Fig 1.
(A) CMs differentiation steps from day 0 (D0), lactate purification at day 13 (D13) and cryopreservation of CMs at day 20 (D20). (B) EHT formation from frozen CMs and different ratios of HCF (0, 1, 3, 5 and 10%) at day 0 (D0); Follow up by data collection of the tissue surface area from day 1 (D1) until day 10 (D10) and contraction analysis carried at day 5 (D5) and day 10 (D10). hPSC = human embryonic stem cell; CMs = Cardiomyocytes; MG = Matrigel; MM = Maturation medium; SF = Serum free; HCF = Human adult cardiac fibroblast. Created with BioRender.com.
Fig 2.
(A) Overview of the software. (B) Automatic contraction analysis option. (C-E) Manual contraction analysis per folder overview(C), selection of the analysis mode on preview contraction wave(automatic or manual) (D) and manually selection of maximums and minimums (E).
Fig 3.
Flow chart representing the workflow of EHT analysis.
(A) Automatic analysis of the required input data inside of a main folder. (B) Tracking the center of the pillars and the surface area, using parallel computing. (C) Contraction waveform post-processing. (D) Output data.
Fig 4.
Contraction analysis by tracking black dots.
(A) Original bright field image of an EHT around pillars with a black tip. (B) Edge detection of the black tip of the pillars from a bright field image. (C) Detection of the center of the pillars black region. (D) Displacement of the pillars over time with resting tension (Blue) and after removing the initial resting tension (Red). (E) Maximum and minimum detection of the contraction motion; complete contraction cycles are with filled marks. (F) Graph of the contraction force with the maximum and minimum. (G) Graph of displacement over time and also indication the moments to achieve 10% and 90% of contraction and relaxation.
Fig 5.
EHT contraction analysis on transparent pillars.
(A) Original bright field image of an EHT around transparent pillars. (B) Region detection of the tip of the pillars from a bright field image. (C) Detection of the regions with low eccentricity. (D) Detection of the center of the pillars. (E) Contour of the EHT. (F) Displacement of the pillars over time with resting tension (Blue) and after removing the initial resting tension (Red). (G) Maximum and minimum detection of the contraction motion; complete contraction cycles are with filled marks. (H) Graph of the contraction force with the maximum and minimum.(I) Graph of displacement over time with the time to achieve 10% and 90% of contraction and relaxation. (J) Graph of force per surface area with maximum and minimum.
Fig 6.
EHT comparison with different ratio of Human adult cardiac fibroblast (HCF).
(A) Success rate of tissue formation using different ratio of HCF. (B) Relative surface area error by comparing the tissue area measured manually using Image J and using the stand alone application. (C) Relative tissue compaction by comparing the initial tissue area at day 0 (10.7 mm2) with the tissue area over time with the different ratio of HCF. (D) Relative tracking error of the pillar’s center by comparing the distance measured manually using Image J and using the stand alone application. (E) Contractile force of EHTs with different ratios of HCF at day 5 and 10. (F) Contractile force of EHTs with different ratios of HCF divided by the surface are of the tissue at day 5 and 10. In E-F, data shown as means, maxima and minima; Two-way ANOVA plus Tukey’s test for comparisons among ratios of HCF; * = p <0.05; ** = p <0.01; *** = p <0.001; **** = p <0.0001,(N = 3).
Fig 7.
Contraction kinetics of EHTs with different ratio of human adult cardiac fibroblast (HCF).
(A-B) Contraction (A) and relaxation (B) velocity of EHTs using different ratio of HCF at day 5 and 10. (C-D) Time to reach 10% (C) and 90% (D) of contraction using different ratio of HCF at day 5 (D5) and day 10 (D10). (E-F) Time to reach 10% (C) and 90% (D) of relaxation using different ratio of HCF at day 5 (D5) and day 10 (D10). Data shown as means, maxima and minima; Two-way ANOVA plus Tukey’s test for comparisons among ratios of HCF; * = p <0.05; ** = p <0.01; *** = p <0.001; **** = p <0.0001, (N = 3).