After differentiation,
beating cardiomyocyte areas were cut with
a scalpel under a microscope and collected. Then, the aggregates were
partially dissociated to loosen the cell-to-cell bonds inside the
aggregate and to better allow the attachment on the hydrogel. Dissociation
was modified from the study of Ahola et al. 2014.11 (link) The enzymatic dissociation buffers were applied to the
cells incubated at 37 °C: First buffer for 45 min, second buffer
for 15 min, and third buffer for 10 min, but no mechanical dissociation
was done. The gentle dissociation treatment loosens the cardiomyocyte
aggregate and makes it more susceptible to attach on to the hydrogel
surface. Four aggregates were plated per well with all coating and
hydrogel preparations (2D and 3D), as described above for fibroblasts.
Cells were cultured with KnockOut-DMEM medium (Thermo Fisher Scientific,
USA) supplemented with 20% FBS, 1% nonessential amino acids (Cambrex,
NJ, USA), 2 mM GlutaMAX (Thermo Fisher Scientific, USA), and 50 U
mL–1 Pen/Strep. The medium was changed every 3 days,
always 1 day before analysis, and the cells were cultured for 7 days
maximum.
beating cardiomyocyte areas were cut with
a scalpel under a microscope and collected. Then, the aggregates were
partially dissociated to loosen the cell-to-cell bonds inside the
aggregate and to better allow the attachment on the hydrogel. Dissociation
was modified from the study of Ahola et al. 2014.11 (link) The enzymatic dissociation buffers were applied to the
cells incubated at 37 °C: First buffer for 45 min, second buffer
for 15 min, and third buffer for 10 min, but no mechanical dissociation
was done. The gentle dissociation treatment loosens the cardiomyocyte
aggregate and makes it more susceptible to attach on to the hydrogel
surface. Four aggregates were plated per well with all coating and
hydrogel preparations (2D and 3D), as described above for fibroblasts.
Cells were cultured with KnockOut-DMEM medium (Thermo Fisher Scientific,
USA) supplemented with 20% FBS, 1% nonessential amino acids (Cambrex,
NJ, USA), 2 mM GlutaMAX (Thermo Fisher Scientific, USA), and 50 U
mL–1 Pen/Strep. The medium was changed every 3 days,
always 1 day before analysis, and the cells were cultured for 7 days
maximum.
