Investigating the physiology of normothermic ex vivo heart perfusion in an isolated slaughterhouse porcine model used for device testing and training
The PhysioHeart™ is a mature acute platform, based isolated slaughterhouse hearts and able to validate cardiac devices and techniques in working mode. Despite perfusion, myocardial edema and time-dependent function degradation are reported. Therefore, monitoring several variables is necessary to identify which of these should be controlled to preserve the heart function. This study presents biochemical, electrophysiological and hemodynamic changes in the PhysioHeart™ to understand the pitfalls of ex vivo slaughterhouse heart hemoperfusion.
Seven porcine hearts were harvested, arrested and revived using the PhysioHeart™. Cardiac output, SaO2, glucose and pH were maintained at physiological levels. Blood analyses were performed hourly and unipolar epicardial electrograms (UEG), pressures and flows were recorded to assess the physiological performance.
Normal cardiac performance was attained in terms of mean cardiac output (5.1 ± 1.7 l/min) and pressures but deteriorated over time. Across the experiments, homeostasis was maintained for 171.4 ± 54 min, osmolarity and blood electrolytes increased significantly between 10 and 80%, heart weight increased by 144 ± 41 g, free fatty acids (− 60%), glucose and lactate diminished, ammonia increased by 273 ± 76% and myocardial necrosis and UEG alterations appeared and aggravated. Progressively deteriorating electrophysiological and hemodynamic functions can be explained by reperfusion injury, waste product intoxication (i.e. hyperammonemia), lack of essential nutrients, ion imbalances and cardiac necrosis as a consequence of hepatological and nephrological plasma clearance absence.
The PhysioHeart™ is an acute model, suitable for cardiac device and therapy assessment, which can precede conventional animal studies. However, observations indicate that ex vivo slaughterhouse hearts resemble cardiac physiology of deteriorating hearts in a multi-organ failure situation and signalize the need for plasma clearance during perfusion to attenuate time-dependent function degradation. The presented study therefore provides an in-dept understanding of the sources and reasons causing the cardiac function loss, as a first step for future effort to prolong cardiac perfusion in the PhysioHeart™. These findings could be also of potential interest for other cardiac platforms.
KeywordsBiomarkers Ex vivo Normothermic perfusion Cardiac physiology Cardiac electrophysiology
Fresh frozen plasma
Right ventricular outflow tract
Unipolar epicardial electrograms
Wave propagation velocity
Isolated perfused hearts have been used for cardiac research since the groundbreaking work of Langendorff  in 1895. Hearts isolated from the body and perfused ex vivo offer results with higher reproducibility, when compared to in vivo counterparts, because they are not affected by systemic influences, such as neurohumoral control and systemic circulation. Extensive work has been done to customize ex vivo heart platforms for precise research purposes and to improve and accelerate the development of cardiac prototypes and interventions. The optimal perfusion with warm oxygenated blood enables realistic device validation, while these setups can be also medical devices themselves (e.g. donor heart transportation) [2, 3]. Nowadays, ex vivo models are available that offer more in-dept research possibilities such as electrophysiological studies [4, 5] and working heart studies [6, 7], in which blood is pumped in a natural way (i.e. the blood enters the heart through the left atrium, it is then pumped to the left ventricle and it is finally ejected through the aorta). This “working mode” allows measurements of pump function, cardiac pressures (i.e. ventricular, aortic, pulmonary) and flows (i.e. aortic, coronary, etc.) and was first described by Neely, Liebermeister  in 1967. As a result of these setup developments, isolated heart preparations are used for a variety of investigations in cardiology, cardiac surgery, physiology and pharmacology to investigate physiological, biochemical, pharmacological and morphological characteristics as well as cardiac function [8, 9, 10, 11, 12, 13].
Pig hearts are appreciated for investigations, which specifically require physiological conditions similar to patient application, as these hearts are a good match to the morphology and physiology of human hearts . However, potential significant differences (i.e. shape, opening of superior and inferior caval veins into the atrium, prominent left azygous vein drainage, number of pulmonary veins, etc.) are known between porcine and human hearts .
Considering the increase of cardiac investigations while bearing in mind the costs and ethical issues related to laboratory animal experiments, isolated slaughterhouse hearts could under carefully chosen circumstances be used for feasibility studies that can precede the conventional and contentious animal testing. Slaughterhouse heart experiments are less cost intense, as experimentation protocols do not need ethical approval, while uncertified teams can perform several experiments a day, due to the abundance of slaughterhouse hearts without sacrificing additional animals for the research conducted. This results in an improved learning curve as investigations can originate faster .
One customized model based on slaughterhouse hearts is the PhysioHeart™, developed by LifeTec Group B.V. (Eindhoven, The Netherlands). Slaughterhouse pig hearts revived in this commercially available isolated heart model have previously shown cardiac output, stroke volume, pressures, valve interactions and dynamic changes that are comparable to those observed in humans . In the last decade, this model has been used to successfully visualize transcatheter aortic valve implantations , to assess computer tomographic myocardial perfusions , to evaluate magnetic resonance imaging-based 4D flow analysis and to study left ventricular assist devices [18, 19], intra-aortic balloon pump support  and coronary autoregulation .
In the face of, extensive work, novelties and decades of experience, isolated heart perfusion remains demanding, in particular in use of slaughterhouse hearts. The unavoidable warm ischemia (between exsanguination of the pig and the cardiac arrest) and the cold transport to the laboratory influence the experimentation outcome negatively. These shortcomings encountered within these experiments, specifically the time-dependent contractility degradation and edema, are under specific aspects similar to those observed within human DCD (donation after circulatory determined death) heart preservation, which are considered for transplantations .
Although the PhysioHeart™ model is well-established for device and therapy testing, less is known about its cardiac metabolic, biochemical and electrical physiology as the experiments progress in time, specifically regarding the source of the abovementioned shortcomings. Therefore, in this study, we report a comprehensive recording of time-dependent metabolic, biochemical, electrical and hemodynamic variables acquired from isolated normothermic, hemoperfused, slaughterhouse porcine hearts. The goal of this study was to create a much as possible complete inventory of the changes over time to identify the causes of the progressive deterioration of cardiac function and development of edema in the PhysioHeart™. This study provides the basis for further investigations and improvements to extend the cardiac function of the slaughterhouse hearts revived in the PhysioHeart™ platform. We envision that this study with its comprehensive recordings could be of potential interest for cardiac normothermic perfusion in other platforms.
Seven hearts were obtained from Dutch Landrace pigs slaughtered for human consumption. Each animal had a weight of approximately 110 Kg. All protocols followed by the slaughterhouse and laboratory were consistent with EC regulations 1069/2009 regarding the use of slaughterhouse animal material for diagnosis and research, supervised by the Dutch Government (Dutch Ministry of Agriculture, Nature and Food Quality) and were approved by the associated legal authorities of animal welfare (Food and Consumer Product Safety Authority).
Isolation and administration of cardioplegia
Modified St. Thomas solution for crystalloid cardioplegia
Mounting isolated hearts onto the circuit
Blood analysis and control
Arterial blood samples were taken from the oxygenator before the heart was connected to the loop, this was called the baseline measurement, and then every 60 min after reperfusion. Blood gas values, temperature, and electrolytes were measured using a VetScan i-STAT 1 (Abaxis, Union City, CA, USA). Based on the i-STAT 1 measurements, the pH, glucose, and ionized calcium were maintained at physiological levels by adding sodium bicarbonate, a glucose/insulin mixture (2 mmol/U) and calcium chloride. For further detailed analysis, full blood was collected in blood collection tubes. Tubes for plasma and serum analysis were centrifuged at 500 g for 10 min. Plasma and full blood samples were stored at 4 °C and serum samples were stored at − 80 °C overnight. The following day, samples were transported for analysis to a clinical laboratory (Máxima Medisch Centrum, Veldhoven, the Netherlands) and were examined with a C8000 analyser (Roche).
Pacing protocols, electrical acquisitions and signal processing and analysis
The seven hearts selected for this study were those that showed initial physiological cardiac hemodynamics which we considered to be at least CO 3 L/min, ATP 10–20 mmHg and AP 60 mmHg at the beginning of the working mode. Statistically significant differences in the mean values among treatment groups were determined by one-way analysis of variance. A paired t-test was used to confirm differences between heart weights before and after the experiment. A p-value ≤0.05 was set as a criterion for significance. All values are presented as the mean ± standard deviation. All the statistical tests were performed with Sigmaplot 11.0.
Cardiac hemodynamics and weight
Cell damage biomarkers
Additional plasma values
Activation maps and wave propagation velocity measurements
The activation maps measured in another experiment included a pacing protocol, in which three different frequencies (at sinus rhythm at 100 bpm, 120 bpm and 150 bpm) have been considered, which can be observed in Fig. 11. These measurements were made using the 6 × 8 electrode grid configuration. The direction of the depolarizing wave during pacing (Fig. 11b-c) was different compared to sinus rhythm (see Fig. 11a), since the natural heart rhythm starts in the sinus node and the pacing is provided at the RVOT, this phenomenon is justified. As before, the region that was monitored by the grid showed no arrhythmic nodes or areas of conduction block during the working mode. Furthermore, as can be observed in Fig. 11b-c changing pacing frequencies had no observable effect on the propagation pattern.
Differences between the activation maps in Fig. 10 and 11 are a consequence of the grid configurations. Activation maps in Fig. 10a-c look more uniform due to the larger grid (11 × 11 electrodes, 55x55mm) compared to Fig. 11a-c (6 × 8 electrodes, 30x48mm), which is also reflected in the shorter travelling time in Fig. 11. Differences in direction are attributable to the different positions of the grids (see Fig. 3).
Mean value (μWPV) and standard deviation (σWPV) of the wave propagation velocities measured during working mode at different pacing frequencies. The table summarizes the acquisitions made across all PhysioHeart™ experiments
− Hypertrophic cardiomyopathy, a general observation in the use of slaughterhouse hearts (before)
− Swelling of the cardiac tissue during reperfusion (after)
− Diminishing cardiac functions, possibly due to the initiation of waste product intoxication, reduction of essential nutrients, ion imbalances, cardiac necrosis and, most likely lastly, reperfusion injury and inflammation
− Hyperpotassemia (b), hypermagnesemia (g) and hyperphosphatemia(c) due to cardiac necrosis and washout of cardioplegia
− Hypersodemia (a) due to sodium bicarbonate administration (pH balancing), while calcium chloride was administered to counterbalance the calcium reduction caused by bicarbonate, resulting in hypercalcemia (f) and hyperchloremia (e)
− Hyperosmolarity (d) due to increase of ions
− General physiological aerobic cardiac metabolism, supported by free fatty acid (c) uptake and lactate (b) as well as glucose (a) metabolism
− Amino acid catabolism, confirmed by rise of ammonia (d)
− Decrease of essential cardiac nutrients (e.g. free fatty acids) and increase of toxic waste products (e.g. ammonia) over time
− Rise of lactate after 180 min signalizes ischemia and acidosis
− Hyperuremia (e)
− Hypercreatininemia (f)
− Static triglycerides (g) verifies the exclusion of fluid evaporation as a cause for the ion increase
− Hypoalbumia (a) due to blood dilution and potential cause for edema
− Slight elevation of free hemoglobin (b) caused by the centrifugal pump
− Hypercalcitriolemia (c)
− Inconspicuous pH (e) and base excess(d) values and atrial oxygenation (f)
− Severe cardiac necrosis possibly due to reperfusion injury and inflammation and, heterogeneous cardioplegia delivery in the slaughterhouse
− Physiological electrical activities of hearts during working mode
− Unaltered electrical conduction pathways
The limited duration of acceptable performance during the isolated heart experiments highlights that the isolated working heart needs to be in an environment that resembles the in-vivo physiology to avoid loss of its morphological and functional integrity. The slaughterhouse pigs used in this study were in general good health and were examined by a veterinarian prior to slaughtering. However, previous research has shown that domestication, selective breeding, scarce physical activity and improved feeding efficiency lead to morphological abnormalities in slaughterhouse-derived porcine hearts ; this was observed in our specimens as hypertrophic cardiomyopathy. Also, the baseline blood measurements revealed elevated levels in damage markers (i.e. CK, ASAT, LDH, troponin and myoglobin). As previous research has shown, elevated damage markers were most likely caused by the limited heart capacity observed in farm animals due to an intensive selection pressure and high stress during regrouping, transport and slaughtering . The baseline measurements also revealed high ion concentrations and hyperosmolarity in blood; as previously reported by Heinze and Mitchell , this was probably a consequence of water accumulation in the intra- and inter-cellular space caused by the electrical stunning. Moreover, it is believed that the hyperkalemia observed at baseline was a consequence of the rapid drop in pH produced by the slaughtering; this drop in pH is known to lead to a cellular intake of H+ and release of K+ as a physiological process of pH balancing . Electrical stunning also produces muscle contraction, which leads to hypoglycemia, hyperlactatemia, elevated creatinine levels and hyperammonemia . These contractions can further lead to acidosis (low pH) and hyperlactatemia, an effect that has previously been reported during epileptic seizures when the muscles suffer from hypoxia [30, 31].
For each heart experiment, blood from different pigs was collected immediately after exsanguination and stored for about 2 h until preparation for reperfusion. Generally, pooling blood leads to transfusion reaction in humans, but the particular characteristics of the porcine hematopoietic system make porcine blood pooling less harmful as it causes transfusion reaction only in very rare cases . However, the storage lesion of erythrocytes, during which glucose is consumed, levels of 2,3-diphosphoglycerate (DPG) and ATP decrease, and ammonia and potassium levels increase [33, 34, 35], is most likely contributing to the pathological blood values observed already at baseline.
Despite the quick harvesting process, warm cardiac ischemia is still expected to occur and to cause cardiac nutrient deficiency, hypoxia, acidosis and necrosis. It is expected that these processes will continue to damage the tissue during the cold storage period. Finally, these already stressed, hypertrophic hearts, were stored in a St. Thomas solution 2, a hypooncotic solution that promotes the influx of water through the endothelial layer into the intracellular space; this causes a further risk of cardiac edema [36, 37]. A more complex composed cardioplegic solutions like Custodiol , Somah , Celsior [40, 41] or UWS , could be of favor during hypothermic storage of slaughterhouse hearts. However, the use of a more complex solution also requires a careful consideration of price and advantages, which are currently under evaluation.
Therefore, the here above described ‘slaughterhouse-associated’ adverse effects should not be ignored when comparing the results with the carefully removed heart. These effects result in an increased chance for a reduced preservation, loss of cardiac tissue and function of the slaughterhouse porcine hearts. Despites these limitations one can learn from this pig heart the following:
Biomarkers and electrolytes
Immediately after cardiac resuscitation, an increase in potassium and magnesium in blood is observed. This is probably due to the washout of the cardioplegic solution from the coronary system. This solution, which is administered during harvesting, contains potassium and magnesium at 16 mmol/L to ensure cardiac arrest during storage. Figure 6g illustrates this wash out on the example of magnesium which experiences its largest increase in the first hour.
Throughout the experiment, we observe a rise in cardiac injury markers caused by reperfusion injury  and inflammatory responses of leukocytes and platelets. It remains speculative, but possible causes for the increasing markers may be heterogeneous cardioplegia delivery to the myocardium, harvest-related thrombosis, air emboli, and/or hypertrophic myocardium. These circumstances vary amongst hearts and therefore result in the observed fluctuating necrosis marker concentrations , initial cardiac outputs and pump functions of slaughterhouse-based hearts.
Hypertrophic hearts are known to be more vulnerable to ischemia and reperfusion injury  due to dilated epicardial coronaries, reduced capillary density and vascular dilatation reserve, which reduces the diffusion of nutrients and oxygen  and could potentially negatively influence the cardiac arrest. The presence of dilated, hyperemic coronaries in beating pig hearts revived in the PhysioHeart™ platform has been recently confirmed by Schampaert, van ‘t Veer  who associated the hyperemia to an endothelial response to the organ harvest and preparation. However, whether the hyperemic circulation is related to these preparation processes or a hypertrophy-related impairment to pharmacological and physiological stimulation, as other works suggest [46, 47], is still not fully understood.
The acidic environment during cardiac storage reduced the pH of the blood pool after cardiac resuscitation. The pH balancing with sodium bicarbonate led to an increase of sodium and reduction of ionized calcium  which was then counter balanced with calcium chloride administration resulting in constant rise of sodium and chloride in the blood. Besides these processes, the revived hearts showed a physiological aerobic cardiac metabolism, supported by free fatty acid uptake and lactate as well as glucose metabolism similar to previous reports [40, 49, 50, 51]. The constant rise of ammonia also confirms an amino acid catabolism.
However, as the cardiac hemoperfusion progresses, essential cardiac nutrients like free fatty acids decrease and toxic waste products like ammonia increase; this is known to cause edema and to disturb oxidative phosphorylation in the mitochondria . This could explain the increasing lactate values and gain of heart weight of more than 20% at the end of the experiments.
The rise of plasma free hemoglobin in our study was not significant. However, in only one experiment free hemoglobin passed 0.08 mmol/L, which occurred already from the beginning of the experiment. That could have resulted from pre-experimental blood handling. We identified the centrifugal pump as the source with the highest risk to induce hemolysis. Finally, the static concentrations of albumin, triglycerides, urea, creatinine, calcitriol but also potassium exclude the possibility that the rise of electrolytes could arise from evaporation of free water in our system.
Epicardial electrical activity during the working mode
Electrical measurements showed physiological electrical activities of hearts revived in the PhysioHeart™ platform and during the working mode. This can be appreciated in the activation patterns presented in Fig. 10 and Fig. 11, which show unaltered electrical conduction pathways with no observable conduction block or ischemic effects in the areas of interest. Also, WPV restitution (i.e. a decrease in wave propagation velocity as the pacing frequency is increased) was observed and presented in Table 2. The analysis of restitution effects is central in the early detection of arrhythmia and in testing anti-arrhythmic drugs and devices; consequently, observing restitution effects in the PhysioHeart™ platform enables its use to investigate these phenomena within the scope of normothermic perfusion.
Although normal physiological behavior was observed during the working mode, all PhysioHeart™ experiments showed abnormally high sodium, potassium and ionized calcium concentrations in blood. These concentrations increased as the experiment progressed, this is evident from Fig. 6. The abnormally high sodium concentration translated, as observed in Table 2 and as supported by previous research , in high wave propagation velocity. Abnormally fast depolarization waves could induce arrhythmias because they may cause re-entrant waves or conduction block. High ionized calcium concentration in blood has also been shown to be related to longer action potentials  and abnormal membrane excitability . Also, the observed hyperkalemia is known to cause elevated resting membrane potentials and reduced cellular excitability  and, consequently, arrhythmia such as atrial fibrillation or ventricular tachycardia. The use of insulin in our experiments may have support these effects as insulin leads to a dose-dependent influx of potassium into the cells . This last fact was also evident because, in some experiments, stimulation protocols induced arrhythmias when pacing higher than sinus rhythm.
These observations put in evidence the importance of, simultaneously, monitoring the ion concentrations in blood and the electrophysiological activity of the cardiac tissue. In particular, the use of electrode grids within normothermic perfusion platforms could enable the detection of localized ischemia and abnormal conduction patterns that could result in arrhythmia during transport. Moreover, the monitoring of the ion concentrations in blood would also enable to determine the causes of any unphysiological electrical behavior, which can result in fast action to prevent the decreased performance of the heart.
Achieving normal cardiac physiology during ex-vivo slaughterhouse heart perfusion
The PhysioHeart™ platform, with its starling resistor as preload and a standard four-element Windkessel model as afterload, generates flow patterns and pressure curves in the revived slaughterhouse hearts that are similar to those measured in humans . For an average of 3 h, physiological and morphological cardiac characteristics, with normal electrical and metabolic activities, can be obtained without any corrective measures. Although not all blood values are physiological prior and during reperfusion, isolated beating slaughterhouse porcine hearts seem to tolerate these pathologies for a limited period. Therefore, it is inferred that the isolated working normothermic heart can be used as a baseline model to study cardiac intervention methods (LVADs, TAVI valve replacements, etc.).
These interventions may be unloading, moderate hypothermia, filtration of plasma for inflammatory components and metabolic waste, addition of nutrients and protective drugs. In view of ethical constraints regarding use of animals for short-term and uncertain-outcome experiments, the platform provides several benefits including availability, low cost, and no ethical objections.
In view of improved and prolonged preservation of the PhysioHeart™ model is the mitigation of the immune response of the pig blood. This can be achieved by separating lymphocytes and platelets, to obtain platelet and lymphocyte-poor blood in combination with administering inflammatory and autoimmune depressing drugs (i.e. dexamethasone, prednisone). The use of antibiotics and fungistatic medication would further serve to avoid infections. In addition, including anti-arrhythmic drugs in the platform will help mitigate the effects of high electrolyte concentrations in blood and, consequently, extend the time in the working mode.
We have identified that the PhyioHeart™ lacks hepatological and nephrological clearances and substance supplementations. In the upcoming future, the blood perfusate should be kept physiological and renewed constantly to maintain metabolomics and proteomic profiles and to remove toxins either with dialysis, new platelet-poor plasma  or a similar complex medium (i.e. HCO3, HEPES, inorganic salts, amino acids, carbohydrates, fatty acids, lipids, vitamins (Cernevit), trace elements, colloids and hormones for vasodilation like Milrinone). Dialysis and hemofiltration may be helpful by removing excess water, toxins and stabilizing the electrolyte and ion balance as the periods of arrhythmia observed in the PhysioHeart™ experiments were associated with high ion concentrations. Finally, these attempts are assumed to attenuate the loss of cardiac function in the platform and would lead to more standardized and improved experimentation.
Limitations of the current PhysioHeart™ experiments
Despite low animal costs, big animal isolated heart experiments remain costly. Therefore, the scope of this study was rather exploratory in order to determine the boundary conditions and attempts needed to prolong physiological cardiac perfusion in our model.
All the hearts had different initial left ventricular function and electrical activity, which result in a ‘normal variation’ that may affect small-number experiments. Therefore, it is important to standardize and optimize the harvest and mounting procedures to avoid outlying negative performance at the start of the experiment. An additional limitation is that the blood pool volumes between experiments were not equal could have influenced blood marker concentrations; a standard initial volume is required to ensure consistency across experimental acquisitions. Additionally, the priming volume for deairing the circuit was not equal between experiments and, most likely, biased the baseline measurements; in future experiments, the priming volume will be considered when analyzing baseline values. Finally, one may consider non-invasive epicardial monitoring such as echocardiography, stress-strain imaging and speckle imaging to detect early signs of ischemia that may result in corrective or supporting actions. Moreover, the use of square grids limits the electrophysiological study to a small region of the heart; the use of more sophisticated electrode arrays, capable of making whole-heart measurements, would enable a more comprehensive electrophysiological study. Finally, micro-puncture histology during and at the end of the experiments may be useful as a hard outcome parameter.
The isolated working slaughterhouse heart is a practical (e.g. abundant numbers, no need to specifically sacrificed laboratory animals, no ethical approval of study protocols, etc.) and cost-efficient model to perform investigative and therapeutic experiments. This study was meant to identify factors limiting these experiments of isolated slaughterhouse porcine heart in the PhysioHeart™ perfusion model. Our findings confirm the viability and function loss of the isolated slaughterhouse hearts are best described by the phenomenon of “time-dependent multi-organ” failure. The blood parameters, biochemical and electrophysiological changes observed in the PhysioHeart™ platform provide a better understanding of the necessary effort to overcome the challenges. Diligent and strictly protocolized harvesting and installation will reduce variation at the start of the PhysioHeart™ platform experiment. This study identified several mechanisms and provided explanations of the potential sources which limit the ex vivo cardiac viability and perfusion time which in turn could be corrected. Perfusate renewal and clearance has been recognized as crucial for prolonged cardiac perfusion in the PhysioHeart™ model which could be of potential interest for other heart platforms.
We thank Dr. Dirk Bakkeren, Anita Groos, Riejean Kuylaars and the rest of the team from the clinical laboratory at the Máxima Medical Centre Veldhoven for the biochemical analysis.
BK, CAL, SvT and MS participated in study design. BK, CAL and SvT carried out the study and prepared the manuscript which was revised and approved by BE, CI, PJT, VD-Z and BAJMdM. BJB and VM assisted with the assessment of the epicardial ECGs. All authors read and approved the final manuscript.
This project has received financial funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No 642612. Bastiaan J. Boukens received financial funding from the Dutch Heart Foundation (2016 T047).
Ethics approval and consent to participate
All protocols followed by the slaughterhouse and laboratory were consistent with EC regulations 1069/2009 regarding the use of slaughterhouse animal material for diagnosis and research, supervised by the Dutch Government (Dutch Ministry of Agriculture, Nature and Food Quality) and were approved by the associated legal authorities of animal welfare (Food and Consumer Product Safety Authority).
Consent for publication
B. Kappler worked with LifeTecGroup B.V. before his employment as PhD researcher at the department of Cardiothoracic Surgery of the Academic Medical Center at the University of Amsterdam. S. van Tuijl. is an employee of the LifeTec Group B.V. M. Stijnen and B.A.J.M. de Mol are founders of LifeTec Group BV and are respectively Head of Medtech Innovation and director of Medical Affairs. Professor B.A.J.M. de Mol was chief of the department of Cardio-thoracic Surgery at the Amsterdam University Medical Center. LifeTec Group B.V. developed the PhysioHeart™ platform and has the proprietary rights. LifeTecGroup BV supported this study with professional laboratory services and provision of materials.
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