Cryopreservation and the age of the allotransplant

For centuries, reconstructive surgeons have restored form and function with autografts. These techniques are highly effective, but they are associated invariably with donor site morbidity. To avoid this, surgeons have long dreamed of using cadaveric sources for reconstructive material. However, allografts have two major limitations: rejection and limited donor tissue. In order to limit rejection, the allograft must be rendered more tolerable to the host or the host must be rendered more tolerant of the allograft. Both strategies have been used with considerable success in recent years. As understanding of the human immune response increases, clinical immunosuppressive regimens will undoubtedly become less morbid, and the indications for allotransplantation will broaden. This will place an even greater burden on the already small donor pool. One way to relieve this burden would be through the development of strategies for the long-term preservation of donated tissues and organs. Cryopreservation has been used clinically for decades, and recent advances in the field have allowed the preservation of an ever widening array of tissues and organs. As cold storage has been shown to reduce the antigenicity of parts, cryopreservation may actually serve to improve the survival rate of transplanted parts, as well as increase their availability. As the era of autotransplantation gives way to the age of allotransplantation, cryopreservation will play an increasingly important role.     

Current techniques and the future of lung preservation

Although lung transplant remains the only option for patients suffering from end-stage lung failure, donor supply is insufficient to meet demand. Static cold preservation is the most common method to preserve lungs in transport to the recipient; however, this method does not improve lung quality and only allows for 8 h of storage. This results in lungs which become available for donation but cannot be used due to failure to meet physiologic criteria or an inability to store them for a sufficient time to find a suitable recipient. Therefore, lungs lost due to failure to meet physiological or compatibility criteria may be mitigated through preservation methods which improve lung function and storage durations. Ex situ lung perfusion (ESLP) is a recently developed method which allows for longer storage times and has been demonstrated to improve lung function such that rejected lungs can be accepted for donation. Although greater use of ESLP will help to improve donor lung utilization, the ability to cryopreserve lungs would allow for organ banking to better utilize donor lungs. However, lung cryopreservation research remains underrepresented in the literature despite its unique advantages for cryopreservation over other organs. Therefore, this review will discuss the current techniques for lung preservation, static cold preservation and ESLP, and provide a review of the cryopreservation challenges and advantages unique to lungs.     

The resurgent landscape of xenotransplantation of pig organs in nonhuman primates

Organ shortage is a major bottleneck in allotransplantation and causes many wait-listed patients to die or become too sick for transplantation. Genetically engineered pigs have been discussed as a potential alternative to allogeneic donor organs. Although xenotransplantation of pig-derived organs in nonhuman primates(NHPs) has shown sequential advances in recent years, there are still underlying problems that need to be completely addressed before clinical applications, including(i) acute humoral xenograft rejection;(ii) acute cellular rejection;(iii) dysregulation of coagulation and inflammation;(iv) physiological incompatibility; and(v) cross-species infection. Moreover, various genetic modifications to the pig donor need to be fully characterized, with the aim of identifying the ideal transgene combination for upcoming clinical trials. In addition, suitable pretransplant screening methods need to be confirmed for optimal donor-recipient matching, ensuring a good outcome from xenotransplantation. Herein, we summarize the understanding of organ xenotransplantation in pigs-to-NHPs and highlight the current status and recent progress in extending the survival time of pig xenografts and recipients. We also discuss practical strategies for overcoming the obstacles to xenotransplantation mentioned above to further advance transplantation of pig organs in the clinic.     

Persufflation (or gaseous oxygen perfusion) as a method of organ preservation

Improved preservation techniques have the potential to improve transplant outcomes by better maintaining donor organ quality and by making more organs available for allotransplantation. Persufflation, (PSF, gaseous oxygen perfusion) is potentially one such technique that has been studied for over a century in a variety of tissues, but has yet to gain wide acceptance for a number of reasons. A principal barrier is the perception that ex vivo PSF will cause in vivo embolization post-transplant. This review summarizes the extensive published work on heart, liver, kidney, small intestine and pancreas PSF, discusses the differences between anterograde and retrograde PSF, and between PSF and other conventional methods of organ preservation (static cold storage, hypothermic machine perfusion). Prospective implications of PSF within the broader field of organ transplantation, and in the specific application with pancreatic islet isolation and transplant are also discussed. Finally, key issues that need to be addressed before PSF becomes a more widely utilized preservation strategy are summarized and discussed.     

From the freezer to the clinic: Antifreeze proteins in the preservation of cells,tissues, and organs

Understanding the mechanisms by which natural anti‐freeze proteins protect cells and tissues from cold could help to improve the availability of donor organs for transplantation.

The first successful organ transplant in humans was performed in 1954 by Joseph Murray, who used a patient’s twin as a kidney donor. Murrays’ breakthrough paved the way for organ transplantation and the number of transplanted organs has grown ever since. For example, in 2017, a total of 139.024 solid organs—mostly kidney, liver, heart, lung, pancreas, and small bowel—were transplanted (Fig 1A). But this number only reflects 10% of the worldwide need; many patients still die of end‐stage organ failure while on a waiting list. The limited number of donor organs contributes only partially to this shortage. Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime. In fact, the percentage of donor organs that are unused is estimated to range from around 25% for kidneys and livers up to 70–80% for hearts and lungs (Giwa et al, 2017); Fig 1B).Open in a separate windowFigure 1Organ transplantation and preservability statusStatistics show a positive correlation between the duration of ex vivo preservation and the number of organ transplants. Number of solid organs transplanted in 2017 (A). Percentage of organs failed to be transplanted (B). Duration of solid organ ex vivo preservation in static cold storage (C). Sources: Data from the Global Observatory on Donation and Transplantation and (Parsons et al, 2014), (Guibert et al, 2011) and (Editorial: Buying time for transplants (2017))

Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime.

To address the shortage of donor organs and decrease the number of organs that go to waste, biobanks could efficiently store viable tissues and organs until transplantation. Yet, the current standard for ex vivo preservation of donor organs is static cold storage (4–8°C) which, depending on the organ, ensures viable conservation for only some hours; hearts are typically viable for a maximum of only 4 h (Fig 1C). In addition, this approach leads to hypothermic damage and to ischemia/reperfusion injury.Hence, there is an urgent need for strategies that prolong the viable preservation of donor organs. Two main strategies have emerged for cryopreservation and subzero storage, both of which cool tissues below the freezing point. While subzero storage just below 0°C may suffice for short‐term preservation, cryopreservation at −80°C or even lower temperatures is required for long‐term storage in biobanks. A proof‐of‐principle study already demonstrated that subzero preservation extended the preservation of rat hearts up to 24 h after collection (Amir et al, 2004); cryopreservation of whole hearts is currently not possible. The main reason is that lowering the temperature below the freezing point of water leads to ice formation, which causes cell damage and destroys tissues. One of the main challenges in biomedical research for organ transplantation is therefore finding non‐toxic and biocompatible antifreeze compounds that enable subzero storage and cryopreservation without causing tissue damage. An additional benefit is a larger time window to perform evaluation in terms of organ size and human leukocyte antigens matching and preparing the recipient patient to increase the chance of a successful transplantation.     

Review of vitreous islet cryopreservation

Transplantation of pancreatic islets for the treatment of diabetes mellitus is widely anticipated to eventually provide a cure once a means for preventing rejection is found without reliance upon global immunosuppression. Long-term storage of islets is crucial for the organization of transplantation, islet banking, tissue matching, organ sharing, immuno-manipulation and multiple donor transplantation. Existing methods of cryopreservation involving freezing are known to be suboptimal providing only about 50% survival. The development of techniques for ice-free cryopreservation of mammalian tissues using both natural and synthetic ice blocking molecules, and the process of vitrification (formation of a glass as opposed to crystalline ice) has been a focus of research during recent years. These approaches have established in other tissues that vitrification can markedly improve survival by circumventing ice-induced injury. Here we review some of the underlying issues that impact the vitrification approach to islet cryopreservation and describe some initial studies to apply these new technologies to the long-term storage of pancreatic islets. These studies were designed to optimize both the pre-vitrification hypothermic exposure conditions using newly developed media and to compare new techniques for ice-free cryopreservation with conventional freezing protocols. Some practical constraints and feasible resolutions are discussed. Eventually the optimized techniques will be applied to clinical allografts and xenografts or genetically-modified islets designed to overcome immune responses in the diabetic host.     

Review of vitreous islet cryopreservation: Some practical issues and their resolution

Transplantation of pancreatic islets for the treatment of diabetes mellitus is widely anticipated to eventually provide a cure once a means for preventing rejection is found without reliance upon global immunosuppression. Long-term storage of islets is crucial for the organization of transplantation, islet banking, tissue matching, organ sharing, immuno-manipulation and multiple donor transplantation. Existing methods of cryopreservation involving freezing are known to be suboptimal providing only about 50% survival. The development of techniques for ice-free cryopreservation of mammalian tissues using both natural and synthetic ice blocking molecules, and the process of vitrification (formation of a glass as opposed to crystalline ice) has been a focus of research during recent years. These approaches have established in other tissues that vitrification can markedly improve survival by circumventing ice-induced injury. Here we review some of the underlying issues that impact the vitrification approach to islet cryopreservation and describe some initial studies to apply these new technologies to the long-term storage of pancreatic islets. These studies were designed to optimize both the pre-vitrification hypothermic exposure conditions using newly developed media and to compare new techniques for ice-free cryopreservation with conventional freezing protocols. Some practical constraints and feasible resolutions are discussed. Eventually the optimized techniques will be applied to clinical allografts and xenografts or genetically-modified islets designed to overcome immune responses in the diabetic host.     

Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs - amputated digits - xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Individuality: the barrier to optimal immunosuppression

Immunosuppressive therapy aims to protect transplanted organs from host responses. Individuals have unique repertoires of responses to foreign antigens and toxic reactions to immunosuppressants; the former determining the type or intensity of rejection reactions and the latter influencing the severity of iatrogenic effects. Because existing agents target molecules that are widely distributed in tissues, new strategies must selectively block lymphoid cells only, disrupt alloresponses but not innate immune responses, interact synergistically with other agents, facilitate the homeostatic process that naturally leads to graft acceptance and ideally only interrupt donor-specific responses. Approaches presently under investigation aim to alter cell trafficking, or selectively deviate the maturation of antigen-presenting cells or inhibit lymphocyte-activation cascades - events that are crucial to rejection responses.     

Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs—amputated digits—xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Face transplantation: where do we stand?

The recent clinical cases of hand and composite tissue allotransplantation opened a new era in the practice of reconstructive surgery. Some have suggested that face (allo)transplantation could be the next step to benefit patients whose conditions cannot be addressed by conventional techniques of reconstructive surgery using autologous tissues. This article reviews the current status of science regarding the prospect of human face transplantation. The main issues fall into three categories: (1) the surgical challenge of the procedure, specifically regarding vascular viability and functional recovery of the graft; (2) the risks of side effects from life-long immunosuppression necessary to prevent graft rejection; and (3) the ethical debate and the effects of the procedure on the population. Although face transplantation could one day be performed and extend the boundaries of reconstructive surgery, there are currently many obstacles that need to be overcome first.     

Twisting immune responses for allogeneic stem cell therapy

Stem cell-derived tissues and organs have the potential to change modern clinical science. However, rejection of allogeneic grafts by the host's immune system is an issue which needs to be addressed before embryonic stem cell-derived cells or tissues can be used as medicines. Mismatches in human leukocyte class I antigens and minor histocompatibility antigens are the central factors that are responsible for various graft-versus-host diseases. Traditional strategies usually involve suppressing the whole immune systems with drugs. There are many side effects associated with these methods. Here, we discuss an emerging strategy for manipulating the central immune tolerance by naturally "introducing" donor antigens to a host so a recipient can acquire tolerance specifically to the donor cells or tissues. This strategy has two distinct stages. The first stage restores the thymic function of adult patients with sex steroid inhibitory drugs (LHRH-A), keratinocyte growth factor (KGF), interleukin 7 (IL-7) and FMS-like tyrosine kinase 3 (FLT3). The second stage introduces hematopoietic stem cells and their downstream progenitors to the restored thymus by direct injection. Hematopoietic stem cells are used to introduce donor antigens because they have priority access to the thymus. We also review several clinical cases to explain this new strategy.     

Vitrification of kidney precursors as a new source for organ transplantation

Kidney transplantation from deceased or living human donors has been limited by donor availability as opposed to the increasing demand, and by the risk of allograft loss rejection and immunosuppressive therapy toxicity. In recent years, xenotransplantation of developed kidney precursor cells has offered a novel solution for the unlimited supply of human donor organs. Specifically, transplantation of kidney precursors in adult hosts showed that intact embryonic kidneys underwent maturation, exhibiting functional properties, and averted humoural rejection post-transplantation from non-immunosuppressed hosts. Even if supply and demand could be balanced using xenotransplants or lab-grown organs from regenerative medicine, the future of these treatments would still be compromised by the ability to physically distribute the organs to patients in need and to produce these products in a way that allows adequate inventory control and quality assurance. Kidney precursors originating from fifteen-day old rabbit embryos were vitrified using Cryotop® as a device and VM3 as vitrification solution. After 3 months of storage in liquid nitrogen, 18 kidney precursors were transplanted into non-immunosuppressed adult hosts by laparoscopy surgery. Twenty-one days after allotransplantation, 9 new kidneys were recovered. All the new kidneys recovered exhibited significant growth and mature glomeruli. Having achieved these encouraging results, we report, for the first time, that it is possible to create a long-term biobank of kidney precursors as an unlimited source of organs for transplantation, facilitating the inventory control and distribution of organs.     

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.     

When oxygen runs short: the microenvironment drives host-pathogen interactions

Pathogens that colonize or infect the human body have to face varying oxygen concentrations within different organs. Inflammation itself promotes oxygen consumption within affected tissues and creates a low oxygen environment. As a consequence, pathogens and the host immune system have to adapt to rapid changes in oxygen availability. Here we summarize recent findings on the adaptation of pathogens, host defense mechanisms and treatment strategies against intracellular pathogens in a low oxygen environment.     

Regenerating the epigenome

The ability of some organisms to regenerate parts of their body has fascinated scientists for decades. The process of regeneration depends on the potential of certain cells to proliferate and contribute to the formation of new tissue. Organisms have evolved two strategies by which to achieve this: the maintenance of adult stem cells and the induction of stem-cell properties in differentiated cells. In both cases, cells must undergo extensive epigenetic reprogramming to attain the specialized functions of the new tissue. Ultimately, the regenerative capacity of a tissue might depend on the plasticity of the cellular epigenome, which determines the ability of the cell to respond to injury-related signals. Understanding this epigenetic plasticity will allow the development of strategies to stimulate the regeneration of damaged tissues and organs in humans.     

Preservation methods for kidney and liver

With the successful testing of the immunosuppressive effects of cyclosporine in transplant patients in 1978, the field of organ transplants began an exponential growth. With that, the field of organ preservation became increasingly important as the need to increase preservation time and improve graft function became paramount. However, for every patient that receives a transplanted organ, there are 4 more on the waiting list. In addition, a patient dies from the lack of a transplant almost every 1½ hour. To alleviate this donor crisis, there is a need to expand the donor pool to marginal donor organs. The main reason these organs are underutilized is because the current method of static preservation, simple cold storage, is ineffective. This article will provide a general review of the methods of preservation including simple cold storage, hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation. In addition, the article will provide a review of how these dynamic preservation methods have improved the recovery and preservation of marginal donor organs including donation after cardiac death and fatty livers.