BACKGROUND:Pulmonary edema due to vascular endothelial injury in donor lungs reduces organ utilization and exacerbates ischemia/reperfusion injury, resulting in poor posttransplant outcomes. Sphingosine-1-phosphate (S1P) improves vascular integrity through S1P receptor 1 (S1PR1) signaling. This study assessed whether S1PR1 agonism during ex vivo lung perfusion (EVLP) reduces vascular permeability and edema formation in human donor lungs. METHODS:S1PR1 agonist CYM5442 was administered to human donor lungs declined for transplant, during 6 h of EVLP, using a paired split-lung model with one lung treated and the other acting as an internal control. Lung physiology, organ weight, vascular endothelial permeability to Evan's blue dye, and direCt Lung Ultrasound Evaluation score for lung water were assessed. Sequential perfusate and tissue samples were collected to evaluate gene and protein expression. RESULTS:During EVLP (n = 7 paired sets), CYM5442 reduced Evan's blue accumulation in bronchoalveolar lavage ( P  = 0.0260) and tissue ( P  = 0.0476). Increases in lung weight were ameliorated and direCt Lung Ultrasound Evaluation scores reduced in the CYM5442-treated group post-EVLP ( P  = 0.0135 and P  = 0.0482, respectively), leading to reduced pulmonary artery and peak airway pressures (interaction P  = 0.0038 and P  < 0.0001). CYM5442 maintained vascular endothelial cadherin expression and reduced interleukin-6 ( P  = 0.0130, 360 min), interleukin-1 beta ( P  = 0.0214, 240 min), and soluble intercellular adhesion molecule-1 ( P  = 0.0067, 360 min) release compared with untreated during EVLP. CONCLUSIONS:Agonism of S1PR1 during EVLP ameliorates pulmonary vascular leak and may be used to reduce the risk of edema formation in donor lungs.

Background & Aim Cellular therapy shows promise in regenerative medicine, but the distribution and fate of transplanted cells is poorly understood. While the literature describes various tracking methods using MRI, PET, SPECT, and Scintigraphy, it is scattered and the terminology inconsistent. To address this, the HESI CT/TRACS committee established the Data Portal for Imaging Cell Therapies (DPICT) database focused on collating the literature. Initial attempts to populate the database by academic, regulatory, and industry experts identified 61 articles. To build on these efforts and ensure a comprehensive collection, we performed a systematic scoping review to collate all the methods of tracking cells that have been used in clinical trials and human whole organ studies where cells are administered in vivo as a therapeutic. Methodology The initial DPICT database papers served as positive controls to validate search terms. In June 2023, we searched English-language papers (post-2001) in Medline Ovid, PubMed, and Scopus using the following structure: (imaging modality) AND (cell label) AND (cell type) AND (humans OR human study OR human studies OR clinical study OR clinical studies OR clinically OR patient* OR perfusion). The titles were screened and excluded followed by the abstracts by two reviewers. Studies deemed potentially suitable for inclusion, or those that could not be definitively excluded based on title and abstract alone, were carried forward for further evaluation. Full-text studies were screened, and any conflicts were resolved collaboratively by members of the CT/TRACS DPICT committee. Results Of 5543 articles identified, 152 were included in the final analysis. The majority of studies tracked CD34+ haematopoietic stem cells (24%), employed MRI (28%) and enrolled patients with a cancer diagnosis (26%) including melanoma, breast cancer and glioblastoma. Key themes included the need for contrast agents with prolonged half-lives for longitudinal studies, high-resolution imaging modalities, and cellular therapies capable of effective target-site migration. Conclusion We developed a custom 'clinical trials filter' as Medline Ovid filters excluded relevant papers. This filter can streamline the population of the DPICT database clearly identifying how this repository of cell tracking articles has been cultivated to benefit the cell-therapy community. This will guide future research by highlighting literature gaps, imaging limitations, and challenges in contrast agents and therapeutic cell action.

Background & Aim Cellular therapy has promising applications in regenerative medicine. As cells respond to their environment, understanding their behaviour in the human body is critical. Novel platforms are required to investigate application of these techniques to human organs, in the pre-clinical setting. In this study, we sought to address this by employing Normothermic Machine Perfusion (NMP) to track 19Fluorine Nanoparticle (19F-NP) labelled THP-1 derived macrophages (THP-1ms) as a positive control for investigation cell biodistribution within a clinical setting. Methodology THP-1 monocytes were incubated in 100ng/ml of Phorbol 12-myristate 13-acetate (PMA) for 48 hrs at 37°C to differentiate into THP-1m macrophages. 20mg/ml 19F-NP (conjugated to a Nile Red fluorophore) was diluted in serum-free RPMI 1640 media and incubated with THP-1ms for 18 hrs at 37°C. 5 × 107 labelled THP-1ms were frozen in liquid nitrogen until a human kidney (which was retrieved for transplant then deemed unsuitable) was accepted for research. The kidney was perfused for 7 hrs using a blood based perfusate at 37°C with biopsies, blood/gas samples, urine measurements and Contrast-Enhanced Ultrasound (CEUS) images being taken throughout. THP-1ms were thawed and arterially infused into the kidney 1 hr into perfusion. Post-perfusion, the kidney was imaged using the In Vivo Imaging System (IVIS) and MRI to further understand cellular localisation. Results The labelling procedure of THP-1ms with 19F-NP is feasible, resulting in high cell viability (>85%), unchanged CD14 expression and strong signal in IF microscopy, IVIS and CEUS. CEUS showed 19F-NP signal within the kidney cortex at all time points after arterial infusion. IVIS confirmed this with increased cortex fluorescent signal post-perfusion. No signal was detected using 19F-MRI. IF microscopy revealed labelled THP-1m within the glomeruli and tubules of kidney tissue throughout the perfusion (figure 1). Conclusion This study demonstrates the feasibility of using 19F-NP labelled THP-1ms for cell tracking in a clinical setting using a NMP model. The findings provide valuable insights into cell fate and biodistribution in an ex vivo human organ context, showcasing the potential of this approach in regenerative medicine research.

Purpose: Single lung transplantation (SLT) for interstitial lung disease (ILD) has dramatically declined globally due to reported inferior long-term outcomes compared to bilateral lung transplantation. However, the impact of this approach on overall population-level waiting list mortality and net transplant benefit is unknown. This study aimed to model potential UK allocation policies with and without prioritising SLT for ILD using a discrete event simulation engine to compare impacts of this approach.

IntroductionConventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200 IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen.MethodsOxygenation of individual islets under three culture conditions, standard low-density, non-GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2 days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, ∼4000 IE/cm2, as measured by DNA) in both GP and non-GP devices.ResultsIn simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non-GP high density cultured islets were anoxic. Similarly, compared to the non-GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non-GP HD cultures.DiscussionCulturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism.

Introduction: Understanding of the pancreas is impacted by its fragility. This in part has led to the under-utilisation of pancreata for transplantation. Gaseous oxygen perfusion (persufflation) is a technique which has shown promise in improving pancreas preservation. However, one key challenge still remains: the lack of knowledge about the interaction between the organ and its preservation. In this pilot study, we describe an unbiased mathematical approach to understand the ScubaTx persufflation device, the pancreas and their interaction to help identify and describe physical and/or biological factors that may impact the quality of the preservation. Method: Using the fully automated ScubaTx device, a pancreas (or suitable analogue), was preserved to gather data regarding key parameters (i.e. flow/pressure). By utilising a combination of lumped parameter modelling, and system identification techniques, we derive equations that describe aspects of the device and pancreas; the amalgamation of which enabled the study of their interaction. Refinement of the equation was performed through experimentation and data-model fusion. The setup used can be seen in Figure 1.Results: Lumped-parameter fluid and grey-box modelling enabled the creation of an accurate steady-state model of the device and the pancreas. The derived equations that describe the system were obtained through specification or experimentation and calibrated to accurately reflect the preservation setup and embedded into a digital model. Changes in the device, organ or the preservation process, such as leaks or blockages in the vasculature or the device’s pneumatics, were accurately detected as anomalies in the model. System internal resistance was derived from the base equations. This gives a combined measure of the pancreas-machine interaction that can provide an indication of the state of the graft’ s vascular biology. A change in resistance is reflected in the model and can allude to changes in the device setup or specific resultant biological interactions. Varying the resistance of an organ analogue allowed for the identification of the device’ s relevant parameters and improved the calibration process and the accuracy of the model. Conclusion: Exploiting techniques from engineering domains allowed us to develop a model that describes the ScubaTx device setup, the pancreas under preservation and their interaction. We are able to observe vascular occlusions such as blockages or leaks and hope that in future works we can generate enough data to classify the various anomalies. We are working further towards a deeper understanding of the vascular-pancreas and how it interacts with preservation devices. Moving forward, we hope to deepen our understanding of the biology of the pancreas to better understand organ preservation, disease and transplant survival. We are grateful to the Poul Due Jensen Foundation, which has supported the establishment of a new Centre for Digital Twin Technology at Aarhus University. EU industrial PhD bursary (IIIP) and the European Regional Development Fund (ERDF). ScubaTx for access to their device, which UK MRC funded the first prototype. Thomas Nielsen for the discussions and thoughts on the setup and brainstorming ideas.

The advent of Machine Perfusion (MP) as a superior form of preservation and assessment for cold storage of both high-risk kidney's and the liver presents opportunities in the field of beta-cell replacement. It is yet unknown whether such techniques, when applied to the pancreas, can increase the pool of suitable donor organs as well as ameliorating the effects of ischemia incurred during the retrieval process. Recent experimental models of pancreatic MP appear promising. Applications of MP to the pancreas, needs refinement regarding perfusion protocols and organ viability assessment criteria. To address the "Role of pancreas machine perfusion to increase the donor pool for beta cell replacement," the European Society for Organ Transplantation (ESOT) assembled a dedicated working group comprising of experts to review literature pertaining to the role of MP as a method of improving donor pancreas quality as well as quantity available for transplant, and to develop guidelines founded on evidence-based reviews in experimental and clinical settings. These were subsequently refined during the Consensus Conference when this took place in Prague.

Currently, there is a shortfall in the number of suitable organs available for transplant resulting in a high number of patients on the active transplant waiting lists worldwide. To address this shortfall and increase the utilization of donor organs, the acceptance criteria for donor organs is gradually expanding including increased use of organs from donation after circulatory death. Use of such extended criteria donors and exposure of organs to more prolonged periods of warm or cold ischaemia also increases the risk of primary graft dysfunction occurring. Normothermic machine perfusion (NMP) offers a unique opportunity to objectively assess donor organ function outside the donor body and potentially recondition those deemed unsuitable on initial evaluation prior to implantation in the recipient. Furthermore, NMP provides a platform to support the use of established and novel therapeutics delivered directly to the organ, without the need to worry about potential deleterious 'off-target' side effects typically considered when treating the whole patient. This review will explore some of the novel therapeutics currently being added to perfusion platforms during NMP experimentally in an attempt to improve organ function and post-transplant outcomes.

Introduction: Acute endocrine cell stress in donor pancreas due to hypoxia and islets >150 µm diameter post-isolation are associated with impaired clinical transplant outcomes1. Cell-cell interactions enhance islet beta-cell function. We aimed to evaluate the use of Kugelmeiers SphericalPlate 5D (SP5D; Kugelmeiers Ltd., Switzerland), a novel 3D microcavity suspension-well system for generation and maintenance of optimally-sized viable engineered islet microtissues and to assess impact on endocrine acute stress and cell-cell interactions. Methods: Primary human islets were isolated using the semi-automated Ricordi method. A tissue biopsy from the head region was taken pre-isolation and isolated islets were sampled 1 hr following isolation (D0). Islets were dissociated into single cell suspensions through addition of Accutase (Sigma-Aldrich, UK) and mechanical agitation in a 37 °C water bath. Cells were seeded at densities of 187,500, 375,000 and 562,500 cells/well into SP5D and maintained for 120 hr. Paired control islets were maintained for 120 hr in non-adherent T75 flasks. Tissues were maintained at 37 °C in 5% CO2 with SP5D and control organoid diameter visually assessed (graticule) and viability estimated following propidium iodide staining (n=50 engineered islet microtissues/ control islets). Samples from pre-isolation biopsy, D0 islets and 120 hr islet microtissues and islets were processed for transmission electron microscopy and imaged following established protocols, with endocrine cells (25 per sample) assessed using the Newcastle Pancreas Endocrine Stress Score (NPESS)2. Results: Following 120 hr preservation, mean engineered islet microtissue diameter of ⁓100 µm was observed in SP5D with none >150 µm (seeding at 562,500 cells/well equivalent to 750 cells/microwell yielded diameter 101.8±7.7 µm). For control islets, mean diameter was significantly higher (132.2±63.1 µm; p=0.001) and less consistent with islets of 300 µm diameter observed. Viability following 120 hr preservation was higher for SP5D (89±8%) vs control (70±22%; p<0.0001). Total NPESS score was improved for SP5D (5.6) vs biopsy (8.4) and D0 (6.5) and was comparable to control (5.22). SP5D had the lowest score for mitochondrial swelling (1.2) vs biopsy (2.7), D0 (1.9), and control (1.2); and cytoplasmic vacuolisation SP5D (0.8) vs biopsy (1.0), D0 (1.1), and control (0.8). Ultrastructural cell-cell interactions were observed in SP5D.Conclusion: We have developed and validated standardised protocols for preparing viable islet microtissues from primary human pancreatic cells using SP5D. Optimal and consistent diameter size of ~100 µm was attained. SP5D demonstrated improved total NPESS score compared to biopsy and D0 islets, with lowest score for mitochondrial swelling and cytoplasmic vacuolisation compared to all other samples. Cell-cell interactions in SP5D further support formation of microtissues mimicking native islets. National Institute for Health and Care Research (NIHR) Blood and Transplant Research Unit in Organ Donation and Transplantation (NIHR203332). References: 1. Lehmann R, Zuellig RA, Kugelmeier P, Baenninger PB, Moritz W, Perren A, Clavien PA, Weber M, Spinas GA. Superiority of small islets in human islet transplantation. Diabetes. 2007 Mar;56(3):594-603 2807/3000 2. Dyson NJ, Kattner N, Honkanen-Scott M, Hunter B, Doyle JA, White K, Davey TS, Ploeg RJ, Bury YA, Tiniakos DG, Shaw JAM, Scott WE 3rd. Development and Application of a Semi quantitative Scoring Method for Ultrastructural Assessment of Acute Stress in Pancreatic Islets. Transplant Direct. 2021 Dec 16;8(1):e1271.

The field of transplantation has witnessed the emergence of Advanced Therapy Medicinal Products (ATMPs) as highly promising solutions to address the challenges associated with organ and tissue transplantation. ATMPs encompass gene therapy, cell therapy, and tissue-engineered products, hold immense potential for breakthroughs in overcoming the obstacles of rejection and the limited availability of donor organs. However, the development and academic research access to ATMPs face significant bottlenecks that hinder progress. This opinion paper emphasizes the importance of addressing bottlenecks in the development and academic research access to ATMPs by implementing several key strategies. These include the establishment of streamlined regulatory processes, securing increased funding for ATMP research, fostering collaborations and partnerships, setting up centralized ATMP facilities, and actively engaging with patient groups. Advocacy at the policy level is essential to provide support for the development and accessibility of ATMPs, thereby driving advancements in transplantation and enhancing patient outcomes. By adopting these strategies, the field of transplantation can pave the way for the introduction of innovative and efficacious ATMP therapies, while simultaneously fostering a nurturing environment for academic research.

BACKGROUND:The association between interleukin-1β (IL-1β) concentrations during ex vivo lung perfusion (EVLP) with donor organ quality and post-lung transplant outcome has been demonstrated in several studies. The mechanism underlying IL-1β-mediated donor lung injury was investigated using a paired single-lung EVLP model. METHODS:Human lung pairs were dissected into individual lungs and perfused on identical separate EVLP circuits, with one lung from each pair receiving a bolus of IL-1β. Fluorescently labeled human neutrophils isolated from a healthy volunteer were infused into both circuits and quantified in perfusate at regular timepoints. Perfusates and tissues were subsequently analyzed, with perfusates also used in functional assays. RESULTS:Neutrophil numbers were significantly lower in perfusate samples collected from the IL-1β-stimulated lungs consistent with increased neutrophil adhesion ( P = 0.042). Stimulated lungs gained significantly more weight than controls ( P = 0.046), which correlated with soluble intercellular adhesion molecule-1 (R 2 = 0.71, P = 0.0043) and von-Willebrand factor (R 2 = 0.39, P = 0.040) in perfusate. RNA expression patterns for inflammatory genes were differentially regulated via IL-1β. Blockade of IL-1β significantly reduced neutrophil adhesion in vitro ( P = 0.025). CONCLUSION:These data illustrate the proinflammatory functions of IL-1β in the context of EVLP, suggesting this pathway may be susceptible to therapeutic modulation before transplantation.

Digital Twins (DTs) are a promising technology for integrating device monitoring and data consumption to improve performance. This technology has seen utilisation in various industries that use cyber-physical systems. An unexpected area is medical devices. In this paper, we explore DTs use for an organ preservation device, which, helps improve transplantation outcomes by actively managing the organ during transport to prevent biological degradation. Whilst reducing the burden on specialists. Digital twinning offers an exciting direction of development for medical devices to improve transplantation outcomes.

Transplantation is an effective treatment for end-stage lung disease but donor organ shortage is a major problem. Ex-vivo lung perfusion (EVLP) of marginal organs enables functional assessment under normothermic conditions to facilitate clinical decision-making around utilisation, but the molecular processes occurring during EVLP, and how they differ between more or less viable lungs, remains to be determined. Here we used RNA sequencing to delineate changes in gene expression occurring in n=10 donor lungs undergoing EVLP, comparing lungs that were deemed transplantable (n=6) to those deemed unusable (n=4). We found that lungs deemed suitable for transplantation following EVLP had reduced induction of a number of innate immune pathways during EVLP, but a greater increase in genes involved in oxidative phosphorylation, a critical ATP-degenerating pathway. Furthermore, SCGB1A1 , a gene encoding an anti-inflammatory secretoglobin CC10, and other club cell genes were significantly increased in transplantable lungs following perfusion, whilst CHIT-1 was decreased. Using a larger validation cohort (n=18), we confirmed that the ratio of CHIT1 and SCGB1A1 protein levels in lung perfusate have potential utility to distinguish transplantable and non-transplantable lungs (AUC 0.81). Together, our data identify novel biomarkers that may assist with pre-transplant lung assessment, as well as pathways that may amenable to therapeutic intervention during EVLP. Single sentence summary Transcriptional changes in lungs undergoing ex vivo normothermic perfusion identify chitinase1 and club cell genes as potential biomarkers to guide utilisation

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2) 1 , could represent a new chemoprophylactic approach for COVID-19 that complements vaccination 2,3 . However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

With the ever-increasing disparity between the number of patients waiting for organ transplants and the number organs available, some patients are unable to receive life-saving transplantation in time. The present, widely-used form of preservation is proving to be incapable of maintaining organ quality during long periods of preservation and meeting the needs of an ever-changing legislative and transplantation landscape. This has led to the need for improved preservation techniques. One such technique that has been extensively researched is gaseous oxygen perfusion or Persufflation (PSF). This method discovered in the early 20th century has shown promise in providing both longer term preservation and organ reconditioning capabilities for multiple organs including the liver, kidneys, and pancreas. PSF utilises the organs own vascular network to provide oxygen to the organ tissue and maintain metabolism during preservation to avoid hypoxic damage. This review delves into the history of this technique, its multiple different approaches and uses, as well as in-depth discussion of work published in the past 15 years. Finally, we discuss exciting commercial developments which may help unlock the potential for this technique to be applied at scale.

ObjectivesIslet transplantation is a treatment option for patients with type 1 diabetes and recurrent life-threatening hypoglycaemia. Pancreatic tissue is dissociated, and islets are purified from exocrine tissue of a donor pancreas through enzymatic and mechanical separation followed by short term culture and transplantation into the portal vein of the recipient. Despite improvements to isolation and transplantation protocols, insulin independence is often not sustained demonstrating stress towards the islets and impaired transplantation outcomes. These stressors include loss of the specific microenvironment and exposure to hypoxia following disconnection from the blood supply. Tissue engineering approaches will be investigated to minimize these stressors.Key findingsDifferent tissue engineering strategies are available to improve islet health and function and therefore outcomes of islet transplantation. Strategies for the replacement of extracellular matrix in the microenvironment of isolated islets should provide cell-matrix contacts and a three-dimensional microenvironment but avoid cyto-toxic components. Strategies for immune protection should shield islets from the immune system whilst enabling sufficient oxygen and mass transfer. Strategies for improved oxygenation of islets should consider in vitro and/or in vivo oxygen requirements. Finally, alternative cell sources of β-cells may provide a standardised and less stressed product, but efficiency, safety, and costs require further improvement.ConclusionThis review summarises the development and composition of islets and their microenvironment in adult pancreata. The impact of peri-transplant stressors including islet isolation and transplantation are explored as well as strategies to minimize these towards enhanced tissue engineered β-cell replacement.