Meeting the oxygen requirements of an isolated perfused rat liver

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.Cataloged from PDF version of thesis.Includes bibliographical references (p. 47-49).Liver perfusion systems can be used as organ culture platforms for metabolic, genetic and systems engineering, tissue regeneration, pharmacokinetics, organ storage and marginal donor reconditioning for transplantation. The primary requirement of such a system is that it maintain ex vivo organ function in a stable manner for indefinite periods of time. The more physiologically relevant the perfusion system is, the lower the likelihood of incurring functional instability, and the greater the clinical relevance of the data obtained. Currently, a major limiting factor in achieving such a design is the absence of organ-specific in vivo data to standardize, evaluate and optimize the state of perfused livers. Oxygen uptake rate, the primary indicator of organ metabolism and therefore a likely important marker of organ viability, was chosen as one such parameter to be investigated. A systematic review and reproduction of numerous oxygenation conditions cited in the literature in addition to in vivo data sampling across the rat liver shows a significant paucity of oxygen uptake in perfusion compared to in vivo. The reasons stem largely from the biological and mechanical flow properties of the perfusate, but also from the altered metabolic state of the organ in perfusion. In the absence of an oxygen carrier, the liver became oxygen starved and lost functionality. The addition of erythrocytes significantly improved oxygen delivery rates while reducing the flow rate and damaging shear stress.(cont.) However, as hematocrit approached physiological values perfusate viscosity became detrimentally high, causing severe structural and ultimately functional damage. Large strains of erythrocytes similarly impacted the liver. The addition of sodium heparin reduced erythrocyte-erythrocyte and erythrocyte-parenchyma interaction, thereby reducing sheer stress on the liver. The optimal oxygen carriers were small heparinized erythrocytes, such as goat or rat erythrocytes, at a hematocrit of 12%-20%. These conditions continued to provide adequate in vivo oxygen delivery rates and sustained organ structure and function.by Maria-Louisa Izamis.S.M

Ex vivo perfusion optimization of donor liver grafts for transplantation and cell isolation

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010.Cataloged from PDF version of thesis.Includes bibliographical references.There is a constant demand for enormous numbers of high quality hepatocytes in the fields of cell transplantation, pharmacotoxicology, tissue engineering, and bioartificial assist devices. The scarcity of viable hepatocytes necessitates the use of suboptimal sources including damaged donor organs that are not transplantable. Many of these organs have potentially reversible pathologies however, that could be treated via ex vivo perfusion thereby increasing their cell yield. With the intent to translate organ recovery by perfusion into the clinic, we engineered a very simple room temperature-operated ex vivo organ perfusion system to test a rat liver model of uncontrolled non-heart beating donors. Seventeen times as many hepatocytes were recovered from livers exposed to an hour of warm ischemia (WI, 34*C) compared to untreated WI livers in only 3 hours of perfusion. Further, fresh liver hepatocyte yields were also increased by 32% postperfusion, demonstrating that both damaged and healthy donor livers could benefit from this methodology. A linear correlation between cell yield and tissue ATP content was established. This enables an accurate prediction of cell recovery during preservation and can be used as a direct measure of organ viability and the trajectory of organ recovery during perfusion resuscitation. Further, a strong correlation between perfusion flow rate and cell yield was also established supporting the use of flow rates as low as possible without causing hypoperfusion or oxygen deprivation. Morphologically and functionally, perfusion-isolated hepatocytes generally performed comparably or better than fresh hepatocytes in cell suspension and plate culture. Cumulatively, these findings strongly support the ubiquitous use of organ perfusion systems in the clinic for optimal enhancement of donor grafts.by Maria-Louisa Izamis.Ph.D

Resuscitation of Ischemic Donor Livers with Normothermic Machine Perfusion: A Metabolic Flux Analysis of Treatment in Rats

Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use

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

Development of Metabolic Indicators of Burn Injury: Very Low Density Lipoprotein (VLDL) and Acetoacetate Are Highly Correlated to Severity of Burn Injury in Rats

Hypermetabolism is a significant sequela to severe trauma such as burns, as well as critical illnesses such as cancer. It persists in parallel to, or beyond, the original pathology for many months as an often-fatal comorbidity. Currently, diagnosis is based solely on clinical observations of increased energy expenditure, severe muscle wasting and progressive organ dysfunction. In order to identify the minimum number of necessary variables, and to develop a rat model of burn injury-induced hypermetabolism, we utilized data mining approaches to identify the metabolic variables that strongly correlate to the severity of injury. A clustering-based algorithm was introduced into a regression model of the extent of burn injury. As a result, a neural network model which employs VLDL and acetoacetate levels was demonstrated to predict the extent of burn injury with 88% accuracy in the rat model. The physiological importance of the identified variables in the context of hypermetabolism, and necessary steps in extension of this preliminary model to a clinically utilizable index of severity of burn injury are outlined

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

Perfusion monitoring by contactless photoplethy smography imaging

The flow of blood, or perfusion, of the skin can be indicative of the local but also systemic health of an individual. The noncontact Photoplethysmography (PPG) imaging is a recently emerging technology able to monitor skin perfusion. Using an off-the-shelf camera and a light source, it is possible to remotely detect the dynamic changes in blood volume beneath the skin and derive a map correlated to the blood perfusion. In this paper we empirically investigate perfusion monitoring by camera-based PPG imaging. Laser Speckle Contrast Analysis (LASCA), a well-known technique for perfusion monitoring, is used as reference. We design an experimental setup that allows simultaneous PPG imaging and laser speckle measurements. We conduct experiments with different local and temporary perfusion perturbation of the skin and show that camera-based PPG imaging can detect the perfusion changes in the tissue. Results correlate well with the laser speckle measurements, suggesting that non-contact PPG imaging is capable of providing a realistic map of skin perfusion

Perfusion monitoring by contactless photoplethy smography imaging

\u3cp\u3eThe flow of blood, or perfusion, of the skin can be indicative of the local but also systemic health of an individual. The noncontact Photoplethysmography (PPG) imaging is a recently emerging technology able to monitor skin perfusion. Using an off-the-shelf camera and a light source, it is possible to remotely detect the dynamic changes in blood volume beneath the skin and derive a map correlated to the blood perfusion. In this paper we empirically investigate perfusion monitoring by camera-based PPG imaging. Laser Speckle Contrast Analysis (LASCA), a well-known technique for perfusion monitoring, is used as reference. We design an experimental setup that allows simultaneous PPG imaging and laser speckle measurements. We conduct experiments with different local and temporary perfusion perturbation of the skin and show that camera-based PPG imaging can detect the perfusion changes in the tissue. Results correlate well with the laser speckle measurements, suggesting that non-contact PPG imaging is capable of providing a realistic map of skin perfusion.\u3c/p\u3