,giving nearly 1.30-fold increase in production.

Screening of metabolite-producing bacteria in microfluidic static droplet array

장성호,이병진,이창수,정규열 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

High-throughput screening is important for combinatorial metabolic engineering to efficiently investigate vast phenotypic diversity. However, current methods place constraints on monitoring of the intracellular concentration of diverse target products at single cell level. Here, we establish a high-throughput screening platform for metabolite production based on microfluidic static droplet array (SDA) and artificial riboswitch. In this system, a genetically encoded riboswitch enables the measurement of the intracellular metabolite concentration through fluorescence. Then, single cells of a library are individually entrapped in SDA to detect and collect highly productive variants. Using this approach, we analyzed L-tryptophan concentrations of Escherichia coli cells. A mutant E. coli library was screened for improved L-tryptophan production and superior strains were obtained exhibiting up to 145% productivity compared to their parental strain.

High-throughput screening strategy for efficient phototosyntheic-convnersion of CO₂ using phototaxis of Chlamydomonas reinhardtii in microfluidic system

최진원,곽호석,김영환,심상준 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.1

Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report high-throughput screening system using phototaxis of Chlamydomonas reinhardtii, for rapid isolation of strains with enhanced photosynthetic efficiencies in microfluidic system. We proved the correlation between phototaxis and PSII efficiency by quantitative analysis of phototaxis. We enriched the strains with improved PSII efficiency by isolating cells showing fast phototactic response from 10,000 mutants. Among 147 strains isolated after screening, 94.6% showed enhanced growth over the parental strain. Two mutants showed much improved performances with up to 1.9- and 8.1-fold increases in growth and lipid production, respectively. Our approach provides a powerful screening tool for rapid improvement of microalgae to enhance photosynthetic efficiency.