• Toxicological Research
• /
• v.35 no.4
• /
• pp.343-351
• /
• 2019

Many in vitro developmental toxicity assays have been proposed over several decades. Since the late 1980s, we have made intermittent attempts to introduce in vitro assays as screening tests for developmental toxicity of inhouse candidate products. Two cell-based assays which were developed two decades apart were intensively studied. One was an assay of inhibitory effects on mouse ascites tumor cell attachment to a concanavalin A-coated plastic sheet surface (MOT assay), which we studied in the early days of assay development. The other was an assay of inhibitory effects on the differentiation of mouse embryonic stem cell to beating heart cells (EST assay), which we assessed more recently. We evaluated the suitability of the assays for screening in-house candidates. The concordance rates with in vivo developmental toxicity were at the 60% level. The EST assay classified chemicals that inhibited cell proliferation as embryo-toxic. Both assays had a significant false positive rate. The assays were generally considered unsuitable for screening the developmental toxicity of our candidate compounds. Recent test systems adopt advanced technologies. Despite such evolution of materials and methods, the concordance rates of the EST and MOT systems were similar. This may suggest that the fundamental predictivity of in vitro developmental toxicity assays has remained basically unchanged for decades. To improve their predictivity, in vitro developmental toxicity assays should be strictly based on elucidated pathogenetic mechanisms of developmental toxicity.

• Animal cells and systems
• /
• v.13 no.2
• /
• pp.97-103
• /
• 2009

Recently, great advance is achieved in the field of genome-wide functional screening using cell-based assay. Here, we briefly introduce well-established and typical cell-based assays of GPCR and some parameters which should be considered for genome-wide functional screening. Because of characters and importance of GPCR as drug targets, several ways of assay systems were devised. Among them, high-content screening (HCS) that is based on the analysis of image by confocal microscope is becoming favorite choice. The advances in this technology have been driven exclusively by industry for their convenience. Now, it is turn for academy to define more detail signaling networks via HCS using cDNA or siRNA libraries at genome-wide level. By isolating novel signaling mediators using cDNA or siRNA library, and postulating them as new candidates for therapeutic target, more understanding about life science and more increased chances to develop therapeutics against human disease will be achieved.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.7 no.3
• /
• pp.178-184
• /
• 2002

High-throughput screening has become a popular method used to identify new “leads”for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of various in-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially glowing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.

• Biomedical Science Letters
• /
• v.15 no.2
• /
• pp.161-166
• /
• 2009

Human erythropoietin(EPO) is a glycoprotein that enhances red blood cell production by stimulating proliferation and differentiation of erythroid progenitor cells in the bone marrow. Patients with chronic kidney disease(CKD) suffer from anemia caused by reduced production of EPO in the kidney. Recombinant human EPO protein has been used successfully for the treatment of anemia associated with CKD. Recently, attention has been paid to the development of side effect of EPO, pure red cell aplasia(PRCA), in some patients with CKD. PRCA is a rare disorder of erythropoiesis that leads to a severe anemia due to an almost complete cessation of red blood cell production. EPO-related PRCA is caused by the production of EPO-neutralizing antibodies(Abs) that eliminate the biological activity of EPO as well as endogenous EPO in patients undergoing therapy. Since 1988, almost 200 cases worldwide have been reported with Ab-positive PRCA after receiving EPO therapeutics. The underlying mechanisms of the breaking of immune tolerance to self-EPO have been investigated. Modification of formulation, organic compounds of container closures, and route of administration has been suggested for the possible mechanism of increased immunogenicity of EPO. A number of assays have been used to detect Abs specific to EPO. These assays are generally grouped into two major categories: binding Ab assays and neutralizing Ab assays(bioassays). There are several types of binding Ab assays, including radioimmunoprecipitation assay, enzyme-linked immunosorbent assay, and the BIAcore biosensor assay. In vitro cell-based bioassays have been utilized for the detection of neutralizing Abs. Finally, the recent experience with anti-EPO Abs may have considerable implications for the future development and approval of EPO preparations. Also, considering that millions of patients are being treated with EPO, clinicians need to be aware of signs and consequences of this rare but severe clinical case.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.2
• /
• pp.213-225
• /
• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Molecules and Cells
• /
• v.27 no.4
• /
• pp.391-396
• /
• 2009

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

• Interdisciplinary Bio Central
• /
• v.1 no.3
• /
• pp.10.1-10.5
• /
• 2009

Toxicity is usually investigated using a set of standardized animal-based studies which, unfortunately, fail to detect all compounds that induce human adverse events and do not provide detailed mechanistic information of observed toxicity. As an alternative to conventional toxicology, toxicogenomics takes advantage of currently advanced technologies in genomics, proteomics, metabolomics, and bioinformatics to gain a molecular level understanding of toxicity and to enhance the predictive power of toxicity testing in drug development and risk/safety assessment. In addition, there has been a renewed interest, particularly in various government agencies, to prioritize and/or supplement animal testing with a battery of mechanistically informative in vitro assays. This article provides a brief summary of the issues, challenges and lessons learned in these fields and discuss the ways forward to further advance toxicology using these technologies.

• 한국생물공학회:학술대회논문집
• /
• 2005.10a
• /
• pp.396-396
• /
• 2005

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2006.04a
• /
• pp.95-105
• /
• 2006

Alzheimer's disease (AD) is an irreversible, progressive brain disorder that is characterized by dementia. Amounts of p25 and cdk5 kinase activity are specifically upregulated in AD patient's brain samples. Considerable evidence now points importance of p25/cdk5 in generation of A$\beta$ peptides and hyperphosphorylation of tau linking amyloid plaques and neurofibrillary tangles, two major pathological hallmarks of AD. We demonstrated that P25/CDK5 phosphorylates BACE1, the first step protease to produce A$\beta$. P25/CDK5 inhibitors to reduce BACE1 phosphorylation and the secretion of A$\beta$ are screened through in silica, in vitro, and cell-based assays. Out of 4.3 million chemicals we finally selected two compounds to have IC50 of 10 microM in cell-based assays. The inhibitors block Tau phosphrorylation as well as BACE1 phosphorylation. In conclusion P25 should be one of the best targets for AD therapeutics.

• Biomolecules & Therapeutics
• /
• v.19 no.1
• /
• pp.1-8
• /
• 2011

G-protein coupled receptors (GPCRs) constitute an important class of drug targets and are involved in every aspect of human physiology including sleep regulation, blood pressure, mood, food intake, perception of pain, control of cancer growth, and immune response. Radiometric assays have been the classic method used during the search for potential therapeutics acting at various GPCRs for most GPCR-based drug discovery research programs. An increasing number of diverse small molecules, together with novel GPCR targets identified from genomics efforts, necessitates the use of high-throughput assays with a good sensitivity and specificity. Currently, a wide array of high-throughput tools for research on GPCRs is available and can be used to study receptor-ligand interaction, receptor driven functional response, receptor-receptor interaction,and receptor internalization. Many of the assay technologies are based on luminescence or fluorescence and can be easily applied in cell based models to reduce gaps between in vitro and in vivo studies for drug discovery processes. Especially, cell based models for GPCR can be efficiently employed to deconvolute the integrated information concerning the ligand-receptor-function axis obtained from label-free detection technology. This review covers various platform technologies used for the research of GPCRs, concentrating on the principal, non-radiometric homogeneous assay technologies. As current technology is rapidly advancing, the combination of probe chemistry, optical instruments, and GPCR biology will provide us with many new technologies to apply in the future.