Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for drug safety and efficacy testing with various techniques, including high content imaging (HCI). Upon drug treatment, a significant number of hiPSC-CMs grown in regular 96-well plates coated with fibronectin detached from the bottom of the plate, complicating data acquisition. Several cell culture configurations were tested to improve cell adherence, and the effects of these configurations on total cell number, separation of feature values between the negative (DMSO 0.1%) and positive (antimycin, staurosporine) controls, scale of feature value differences, and data variability were statistically calculated. hiPSC-CMs were plated on fibronectin- (in "blanket" configuration) or MaxGel- (in "sandwich" configuration) coated plates and covered with a layer of either HydroMatrix or MaxGel 2, 7, or 11d after plating. After a total of 14d in culture, cells were treated with compounds, labeled with four fluorescent dyes (Hoechst, TMRM, NucView, and RedDot), and imaged with GE INCell2000. Based on the statistical parameters calculated, the MaxGel 25% 7d "sandwich" was superior to all other tested conditions when the cells were treated with 0.3 μM antimycin for 2 h and test compounds 10 μM crizotinib and 30 μM amiodarone for 48 h. For staurosporine treatment, the best culturing condition varied between MaxGel "sandwich" systems, depending on which parameters were under consideration. Thus, cell culturing conditions can significantly affect the ability of high content imaging to detect changes in cellular features during compound treatment and should be thoroughly evaluated before committing to compound testing.

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