A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety

Molecules

Kyeong Hwan Ahn¹  Sooil Kim¹  Dong Woo Lee¹  Mihi Yang² 

[1] Department of Biomedical Engineering, Konyang University, Daejeon 35365, Korea;Department of Toxicology, College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Korea;

关键词: pillar strip;    myogenic differentiation;    developmental toxicity;    C2C12;    Matrigel coating plate;   

DOI  :  10.3390/molecules26195805

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown