A Fitness Index for Transplantation of Machine-Perfused Cadaveric Rat Livers

Background: The 110,000 patients currently on the transplant waiting list reflect the critical shortage of viable donor organs. However, a large pool of unused organs, from donors after cardiac death (DCD) that are disqualified because of extensive ischemic injury, may prove transplantable after machine perfusion treatment, fundamentally impacting the availability of treatment for end-stage organ failure. Machine perfusion is an ex-vivo organ preservation and treatment procedure that has the capacity to quantitatively evaluate and resuscitate cadaveric organs for transplantation. Methods To diagnose whether an organ was fresh or ischemic, an initial assessment of liver quality was conducted via dynamic discriminant analysis. Subsequently, to determine whether the organs were sufficiently viable for successful implantation, fitness indices for transplantation were calculated based on squared prediction errors (SPE) for fresh and ischemic livers. Results: With just three perfusate metabolites, glucose, urea and lactate, the developed MPLSDA model distinguished livers as fresh or ischemic with 90% specificity. The SPE analyses revealed that fresh livers with SPEF < 10.03 and WI livers with SPEWI < 3.92 yield successful transplantation with 95% specificity. Conclusions: The statistical methods used here can discriminate between fresh and ischemic livers based on simple metabolic indicators measured during perfusion. The result is a predictive fitness index for transplantation of rat livers procured after cardiac death. The translational implications of this study are that any donor organ procured from controlled, but most especially from uncontrolled cardiac death donors, will be objectively assessed and its recovery monitored over time, minimizing the critical loss of otherwise viable organs

Successful Supercooled Liver Storage for 4 Days

The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials

Immune responses to bioengineered organs

Purpose of review Organ donation in the United States registered 9079 deceased organ donors in 2015. This high percentage of donations allowed organ transplantation in 29 851 recipients. Despite increasing numbers of transplants performed in comparison with previous years, the numbers of patients that are in need for a transplant increase every year at a higher rate. This reveals that the discrepancy between the demand and availability of organs remains fundamental problem in organ transplantation. Recent findings Development of bioengineered organs represents a promising approach to increase the pool of organs for transplantation. The technology involves obtaining complex three-dimensional scaffolds that support cellular activity and functional remodeling though tissue recellularization protocols using progenitor cells. This innovative approach integrates cross-thematic approaches from specific areas of transplant immunology, tissue engineering and stem cell biology, to potentially manufacture an unlimited source of donor organs for transplantation. Summary Although bioengineered organs are thought to escape immune recognition, the potential immune reactivity toward each of its components has not been studied in detail. Here, we summarize the host immune response toward different progenitor cells and discuss the potential implications of using nonself biological scaffolds to develop bioengineered organs

Effects of Immobilized Glycosaminoglycans on the Proliferation and Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are adult stem cells with potential for multilineage differentiation. They represent an attractive cell source alternative to embryonic stem cells for therapeutic applications. Optimal utilization of MSCs for tissue engineering requires improved biomaterials that can enhance their growth and direct differentiation. The biological activity of glycosaminoglycans (GAGs) has been previously exploited for use in tissue engineering applications. In this study, MSC proliferation and differentiation was studied on GAG-derivatized chitosan membranes. The GAGs included heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid. The covalent GAG immobilization method and amount of immobilized GAG were varied. It was found that MSC growth increased as much as fivefold on GAG-immobilized surfaces compared to tissue culture plastic and chitosan-only controls. The MSC growth rates increased significantly with increasing GAG density on the culture surfaces. The MSC proliferation rates on heparin, heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate exhibited nonlinear increases with the level of fibronectin binding on these surfaces. In contrast, MSC proliferation on hyaluronic acid and chondroitin 4-sulfate was found to be independent of fibronectin or vitronectin binding on the surfaces, suggesting that these GAGs influenced MSC proliferation through different mechanisms. In conclusion, the results indicate that GAG immobilization on chitosan scaffolds provides an effective means of manipulating MSC proliferation and has promising potential for directing MSC differentiation in tissue engineering applications employing chitosan

Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by “electric field sinks”

Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for “electric field sinks” in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures