**2.1 Cell isolation**

Total heart cells (excluding the atria) were isolated from neonatal Wistar rats (postnatal day 0 to 3) by a fractionated DNase/Trypsin digestion protocol as previously described (Eschenhagen & Zimmermann, 2005). The resulting cell populations were immediately subjected to FBME generation. Experimental procedures were reviewed and approved by Ethics Committee, Hamburg University.

#### **2.2 Manufacturing teflon spacers and sylgard posts racks**

For the generation of fibrin-based mini-EHTs (FBMEs), Teflon spacer and silicone post racks were used. The Teflon spacers were important for the casting molds. They had the following geometry (Figure: 1B): length 12 mm, width 3 mm, height 13.5 mm. Sylgard 184 silicone elastomer (Dow Corning) was used for the production of silicone post racks, which were needed for culturing the FBMEs. The silicone post racks were made in custom-made Teflon casting molds. According to the manufacture's instructions, the 2-component Sylgard 184 was degassed under vacuum conditions before casting. The final silicone post racks consisted of 4 pairs of posts, having a little plate at their end. The racks had the following geometry (Figure 1A): length/width of rack: 79x18.5 mm, length of posts 12 mm, diameter 1 mm, plate diameter 2 mm, distance (center-center) 8.5 mm. They were initially self-made and currently industrialmade. Silicone post racks can be autoclaved and reused for several times.

### **2.3 Generation of fibrin-based mini-EHTs**

The reconstitution mixture for the generation of fibrin-based mini-EHTs was prepared on ice as follows (final concentration): 4.1x106 cells/ml, 5 mg/ml bovine fibrinogen (Sigma F4753, stock solution: 200 mg/ml in 0.9% NaCl supplemented with 0.5 µg/mg aprotinin), 3 U/ml bovine thrombin (Sigma T7513, stock solution: 100 U/ml). To ensure isotonic conditions, one additional fibrinogen and thrombin volume of 2x DMEM was added. Ordinary 24-well cell culture plates were used as casting molds. After 1.6 ml of sterile 2% agarose (Invitrogen 15510-027) in PBS was pipetted into each well, the Teflon spacers could be placed. After the agarose was solidified, the Teflon spacers were removed. The silicone posts racks were placed onto the cell culture dish with each pair of silicone posts reaching into one of the preformed casting molds (geometry: 12x3x4 mm). The reconstitution mix was carefully resuspended. For each FBME 100 µl of the mixture was mixed briefly with an appropriate volume of thrombin and pipetted into an agarose slot. To ensure complete polymerization of the fibrinogen, the constructs were placed into a humidified cell culture incubator (37 °C, 7% CO2, 40% O2) for 2 hours. Before transferring the silicone posts racks to

Multi-Well Engineered Heart Tissue for Drug Screening and Predictive Toxicology 75

equation (Vandenburgh et al. 2008). The recorded contractions are filtered and identified as such by certain peak criteria (e.g. threshold value, minimal force and minimum relaxation). Besides average force, the software calculates values for frequency, fractional shortening, contraction- and relaxation time (bpm, T1 and T2, respectively) based on the recognized contraction peaks. T1 and T2 were determined at 20% of peak maximum. Reports with an overview of the environmental information (temperature, gas, humidity) in the cell culture like unit plus all calculated parameters are automatically generated after each run. These underlie two levels of quality control: pictures are taken at the beginning and the end of each recording. Blue squares indicate top and bottom end of each respective FBME where the software placed the points for the measurement (Figure 2D). Contractions are recorded as force development over time. Identified peaks, which are included in the calculation, are marked with green squares (Figure 2C). The effort to analyse a 24-well plate with contracting muscle strips is limited to defining the XYZ-coordinates for each well once

**B** 

Fig. 2. Illustration of the experimental setup for video-optical recordings of FBME, a 24 well cell culture dish with FBMEs, a magnified view of a single FBME as recorded by the video camera and an example of a contraction pattern (one FBME for 60 s). A shows a schematic picture of the experimental setup for video-optical recordings. In B a 24 well cell culture dish with FBMEs is shown (view from above). The original contraction pattern of one FBME over time (60 s, C). Image of a contractile muscle strip from an automatically generated report (D). Blue squares indicate positions automatically recognized by the software at top and bottom end of the FBME. Green squares mark the recognized contraction peaks which

**D** 

are included for the calculation of several parameters (Hansen et al. 2010).

before starting a series of measurements.

**C** 

**A** 

a new medium-filled culture plate, every construct was covered with DMEM (300 µl) to ease the removal. FBMEs were maintained in 37 °C, 7% CO2, 40% O2 in a humidified cell culture incubator. Media was changed on Mondays, Wednesdays and Fridays. FBME medium consisted of DMEM (Biochrom F0415) supplemented with 10% horse serum (Gibco 26050), 2% chick embryo extract (self-made), 1% penicillin/streptomycin (Gibco 15140), insulin (Sigma I9278, 10 µg/ml) and aprotinin (Sigma A1153, 33 µg/ml).

Fig. 1. Illustration of the experimental setup for casting and cultivation and photography of a silicone post rack with four FBMEs. Silicone post rack with four FBMEs (turned upside down, scale in millimetres; A). Teflon spacer for the generation of agarose casting molds (turned upside down, scale in millimetres; B). Illustration of FBME generation (C). First lane: Casting molds are made using Teflon spacers and agarose in a 24-well cell culture dish. Silicone posts racks are placed onto the culture dish, with each pair of posts reaching into a mold. Second lane: Mastermix is pipetted into each mold. Third lane: After 2 h the fibrin is polymerized and the silicone posts are embedded in the hydrogel. FBMEs can be transferred into a new medium-filled 24-well culture plate. Fourth lane: FBMEs are maintained in culture for 15 to 30 days (Hansen et al. 2010).

#### **2.4 Video-optical analyses**

The setup for video-optical analyses consisted of a cell culture incubator-like unit, in which gas conditions, humidity and temperature could be controlled. This device was equipped with a glass roof for monitoring purposes. A Basler CCD-camera (Type A 602f-2) was attached to an XYZ-device (IAI Corporation) and positioned above the glassroof in a PCcontrolled manner. Light-emitting diodes (LEDs) were placed underneath the cell culture dish. Illumination of a single LED was synchronized with the video-optical recording procedure in order to minimize heating of the cell culture medium by LED waste heat. Figure 2A shows a schematic picture of the whole setup and 2B shows a 24 well cell culture plate with six silicone posts racks and with one FBME in every well (view from above). For the video-optical analyses a customized software package developed by Consulting Team Machine Vision (ctmv.de; Pforzheim, Germany) was used. This software is based on figure recognition and is able to identify the FBME's shape in a fully automated manner. In brief, the system automatically places measuring points at the top and bottom end of the contracting muscle strip. Due to the contraction, the distance in between the moving silicone posts changes. These changes are determined and recorded by the software over time. Based on post geometry, elastic modulus of the Sylgard 184 (2.6 kPa) and the delta value of post distance (post deflection), the developed force was calculated based on a recently published

a new medium-filled culture plate, every construct was covered with DMEM (300 µl) to ease the removal. FBMEs were maintained in 37 °C, 7% CO2, 40% O2 in a humidified cell culture incubator. Media was changed on Mondays, Wednesdays and Fridays. FBME medium consisted of DMEM (Biochrom F0415) supplemented with 10% horse serum (Gibco 26050), 2% chick embryo extract (self-made), 1% penicillin/streptomycin (Gibco 15140), insulin

**C** 

Fig. 1. Illustration of the experimental setup for casting and cultivation and photography of a silicone post rack with four FBMEs. Silicone post rack with four FBMEs (turned upside down, scale in millimetres; A). Teflon spacer for the generation of agarose casting molds (turned upside down, scale in millimetres; B). Illustration of FBME generation (C). First lane: Casting molds are made using Teflon spacers and agarose in a 24-well cell culture dish. Silicone posts racks are placed onto the culture dish, with each pair of posts reaching into a mold. Second lane: Mastermix is pipetted into each mold. Third lane: After 2 h the fibrin is polymerized and

the silicone posts are embedded in the hydrogel. FBMEs can be transferred into a new

days (Hansen et al. 2010).

**A** 

**B** 

**2.4 Video-optical analyses** 

medium-filled 24-well culture plate. Fourth lane: FBMEs are maintained in culture for 15 to 30

The setup for video-optical analyses consisted of a cell culture incubator-like unit, in which gas conditions, humidity and temperature could be controlled. This device was equipped with a glass roof for monitoring purposes. A Basler CCD-camera (Type A 602f-2) was attached to an XYZ-device (IAI Corporation) and positioned above the glassroof in a PCcontrolled manner. Light-emitting diodes (LEDs) were placed underneath the cell culture dish. Illumination of a single LED was synchronized with the video-optical recording procedure in order to minimize heating of the cell culture medium by LED waste heat. Figure 2A shows a schematic picture of the whole setup and 2B shows a 24 well cell culture plate with six silicone posts racks and with one FBME in every well (view from above). For the video-optical analyses a customized software package developed by Consulting Team Machine Vision (ctmv.de; Pforzheim, Germany) was used. This software is based on figure recognition and is able to identify the FBME's shape in a fully automated manner. In brief, the system automatically places measuring points at the top and bottom end of the contracting muscle strip. Due to the contraction, the distance in between the moving silicone posts changes. These changes are determined and recorded by the software over time. Based on post geometry, elastic modulus of the Sylgard 184 (2.6 kPa) and the delta value of post distance (post deflection), the developed force was calculated based on a recently published

(Sigma I9278, 10 µg/ml) and aprotinin (Sigma A1153, 33 µg/ml).

equation (Vandenburgh et al. 2008). The recorded contractions are filtered and identified as such by certain peak criteria (e.g. threshold value, minimal force and minimum relaxation). Besides average force, the software calculates values for frequency, fractional shortening, contraction- and relaxation time (bpm, T1 and T2, respectively) based on the recognized contraction peaks. T1 and T2 were determined at 20% of peak maximum. Reports with an overview of the environmental information (temperature, gas, humidity) in the cell culture like unit plus all calculated parameters are automatically generated after each run. These underlie two levels of quality control: pictures are taken at the beginning and the end of each recording. Blue squares indicate top and bottom end of each respective FBME where the software placed the points for the measurement (Figure 2D). Contractions are recorded as force development over time. Identified peaks, which are included in the calculation, are marked with green squares (Figure 2C). The effort to analyse a 24-well plate with contracting muscle strips is limited to defining the XYZ-coordinates for each well once before starting a series of measurements.

Fig. 2. Illustration of the experimental setup for video-optical recordings of FBME, a 24 well cell culture dish with FBMEs, a magnified view of a single FBME as recorded by the video camera and an example of a contraction pattern (one FBME for 60 s). A shows a schematic picture of the experimental setup for video-optical recordings. In B a 24 well cell culture dish with FBMEs is shown (view from above). The original contraction pattern of one FBME over time (60 s, C). Image of a contractile muscle strip from an automatically generated report (D). Blue squares indicate positions automatically recognized by the software at top and bottom end of the FBME. Green squares mark the recognized contraction peaks which are included for the calculation of several parameters (Hansen et al. 2010).

We developed a new technique for the generation and evaluation of contractile cardiac tissue from neonatal rat heart cells *in vitro* (Hansen et al. 2010). In this method, isolated heart cells are mixed with fibrinogen, thrombin and medium and pipetted into rectangular casting moulds made from 2%-agarose in ordinary 24-well cell culture plates. Due to the polymerisation of the fibrin, the gel is fixed to both silicone posts. After 2 h at 37 °C the constructs can be transferred to new culture dishes and maintained under cell culture conditions for several days. Figure 1C demonstrates this procedure in a schematic way. During cultivation, the cells inside the gel spread along the force lines and form extensive cell-cell contacts, the hydrogel is remodelled and degraded. These processes are accompanied by marked condensation of the constructs and deflection of the silicone posts towards each other. The initial length of a FBME directly after casting is 8.5 mm and the mean final length 6.5 mm. The post deflection differs between individual FBMEs, likely reflecting their degree of cardiac tissue development. In consequence, each FBME is exposed to an "individually optimized preload". With this simplified method, 48-72 FBMEs can be routinely generated out of one cell preparation (30 rat hearts). Figure 2B illustrates a typical 24 well plate with silicone posts racks and FBMEs. Fibrin is affected by proteases in the culture medium. To decrease degradation, the protease inhibitor aprotinin at a concentration of 33 µg/ml is added to the medium. This inhibitor markedly reduces fibrinolysis but cannot entirely stop it. To further protect the hydrogel from proteolysis tranexamic acid, another protease inhibitor, can be added to the medium. The combination of both inhibitors results in improved stability and allows longer cultivation periods. Tranexamic acid-treated FBMEs have a markedly increased diameter (final width 1.3 to 1.4 mm [Figure 2C and 8D] instead of 0.2 to 1.0 mm in its absence [Figure 4A]).
