Methods for evaluating cardiac safety of drug using cardiomyocytes derived from human stem cells

A method of assessing drug cardiac safety using human stem cell-derived cardiomyocytes, the method comprising:

• step (A) of preparing cardiomyocytes by culturing human stem cells to differentiate into cardiomyocytes;
• step (B) of diluting fibronectin in phosphate buffered saline (PBS) to a concentration of 50 μg/ml, wherein the step (B) comprises:
• step (B-1) of preparing a fibronectin stock by dissolving fibronectin in the PBS at a concentration of 1 mg/ml, and
• step (B-2) of diluting the fibronectin prepared in the step (B-1) in the PBS to a concentration of 50 μg/ml;
• step (C) of adding the fibronectin solution obtained in the step (B-2) so as to cover all dots of electrodes disposed in a center of each of a plurality of wells arranged in a multielectrode array (MEA) plate, and adding the PBS to spaces between the wells of the MEA plate to fill the spaces;
• step (D) of placing the MEA plate, to which the fibronectin solution and the PBS have been added through the step (C), in a cell incubator maintained at 37° C. under 5% CO2 for 50-60 minutes to coat the wells;
• step (E) of removing the fibronectin solution from specific wells of the MEA plate coated in the step (D), and then seeding the cardiomyocytes prepared in the step (A) in the specific wells so as to cover all dots of the electrodes, wherein the step (E) comprises:
• step (E-1) of adding the human stem cell-derived cardiomyocytes prepared in the step (A) to a plating medium, centrifuging the mixture, removing the natant and adding fresh plating medium to prepare a cell suspension;
• and
• step (E-2) of removing the fibronectin solution from each of the specific wells of the MEA plate and seeding, in each specific well, a portion of the cell suspension prepared in the step (E-1) containing 5×104 human stem cell-derived cardiomyocytes, so as to cover all dots of the electrodes;
• step (F) of adding a predetermined medium to the specific wells of the MEA plate that have been seeded with the cardiomyocytes through the step (E), and then incubating the MEA plate in a cell incubator, wherein the step (F) comprises:
• step (F-1) of incubating the MEA plate, seeded with the cardiomyocytes in the step (F-2), at 20° C. to 25° C. for 60 minutes as a first incubation;
• step (F-2) of after the first incubation of the step (F-1) is complete, adding a plating medium to each of the specific wells and then placing the MEA plate in a cell culture incubator maintained at 37° C. under 5% CO2 for 23-24 hours as a second incubation;
• and
• step (F-3) of after the second incubation of the step (F-2) is complete, replacing the plating medium in each of the specific wells with a maintenance medium, and then incubating the MEA plate at 37° C. under 5% CO2 for 6-7 days as a third incubation;
• step (G) of measuring changes in information values for beat rate, spike amplitude, and field potential duration in the specific wells of the MEA plate, which have been seeded with the cardiomyocytes and incubated for a predetermined period in the step (F), depending on whether a drug to be assessed has been added and on the concentration of the added drug, by using a multielectrode array (MEA) measurement device electrically connected to the electrodes disposed in each well of the MEA plate, and assessing the cardiac safety of the drug based on the measured information values;
• and
• step (H) of providing an application of the measurement data by transmitting the measured information values and the results of the cardiac safety assessment from the step (G) to a drug evaluation system for further analysis of the drug's safety.