WO2022233642A1

WO2022233642A1 - Methods for removing leucocytes from isolated organs

Info

Publication number: WO2022233642A1
Application number: PCT/EP2022/061048
Authority: WO
Inventors: Anne-Li SIGVARDSSON
Original assignee: Xvivo Perfusion Ab
Priority date: 2021-05-06
Filing date: 2022-04-26
Publication date: 2022-11-10

Abstract

The present invention provides a method of removing leucocytes from an isolated organ, the method comprising the step of flushing the isolated organ with a flush solution, wherein the flush solution is at a temperature of 34°C to 42°C. The present invention makes uses of a previously unknown temperature dependent relationship for interaction between leucocytes and vascular endothelium in isolated organs.

Description

Methods for Removing Leucocytes from Isolated Organs

Field of Invention

The present invention relates to the field of organ transplantation. The inventors have discovered a new technique for removing leucocytes from an organ before it is transplanted. This technique has the potential to improve post-transplantation immunologic outcomes.

Background to the Invention

Immune cells, also known as leucocytes, are present in most types of organs and tissues, both as resident cells within the tissue and as cells attached to the vascular bed. During standard cold organ flushing in an organ donor, some of the vascularised cells are removed, but many remain. The remaining cells will follow the organ to the recipient. Hence organ transplantation involves the immune responses of leucocytes from two individuals, the donor from which the organ has been retrieved, and the recipient into which it is transplanted. The natural mechanisms for each of them is to fight the other as the other is considered a foreign invader. In other words, once the leucocytes from the donated organ are in the recipient they are capable of inducing a graft versus host response. Such a reaction is rarely noticed during solid organ transplantation but is commonly seen during bone marrow transplants. However, even if the graft versus host response is not clinically noticed during solid organ transplantation, it most likely occurs to a degree and impacts the future success of the organ transplantation.

Ex vivo organ perfusion (EVOP) has been utilized for more than 20 years to evaluate organs before transplantation. An example of this is ex vivo lung perfusion (EVLP) that has been in clinical use since early 2000. During EVLP, lungs are gradually warmed up through perfusion with a heated perfusate to normothermia or near normothermia i.e. 35-37°C with a plasma-like perfusate such as STEEN Solution (TM), which is described in WO2002/35929 Al. When the lungs reach a temperature of about 32°C, ventilation of the lungs is initiated. Usually EVLP lasts for about 2-6 hours, during which time the function of the lung is evaluated. At end of EVLP, the procedure is finalised with a cold flush, which involves flushing the organ with a solution at a temperature of about 2- 8°C. The cold preservation solution is typically one such as Perfadex Plus (TM) which is described in WO2018/133921 Al. The cold flush can immediately follow perfusion with the heated perfusate. Alternatively and more commonly the temperature of the system is reduced until the lungs have cooled down to between about 15-32°C, depending on the protocol used during continued perfusion and ventilation, before they are finally flushed with cold solution at about 2-8°C.

Organs other than lungs, especially kidneys, are more commonly perfused at hypothermic conditions at about 4-15°C. As such they are perfused with oxygenated fluid during transportation as a means to avoid ischemic damage to the organ. Although the energy demand of the organ is reduced at the hypothermic condition, continuous oxygenation has been considered important to maintain basic cellular metabolism. Normothermic or near normothermic perfusion can be used to evaluate kidneys and other organs such as livers and hearts that are commonly perfused cooled during transportation and then warmed up for evaluation purposes.

Leucocytes are a large heterogeneous group of cells with various immune functions. For instance, many cells, including monocytes, neutrophils and B cells, have antigen- presenting capacity (APC). These passenger APC cells can cause direct presentation and stimulate the recipient T cells after organ transplantation. The effect of “passenger” leucocytes, which are transplanted with an organ, is not well understood. Some types of passenger leucocytes have been shown to promote tolerance to the organ. Tolerance or adaption has mainly been related to recipients receiving kidney transplantations. Regulatory T-cells have been postulated to participate in promoting tolerance. Interestingly, those that have received kidney plus heart transplantation from the same donor can also develop tolerance, whereas those that only have received a heart transplantation rarely do so. Only anecdotal cases of tolerance have been reported among lung transplantation recipients. This is likely related to the exposed position of the lungs. Lungs are in essence in direct contact with the outer world and are constantly affected by microorganisms, toxins and pollution in the air, resulting in a more or less constant state of inflammation. This constant attack on the lungs from external elements results in a continuous state of defence and makes the lungs the solid organ that is most vulnerable to rejection. The lung contains a large pool of immune cells, and subclasses of donor leucocytes are fundamentally involved in acute allograft rejection. Rejection is the state when the recipient’ s immune response attacks the transplanted organ or in other words there is a host versus graft response.

Leucocyte filters are commonly used in the EVOP circuit, but are largely insufficient and bind only a small proportion of the released leucocytes.

Rejection of donated organs to any extent is a major problem, and in some cases can cause the transplant to fail completely. The aim of the present invention is to improve post-transplantation immunologic outcomes, i.e. to promote optimal acceptance of a transplanted organ, and reduce the likelihood of an organ being rejected.

Summary of the Invention

According to a first aspect, the present invention provides a method of removing leucocytes from an isolated organ, the method comprising the step of flushing the isolated organ with a flush solution, wherein the flush solution is at a temperature of 34°C to 42°C.

According to a second aspect, the present invention provides a method of preparing an isolated organ for transplantation, the method comprising the steps of: perfusing the isolated organ with a perfusate; flushing the isolated organ with a flush solution which is at a temperature of 34°C to 42°C; and then flushing the isolated organ with another flush solution which is at a temperature of 2°C to 8°C.

According to a third aspect, the present invention provides the use of a warm flush for removing leucocytes from an isolated organ, wherein the warm flush comprises flushing the isolated organ with a flush solution, wherein the flush solution is at a temperature of 34°C to 42°C.

In all aspects the present invention provides a new means to remove leucocytes, particularly activated leucocytes, from an organ. The means is a flushing step, in particular flushing the isolated organ with a flush solution at a temperature of between 34°C and 42°C. This is hereinafter referred to as a warm flush. The present invention makes uses of a previously unknown temperature dependent relationship for interaction between leucocytes and vascular endothelium. The definition of a flush is that the solution performs only one passage through the organ before it is removed from circulation. This in contrast to perfusion, where the solution is recirculated.

As discussed above, during transplantation of solid organs, not only the organ but also passenger leucocytes are transplanted. These transplanted leucocytes affect the immune response between the transplanted organ and the recipient. The multifactorial interactions between graft and host make the system complex and a total understanding is far from reached. However, some leucocyte cell types, and especially “activated” leucocytes that have been circulating in the perfusate during organ perfusion for hours, could be characterised as solely negative when transplanted along with the organ.

Leucocytes can be “activated” before or during the transplantation process, on retrieval, or when they spend time in the circulating perfusate. The whole procedure inevitably involves cell damage due to surgical and ischemic injury, and the accumulation of damaged cells and molecules in such a closed system induces immune activation. Moreover, the ex vivo perfusion circuit consists of artificial surfaces, and the foreign materials are known agonists of leucocyte activation. Sensing that they are in a dangerous or foreign environment, for example through interactions with surfaces of the EVOP equipment, or with the perfusate, the leucocytes become primed to mount an immune response. The extracorporeal treatment leads to systematic inflammatory responses via profound leucocytes activation and cytokine secretion. So called “activated” leucocytes are more aggressive than non-activated leucocytes, and therefore more likely to initiate a damaging graft versus host response immediately or shortly after transplantation, which in turn can lead a further damaging host versus graft response, leading to rejection episodes. Removal of as many of those cells as possible before transplantation should improve transplantation outcomes and reduce early and long-term rejection episodes.

Taking the example of lungs, these are very immune-active organs and comprise billions of passenger leucocytes upon retrieval. During EVLP, the lungs are usually perfused for between two and six hours at around 35-37°C. During this time, the perfusate accumulates about 1-4 billion leucocytes, sometimes more, that are released from the lungs into the perfusate. The numbers of leukocytes accumulating are largely dependent on the size of the lungs, where larger lungs results in more leucocytes per ml. These leucocytes become activated as above. Normally an EVLP is finalised through lowering the temperature on the circuit to prepare the lungs for transplantation. Before being transplanted the lungs are then flushed with cold flush solutions at about 2-8°C. This is to reduce warm ischemia during the transplant operation.

The inventors have discovered that there is a temperature dependency for leucocyte interactions with the vascular endothelium during ex vivo perfusion of an isolated organ. The result of this is that cooling to around 20°C causes 80-90% of the leucocytes released in the perfusate to return back to the organ, a phenomenon termed “homing” by the inventors. The leucocytes stay “homed” in the organ until the temperature is once again increased, which would be at reperfusion in the recipient.

The homing effect is very fast and so during the cooling typically done at the end of EVOP to around 20°C it is thought that 80-90% of all the leucocytes that had been released in the perfusate return back to the organ. Even if cooling is only done down to 32°C before a cold flush is used about 30-40% of the leucocytes present in the perfusate are estimated to home back to lung. Considering that there are about 1-4 billion leucocytes present in the perfusate after an EVLP, this means that a significant population of leucocytes that have been activated during EVLP home back to the lung. Although lower absolute numbers are expected for liver, hearts and kidneys, a significant population of cells would still home back to the organ after being activated during EVOP.

Accordingly, the present invention makes use of a warm flush at 34 - 42°C, before any final cold flush or cooling on the circuit is done. Since the warm flush does not cause homing, the billion or so activated leucocytes present in the perfusate are permanently removed from the organ, and are not transplanted into the recipient. Although the example of lungs has been used, it is thought that the homing effect would be present in all organs, so the warm flush of the invention would be equally effective for other organs. This deceptively simple step, which is easy and inexpensive to introduce, reduces the likelihood of a damaging host versus graft response, improves post transplantation immunologic outcomes and could reduce rejection episodes.

The leucocytes comprise a wide flora of different cell types including T cells and immature neutrophils. Most have mainly inflammatory purposes, but some are more immune-silencing. For example, neutrophils and cytotoxic T cells are pro- inflammatory, whereas regulatory T cells are more immune silencing and considered important for development of tolerance to a transplanted organ. Immature neutrophils are persistent leucocytes with an aggressive potential. Ideally, a transplanted organ should maintain regulatory T-cells to promote tolerance but have as many of the cytotoxic T cells removed as possible to evade an immediate graft versus host response. If the organ is a lung(s) it might be important to maintain also other CD45+ cell types such as macrophages and eosinophils to protect the lungs from infections and pollution post-transplantation.

The present inventors have found that T cells are the most abundant type recovered in the perfusate during EVLP. Whilst the immune-modulating types like helper T cells and regulatory T cells are detected in the perfusate, the largest T cell proportion seen are the effector cytotoxic T cells. These cells carry out direct killing function of any target, playing an important role against viral infection. Moreover, they would also take part in immune modulation via production of cytokines and chemokines which induce a direct immune response post-transplantation. In addition to T cells, the inventors have found that neutrophils also make up a large number of cells present in the EVLP perfusate. They are pro-inflammatory, and play an important role in innate immune response. In particular, the immature neutrophils which are detected as the dominant type in EVLP, generally have a longer life span and resistance to apoptosis. The “maturity” can be gained ex vivo, e.g., exhibiting different levels of receptors and granules.

The inventors have discovered that the CD45+ cell composition in the perfusate is mainly pro-inflammatory cells. Permanent removal of these, while largely maintaining the immune silencing ability and the natural immune defences, particularly in lungs, is considered an important advantage of the invention.

Advantageously use of a warm flush at end of EVOP could replace the need for a leucocyte filter as the flush would remove at least as many activated leucocytes from the perfusate as a leucocyte filter.

In addition to leucocytes, other cell types, such as for example endothelial cells that are released from the perfused tissue into the perfusate, can induce a host immune response upon transplantation. Therefore, these cells should preferably not be allowed as passengers in the organ during transplantation, to reduce the risk of rejection episodes. As is shown in example 4, a warm flush is about five times more efficient in removing cells from the perfusate compared to a cold flush only.

Description of the Figures

Fig 1 is a graph that reflects the results from Example 1 and shows the linear relationship between perfusate temperature and the CD45+ cell count in the perfusate as well as the return of the cells to the perfusate when temperature is again increased. Fig 2 is a graph that reflects the results from Example 2 and shows that cooling of the lungs on the machine (T2 to T3), results in a 70-80% decrease of the CD45+ cell count in the perfusate. The cells do not reappear after removal of the lung and upon increase of the temperature of the circuit without the lungs (T4 to T5).

Fig 3 is a graph that reflects the results from Example 3 and shows that if removal of the lung is done before cooling of the system, there is no change to the CD+45 cell count in the perfusate upon cooling, showing that indeed the CD45+ cells are homing to the lung during cooling instead of sticking to the artificial circuit.

Fig 4. indicates the main subsets of CD45+ cells present in the perfusate. As can be seen T-cells are the dominant cell type. These cells are further characterised to predominantly be cytotoxic T-cells. Among the granulocytes the immature neutrophils are dominating.

Detailed Description

The present invention makes use of a previously unknown temperature dependency of leucocyte interaction with isolated organs. This is that at approximately normothermic conditions (34°C to 42°C), billions of leucocytes will leave the isolated organ when it is being perfused and circulate freely in the perfusate. However, the inventors have discovered a homing effect which occurs when the temperature is lowered and causes the leucocytes to progressively return to the organ. The inventors have discovered that when the temperature is lowered to 20°C, most, usually around 80%, of the billion(s) of leucocytes that have been released to the perfusate return to the organ.

The leucocytes circulating in the perfusate are in contact with artificial surfaces such as plastic tubing, and oxygenator membranes etc. for hours and are therefore most likely to have become activated. With activated status they might pose an immediate threat to the recipient, by initiating a devastating counterattack from the recipient’s immune response to the transplanted organ, which could eventually lead to rejection of the transplanted organ. Some of these cells are also antigen presenting cells that could assist the recipient’s immune system in a host versus graft rejection response. Removal of these activated leucocytes cells before transplantation could improve post transplantation immunological outcomes, improving graft survival and well-being in the recipient.

In the present invention this removal is achieved through the simple step of a warm flush. By warm we mean that the isolated organ is flushed with a flush solution at a temperature of 34°C to 42°C, preferably 37°C to 42°C most preferably 37.5 °C to 39°C. By flush we mean that the flush solution performs only one passage through the organ before it is removed from the circulation. This in contrast to perfusion, where the solution is recirculated. This means that the flush solution is able to carry away the leucocytes that have been released during continuous perfusion. It is estimated that about 0.2 - 4 litres, usually 0.5 to 3, 1 to 3 or 2 to 3 litres of warm flush solution would be sufficient, depending on the organ size and characteristics.

The warm flush could be implemented by itself as a standalone way to remove leucocytes from an isolated organ, for example it may be performed directly after organ procurement to improve removal of leucocytes activated in the donor. Alternatively the warm flush can be used as part of a method of preparing an isolated organ for transplantation. Usually it will be used in combination with an EVOP process, such as EVLP. This will involve an initial step of perfusing the isolated organ with a perfusate. The EVOP perfusion system might be operated at hypothermic (about 4- 15°C), sub- normothermic (about 15-30°C), near normothermic (about 30-36°C) or normothermic (about 37°C) conditions. In one embodiment of the present invention, hyperthermic conditions between 37-42 °C is used during whole or part of ex vivo organ perfusion to release more cells into the perfusate before the warm flush. In this embodiment the perfusate is preferably at 37°C to 42°C, more preferably 37.5°C to 39°C. The invention is particularly important after a period of continuous isolated organ perfusion as the recirculation will activate the leucocytes and it is in particular those leucocytes that the invention aims to remove. Before the conventional step of cooling the organ on the perfusion circuit or through a cold flush, in the invention a warm flush is used to remove a high proportion of the activated leucocytes from the isolated organ. After the warm flush, the usual cooling steps can be carried out, including that the isolated organ is flushed with another flush solution at a temperature of between 2°C and 8°C, i.e. a cold flush, to make the organ ready for storage and to transplant. An intermediate tempered flush with a solution at about 15-30°C or more preferably 18-25°C might be used between the warm flush and cold flush. Cooling down the organ with a cooled flush might induce vasoconstriction that prevents removal of the activated CD45+ cells as effectively as a warm flush can. Suitable cold flush solutions are known, such as Perfadex Plus (TM) which is available from XVIVO Perfusion (TM). The cold flush solution can be the same as the solution used in the warm flush, but is usually different, as solutions have been developed that are adapted to warm and cold conditions, for example in relation to temperature dependency of pH, so it is optimal to use the corresponding solution.

The invention will work with any suitable organ. Usually the organ will be a heart, lung(s), liver, kidney, pancreas or all or part of a small bowel. It might also be used for tissue perfusion as for example during limb or facial perfusion. The organ will have been retrieved from a donor and usually be intended for organ transplantation. In a preferred embodiment the organ is a lung or lungs.

By isolated organ we mean an organ that has been isolated from a donor in the usual manner. The isolated organ is usually ex vivo. However, it is also possible for the organ to be circulatory isolated in vivo. The isolated organ is generally not transplanted into the body from which it was retrieved, but into another individual, in the conventional manner. In another embodiment the isolated organ, tissue or limb might be circulatory isolated in a patient during localised treatment as for example during isolated perfusion used in cancer treatment or for local treatments of severe infections.

Any solution adapted for use at normothermic or hyperthermic temperature can be used as the warm flush solution. The warm flush solution is typically the same as the perfusate which is used in the near normothermic or normothermic perfusion step. Suitable solutions have been used for decades and are well known to the skilled person. An example of a suitable solution is STEEN Solution (TM) which is commercially available from XVIVO Perfusion (TM). STEEN solution comprises human serum albumin and dextran 40 together with glucose and balanced extracellular electrolytes. Another example might be physiologically buffered XVIVO Heart Solution (TM) which is a cardioplegic solution with increased K+ to cease the heart beats and Mg2+ to protect the vascular endothelium from the increased K+ concentration. This solution is currently in clinical trials. Although, a serum albumin and dextran solution is considered most suitable for the invention in order to preserve the vascular endothelium, other suitable solutions might be used. Generally, cold perfusion and preservation solutions are not optimal for warm flush unless the pH is temperature adjusted for the temperature of use.

In a preferred embodiment of the invention, fucoidan, a specific immune cell- endothelium blocker agent is added to the warm flush solution. Fucoidan is shown to inhibit leucocyte adhesion and accumulation in the vasculature by binding to the selectin proteins. Thereby, the “homing” of the leucocytes would be hampered in the presence of fucoidan.

Blocking the “homing” effect might be enhanced through use of increased levels of Dextran 40 in the flush solution compared to the concentration in the STEEN Solution (5 g/1) or in the buffered XVIVO Heart Solution (1 g/1). The dextran 40 concentration might be increased to 10-80 g/1 or more preferably to 20 to 60 g/1, or 20-50 g/1 or most preferably between 20-40 g/1 based on the total flush solution. While increasing the Dextran 40 concentration, the human serum albumin concentration might be decreased to avoid an extreme hyper-oncotic pressure in the solution. A solution only using Dextran 40 as the macromolecule might also be used. In that case the Dextran concentration would be between 20-60 g/1, depending on the organ to perfuse. The warm flush solution might also be free from ions, such as Ca2+, Mg2+ and Mn2+ which promote the CD45+ cell-endothelial cell interaction; or include Ca, Mg and Mn ion chelators, as well as agents that antagonise the cell-cell interaction as discussed above.

Advantageously the main population of CD45+ cells removed with the invention are pro-inflammatory cells, particularly cytotoxic T cells and immature neutrophils. Usually these cytotoxic T cells and immature neutrophils have been activated during EVOP.

In a preferred embodiment the number of leucocytes are measured in the perfusate and then at least once in the flush solution, to indicate that at end of the flush there is at least a 75% reduction, preferably a 90% reduction of leucocytes per volume in the last flush solution effluent compared to in the perfusate, indicating that the procedure has been performed with a sufficient amount of flush solution.

Examples Example 1

Temperature dependency of interaction between leucocytes in the perfusate and the vascular endothelium was evaluated at end of a four-hour EVLP experiment.

The flow cytometry method was used to count the total live leucocyte numbers by staining the cell surface protein CD45 (leucocyte common antigen). Leucocytes are referred to in the Examples as CD45+ cells.

The EVLP was mn with porcine lungs on the XPS (TM) EVLP platform commercially available from XVIVO Perfusion (TM), using the protocol according to the XPS instruction for use. The lungs were perfused with about 2 litres of STEEN solution (TM), which is a perfusate comprising human serum albumin, dextran 40, glucose and electrolytes. A partial perfusate exchange was performed after the first hour of EVLP or after finalization of the warm-up phase. EVLP was n at around 36°C, with the heater/cooler unit set at 38°C. The lengths of the tubing always adjust the temperature somewhat towards ambient temperature.

At end of EVLP, the temperature of the heater/cooler unit was decreased to 15°C. The temperature of the effluent from the lungs, measured in the solution close to the pulmonary veins, was monitored as it was decreasing and perfusate samples were continuously taken to use for CD45+ cell count. It took about 10 minutes to lower the temperature to 26°C. At that point, the temperature setting was once again increased to 38 °C. It took about 10 minutes to reheat the effluent of the perfusate to 36°C and a new sample was drawn for CD45+ cell counting. The results, which are displayed in Figure 1, surprisingly showed a linear relationship between temperature in the perfusate and the number of CD45+ cells found in the perfusate. The results also showed that the reduction in the cell count is completely reversible, as the cell number reached to the same level when perfusate temperature reset to pre-cooling (36°C). The total cell-count is shown per ml perfusate and the total volume of perfusate is 2 liters.

Example 2

To investigate if the CD45+ cells in the perfusate were attached to any part of the perfusion system other than the lungs, four circuits were re-heated after removal of the lungs from the circuit. The mean results are shown in Figure 2. The EVFPs were run according to Example 1, with TO being about 35°C, T1 being about 36°C, T2 being about 36°C, and T3 being about 22°C. The lungs were left on the circuit with cooled perfusate down to between 20-24°C for an hour, between T3 and T4 before removal of the lungs, at T4. When the temperature was increased without the lungs present (T5 is about 37°C), there was no re-introduction of the CD45+ cells into the perfusate, indicating that it is indeed the lungs per se that are the harbouring sites for the CD45+ cells.

In summary Figure 2 shows that cooling of the lungs on the machine at (T2 to T3), results in a 70-80% decrease of the CD45+ cell count in the perfusate. The reduced cells do not reappear upon increase of the temperature of the circuit (T4 to T5) after the lung was removed at T4, so must have been homed in the lungs, rather than in the EVLP equipment.

The total cell-count is shown per ml perfusate and the total volume of perfusate is 2 liters.

Example 3

To further investigate whether or not the circuit is responsible for removal of CD45+ cells from the perfusate, the temperature was lowered after removal of the lungs from the circuit. The EVLPs were run for five hours according to Example 1, apart from that the lungs were removed at T4.

The temperature of the heater/cooler unit was set to 38°C at TO, resulting in a perfusate temperature of about 36°C. This temperature was maintained until removal of the lung at T4 after which the temperature of the heater/cooler were set to 15°C resulting in a temperature at T5 of about 20-24°C. When the temperature of the perfusate was lowered after removal of the lungs from the system, there was no decrease of the cells, also confirming that the disappearance of the CD45+ cells from the perfusate is due to homing of the cells to lung. (See Figure 3)

The total cell-count is shown per ml perfusate and the total volume of perfusate is 2 liters. Example 4

In a fourth example the use of a warm flush in addition to a cold flush compared to a cold flush only was investigated.

Method Porcine lungs were retrieved from Swedish domestic pigs and were flushed with and stored in cold Perfadex Plus (XVIVO Perfusion AB) for one hour. The lungs were placed on LS machine (XVIVO Perfusion AB). The lungs were gradually rewarmed on the circuit for one hour and after that a physiological pulmonary artery pressure and flow were used. Three liters of a STEEN Solution like perfusate (See WO2002/35929 Al) were used in the circuit. After one hour of full flow EVLP, 500 ml of perfusate was removed. The removed perfusate was divided into ten 50 ml Falcon tubes and centrifuged. The perfusate was saved and the cells in the pellet were stained using CellTracer violet 1:4000. The cells were incubated with the dye for 20 minutes at 37°C. After additional centrifugation step the cells were resuspended in 250 ml of the removed perfusate. The EVLP was run for an additional hour to allow the traced cells to disseminate throughout the tissue and system. At end of EVLP the lungs were flushed either with cold Perfadex Plus or warm STEEN like perfusate followed by cold flush with Perfadex Plus. CD45+ cells and traced cells were counted in the whole perfusate, the flush solutions and in the leucocyte filter of the circuit The removal of cells from the leucocyte filter utilised Trypsin digestion followed by rinsing.

The cells were counted using flow cytometry. The samples were pre-treated with red blood cell lysis buffer before staining to remove interference with residual red blood cells in the perfusate. Results

Discussion

The actual numbers of leucocytes differ from lungs to lungs. In general terms, the numbers found in the perfusate is about 1 billion CD45+ cells. Not only the actual number but also the properties differ from different lungs, depending on for example ongoing infections before lung retrieval. Therefore, the counting of traced cells is important. These cells are not only CD45+ cells, but also other cells like endothelial cells that have been released from the tissue during perfusion. All donor cells that are released from the tissue upon transplantation might induce immune response in the recipient, making their removal important to reduce the risk of rejection. The data from this example indicate that flushing the lungs at 37°C is much more efficient than the leucocyte filter in removing both CD45+ cells and the traced cells. The data also indicates that a warm flush is more efficient in removing CD45+ cells and even more so in removing traced cells, that have been released from the tissue during the perfusion than a cold flush is. Example 5

During a series of experiments the CD45+ cells present in the perfusate after about 1 hour of warm-up and two hours of EVLP at about 36°C, were further characterised using flow cytometry. Several fluorophore-conjugated antibodies were used to characterize different leucocyte types, including T cells (CD3+), cytotoxic T cells (CD3+CD8+), helper T cells (CD3+CD4+), regulatory T cells (CD4+CD25+), monocytes (CD14+), immature neutrophils (6D10+2B2-) and mature neutrophils (6D10+2B2+). The gating strategies and analysis were performed using FlowJo. As shown in Figure 4, it is clear to see that among all CD45+ cells, the largest population seen in the perfusate was T cells, of which cytotoxic T cells were the most abundant. The total cell-count is shown per ml perfusate and the total volume of perfusate is 2 liters.

Conclusion The present inventors have shown that during EVLP, about 80% of the billion(s) of CD45+ cells that are released during EVLP are returned to the lung when the temperature is lowered on the machine to about 20°C. The absolute level of cells varies between the Examples due to different size lungs being used, but the homing effect is the same. The relationship appears to be strictly moderated by temperature and if the temperature is again increased in the perfusate, the CD45 + cell count returns to pre cooling values (see Fig 1). The inventors have confirmed that the CD45+ cells do not stick to the EVLP circuit as the CD45+ cells do stay in the perfusate if the temperature is lowered after the lungs have been removed (see Figs 2 and 3).

Claims

1. A method of removing leucocytes from an isolated organ, the method comprising the step of flushing the isolated organ with a flush solution, wherein the flush solution is at a temperature of 34°C to 42°C.

2. The method according to claim 1, wherein the flush solution is at a temperature of

37°C to 42°C, preferably 37.5°C to 39°C.

3. The method according to claim 1 or 2, wherein the isolated organ is a liver, a heart, a lung, lungs, a kidney, a pancreas or small bowel, preferably wherein the isolated organ is a lung or lungs.

4. The method according to any preceding claim, wherein the flush solution comprises human serum albumin, dextran 40, glucose and electrolytes.

5. The method according to any preceding claim, wherein the flush solution comprises dextran 40 in an amount of 10-80 g/1, preferably to 20-50 g/1, most preferably 20-40 g/1 based on the total flush solution.

6. The method according to any preceding claim, wherein the isolated organ is isolated ex vivo.

7. The method according to any preceding claim, wherein the isolated organ is not transplanted into the body from which it was retrieved, but into another individual.

8. The method according to any preceding claim, wherein there is at least a 75% reduction, preferably a 90% reduction of leucocytes per volume in the last flush solution effluent compared to in the perfusate.

9. The method according to any preceding claim, wherein the flush solution additionally comprises an immune cell endothelium blocker agent, preferably wherein the immune cell endothelium blocker agent is fucoidan.

10. A method of preparing an isolated organ for transplantation, the method comprising the steps of: perfusing the isolated organ with a perfusate; flushing the isolated organ with a flush solution which is at a temperature of 34°C to 42°C; and then flushing the isolated organ with another flush solution, which is at a temperature of 2°C to 8°C.

11. The method according to claim 10, wherein during all or part of the perfusion step, the perfusate is at a temperature of 34°C to 42°C, preferably 37°C to 42°C, most preferably 37.5°C to 39°C.

12. Use of a warm flush for removing leucocytes from an isolated organ, wherein the warm flush comprises flushing the isolated organ with a flush solution, wherein the flush solution is at a temperature of 34°C to 42°C.

13. Use according to claim 12, wherein the leucocytes have been released from the isolated organ during perfusion.

14. Use according to claim 12 or 13, wherein the isolated organ is a lung or lungs, and the warm flush is used to remove leucocytes that have been released into the perfusate during ex vivo lung perfusion.