A method for evaluating the safety of a drug on a heart using human stem cell-derived cardiomyocytes

Abstract

The method for assessing the cardiac safety of drugs using human stem cell-derived cardiomyocytes according to the present invention includes: Step A: culturing human stem cells to differentiate them into cardiomyocytes for preparation; Step B: diluting fibronectin to a concentration of 50 μg / ml using Dulbecco's phosphate buffered saline (DPBS); Step C: adding the 50 μg / ml fibronectin dilution obtained in Step B to a MEA (Multielectrode) solution. The MEA plate is arranged in multiple columns of wells, and all the dots of the electrodes located in the center of each well are covered. Then, DPBS is filled into the spaces between the wells of the MEA plate. Step D: The MEA plate, which has been prepared in step C with fibroin dilution and DPBS added to each well and the spaces between each well, is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment of 50 to 60 minutes. Step E: The human stem cell-derived myocardial cells prepared in step A are then spread in the MEA plate. Cells are seeded into specific wells of the MEA plate coated in step D above, covering all the dots of the electrodes in the specific wells of the MEA plate with the fibroin-removing diluent; Step F: A certain amount of culture medium is added to the specific wells of the MEA plate seeded with human stem cell-derived cardiomyocytes in step E above, and then the MEA plate is placed in a cell culture machine for culture; and Step G: Using a MEA (Multielectrode arrays) measuring instrument electrically connected to the electrodes in each well of the MEA plate, the addition of the drug to be evaluated in each well of the MEA plate seeded with human stem cell-derived cardiomyocytes cultured for a certain period of time in step F is measured, and the changes in the Beat Rate, Spike Amplitude, and Field Potential Duration values ​​based on the added concentration are assessed, and the cardiac safety of the drug to be evaluated is assessed based on the measured values.

Description

Technical Field

[0001] This invention relates to a method for assessing the cardiac safety of drugs using human stem cell-derived cardiomyocytes. Background Technology

[0002] The QT interval on an electrocardiogram (ECG) measures the duration of depolarization and repolarization. If the QT interval is prolonged, serious rapid supraventricular arrhythmias may occur, such as torsades de pointes (TdP).

[0003] In response, during the new drug development stage, to prevent drugs that may cause arrhythmias upon ingestion from being approved, the hERG Assay test is implemented in non-clinical studies to assess the ability to block hERG (human ether-a-go-go related gene) ion channels.

[0004] For ongoing research and experimentation, this hERG assay is used to assess drug-induced cardiotoxicity, with human stem cell-derived cardiomyocytes used as clinical derivatives.

[0005] In this regard, the existing technology for evaluating drug-induced cardiotoxicity by differentiating human pluripotent stem cells into cardiomyocytes and then performing the evaluation in an electrophysiological manner is Korean Patent Publication No. 10-1916393, entitled "In vitro cardiotoxicity evaluation technology using cardiomyocytes derived from human pluripotent stem cells" (hereinafter referred to as "the prior art").

[0006] However, previous methods for assessing cardiac safety using cardiomyocytes, including existing technologies, have high sensitivity but low specificity because they are based on the hERG assay. Consequently, focusing on one of the multiple pathways interfering with cardiac action potentials results in a relatively low degree of similarity in inducing TdP.

[0007] More specifically, previous methods for assessing the cardiac safety of drugs using cardiomyocytes, including existing technologies, based on hERG assays have reported discrepancies of up to 30% with non-clinical trial results that actually assess the risk of arrhythmias. Even for non-cardiotoxic drugs, extensive electrocardiogram studies have been conducted to obtain approval, thus increasing clinical development costs. In other words, drugs that may affect hERG and QT prolongation but actually pose little risk of TdP may be deprived of or excluded from further development.

[0008] Furthermore, even if these drugs are approved, their value may be reduced, for example, by restricting clinical use by inserting precautions on product labels. Therefore, there is a need to develop a new model for assessing the likelihood of TdP. To overcome this problem, the United States, Japan, and other countries are currently leading a movement to revise international guidelines for assessing new cardiac pharmacological toxicities. Summary of the Invention

[0009] The problem to be solved

[0010] The present invention is proposed to solve the above-mentioned problems. The purpose of the present invention is to provide a method for improving the shortcomings of the current hERG assay for assessing cardiac pharmacological toxicity throughout the clinical stage, and to provide a more accurate and comprehensive method for assessing cardiac pharmacological toxicity in vitro.

[0011] Problem Solving Methods

[0012] To achieve the above objectives, the present invention provides a method for assessing the cardiac safety of drugs using human stem cell-derived cardiomyocytes, comprising: Step A: culturing human stem cells to differentiate them into cardiomyocytes for later use; Step B: diluting fibronectin to a concentration of 50 μg / ml using Dulbecco's phosphate buffered saline (DPBS); Step C: adding the 50 μg / ml fibronectin dilution obtained in Step B to a MEA (Multielectrode) solution. The MEA plate is arranged in multiple columns of wells, and all the dots of the electrodes located in the center of each well are covered. Then, DPBS is filled into the spaces between the wells of the MEA plate. Step D: The MEA plate, which has been prepared in step C with fibroin dilution and DPBS added to each well and the spaces between each well, is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment of 50 to 60 minutes. Step E: The human stem cell-derived heart cells prepared in step A are then used for further processing. Myocardial cells are seeded into specific wells of the MEA plate coated with a fibroin-removing diluent as described in step D above, covering all the dots of the electrodes in the specific wells of the MEA plate; Step F: A certain amount of culture medium is added to the specific wells of the MEA plate seeded with human stem cell-derived cardiomyocytes as described in step E above, and then the MEA plate is placed in a cell culture machine for culture; and Step G: Using a MEA (Multielectrode arrays) measuring instrument electrically connected to the electrodes in each well of the MEA plate, the addition of the drug to be evaluated in each well of the MEA plate seeded with human stem cell-derived cardiomyocytes cultured for a certain period of time as described in step F is measured, along with changes in the Beat Rate, Spike Amplitude, and Field Potential Duration values ​​based on the added concentration, and the cardiac safety of the drug to be evaluated is assessed based on the measured values.

[0013] Among them, the human stem cells that differentiate into cardiomyocytes through step A are either human pluripotent stem cells (hPSC) or human embryonic stem cells (hESC).

[0014] Step B above includes: Step B-1: Dissolving fibroin with DPBS to prepare a fibroin stock with a concentration of 1 mg / ml; Step B-2: Dissolving the fibroin stock with a concentration of 1 mg / ml prepared by Step B-1 with DPBS and diluting it to a concentration of 50 μg / ml.

[0015] Step E above includes: Step E-1: placing the human stem cell-derived cardiomyocytes obtained in step A on a plate culture medium, centrifuging, removing the supernatant, and adding fresh plate culture medium to prepare a cell suspension; and Step E-2: adding 5 × 10⁻⁶ cells to the cell suspension prepared in step E-1. 4 A portion of the number of cardiomyocytes derived from human stem cells is seeded into specific wells of the MEA plate after removing the fibroin dilution, and seeded in a manner that covers all the dots of the electrode.

[0016] Step F above includes: Step F-1: The MEA plate containing a portion of the cell suspension of human stem cell-derived cardiomyocytes, which was seeded in Step E-2 above, is cultured for the first time for 60 minutes; Step F-2: After adding plate culture medium to specific wells of the MEA plate that has undergone the first culture in Step F-1 above, the MEA plate is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment for the second culture for 23 to 24 hours; and Step F-3: After replacing the plate culture medium added to the specific wells of the MEA plate that has undergone the second culture in Step F-2 above with maintenance medium, the MEA plate with the replaced culture medium is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment for the third culture for 7 days.

[0017] The F-3 step described above involves replacing the maintenance medium every 2 days during the third 7-day culture process in each specific well of the MEA plate with a new maintenance medium.

[0018] Step G above includes: Step G-1: After replacing the maintenance medium added to each well of the MEA plate that has undergone three culture cycles in Step F-3 with new maintenance medium, the MEA plate with the replacement medium is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment to stabilize the cells for 3 to 5 hours; Step G-2: Using the MEA measuring instrument, the Beat Rate, Spike Amplitude, and Field Potential of at least one human stem cell-derived cardiomyocytes seeded in multiple specific wells of the MEA plate that have undergone cell stabilization in Step G-1 are measured. The relevant values ​​of duration are calculated, and first measurement information is generated; Step G-3: From at least one well of the multiple specific wells of the MEA plate seeded through the cell stabilization implemented in Step G-1, a portion of the maintenance medium equivalent to 1 / 10 of the total weight of the maintenance medium is removed. Then, the drug to be evaluated, with an equivalent weight of the removed maintenance medium, is added at a certain concentration. After 30 to 40 minutes, the Beat Rate, Spike Amplitude, and Field Potential Duration of human stem cell-derived cardiomyocytes seeded in the wells containing the drug to be evaluated are measured using the MEA measuring instrument, and second measurement information is generated; and Step G-4: Based on the first and second measurement information generated through Steps G-2 and G-3, the FPDcF (Field Potential Duration corrected by Fridericia's formula) and Spike Amplitude are calculated according to the concentration of the drug to be evaluated. The variation pattern of amplitude is used to assess the cardiac safety of the drug under evaluation. The G-3 step involves taking different concentrations of the drug under evaluation and repeating the process at least twice to generate two or more second measurement results.

[0019] The effects of the invention

[0020] The effects of this invention are as follows:

[0021] First, unlike existing hERG assay-based assessment methods, this paper proposes a novel method for comprehensive in vitro cardiac pharmacological toxicity assessment based on multielectrode arrays (MEA).

[0022] Secondly, by measuring more comprehensive and diverse parameter information than existing technologies through the measurement of human stem cell-derived cardiomyocytes seeded in each well of the MEA (Multielectrode arrays) plate, such as Beat Rate, Spike Amplitude, and Field Potential Duration, it is possible to achieve cardiac safety assessment of drugs using this technology.

[0023] Third, through efficient safety assessment methods, cardiac safety assessments of drugs can be conducted more easily and accurately. Moreover, the use of human stem cell-derived cardiomyocytes in these experiments reduces the use of experimental animals. This allows for the implementation of long-term drug risk assessment trials and the identification of potential differences in the electrophysiological effects of cells that may affect the assessment of ventricular arrhythmia risk, enabling further evaluation. Attached Figure Description

[0024] Figure 1 This is a sequence diagram of the drug cardiac safety assessment method using human stem cell-derived cardiomyocytes according to the present invention;

[0025] Figure 2 A photograph of the morphology of the MEA plate used in the MEA plate preparation step in the drug cardiac safety assessment method using human stem cell-derived cardiomyocytes according to the present invention.

[0026] Figure 3 and Figure 4 This is a conceptual illustration of an implementation method for adding substances or seeding cells into the MEA plate used in the MEA plate preparation step in the drug cardiac safety assessment method using human stem cell-derived cardiomyocytes according to the present invention.

[0027] Figure 5 This invention relates to a method for evaluating the cardiac safety of drugs using human stem cell-derived cardiomyocytes. The method involves seeding human stem cell-derived cardiomyocytes onto electrodes within the wells of a MEA plate used in the MEA plate preparation step, and observing the changes in their state over time.

[0028] Figure 6 In the method for evaluating the cardiac safety of drugs using human stem cell-derived cardiomyocytes according to the present invention, a morphological curve is plotted based on the measurement information of human stem cell-derived cardiomyocytes seeded in each well through an electrode provided in the well of an MEA plate, in accordance with the cardiac safety evaluation steps.

[0029] Figure 7In the method for assessing the cardiac safety of drugs using human stem cell-derived cardiomyocytes according to the present invention, the method includes, according to the cardiac safety assessment steps, evaluating the change patterns of FPDcF (Field potential duration corrected by Fridericia's formula) and Spike Amplitude based on the concentration changes of each drug. Detailed Implementation

[0030] Preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings. For the sake of brevity, well-known technical aspects will be omitted or simplified.

[0031] <Description of a Method for Drug Cardiac Safety Assessment Using Human Stem Cell-Derived Cardiomyocytes>

[0032] The following is for reference Figure 1 The preparation process of cardiomyocytes derived from human pluripotent stem cells according to the present invention is described in detail.

[0033] (1) Cardiomyocyte Preparation Step <S110, Step A>

[0034] The process performed in this step (S110) is as follows: human stem cells are cultured to differentiate them into cardiomyocytes for later use.

[0035] Here, human stem cells refer to one of the following: human pluripotent stem cells (hPSC) or human embryonic stem cells (hESC), which differentiate into cardiomyocytes through a predetermined culture process and under specific conditions.

[0036] Specifically, it is preferred to use human stem cell-derived cardiomyocytes derived from the prior registered patent No. 10-1994035 of this invention, "Method for preparing functional cardiomyocytes derived from human pluripotent stem cells through cell differentiation and maturation and functional cardiomyocytes derived from human pluripotent stem cells prepared by the method".

[0037] Furthermore, the differentiated human stem cell-derived cardiomyocytes are first frozen and stored in a liquid nitrogen tank for later use until the subsequent inoculation process (S150). Before use, they are taken out and thawed.

[0038] (2) Fibronectin Diluent Preparation Step <S120, Step B>

[0039] The procedure performed in this step (S120) is as follows: Fibronectin is diluted to a concentration of 50 μg / ml using DPBS (Dulbecco's phosphate buffered saline).

[0040] Specifically, it is divided into two steps and carried out sequentially. First, fibroin is dissolved using DPBS to prepare a fibroin stock with a concentration of 1 mg / ml, and then it is refrigerated (step B-1).

[0041] Then, remove the fibroin stock (stock) with a concentration of 1 mg / ml prepared by the above step (step B-1) from the refrigerated state, dissolve and dilute it with DPBS to a concentration of 50 μg / ml, and set it aside for later use.

[0042] (3) MEA Plate Preparation Step <S130, Step C>

[0043] The process implemented in this step (S130) is as follows: the fibroin diluent with a concentration of 50 μg / ml prepared by the above-mentioned fibroin diluent preparation step (S120) is added to each well arranged in multiple columns in the MEA (Multielectrode arrays) plate, so that the fibroin diluent covers all the dots of the electrodes in the center of each well arranged in multiple columns in the MEA (Multielectrode arrays) plate, and DPBS is filled in the space between each well of the MEA plate.

[0044] Here, the MEA (Multielectrode Arrays) plate is as follows: Figure 2 As shown, it has multiple wells composed of rows and columns, and each well has a multi-electrode formed by multiple dots in its central part, such as... Figure 3 As shown.

[0045] Furthermore, the MEA (Multielectrodearrays) measuring instrument, which is electrically connected to multiple dots of the multielectrode, is used to measure the extracellular field potential generated by microcurrents in seeded cardiomyocytes that are in contact with the electrodes whenever an action potential is induced.

[0046] The aforementioned fibronectin dilution solution was added to the MEA (Multielectrode Arrays) plate at 5 μl increments, covering all the dots of the electrodes located in the center of each well arranged in multiple columns within the MEA plate. This process was as follows: Figure 3 As shown; and to prevent the MEA plate from drying out, the space between the wells of the MEA plate is filled with DPBS, as follows. Figure 4 As shown.

[0047] (4) First Culture Treatment Step <S140, Step D>

[0048] The process implemented in this step (S140) is as follows: the MEA plate containing fibroin dilution and DPBS added to the spaces between each well in the MEA plate preparation step (S130) is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment of 5% and cultured for 50 to 60 minutes.

[0049] More specifically, MEA plates containing fibroin dilution to cover all multi-electrodes and DPBS to fill the spaces between wells were placed in a cell culture machine with a temperature of 37°C and a CO2 environment of 5% for 60 minutes to achieve good coating.

[0050] (5) Seeding Treatment Step <S150, Step E>

[0051] The process implemented in this step (S150) is as follows: after removing the fibroin diluent from the specific wells of the MEA plate coated in step D, the human stem cell-derived cardiomyocytes prepared in the cardiomyocyte preparation step (S110) are seeded into the specific wells and cover all the dots of the electrode.

[0052] More specifically, this step (S150) is divided into two steps to be performed in sequence. First, the process of preparing the cell suspension (step E-1) is carried out, which is as follows: human stem cell-derived cardiomyocytes prepared by the cardiomyocyte preparation step (S110) are placed in a plate culture medium for centrifugation, and after the supernatant is removed, plate culture medium is added again to prepare the cell suspension.

[0053] Among them, the plate medium can be Fujifilm Cellular Dynamics, Inc.'s 'iCells', R1057, etc., but is not limited to this, and can be selectively used alternately from publicly available plate mediums that can be used for the differentiation and culture of cardiomyocytes.

[0054] Most preferably, the plate medium actually uses Nexel Cardiosight-S Media containing RPMI 1640 and B27 supplement without vitamin A.

[0055] Furthermore, the human stem cell-derived cardiomyocytes prepared in the cardiomyocyte preparation step (S110) are taken out of the liquid nitrogen tank, thawed in a water bath at 37°C to dissolve them, and then the cell solution is added dropwise to a plate medium to dilute it. The medium is then placed in a centrifuge and centrifuged at 180g for 3 minutes.

[0056] Specifically, after adding the thawed cell solution to 8 ml of plating medium and mixing well, 1 ml of plating medium was used to wash the inside of the cell vial to recover any remaining cells, thereby diluting a total of 10 ml of plating medium and human stem cell-derived cardiomyocytes, and then centrifuging them.

[0057] Subsequently, the supernatant separated by centrifugation was removed, and 1 ml of fresh plate culture medium was added to complete the preparation of the cell suspension.

[0058] Next, a seeding process (step E-2) is performed to cover all the dots for the electrodes. This involves taking a sample of the cell suspension prepared in the above process (step E-1) containing 5 × 10⁻⁶ cells. 4 A cell suspension of individual human stem cell-derived cardiomyocytes was seeded into each specific well of an MEA plate after removing the fibroin dilution to cover all the dots of the electrode.

[0059] Here, after completely removing the fibronectin diluent added to each specific well of the MEA plate, the optimal seeding density of human stem cell-derived cardiomyocytes per well is 5 × 10⁻⁶. 4 There are 1, and they cover all the small dots (Dots) of the electrode.

[0060] Most preferably, 10 μl of the cell suspension containing 1 ml of plate culture medium is taken and mixed with 10 μl of trypan blue. The total number of cells is counted using a cell counter, and enough human stem cell-derived cardiomyocytes to be seeded into each well is taken from the cell suspension to reach a number sufficient to seed 5 × 10⁶ cells per well. 4 The number of cells was measured and centrifuged, then the supernatant was removed and 5 μl was dispensed into each well.

[0061] (6) Second Culture Treatment Step <S160, Step F>

[0062] The process performed in this step (S160) is as follows: a certain amount of plating medium is added to specific wells of the MEA plate inoculated with human stem cell-derived cardiomyocytes in the above-mentioned inoculation step (S150), and then the MEA plate is placed in a cell culture machine for culture.

[0063] Specifically, the second culture treatment step (S160) is subdivided into three steps, which are carried out sequentially under different culture conditions. First, the first culture process (F-1 step) is carried out, which is: the seeding process (E-2 step) of human stem cell-derived cardiomyocytes covered by cell suspension to cover all the dots of the electrode. A portion of the MEA plate containing human stem cell-derived cardiomyocytes seeded in cell suspension is placed at room temperature (20°C to 25°C) and cultured for 60 minutes.

[0064] Through this process (F-1 step), the human stem cell-derived cardiomyocytes seeded with the cell suspension used to cover all the dots of the electrode will more easily attach to the center of the pore.

[0065] Secondly, a second culture process (F-2 step) is carried out, which involves adding plate culture medium to each specific well of the MEA plate after the first culture (F-1 step), and then placing the MEA plate in a cell culture machine with a temperature environment of 37°C and a CO2 environment of 5% for 23 to 24 hours (preferably 1 day).

[0066] Finally, the third culture process (F-3 step) is carried out, which is as follows: the plate culture medium added to each specific well of the MEA plate that has completed the second culture (F-2 step) is replaced with maintenance medium, and then the MEA plate with the replaced medium is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment, and cultured for 6 to 7 days (preferably 6 days).

[0067] Among them, the plate medium can be Fujifilm Cellular Dynamics, Inc.'s 'iCells', R1057, etc., but is not limited to this, and can be selectively used alternately from publicly available plate mediums that can be used for the differentiation and culture of cardiomyocytes.

[0068] Finally, preferably, the entire specific culture process, from the first to the third, is completed in the second culture treatment step (S160) over a total of 7 days. In particular, during the third culture (F-3 step), the maintenance medium, which is alternately added to each specific well of the MEA plate, should be replaced with a new maintenance medium every two days during the three cultures over the seven days.

[0069] Thus, the entire specific culture process, from the first to the third stage, is carried out within the second culture treatment step (S160) over 7 days. Figure 5 As shown, as the process progresses, it can be confirmed that the myocardial cells placed on the electrodes clump together and their beating becomes increasingly intense.

[0070] (7) Cardiac Safety Assessment Step <S170, Step G>

[0071] The process implemented in this step (S170) is as follows: using a MEA (Multielectrode arrays) measuring instrument electrically connected to electrodes in each well of the MEA plate, the addition of the drug to be evaluated in each well of the MEA plate seeded with human stem cell-derived cardiomyocytes cultured for a certain period of time in the second culture treatment step (S160) is measured, and the changes in the relevant values ​​of Beat Rate, Spike Amplitude, and FieldPotential Duration based on the added concentration are measured, and the cardiac safety of the drug to be evaluated is assessed based on the measured values.

[0072] More specifically, the cardiac safety assessment procedure is divided into several steps and implemented sequentially. First, a cell stabilization process (G-1 step) is implemented, which involves replacing the maintenance medium added to each specific well of the MEA plate that has completed three cultures (F-3 step) with new maintenance medium. Then, the MEA plate with the replaced medium is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment of 5% and cultured for 3 to 5 hours (preferably 3 hours).

[0073] Next, the first measurement information generation process (G-2 step) is implemented, which involves using an MEA measuring instrument to measure the relevant values ​​of Beat Rate, Spike Amplitude, and FieldPotential Duration of at least one human stem cell-derived cardiomyocytes seeded in each specific well of the MEA plate that has undergone cell stabilization through the cell stabilization process (G-1 step) to generate the first measurement information (G-2 step).

[0074] The first measurement information generated using the MEA measuring instrument corresponds to a part of a series of parameters used as the basis for drug cardiac safety assessment, showing the baseline measurement results for cells not treated with the drug to be evaluated.

[0075] To reduce errors caused by pipetting, a 10-minute stabilization process is required after pipetting the multi-well before recording the baseline measurement. The measurement process should be recorded over a period of 5 minutes.

[0076] Secondly, after completing the first assay information generation process (G-2 step), in at least one of the specific wells of the MEA plate that has completed cell stabilization through the cell stabilization process (G-1 step), a portion of the maintenance medium equivalent to 1 / 10 of the total volume of the maintenance medium is removed.

[0077] Next, the second measurement information generation process (G-3 step) is implemented, which involves adding a certain concentration of the drug to be evaluated at the same weight as the removed maintenance culture medium. After 30 to 40 minutes (preferably 30 minutes), the Beat Rate, Spike Amplitude, and Field Potential Duration values ​​of human stem cell-derived cardiomyocytes seeded in the wells containing the drug to be evaluated are measured using an MEA measuring instrument to generate the second measurement information (G-3 step).

[0078] The second measurement information, generated using the MEA measuring instrument, corresponds to a part of a series of parameter information used as the basis for drug cardiac safety assessment. It shows the measurement results of the line where cells are treated with a certain concentration of the drug to be evaluated.

[0079] Therefore, the process of generating the second measurement information (step G-3) is preferably to take different concentrations of the drug to be evaluated and repeat the process at least twice to generate more than two sets of second measurement information.

[0080] Furthermore, the process of generating the second measurement information (step G-3) is preferably to start the measurement 30 minutes after adding different concentrations of the drug to be evaluated, and to take 5 minutes to generate the second measurement information.

[0081] For example, after initially treating the drug to be evaluated with a concentration of 10X to complete the second assay information generation process (G-3 step), the second assay information generation process (G-3 step) is generated by gradually increasing the concentration, and the process is repeated to obtain multiple second assay information.

[0082] Finally, the process of determining the cardiac safety of the drug under evaluation using an MEA measuring instrument (G-4 step) is implemented, which is as follows: based on the first and second measurement information generated through the first measurement information generation process (G-2 step) and at least one other second measurement information generation process (G-3 step), the process of determining the cardiac safety of the drug under evaluation using an MEA measuring instrument is performed using the change patterns of FPDcF (Fieldpotential duration corrected by Fridericia's formula) and Spike Amplitude according to the concentration change of the drug under evaluation (G-4 step).

[0083] Here, after retrieving the raw file stored in the MEA measuring instrument's program, the data to be extracted is re-recorded and saved as a CSV file. The CSV file is opened, and based on the measured first and second measurement information (beat interval, spike amplitude, field potential duration (FPD), the rate of change of parameters measuring the responsiveness to different drug concentrations is calculated to assess the impact of the test substance concentration on the field potential of cardiomyocytes.

[0084] Specifically, such as Figure 6 As shown, when using a MEA board consisting of 16 electrodes, the beats and field potentials recorded by the 16 electrodes located in each hole can be confirmed simultaneously and as a whole. Figure 6 As shown in the diagram on the left, which is a patterned representation of the field potential of a single beat, the beat interval, spike amplitude, and field potential duration (FPD) can be analyzed and utilized as parameter measurements through the curve pattern.

[0085] Here, since Field Potential Duration (FPD) is an indicator of QT interval and it varies with the beat of QT interval, it is particularly important to correct for changes in the beat ratio by calculating the Field Potential Duration corrected by Fridericia's formula (FPDcF) in order to reasonably explain the direct impact on FPD or QT. Furthermore, the cardiotoxicity of a drug can be determined by analyzing the changes in FPDcF and spike amplitude.

[0086] Therefore, the Field Potential Duration corrected by Fridericia's formula (FPDcF) is defined as 'FPDcF = Field Potential Duration / Beat Rate (Interval)'. 1 / 3 It is derived from Fridericia's formula.

[0087] As an example, representative ion channel inhibitors (Calcium channel blocker (Nifedipine), Potassium channel blocker (E4031), and Sodium channel blocker (Mexiletine)) that affect changes in heartbeat were selected as drugs to be evaluated. The inventors implemented a drug cardiac safety assessment method using human stem cell-derived cardiomyocytes. The cardiac safety assessment results of the drugs to be evaluated, derived using an MEA analyzer, are as follows: Figure 7 As shown.

[0088] Available from Figure 7 It was found that the changes in FPDcF and spike amplitude values ​​in different wells of MEA seeded with human stem cell-derived cardiomyocytes when treated with different concentrations of ion channel inhibitors were investigated. When DMSO, used as a negative control, was applied to human stem cell-derived cardiomyocytes, the FPDcF value did not change. In contrast, the groups treated with Nifedipine and E4031 showed drug responsiveness with increases or decreases in FPDcF value according to the concentration treated, and the group treated with mexiletine showed a decrease in spike amplitude according to the concentration treated.

[0089] Most preferably, the procedure involves assessing the cardiac safety of the drug under evaluation using the changes in FPDcF (Field potential duration corrected by Fridericia's formula) and Spike Amplitude as the concentration of the drug changes, and using an MEA analyzer (G-4). The paper 'Gintant, G et al., Regul Toxicol Pharmacol. 2020 Aug 19; 117:104756.' states that "as a result of the CiPA study, a significance (n=3) can be defined when the drug's FDPcF changes by more than 20%, and the degree of cardiac pharmacological safety can be distinguished." Based on this published content, an assessment benchmark for the cardiac safety of the drug to be evaluated using an MEA analyzer has been established, and the result data has been generated and output based on this design.

[0090] Accordingly, the cardiac safety assessment method for drugs using human stem cell-derived cardiomyocytes of the present invention improves upon the shortcomings of the hERG assay currently used for cardiac pharmacological toxicity assessment throughout the entire clinical phase of new drug development, and provides a more accurate and comprehensive method for in vitro cardiac pharmacological toxicity assessment.

[0091] The embodiments shown in this invention are for illustrative purposes only and are not intended to limit the scope of the invention's technical concept. The scope of protection should be interpreted according to the scope of the appended claims, and all technical concepts within the equivalent scope should fall within the scope of this invention.

Claims

1. A method for in vitro cardiac safety assessment of drugs using cardiomyocytes differentiated from human stem cells, characterized in that, include: Step A: Culture human stem cells to differentiate them into cardiomyocytes in preparation; Step B: Dilute Fibronectin to a concentration of 50 μg / ml using DPBS (Dulbecco's phosphate buffered saline); Step C: Add the 50 μg / ml fibroin dilution solution obtained in Step B to each well in the MEA (Multielectrode arrays) plate, covering all the dots of the electrodes located in the center of each well. Then fill the space between the wells of the MEA plate with DPBS. Step D: Place the MEA plate containing fibroin dilution and DPBS in the spaces between the wells from Step C into a cell culture machine with a temperature environment of 37°C and a CO2 environment of 5% and spread it for 50 to 60 minutes. Step E: Seed the human stem cell differentiated cardiomyocytes prepared in Step A into the wells of the MEA plate coated in Step D with the fibroin-removed diluent, and cover all the dots of the electrodes in the wells of the MEA plate. Step E includes: Step E-1: The cardiomyocytes differentiated from human stem cells obtained in Step A are placed in a plate culture medium, centrifuged, and after removing the supernatant, fresh plate culture medium is added to prepare a cell suspension; and Step E-2: Take a portion of the cell suspension prepared in step E-1 containing 5×10 4 A cell suspension of cardiomyocytes differentiated from human stem cells was seeded into each well of the MEA plate with a defibrin-removed dilution, and seeded in a manner that covered all the electrodes (dots). Step F: After adding a certain amount of culture medium to the wells of the MEA plate inoculated with cardiomyocytes differentiated from human stem cells in step E, the MEA plate is placed in a cell culture machine for culture. Step F includes: Step F-1: The MEA plate, which has been seeded with a portion of the cell suspension containing cardiomyocytes differentiated from human stem cells through step E-2, is placed in a temperature environment of 20°C to 25°C for the first culture, which lasts for 60 minutes. Step F-2: After adding plate culture medium to each well of the MEA plate that has completed the first culture in Step F-1, the MEA plate is placed in a cell culture machine with a temperature environment of 37°C and a CO2 environment for a second culture, which lasts for 23 to 24 hours; Step F-3: After replacing the plate culture medium added to each well of the MEA plate that has undergone the second culture in Step F-2 with maintenance medium, place the MEA plate with the replaced medium in a cell culture machine with a temperature environment of 37°C and a CO2 environment for a third culture, which lasts for 6 to 7 days; Step G: Using a MEA (Multielectrode Arrays) measuring instrument electrically connected to electrodes in each well of the MEA plate, the addition or absence of the drug to be evaluated in each well of the MEA plate inoculated with cardiomyocytes differentiated from human stem cells cultured for a certain period of time in Step F is measured, along with changes in Beat Rate, Spike Amplitude, and Field Potential Duration based on the added concentration. The cardiac safety of the drug to be evaluated is assessed based on the measured values. Specifically, after adding the drug to be evaluated and waiting 30 to 40 minutes, the Beat Rate, Spike Amplitude, and Field Potential Duration of the cardiomyocytes differentiated from human stem cells inoculated with the drug to be evaluated are measured using the MEA measuring instrument.

2. The method for in vitro cardiac safety assessment of drugs using cardiomyocytes differentiated from human stem cells according to claim 1, characterized in that, Human stem cells that differentiate into cardiomyocytes through step A are called human pluripotent stem cells (hPSCs).

3. The method for in vitro cardiac safety assessment of drugs using cardiomyocytes differentiated from human stem cells according to claim 1, characterized in that, Step B includes: Step B-1: Dissolve fibroin in DPBS to prepare a 1 mg / ml fibroin stock; and Step B-2: Dissolve the fibroin stock (stock) prepared in step B-1 at a concentration of 1 mg / ml using DPBS and dilute it to a concentration of 50 μg / ml.

4. The method for in vitro cardiac safety assessment of drugs using cardiomyocytes differentiated from human stem cells according to claim 1, characterized in that, Step F-3 involves replacing the maintenance medium added to each well of the MEA plate for a third culture process of 6 to 7 days, with a cycle of replacing the maintenance medium every 2 days.

5. The method for in vitro cardiac safety assessment of drugs using cardiomyocytes differentiated from human stem cells according to claim 1, characterized in that, Step G includes: Step G-1: After replacing the maintenance medium added to each well of the MEA plate that has completed three cultures in step F-3 with new maintenance medium, place the MEA plate with the replaced medium in a cell culture machine with a temperature environment of 37°C and a CO2 environment to stabilize the cells for 3 to 5 hours. Step G-2: Using the MEA measuring instrument, measure the Beat Rate, Spike Amplitude, and Field Potential Duration of at least one human stem cell-differentiated cardiomyocytes seeded in multiple wells of the MEA plate through cell stabilization implemented in step G-1, and generate first measurement information. Step G-3: From at least one well of the MEA plate seeded with cells stabilized in step G-1, a portion of maintenance medium equivalent to 1 / 10 of the total weight of the maintenance medium is removed. Then, an equivalent weight of the drug to be evaluated is added at a specific concentration. After 30 to 40 minutes, the Beat Rate, Spike Amplitude, and Field Potential Duration of human stem cell-differentiated cardiomyocytes seeded in the wells containing the added drug are measured using the MEA instrument, and second measurement information is generated. Step G-4: Based on the first and second measurement information generated through steps G-2 and G-3, the cardiac safety of the drug under evaluation is assessed using the variation patterns of FPDcF (Field potential duration corrected by Fridricia's formula) and Spike Amplitude according to the concentration of the drug under evaluation. The G-3 step involves treating the drug to be evaluated with different concentrations at least twice to generate two or more second assay results.

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