Investigating the physiology of normothermic ex vivo heart perfusion in an isolated slaughterhouse porcine model used for device testing and training.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The cytoprotective capacity of processed human cardiac extracellular matrix.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 µm (15-157 µm) by laser diffraction, and 57 µm (20-182 µm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, -72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Normothermic Ex Vivo Liver Platform Using Porcine Slaughterhouse Livers for Disease Modeling.

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

Attenuated cardiac function degradation in ex vivo pig hearts.

Isolated hearts offer the opportunity to evaluate heart function, treatments, and diagnostic tools without in vivo factor interference. However, the early loss of cardiac function and edema occur over time and do limit the duration of the experiment. This research focuses on delaying these limitations using optimal blood control. This study examines whether blood conditioning by means of the combination of blood predilution and hemodialysis can significantly reduce cardiac function degradation. Slaughterhouse porcine hearts were revived in the PhysioHeart™ platform to restore physiological cardiac performance. Twelve hearts were divided into a control group and a dialysis group; in the latter group, hemodialysis was attached to the blood reservoir. Cardiac hemodynamics and blood parameters were recorded and evaluated. Blood conditioning significantly reduced the loss of cardiac pump function (control group vs dialysis group, -14.9 ± 6.3%/h vs -9.7 ± 2.7%/h) and loss of cardiac output (control group vs dialysis group, -11.8 ± 3.4%/h vs -5.9 ± 2.0%/h). Hemodialysis resulted in physiological and stable blood parameters, whereas in the control group ions reached pathological values, while interstitial edema still occurred. The combination of blood predilution and hemodialysis significantly attenuated ex vivo cardiac function degradation and delayed the loss of cardiac hemodynamics. We hypothesized that besides electrolyte and metabolic control, the hemodialysis-accompanied increase in hematocrit resulted in improved oxygen transport. This could have temporarily compensated the deleterious effect of an increased oxygen-diffusion distance due to edema in the dialysis group and resulted in less progression of cell decay. Clinically validated measures delaying edema might improve the effectiveness of the PhysioHeart™ platform.

Measurement of in vitro cardiac deformation by means of 3D digital image correlation and ultrasound 2D speckle-tracking echocardiography.

Ultrasound-based 2D speckle-tracking echocardiography (US-2D-STE) is increasingly used to assess the functionality of the heart. In particular, the analysis of cardiac strain plays an important role in the identification of several cardiovascular diseases. However, this imaging technique presents some limitations associated with its operating principle that result in low accuracy and reproducibility of the measurement. In this study, an experimental framework for multimodal strain imaging in an in vitro porcine heart was developed. Specifically, the aim of this work was to analyse displacement and strain in the heart by means of 3D digital image correlation (3D-DIC) and US-2D-STE. Over a single cardiac cycle, displacement values obtained from the two techniques were in strong correlation, although systematically larger displacements were observed with 3D-DIC. Notwithstanding an absolute comparison of the strain measurements was not possible to achieve between the two methods, maximum principal strain directions computed with 3D-DIC were consistent with the longitudinal and circumferential strain distribution measured with US-2D-STE. 3D-DIC confirmed its high repeatability in quantifying displacement and strain over multiple cardiac cycles, unlike US-2D-STE which is affected by accumulated errors over time (i.e. drift). To conclude, this study demonstrates the potential of 3D-DIC to perform dynamic measurement of displacement and strain during heart deformations and supports future applications of this method in ex vivo beating heart platforms, which replicate more fully the complex contraction of the heart.

Feasibility of mapping and cannulation of the porcine epicardial lymphatic system for sampling and decompression in heart failure research.

BACKGROUND AND AIM: The cardiac lymphatic system drains excess fluid from the cardiac interstitium. Any impairment or dysfunction of the lymph structures can result in the accumulation of interstitial fluid, and may lead to edema and eventually cardiac dysfunction. Lymph originates directly from the interstitium and carries real-time information about the metabolic state of cells in specific regions of the heart. The detailed anatomy of the epicardial lymphatic system in individuals is broadly unknown. Generally, the epicardial lymphatic system is not taken into consideration during heart surgery. This study investigates the feasibility of detailed mapping and cannulation of the porcine epicardial lymphatic system for use in preservation of explanted hearts and heart failure studies in pigs and humans. METHODS: The anatomy of the epicardial lymphatic systems of forty pig hearts was studied and documented. Using a 27 G needle, India ink was introduced directly into the epicardial lymphatic vessels in order to visualise them. Based on the anatomical findings thus obtained, two cannulation regions for the left and right principal trunks were identified. These regions were cannulated with a 26 G intravenous Venflon cannula-over-needle, and a Galeo Hydro Guide F014 wire was used to verify that the lumen was patent. RESULTS: The main epicardial lymphatic collectors were found to follow the main coronary arteries. Most of the lymph vessels drained into the left ventricular trunk, which evacuates fluid from the left heart and also partially from the right heart. The right trunk was often found to drain into the left trunk anterior basally. Right heart drainage was highly variable compared to the left. In addition, the overall cannulation success rate of the selected cannulation sites was only 57%. CONSLUSIONS: Mapping of the porcine epicardial lymphatic anatomy is feasible. The right ventricular drainage system had a higher degree of variability than the left, and the right cardiac lymph system was found to be partially cleared through the left lymphatic trunk. To improve cannulation success rate, we proposed two sites for cannulation based on these findings and the use of Venflon cannulas (26 G) for cannulation and lymph collection. This method might be helpful for future studies that focus on biochemical sample analysis and decompression. RELEVANCE FOR PATIENTS: Real-time biochemical assessment and decompression of lymph may contribute to the understanding of heart failure and eventually result in preventive measures. First its relevance should be established by additional research in both arrested and working porcine hearts. Imaging and mapping of the epicardial lymphatics may enable sampling and drainage and contribute to the prevention or treatment of heart failure. We envision that this approach may be considered in patients with a high risk of postoperative left and right heart failure during open-heart surgery.

Full-field analysis of epicardial strain in an in vitro porcine heart platform.

The quantitative assessment of cardiac strain is increasingly performed to provide valuable insights on heart function. Currently, the most frequently used technique in the clinic is ultrasound-based speckle tracking echocardiography (STE). However, verification and validation of this modality are still under investigation and further reference measurements are required to support this activity. The aim of this work was to enable these reference measurements using a dynamic beating heart simulator to ensure reproducible, controlled, and realistic haemodynamic conditions and to validate the reliability of optical-based three-dimensional digital image correlation (3D-DIC) for a dynamic full-field analysis of epicardial strain. Specifically, performance assessment of 3D-DIC was carried out by evaluating the accuracy and repeatability of the strain measurements across multiple cardiac cycles in a single heart and between five hearts. Moreover, the ability of this optical method to differentiate strain variations when different haemodynamic conditions were imposed in the same heart was examined. Strain measurements were successfully accomplished in a region of the lateral left ventricle surface. Results were highly repeatable over heartbeats and across hearts (intraclass correlation coefficient = 0.99), whilst strain magnitude was significantly different between hearts, due to change in anatomy and wall thickness. Within an individual heart, strain variations between different haemodynamic scenarios were greater than the estimated error of the measurement technique. This study demonstrated the feasibility of applying 3D-DIC in a dynamic passive heart simulator. Most importantly, non-contact measurements were obtained at a high spatial resolution (~ 1.5 mm) allowing resolution of local variation of strain on the epicardial surface during ventricular filling. The experimental framework developed in this paper provides detailed measurement of cardiac strains under controlled conditions, as a reference for validation of clinical cardiac strain imaging modalities.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.