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A three-dimensional multicellular organism maintains the biological functions of life support by using the blood circulation to transport oxygen and nutrients and to regulate body temperature for intracellular enzymatic reactions. Donor organ transplantation using low-temperature storage is used as the fundamental treatment for dysfunctional organs.
However, this approach has a serious problem in that donor organs maintain healthy conditions only during short-term storage. In this study, we developed a novel liver perfusion culture system based on biological metabolism that can maintain physiological functions, including albumin synthesis, bile secretion and urea production. This system also allows for the resurrection of a severely ischaemic liver. This study represents a significant advance for the development of an ex vivo organ perfusion system based on biological metabolism.
It can be used not only to address donor organ shortages but also as the basis of future regenerative organ replacement therapy. Multicellular organisms are composed of various organs and tissues comprising specialised functional cells that have a precise three-dimensional (3D) arrangement in the living body. The organ systems include the nervous system, digestive system and circulatory system and they are essential for maintaining fully functional networks in a living body. The nervous system, with its sympathetic and parasympathetic nerves, antagonistically regulates organ functions such as the heartbeat, gastrointestinal motility and thermoregulation. To maintain homeostasis, the digestive system plays crucial roles in the digestion and absorption of various nutrients. Multicellular organisms can be supported by oxygen immobilised in erythrocytes and by nutrients, hormones and biological materials solubilised in the blood serum through the circulatory system, including the heart, lungs and sterical vascular network.
The materials that are transported through the organ network systems are essential for cell proliferation and the physiological functions of various cells.Vascular networks, comprising arteriovenous vessels and microvessels, contribute to 3D tissue formation by supplying blood containing oxygen and nutrients. Blood contains various cell types, including erythrocytes and immune-competent cells for immunological defence and these cells are involved in life support and homeostasis. Blood serum plays essential roles in various biological functions, including the transportation of nutrients and bioactive factors and the transitional regulation of colloidal osmotic pressure by serum proteins. The blood and vessels are responsible for heat dissipation via vasoconstriction and vasodilatation in peripheral regions such as the fingers, palms and ear lobes and they also contribute to thermal regulation, which strongly affects cellular proliferation and functions. Decreased body temperature, or hypothermia, causes depression of the heartbeat and respiration and life ends due to the degradation of various metabolic factors. Therapeutic hypothermia, which induces metabolic suppression in response to temperatures between 30 and 33°C, has been used to treat patients experiencing subarachnoid haemorrhage and cerebral infarction. Severe hypothermia, which occurs at temperatures between 20 and 28°C, causes a reduction of physiological functions such as heart rate, respiratory rate and blood pressure.
However, hypothermia below a body temperature of 20°C is lethal. In severe and lethal hypothermia, reduced levels of adenosine triphosphate (ATP), which is generated by glycolysis and plays an essential role in the support of cell activities, have been reported. However, the fundamental factors that regulate the clinical outcomes of severe and lethal hypothermia have not yet been clarified.Organ transplantation is currently used to replace a dysfunctional organ and to restore organ function in vivo. The donor organs are stored using static cold preservation systems using various organ preservation solutions including intracellular- or extracellular-type fluids during organ transportation and pre-treatment of the recipient.
Although these organ preservation methods have been developed, there is a worldwide donor organ shortage that remains an unresolved problem. To effectively use organs from the limited number of donors, new approaches are being developed to prolong the organ preservation time by storing organs at sub-zero temperatures.
Techniques are currently being developed to increase the number of donor organs by expanding the use of organs from extended criteria donors (ECDs) and donation after cardiac death (DCD). Thus, the development of organ preservation methods and resuscitation methods for successful and safe transplantation of marginal donor organs such as ECDs and DCDs are greatly needed. Recently, various preservation methods using machine perfusion systems combined with organ preservation solutions, which are modified using culture medium and blood components under cold or normothermic temperatures, have also been developed to decrease the risk of serious post-operative complications associated with graft survival and dysfunction. However, physiological activities of the organ were strongly depressed under static cold storage and various metabolites that are necessary for cellular activities, such as amino acids, proteins and ATP, were deficient in the preserved organ. Additional novel concepts and technological developments for organ preservation based on biological responses and mechanisms have also been expected for donor expansion in organ transplantation therapy.In this study, we demonstrate the essential factors, including oxygen supply using erythrocytes and hypothermic regulation at 22°C, necessary for the survival and restoration of organ functions. Our tests are based on biological responses such as cell activity, cellular disorders and metabolism using our developed 3D liver perfusion culture system. In a metabolome analysis using cultured hepatocytes, we determined that a biological window for cell survival, but not cell proliferation and function, exists in the hypothermic zone of approximately 22°C and we found that hypothermic temperature could regulate the biological factors involved in cell survival, such as ATP synthesis, amino acid synthesis and glucose metabolism.
This 3D perfusion culture based on biological responses was able to preserve a donor liver in vitro and could be used to resuscitate a DCD liver. It also has the potential to replace donor organ transplantation and regeneration in vivo. This study thus represents a concept that has considerable potential as a novel organ preservation and restoration method for the next generation of 3D organ culture. Optimisation of a 3D liver perfusion culture system using erythrocytes at hypothermic temperatureTo establish a 3D organ perfusion culture system, we designed an extracorporeal liver perfusion circuit that regulates the oxygen supply and culture temperature. The circuit was connected to the portal vein and hepatic upper inferior vena cava of an isolated rat liver.
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The isolated liver was placed in a culture device filled with culture medium to achieve sufficient perfusion flow and had similar intrahepatic hemodynamic to natural liver. This hanging method successfully maintained the flow of culture medium so that it was well perfused into all lobes through vascular networks of the cultured liver. Such perfusion of the cultured liver was not achievable with the putting liver as represented by the angiogram image and trypan blue infusion data shown in. We first examined how the culture temperature affected the physiological functions of the liver cultured in this system. At temperatures of 4, 10, 22, 33 and 37°C, we evaluated the concentration of alanine aminotransferase (ALT) as a hepatic disorder marker and we measured albumin and bile as variables representing liver-specific physiological functions. After perfusion at 37°C for 20 hours (hrs) and 33°C for 32 hrs, ALT concentrations of 121 IU/L and 81 IU/L were observed, respectively. By contrast, the ALT concentrations of the perfusion cultures at 4, 10 and 22°C remained low (, left).
Continuous high-level albumin and bile synthesis was observed only in the culture at 22°C after 48 hrs, not in cultures at 4 and 10°C (, centre and right). Swelling and disorganisation of the sinusoidal structure were observed in the cultures at 37 and 33°C after 48 hrs.
By contrast, the sinusoidal structures were successfully maintained and were equivalent to those in a living liver in the livers cultured at 4, 10 and 22°C. Establishment and optimisation of the liver perfusion culture system.(a), Diagram of the ex vivo organ perfusion culture circuit. (b), Photograph of isolated liver placement by our hanging method in the organ chamber. (c), Assessments of ALT activity ( left), albumin synthesis ( centre) and bile production ( right) during liver perfusion culture at 22°C with 5.0 × 10 11 cells/L erythrocytes. The coloured lines represent temperatures of 37°C (black), 33°C (green), 22°C (red), 10°C (purple) and 4°C (blue).
(d), Photographs ( top) and histological images ( middle and bottom) of the cultured livers at different temperatures. Higher-magnification images are shown in the boxed area ( bottom). Scale bars, 100 μm. (e), Assessments of ALT activity ( left), albumin synthesis ( centre left), bile production ( centre right) and urea synthesis ( right) during liver perfusion culture at 22°C with/without erythrocytes. These data represent erythrocyte concentrations of 0.5 × 10 11 cells/L (green), 2.0 × 10 11 cells/L (orange), with 5.0 × 10 11 cells/L (red) and no erythrocytes (blue).
(f), 3D images of sinusoidal structure in natural ( top column) and cultured livers after 48 hr of perfusion at 22°C with/without erythrocyte ( middle column and bottom column). The sagittal section ( top) and horizontal section ( bottom) are shown in a hepatic lobule between the central vein (CV) and the portal vein (PV). The vascular structure is represented by FITC (green) and dead cells (red) are indicated by the propidium iodide staining.
All sections are counterstained with Hoechst 33342 (blue). Scale bars: 100 μm.
(g), The numbers of total cells (white) and dead cells (black) in the natural ( left) and cultured livers after 48 hrs at 22°C with/without erythrocytes ( centre and right). These cells are counted in the optional areas evenly between CV and PV in a range of 100 μm (wide) × 200 μm (depth) × 100 μm (height). (h), Histological analysis of natural ( left) and cultured livers after 48 hrs at 22°C with/without erythrocytes ( centre and right). Higher-magnification images are shown in the boxed area ( bottom). Scale bars, 100 μm. We next used erythrocytes to analyse the effect of oxygen supply on liver damage and physiological functions.
With the perfusion culture using L-15 medium containing 10% FCS with a dissolved oxygen concentration at 6.77 ppm without erythrocytes, the ALT concentration drastically increased after 10 hrs. By contrast, ALT release was supressed by the addition of erythrocytes in a dose-dependent manner and the cultured liver with erythrocytes at a density of 5 × 10 11 cells/L was sufficiently maintained after 48 hrs of culture in terms of not only the ALT concentration but also the physiological functions, such as the changing of the pH of the medium, albumin and bile secretion and urea synthesis rate ( and ).
Histological analysis also revealed the effects of the erythrocytes in the 22°C culture condition on the maintenance of the cultured liver. To further evaluate the preservative effect of erythrocytes in the 22°C culture condition, we performed 3D image analysis, in which both the structural maintenance of the sinusoidal network and hepatocyte survival could be represented using a fluorescein 5-isothiocyanate (FITC)-conjugated gelatine and propidium iodide (PI) in a hepatic lobule. The PI-positive dead hepatocytes (red to orange) and the destruction of the sinusoidal network by leaking of FITC conjugated gelatine were clearly observed around the central vein but not the hepatic portal vein in the culture without erythrocytes.
By contrast, the sinusoidal network and hepatocyte survival of the cultured liver cultured with erythrocytes were successfully maintained at equivalent states to those of the natural liver. These findings indicate that our perfusion culture system optimised the use of erythrocytes and that the 22°C culture condition shows potential for long-term organ preservation. Evaluation of cellular physiological activity due to culture temperature changeTo investigate the effect of the culture temperature on cellular activities including cell proliferation, albumin synthesis and glucose metabolism of the liver, we analysed the responses of a human hepatocellular cell line, Huh7, under various culture temperature conditions and we used these cells as a model of the hepatocytes that constitute the majority of the liver. Cell proliferation at 37 and 33°C was clearly temperature-dependent, whereas proliferation below 22°C could not be observed (, top and ). The cells cultured at 22°C, but not those at 4 and 10°C, could successfully maintain the ability to proliferate in response to rewarming to 37°C for at least 48 hrs (, centre and bottom). Albumin synthesis and glucose metabolism, which produces lactate from glucose and is related to cell proliferation and survival, were also observed in a temperature-dependent manner at 33 and 37°C, according to the results of the cell proliferation assay.
These results indicate that the culture temperature affects cellular activities and the marginal temperature for cell survival might occur at approximately 22°C. Affects on cellular activity and intracellular metabolism due to temperature change.(a), Assessments of proliferative activity in Huh7 cells at various culture temperatures ( top) and re-warming to 37°C from each culture temperature at 24 hrs ( middle) or 48 hrs ( bottom). The coloured lines represent temperatures of 37°C (black), 33°C (green), 22°C (red), 10°C (purple) and 4°C (blue). (b), Measurements of albumin synthesis ( upper), glucose concentration ( middle) and lactate concentration ( bottom) in the culture supernatant. The coloured lines represent temperatures of 37°C (black), 33°C (green), 22°C (red), 10°C (purple) and 4°C (blue). (c), Schematic representation of the intercellular metabolic pathways in primary hepatocytes at culture temperatures of 4, 10, 22, 33 and 37°C. The accumulation of each metabolite is indicated by coloured dots, which are highly expressed in the lower temperature condition (4, 10°C; blue), the hypothermic and body temperature condition (22, 33, 37°C; red).
The dependence with temperature increases (green). (d), Relative levels of ATP ( top left), ADP ( middle left), AMP ( bottom left), adenosine ( top right) and uric acid ( middle right) in primary hepatocytes under various culture temperatures. The coloured bars represent temperatures of 37°C (black), 33°C (green), 22°C (red), 10°C (purple) and 4°C (blue).
Functional analysis of intracellular metabolism changes due to temperature changeTo analyse the intracellular metabolism as a function of culture temperature, we performed a metabolome analysis of primary cultured hepatocytes under various culture temperatures. In this study, the major metabolites relevant to hepatocellular metabolism were analysed.
In a heat-map analysis of intracellular metabolites under various culture conditions, the metabolome profiles of the 4 and 10°C culture conditions were clearly different to those of the other temperature conditions. The relative concentrations of metabolites in all culture conditions were analysed statistically and then divided into four groups. Group 1 included the higher metabolite accumulation at the 22, 33 and 37°C culture conditions compared with the 4 and 10°C cultures.
Group 2 included the higher metabolite accumulation at the 4 and 10°C culture conditions compared with the 22, 33 and 37°C cultures. Group 3 included the accumulated metabolites dependent on the temperature rise. And Group 4 included the metabolites with relative concentrations that were not detectable at each culture temperature.The categorised metabolites in Groups 1 to 3 are summarised in and shown in the representative metabolic pathway maps in.
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