Normothermic perfusion has been applied in several organ-preservation studies such as those involving the heart [13], liver [14], kidney [15], and lung [16]. Furthermore, successful transplantation has been performed after ex vivo normothermic perfusion of organs for 12 h [13, 16] or 20 h [15]. However, to our knowledge, no previous investigations have reported longer term (i.e., > 24 h) organ preservation with normothermic perfusion. In the present study, we evaluated whether a newly developed bioreactor system that intermittently applied external positive pressure would have a beneficial effect on the viability of organs perfused under normothermic conditions. We found that intermittent application of external pressure generated pulsatile flow in the vascular system of the small intestine. Furthermore, intermittent positive pressure improved the uniformity of perfusion in small intestine preparations and the perfusion ratio in skeletal muscle preparations. In addition, intermittent positive pressure helped to preserve vascular structures and surrounding tissue. Therefore, our novel findings demonstrate that long-term organ preservation can be achieved successfully by the use of intermittent pressurization to generate pulsatile vascular flow during normothermic perfusion.
Pulsatile flow has been reported to have several advantages over constant flow in ex vivo studies of the isolated kidney, including positive effects on vascular endothelial function (such as greater NO release from endothelial cells), blood vessel function, and renal viability (indicated by a higher renal oxygen demand) [17]. However, the pulsatile flow created by the heart in vivo is attenuated in the peripheral vascular system [18]. Therefore, when organs are cultured ex vivo, it is possible that pulsatile flow generated by an infusion pump might also be attenuated from the proximal end to the distal end of the inlet tube. In this study, pulsatile flow was generated by intermittent external positive pressurization in a bioreactor system. Pulsatile flow of medium to the entire vascular system, including peripheral blood vessels, was successfully achieved and led to long-term organ preservation (i.e., 14 days).
A pressure of 10 mmHg was utilized in the present study, because it is likely within the physiologic range of external pressures for the organs used. For example, intraperitoneal pressure in vivo can vary widely from 0.7 to 14 mmHg depending on body position and activity [19], and intramuscular pressure can range from 5.7 to 12 mmHg during the resting phase [20]. The pressure within the blood vessels of the small intestine varied with intermittent pressurization, indicating that the external pressure changes in our system affected not only the organ surface, but also its vascular system. Furthermore, when compared with no pressurization, intermittent external pressurization resulted in more uniform perfusion of the small intestine preparation and a better perfusion ratio in the skeletal muscle preparation. We speculate that intermittent compression of the microvascular system may improve the perfusion of medium through the vascular system by reducing stagnation of flow.
In this study, the external pressure changes were achieved using a gas-driven pressurizing bioreactor designed and built in-house. The pulsatile pressure generator had a pressure regulating path that determined the upper limit of pressure inside the organ chamber and a gas release path that enabled depressurization of the chamber. The chamber was sealed within a confined dead space, allowing the bioreactor system to accurately pressurize the organ to a predefined level by injecting gas into the chamber at a slow flow rate (i.e., 150–200 mL/min). Our experiments confirmed that the inner chamber pressure was reproducibly pressurized to 10 mmHg within 20 s and depressurized to 0 mmHg (relative to atmospheric pressure) within 20 s.
The perfusion ratio was defined as the ratio of the mass of medium leaving the organ via its vein to that of the medium infused via the artery. During ex vivo perfusion, some of the perfused medium leaks from the vascular system into the surrounding tissues and is lost from the surface of the organ. Previous studies have reported perfusion ratios of 40 ± 7% in skeletal muscle preparations perfused for 3 days [5] and 55.4 ± 4.4% in engineered skin flaps perfused for 3 days [21]. In our study, the perfusion ratio in the non-pressurized (control) group was 24.9 ± 15.0% over the course of 3 days, which is somewhat lower than the values obtained by the investigations mentioned above. One possible reason for this discrepancy is that our perfusion medium did not contain growth factors, whereas the previous studies included fibroblast growth factor-2 (FGF-2) in the medium perfusing skeletal muscle [5] and epidermal growth factor (EGF) in the medium used for skin flap perfusion [21]. FGF-2 induces the reorganization of endothelial junctions and deposition of extracellular matrix [22], and EGF modulates vascular tone and tissue homeostasis [23]. Since these growth factors were included to maintain the viability of the vascular system, it is reasonable to assume that they would increase the perfusion ratio by reducing the loss of medium from the vasculature. Although the medium that we used did not contain any growth factors, it was notable that the perfusion ratio in the pressurized group (63.1 ± 6.3%) was higher than the values reported by previous studies [5, 21]. Indeed, the perfusion ratio for the skeletal muscle preparation was substantially higher in the pressurized group than in the control group at day 3 (63.1 ± 6.3% vs. 24.9 ± 15.0%) and day 14 (41.7 ± 6.8% vs. 8.0 ± 5.2%). One likely mechanism underlying the differences between groups is that the vascular structures in the organ showed substantial deterioration in the non-pressurized group, but were relatively well maintained in the pressurized group. Consistent with our observations, von Horn et al. reported that pulsatile flow helped to sustain vascular function, reduce the leakage of medium, and maintain the perfusion ratio in the isolated perfused kidney [17]. In addition, it is likely that the smaller difference between internal vascular pressure and chamber pressure in the pressurized group suppressed the leakage of medium from the vessels to the surrounding tissue spaces.
The present study used bioluminescence imaging after perfusion with luciferin to evaluate the viability of femoral muscle harvested from luciferase-expressing rats [12]. After 14 days of ex vivo perfusion and culture, the bioluminescence intensity was observed to be higher in the pressurized group than in the control group, suggesting that organ viability had been better maintained in the pressurized group. This conclusion was supported by the findings of the histology experiments, which revealed less necrosis and better maintenance of vascular structures and muscle cell nuclei in the pressurized group. It is likely that maintenance of vascular structures in the pressurized group helped to sustain adequate levels of nutrient provision and waste product removal, thereby preserving cell viability.
Although it was beyond the scope of the present study to investigate the mechanisms of intermittent external pressurization at the cellular and molecular level, the previous research has yielded some insight into the effects of increased pressure and mechanical stimulation on various cell types. Ando et al. reported that shear stress promoted the differentiation of immature endothelial cells to mature endothelial cells that secreted factors such as NO [24, 25]. Jeong et al. found that vascular smooth muscle cells stimulated mechanically by pulsatile flow differentiated into a mature phenotype [26]. Wann et al. observed that an elevation in hydrostatic pressure increased the duration of the action potential in excitable cells by slowing both the peak depolarization and repolarization rates [27]. In addition, Wong et al. reported that cyclic hydrostatic pressure affected chondrocyte gene expression, slowed cartilage differentiation, and exerted an anti-angiogenic effect [28]. However, the relevance of these previously published findings to the present study is unclear, because much higher pressures were used than in our experiments (7600 to 76000-fold higher in the study of Wann et al. and 3800-fold higher in the study of Wong et al.). Notably, in our study, the perfusion ratio for the skeletal muscle preparation was significantly higher in the pressurized group than in the control group at day 0–1. This short-term outcome is more likely to be due to the effects of physical pressure at the organ level rather than direct actions at the cellular level involving changes in cell membrane properties due to altered gene expression. Therefore, as noted earlier, we speculate that the main beneficial effects of intermittent external pressurization on organ preservation are achieved through two mechanisms that cooperate to maintain the viability of cells in the tissue. First, intermittent external pressurization generates pulsatile flow of medium to the entire vascular system of the organ without attenuation of the pulsatile effect in peripheral vessels, and the pulsatile flow contributes to less stagnation of medium flow. Second, intermittent physical compression of the microvascular system may reduce the leakage of medium from the vessels to the tissue spaces. Nevertheless, it remains possible that low pressures such as those used in the present study might act directly on cells to affect their long-term viability. Further mechanobiological research will be needed to elucidate the mechanisms that contributed to the effects of intermittent pressurization observed in the present study.
This study has a limitation that should be noted. Our objective was to investigate whether the intermittent application of external positive pressure within the physiological range might have beneficial effects during ex vivo organ perfusion and culture, but our proof-of-concept study only utilized a pressure of 10 mmHg. It is possible that a pressure of 10 mmHg may not have been optimal and that more effective organ preservation might be achieved by the application of a different pressure. Further research utilizing a range of pressures will be needed to optimize the conditions for intermittent application of external positive pressure. Nonetheless, the data described in this manuscript provide strong evidence that the intermittently pressurized bioreactor system could potentially be used to facilitate the long-term preservation of donor organs or maturation of bioengineered tissues or organs.