Experimental work performed by Bharathi Balasubramanian and Elena Trepakova
Merck & Co, New Jersey, USA.
CDI iCells cardiomyocytes were fixed and stained with antibodies for f-actin (orange red) and integrin beta-1 (green), and imaged with GE InCell 2000 Analyzer with 20X objective. Blue: Nuclei (Hoechst staining). Scale bar = 50 μm.
In Mimetix aligned multiwell plates (PLLA fibres), f-actin myofibrils are aligned along the polymer fibres with noticeable Z-lines, whereas in 2D plates f-actin fibres are randomly oriented. Integrin beta-1 expression is more pronounced along the fibres. The combined image shows the colocalization of the two proteins.
hiPSC-CMs grown on 3D-aligned Mimetix® plates resembled a native human cardiac tissue, and the inner cell structures shared closer similarity to the native CMs, with myofibrils aligned along the long cell axis and clearly visible Z-lines.
Effects of 4 drugs on hiPSC-CMs after 12 days of culture. Cells were stained with Annexin V (green), TMRM (orange), RedDot (red) and Hoechst (blue). Green mask : cytoplasm outline; red mask : dead cells (excluded from analysis); cells without mask : out of focus (excluded from analysis). Scale bar = 50 μm.
Treatment with Antimycin Staurosporine and Sunitinib , 3 potent mitochondria and kinase inhibitors, contributed to significant apoptosis whereas Haloperidol, an antipsychotic drug, only caused slight changes. These tests couldn’t be performed in 2D as the cells detached upon compound addition.
Calcium transient parameters from multiple wells across replicate plates (1 – 3) were averaged for each plating condition. Comparison circles to the right of each box plot show statistical significance of differences (α=0.01), with no overlap indicating statistical significance.
Rising slopes in the PLLA plates (orange and purple) are higher than 2D (red). This suggests faster kinetics of calcium response, as expected in more mature cells.
All four 3D aligned plate conditions showed statistically significantly smaller amplitudes, as well as smaller peak width durations, thus making the total area under the curve smaller. This implies a more rapid clearance of calcium in the aligned cells as compared to 2D.
MATERIAL AND METHODS
Human iPSC-cardiomyocytes (hiPSC-CMs) were obtained from CDI (Madison, WI) and seeded onto 96-well cell culture plates (Greiner Bio-one and Mimetix® plates) at 10,000 cells/well (or 30,000 cells/well for functional assays) coated with fibronectin (10 μg/mL). Cells were maintained for a period of 14 days with media exchanged every 2 days.
3D cell culture
Four different 3D conditions were investigated, all based on the Mimetix aligned PLLA fibres in 96-well format. In addition to PLLA, 2 different polymers with various stiffnesses were tested : PLCL (Purac) and Bionate® II80A (DSM). All plates were rinsed for 5 min with 20% ethanol prior to seeding. Mimetix plates containing wavy PLLA fibres were obtained after Mimetix aligned 96-well plates were pre-treated with a harsher ethanol wash (70% ethanol for 30 min) before fibronectin coating (see below).
hiPSC-CMs grown on Mimetix aligned scaffold structurally resemble native adult human cardiomyocytes
Calcium transients measured in Mimetix aligned scaffold suggest that cells are more mature