Editorial Board

A Sylgard 184 silicone elastomer base and a Sylgard 184 silicone elastomer curing agent manufactured by Dow Corning (Midland, MI) were used to create the PDMS substrate. In this study, we used a series of PDMS samples with different base/curing agent mass ratios to explore the relationship between the elastic modulus and a different amount of crosslinking of PDMS network, whose base/agent mass ratios were 10:1 and 20:1. Moreover, PS was maintained as a reference sample to compare the contractile properties of cardiomyocytes with a standard stiff substrate. The schematic is shown in Fig. 1 (a). PDMS substrate (2 mm thick) was created by thoroughly mixing the corresponding elastomer base and the curing agent mixtures before pouring the mixture into a flat bottom square polystyrene dish and degassing the PDMS under vacuum to remove the air bubbles. The PDMS was cured on a hot plate at 80°C for 2 h. Following the curing, the PDMS substrate samples were cut into square shapes of 20×20 mm using a medical knife.

Fig. 1. 
(a) Schematic of the cardiomyocytes cultured on PS, PDMS (10:1), and PDMS (20:1) substrates used for analysis, (b) Schematic of the setup used for recording the motion of the cardiomyocytes.

The relationship between stress and strain was measured using a tabletop universal testing instrument (EZ-L 500N Shimadzu, Japan). The Young’s modulus of elasticity was then calculated from the tensile testing data.

The ventricles were harvested from 1-3-day old neonatal Sprague-Dawley rats, which was approved by the Animal Ethics Committee of Chonnam National University. The ventricular tissue was digested in a mixture of collagenase (0.4 mg/mL) and pancreatin (0.6 mg/mL) to separate the cardiomyocytes from the tissues. Once the cardiomyocytes and the fibroblast layer were separated with centrifugal separation using Purcell, the cardiomyocytes with high purity were collected. The prepared cardiomyocytes were seeded at the PS and PDMS (10:1, 20:1) substrates with 1000 cells per mm² and cultured in an incubator at 37°C with 5% carbon dioxide. The culture medium was refreshed once every three days.

An inverted microscope (Eclipse TS100) was used to optically measure the contractility of the cells cultured on the different PDMS substrate well plate, as shown in Fig. 1 (b). The focal length of the microscope was set to 40x to record the video (Fig. 2 (a)). The recorded video was utilized to evaluate the contractile properties of the cultured cardiomyocytes. The video was then processed using the Tracker free video analysis and modeling tool developed using the Open-Source Physics (OSP) Java framework to show the target and select the desired cell on the culture substrate. Subsequently, the processed video was read frame by frame to track the selected cells on the substrate set by the user. The displacement data of the cell were derived from the tracking information, which contained the change in position of the cell for each frame (Fig. 2 (b)). The data was processed to convert the frame pixel into contractile distance. The step-by-step procedure for analysis using the Tracker software is presented in Fig. 2 (c).

Fig. 2. 
(a) Schematic of the video recording and cell selection, (b) Concept of freely available Tracker software for analysis, (c) Flow chart for using the Tracker software with a screenshot of its dashboard.

Drug toxicity experiments were performed using Verapamil (Sigma-Aldrich), an L-type Ca2+ channel blocker. The drug was diluted in ethanol, and the total ethanol concentration was maintained below 0.1 % v/v. Drug concentrations were selected based on the IC50 values approved by the Food and Drug Administration. Contractile changes were assessed at various verapamil concentrations: 10 nM, 100 nM, 300 nM, 500 nM and 1000 nM.

In this study, results are presented as mean ± standard deviation (S.D.). All statistical analyses were performed using the GraphPad Prism 7 software. Statistical significance of the data was determined using one-way ANOVA followed by Tukey’s honest significant difference test, with significant differences defined by *p < 0.05 and **p < 0.001.

First, the fabricated PDMS substrates were characterized to determine their mechanical properties. A 5 N force was applied up to PDMS (10:1) and a 3.5 N force was applied up to PDMS (20:1). The stress analysis with respect to displacement is shown in Fig. 3. PDMS (10:1) experienced stress up to 0.16 N/mm², while PDMS (20:1) experienced stress up to 0.07 N/mm². The corresponding Young’s moduli for PDMS (10:1) and (20:1) were 1330 kPa and 706 kPa, respectively. The details are described in Table S1 in the supplementary information. The data for PDMS (10:1) indicate that the polymer has at least two distinct tangent moduli, and lower stiffness was observed at lower strain levels than at higher strains, and the particular transition was not observed in PDMS (20:1), although it is likely that this was because the samples failed before they reached the strain level at which the transition occurred.

Fig. 3. 
Stress-strain profiles of (a) 10:1 PDMS, (b) 20:1 PDMS substrates.

The cardiac tissue adhered and stabilized once the NRVM were cultured on the PS, PDMS (10:1) and PDMS (20:1), and started displaying beating activity on day 3 after cell culture. From day 3 to day 8, the contractile profile was recorded under the microscope and subsequently analyzed using the Tracker software. Fig. 4 (a) shows the cell condition on the three substrates on day 6 after cell culture. Fig. 4 (b, c) shows the day-wise contractility and beating rate trends. The contractile displacement for PS, PDMS (10:1), and PDMS (20:1) on day 3 were 0.466 ± 0.042 μm, 0.86 ± 0.065 μm, and 0.932 ± 0.152 μm, respectively, and by day 8, it had increased to 2.4 ± 0.089 μm, 2.49 ± 0.085 μm, and 2.936 ± 0.270 μm, respectively. When compared among the three substrates, contractility in PDMS (20:1) was highest followed by PDMS (10:1) followed by PS. The difference between PS and PDMS (10:1), and PS and PDMS (20:1) on days 3 and 4 was statistically significant. The contractile difference between PS and PDMS (10:1) was not significant on days 7 and 8, while the difference between PS and PDMS (20:1) remained significant throughout the culture period.

Fig. 4. 
(a) Optical images of cardiomyocytes on the different substrates on day 6 after cell culture (scale bar = 100 mm). Day-wise changes in (b) contractility and (c) beat rate as measured by Tracker. Results are displayed as mean ± S.D with n = 5. Statistical significance of data was determined using one-way ANOVA followed by Tukey’s honest significant difference test, with significant differences defined by *p < 0.05 and **p < 0.001.

Meanwhile, the beating rate reduced from 2.5 ± 0.061 Hz, 2.2 ± 0.132 Hz, and 2.152 ± 0.167 Hz on day 3 to 1.6 ± 0.160 Hz, 1.5 ± 0.241 Hz, and 1.48 ± 0.272 Hz for PS, PDMS (10:1), and PDMS (20:1), respectively. Regarding the beating rate, the mean beating rate for PDMS (20:1) was lowest, which was followed by PDMS (10:1) followed by PS. On day 3, the difference between PS and PDMS (10:1), and PS and PDMS (20:1) was statistically significant, while on day 4, only the difference between PS and PDMS (20:1) was significant. The difference among the three substrates was not significant over the rest of the culture period. PDMS (20:1), having the lowest Young’s modulus, exhibited a larger contractility on a daily basis as the culture grew. Substrates with lower Young’s modulus are conducive for better cellular growth and adhesion, resulting in larger contractile displacements and lower beating rates [16-17].

Following cell culture, drug toxicity experiments were performed on day 9 on the three substrates using verapamil, which is an L-type Ca2+ channel blocker. The contractile patterns of the cardiomyocytes were recorded after adding sequentially increasing drug concentrations from 10 nM to 1000 nM from the three types of substrates with varying elastic moduli. The representative contractile motions as recorded by the Tracker software for control, 300 nM, and 1000 nM of verapamil for PS, PDMS (10:1), and PDMS (20:1) are depicted in Fig. 5. It is evident that both the contractility and beat rate are decreasing at higher concentrations of the drug.

Fig. 5. 
Real-time changes of contractility as measured by Tracker in response to drug toxicity using verapamil. (a), (b), (c) display PS contractility, (d), (e), (f) illustrate PDMS (10:1) contractility, and (g), (h), (i) illustrate PDMS (20:1) contractility. The three concentrations showed are control, 300 nM, and 1000 nM of verapamil.

Fig. 6 illustrates a detailed analysis of the contractility and beat rate changes after the drug was added. The contractility of all three substrates reduced after adding verapamil; however, there was no statistical significance between PS, PDMS (10:1) and PDMS (20:1) up to 100 nM. At 300 nM, PS, PDMS (10:1) and PDMS (20:1) reduced to 70.79%, 77.28%, and 79.64% of control, and the change was statistically significant between PS and PDMS (20:1). The reduction after adding higher doses of verapamil was also statistically significant, with 49.88%, 58.98%, and 67.21% at 1000 nM for PS, PDMS (10:1), and PDMS (20:1), respectively. Moreover, the reduction in contractility was observed to be the highest for PS and lower for PDMS 20:1. In all substrates, the beating rate reduced in a concentration-dependent manner; however, there was no significant difference in the reduction among different substrates, except at a high concentration of 1000 nM. At 1000 nM, there was a reduction of 53.12% for PS, 52.22% for PDMS (10:1), and 32.84% for PDMS (20:1), which was statistically significant.

Fig. 6. 
Concentration-wise response to (a) normalized contractility and (b) normalized beating rate on addition of verapamil. Results are displayed as mean ± S.D with n = 5. Statistical significance of data was determined using one-way ANOVA followed by Tukey’s honest significant difference test, with significant differences defined by *p < 0.05 and **p < 0.001.

Generally, Verapamil is used to prevent supraventricular tachycardia by controlling the ventricular rate, particularly as the contraction and beat rate reduce [18]. As seen in Fig. 6, the cells predictably displayed negative chronotropy and negative inotropy in response to verapamil, while the rate of reduction changed with each substrate. Cells on the most flexible substrate PDMS (20:1) exhibited the lowest change in contractility, while the stiff substrate PS had the highest change. Accordingly, it could be inferred that the cellular maturation on PDMS (20:1) was the highest and PS was the lowest. Consequently, verapamil proved to be more toxic to the cells on PS, primarily owing to lesser maturity. Meanwhile, a more flexible substrate like PDMS (20:1) aided in higher maturation of cardiomyocytes. This was seconded by the day-wise trends (Fig. 4), where cells on PDMS (20:1) possessed the highest contractile motion and lowest beating rate.

The above data of the day-wise cellular trends and response to cardiotoxicity was successfully accomplished using the Tracker software. Tracker proved to be an easy-to-use video-based analysis tool to assess the cardiac contractility. Until now, this software was primarily used for motion tracking of macro-sized objects. The present study is the first successful demonstration of Tracker for cellular activity. Tracker can be further investigated for other cellular motion and behaviors.