A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety
High-throughput, pillar-strip-based assays have been proposed as a drug-safety screening tool for developmental toxicity. In the assay described here, muscle cell culture and differentiation were allowed to occur at the end of a pillar strip (eight pillars) compatible with commercially available 96-...
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| Format: | Article |
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MDPI AG
2021-09-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/19/5805 |
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| author | Kyeong Hwan Ahn Sooil Kim Mihi Yang Dong Woo Lee |
| author_facet | Kyeong Hwan Ahn Sooil Kim Mihi Yang Dong Woo Lee |
| author_sort | Kyeong Hwan Ahn |
| collection | DOAJ |
| description | High-throughput, pillar-strip-based assays have been proposed as a drug-safety screening tool for developmental toxicity. In the assay described here, muscle cell culture and differentiation were allowed to occur at the end of a pillar strip (eight pillars) compatible with commercially available 96-well plates. Previous approaches to characterize cellular differentiation with immunostaining required a burdensome number of washing steps; these multiple washes also resulted in a high proportion of cellular loss resulting in poor yield. To overcome these limitations, the approach described here utilizes cell growth by easily moving the pillars for washing and immunostaining without significant loss of cells. Thus, the present pillar-strip approach is deemed suitable for monitoring high-throughput myogenic differentiation. Using this experimental high-throughput approach, eight drugs (including two well-known myogenic inhibitory drugs) were tested at six doses in triplicate, which allows for the generation of dose–response curves of nuclei and myotubes in a 96-well platform. As a result of comparing these F-actin (an actin-cytoskeleton protein), nucleus, and myotube data, two proposed differentiation indices—curve-area-based differentiation index (CA-DI) and maximum-point-based differentiation index (MP-DI) were generated. Both indices successfully allowed for screening of high-myogenic inhibitory drugs, and the maximum-point-based differentiation index (MP-DI) experimentally demonstrated sensitivity for quantifying drugs that inhibited myogenic differentiation. |
| first_indexed | 2024-03-10T06:55:21Z |
| format | Article |
| id | doaj.art-c140b823df454d5182c3b1abbcf842ba |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T06:55:21Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-c140b823df454d5182c3b1abbcf842ba2023-11-22T16:32:55ZengMDPI AGMolecules1420-30492021-09-012619580510.3390/molecules26195805A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug SafetyKyeong Hwan Ahn0Sooil Kim1Mihi Yang2Dong Woo Lee3Department of Biomedical Engineering, Konyang University, Daejeon 35365, KoreaDepartment of Biomedical Engineering, Konyang University, Daejeon 35365, KoreaDepartment of Toxicology, College of Pharmacy, Sookmyung Women’s University, Seoul 04310, KoreaDepartment of Biomedical Engineering, Konyang University, Daejeon 35365, KoreaHigh-throughput, pillar-strip-based assays have been proposed as a drug-safety screening tool for developmental toxicity. In the assay described here, muscle cell culture and differentiation were allowed to occur at the end of a pillar strip (eight pillars) compatible with commercially available 96-well plates. Previous approaches to characterize cellular differentiation with immunostaining required a burdensome number of washing steps; these multiple washes also resulted in a high proportion of cellular loss resulting in poor yield. To overcome these limitations, the approach described here utilizes cell growth by easily moving the pillars for washing and immunostaining without significant loss of cells. Thus, the present pillar-strip approach is deemed suitable for monitoring high-throughput myogenic differentiation. Using this experimental high-throughput approach, eight drugs (including two well-known myogenic inhibitory drugs) were tested at six doses in triplicate, which allows for the generation of dose–response curves of nuclei and myotubes in a 96-well platform. As a result of comparing these F-actin (an actin-cytoskeleton protein), nucleus, and myotube data, two proposed differentiation indices—curve-area-based differentiation index (CA-DI) and maximum-point-based differentiation index (MP-DI) were generated. Both indices successfully allowed for screening of high-myogenic inhibitory drugs, and the maximum-point-based differentiation index (MP-DI) experimentally demonstrated sensitivity for quantifying drugs that inhibited myogenic differentiation.https://www.mdpi.com/1420-3049/26/19/5805pillar stripmyogenic differentiationdevelopmental toxicityC2C12Matrigel coating plate |
| spellingShingle | Kyeong Hwan Ahn Sooil Kim Mihi Yang Dong Woo Lee A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety Molecules pillar strip myogenic differentiation developmental toxicity C2C12 Matrigel coating plate |
| title | A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety |
| title_full | A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety |
| title_fullStr | A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety |
| title_full_unstemmed | A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety |
| title_short | A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety |
| title_sort | pillar based high throughput myogenic differentiation assay to assess drug safety |
| topic | pillar strip myogenic differentiation developmental toxicity C2C12 Matrigel coating plate |
| url | https://www.mdpi.com/1420-3049/26/19/5805 |
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