Performance

Four vials of Cellartis cardiomyocytes were thawed and yielded 1.5 x 10⁷ viable cells. With 10% loss during pipetting, this accounted for 14 Cellartis EHTs. Within nine days, the spontaneously beating cells formed a syncytium and macroscopic contractions of the EHT was measured with the EHT analysis instrument (Figure 1).

Figure 1. Contraction analysis of Cellartis-derived engineered heart tissue (EHT). Left: live image of an EHT during contraction analysis. The EHT is spanning between two flexible silicone posts where optical figure recognition (blue crosses) is based and illuminated by an LED. Scale bar = 1 mm. Right: exemplary contraction peaks of a spontaneously beating EHT showing contraction force over time.  

As known from other EHT studies (Hansen et al. 2010, Mannhardt et al. 2016), EHT contractions became stronger during culture. After three weeks in culture, the tissues were spontaneously beating with 43 ± 7 beats per minute, 0.15 ± 0.03 mN in contraction forces, a contraction time (T1) of 176 ± 5 ms, a relaxation time (T2) of 401 ± 42 ms, a contraction velocity of 1.3 ± 0.2 mN/sec, and a relaxation velocity of 0.7 ± 0.1 mN/sec (data are mean ± SD and n=13; Figure 2). The beating pattern was highly regular (low RR' index; Figure 2). 

Characterization

Whole mount immunofluorescence staining of Cellartis EHTs revealed elongation of the cells with good sarcomeric organization and distinct actinin and myosin light chain 2v (MLC2v) patterning (Figure 3).

Figure 3.  Immunofluorescence characterization of Cellartis cardiomyocytes cultivated into EHT. Green: anti-α-actinin; red: anti-MLC2v; and blue: DRAQ5. Scale bar = 5 µm.

Cellartis cardiomyocytes are well suited for the generation of EHT. Tissue development was fast and contractions strong and regular. Histological structure revealed very good sarcomere organization and predominant expression of MLC2v, a marker of ventricular cardiomyocytes. 

Cardiomyocyte preparation

Four vials of hiPSC-derived cardiomyocytes (sold one each as a part of the Cellartis Cardiomyocytes [from ChiPSC22] Kit) were thawed by dropwise addition of Cellartis CM Thawing Base. The thawing base contained 45 ml of Cellartis CM Thawing Base supplemented with 20% heat-inactivated fetal calf serum (Gibco). After centrifugation (60g, 15 min), cells were resuspended in DMEM (Biochrom) supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine (Gibco), and 1% penicillin/streptomycin (Gibco); next, the cells were stored on ice. Reconstitution mix for EHT generation was prepared as recently published with 1 x 10⁶ cells per EHT (Mannhardt et al. 2016). In brief, cells were mixed with fibrinogen (5 mg/ml; Sigma), 10% matrigel (BD Biosciences), and 2X DMEM for isotonisation.

Scaffold construction

Agarose casting molds were prepared in 24-well plates (Nunc) with PTFE spacers (EHT Technologies GmbH), and PDMS racks (EHT Technologies GmbH) were positioned with two posts in each casting mold. For each tissue, 100 µl of this reconstitution mix was mixed with 3 µl of thrombin and pipetted into the casting molds around the posts of the PDMS racks. After polymerization of the fibrin matrix (2 hr at 37°C), EHTs were transferred to a 24-well plate with EHT medium, consisting of DMEM, 10% heat-inactivated horse serum (Life technologies, Cat. # 26050088), 1% penicillin/streptomycin, 10 µg/ml insulin (Sigma) and 33 µg/ml aprotinin (Sigma). EHTs were cultivated at 37°C, 40% O₂, and 7% CO₂, and the medium was changed Mondays, Wednesdays, and Fridays.

Performance analysis

Contraction analysis of Cellartis EHTs was performed in the EHT analysis instrument (EHT Technologies GmbH). Over a period of three weeks, EHT contractions were analyzed regarding beating frequency, contraction force, contraction kinetics (contraction time [T1] and relaxation time [T2]), and regularity of beating (RR'). At Day 22, Cellartis EHTs were fixed in paraformaldehyde and subjected to immunofluorescence staining as described previously (Mannhardt et al. 2016).

Hansen, A. et al. Development of a drug screening platform based on engineered heart tissue. Circulation research 107, 35–44 (2010).

Mannhardt, I. et al. Human Engineered Heart Tissue: Analysis of Contractile Force. Stem Cell Reports 7, 29–42 (2016).

Moretti, A. et al. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N. Engl. J. Med 363, 1397–1409 (2010).

Weinberger, F. et al. Cardiac repair with engineered heart tissue derived from human induced pluripotent stem cells. Sci Transl Med. 8, 363ra148 (2016).