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Developmental Neurotoxicity in vitro Screening Test Method that Measures Cell Viability and Proliferation using Human Neuroprogenitor Cells

김은지|김태성|윤혜성|강남희|윤남희|김학|안준익
한국동물실험대체법학회 (Korean Society for Alternatives to Animal Experiments)|2022.12|한국동물실험대체법학회지 (Journal of Alternatives to Animal Experiments)|제16권 제1호|pp. 25-34

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키워드

ProliferationDevelopmental neurotoxicityIn vitro screening testHuman neural progenitor cellsAlternative test

초록

Current developmental neurotoxicity tests are based on in vivo tests, and many kinds of chemicals cannot be evaluated due to restrictions such as the number, cost, and time of animals used in the test. Recently, in vitro test methods for screening test chemicals that may damage the nervous system are being studied. As an alternative test for developmental neurotoxicity, an in vitro test method that measures the endpoint of neurogenesis process is being studied; however, a single in vitro assay cannot completely cover all neurogenesis processes. Therefore, studies are being carried out to combine test methods as endpoints for key events in neurogenesis process and test battery study. In this study, we used human neuroprogenitor cells, ReNcell™ CX, to evaluate developmental neurotoxicity of test substances on cell viability (MTT assay) and proliferation (BrdU assay) by ELISA method. This method has the advantage of being able to quickly and easily evaluates many substances compared to previous methods using fluorescence microscope. First, we tested to optimize the test method using known antiproliferative/ non-antiproliferative chemicals, we were able to obtain 100% accuracy compared to the previous study. This was verified by confirming that it was appropriate to evaluate cell viability and proliferation using a human neural progenitor cells (ReNcell™ CX). In the utilization study, 18 test substances were evaluated. As a result, 11 test substances (Acrylamide, Bisphenol A, Carbamazepine, Chlorpyrifos, Diethylstilbestrol, Perfluorooctanoic Acid, Sodium arsenite, 3,3’,5,5 ‘-Tetrabromobisphenol A, Ibuprofen, Omeprazole, and Verapamil) were judged as substances with developmental neurotoxicity potential, and 7 test substances (Di (2-ethylhexyl) phthalate, Fluconazole, Permethrin, Atropine, Captopril, D-Mannitol, and Dinotefuran) were judged as non-developmental neurotoxicity substances. The sensitivity was 70% (7/10), specificity 50% (4/8), accuracy 61% (11/18), false positive rate 50% (4/8), and false negative rate 30% (3/10). These results show that it can be used as an in vitro screening test for developmental neurotoxicity that can measure and evaluate many substances within a short time compared to the existing in vivo tests. In addition, this test is a single in vitro screening test, and further studies are needed to evaluate it in combination with other in vitro test method.

Abstract

Current developmental neurotoxicity tests are based on in vivo tests, and many kinds of chemicals cannot be evaluated due to restrictions such as the number, cost, and time of animals used in the test. Recently, in vitro test methods for screening test chemicals that may damage the nervous system are being studied. As an alternative test for developmental neurotoxicity, an in vitro test method that measures the endpoint of neurogenesis process is being studied; however, a single in vitro assay cannot completely cover all neurogenesis processes. Therefore, studies are being carried out to combine test methods as endpoints for key events in neurogenesis process and test battery study. In this study, we used human neuroprogenitor cells, ReNcell™ CX, to evaluate developmental neurotoxicity of test substances on cell viability (MTT assay) and proliferation (BrdU assay) by ELISA method. This method has the advantage of being able to quickly and easily evaluates many substances compared to previous methods using fluorescence microscope. First, we tested to optimize the test method using known antiproliferative/ non-antiproliferative chemicals, we were able to obtain 100% accuracy compared to the previous study. This was verified by confirming that it was appropriate to evaluate cell viability and proliferation using a human neural progenitor cells (ReNcell™ CX). In the utilization study, 18 test substances were evaluated. As a result, 11 test substances (Acrylamide, Bisphenol A, Carbamazepine, Chlorpyrifos, Diethylstilbestrol, Perfluorooctanoic Acid, Sodium arsenite, 3,3’,5,5 ‘-Tetrabromobisphenol A, Ibuprofen, Omeprazole, and Verapamil) were judged as substances with developmental neurotoxicity potential, and 7 test substances (Di (2-ethylhexyl) phthalate, Fluconazole, Permethrin, Atropine, Captopril, D-Mannitol, and Dinotefuran) were judged as non-developmental neurotoxicity substances. The sensitivity was 70% (7/10), specificity 50% (4/8), accuracy 61% (11/18), false positive rate 50% (4/8), and false negative rate 30% (3/10). These results show that it can be used as an in vitro screening test for developmental neurotoxicity that can measure and evaluate many substances within a short time compared to the existing in vivo tests. In addition, this test is a single in vitro screening test, and further studies are needed to evaluate it in combination with other in vitro test method.

참고문헌 (36)

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