Claudy Mullon

The Isolation and Function of Porcine Islets from Market Weight Pigs

The efficacy of clinical islet transplantation has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is confounded by the necessity of immunosuppression, the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. Porcine islet xenotransplantation, together with a successful immune intervention strategy, may provide the necessary clinical alternative. However, a major obstacle in evaluating this approach has been the difficulty of obtaining adequate volumes of functional islet tissue from pigs. Donors of market weight are preferable to retired breeders due to their abundance, lower animal and husbandry costs, and are more suitable to meet regulatory guidelines for donor tissue for xenotransplantation. We describe a simple isolation procedure that following purification yields a mean of 350,000 IE, corresponding to 179 units of insulin and 1.8 mg of DNA with an islet purity and viability in excess of 85% (n = 317 isolations). In both short- and long-term cell cultures, porcine islets demonstrated glucose-responsive insulin secretion. However, this secretion is density dependent, which may have significant consequences in the development of immunoisolation technologies to support porcine islet xenotransplantation. Following implantation into diabetic nude mice, porcine islets remained functional in excess of 1 year. Implantation of a bioartificial pancreas containing porcine islets into pancreatectomized dogs provided significant clinical benefit with an improved diabetic condition. Finally, secretagogue-induced insulin release was demonstrated in vitro from these devices after removal from immunocompetent recipients. Immunohistochemical staining identified well-granulated islets following long-term implantation in both the rodent and canine models. This study demonstrates the ability to isolate porcine islets in clinically relevant numbers from market animals, which survive and remain functional for prolonged periods of time in an immune-deficient or immunoprotected environment.

Overview of Extracorporeal Liver Support Systems and Clinical Results

ABSTRACT: Patients with acute liver failure (ALF) continue to have an almost 50% mortality raate despite improvements associated with the use of orthotopic liver transplantation (OLT). Numerous ex vivo methods have been developed in attempts to improve patient survival. These methods scan be divided into three groups: detoxification (e.g., dialysis adsorption, plasma exchange), which only provides excretory function; ex vivo liver perfusion (e.g., whole organ or tissue perfusion), which provides some metabolic function; and bioartificial or cell‐based systems, which combine elements of the first two methods. Clinical trials have shown minimal efficacy of the various detoxification methods in terms of ALF patient survival, while the relative sucess of OLT has shown the importance of providing metabolic as well as excretory functions. Attempts to provide those additional functions with ex vivo tissue perfusion have been fraught with complications such as clothing and acute tissue rejection, leading to the conceptual development of cell‐based bioreactor sytems. A number of these bioartificial systems have been clinically evaluated, and the preliminary patient survival rates have encouraged further work in this area.

Comparison of Analytical Methods for Quantitation of Isolated Porcine Hepatocyte Yields

As cell-based therapies receive approval for clinical evaluation and use, the development of reliable methods to quantify cell number and control the dose of therapy delivered is becoming increasingly important. An example is the determination of the number and volume of primary porcine hepatocytes used in an extracorporeal treatment for patients with liver disease. Conventional cell counting using optical microscopy was compared against two alternate methods to quantify isolated porcine hepatocytes: (1) automated cell counting using a commercially available particle characterization instrument, and (2) quantitation by cell mass. Methods were compared based on accuracy, precision, specificity, linear range, and ruggedness. The automated method delivered substantially improved accuracy, precision, and ruggedness when compared to the conventional optical method. It also provided valuable information about the size distribution of cell preparations, which often contained clumps of cells, and showed that processing steps such as cryopreservation can alter the size characteristics of a cell population. The automated method was also faster, and was well suited to use in a commercial manufacturing process. The mass-based method was simple and inexpensive, but suffered from nonlinearity at low cell concentrations. Automated cell quantitation using a commercially available particle characterization instrument proved to be the preferred method for obtaining accurate and consistent porcine hepatocyte counts in a timely manner.

Simulation of bilirubin detoxification in the newborn using an extracorporeal bilirubin oxidase reactor.

ABSTRACT: Jaundice, which is characterized by an excessive accumulation of bilirubin in the blood and tissues, occurs in 13% of newborns. The common treatments for neonatal jaundice are phototherapy and blood exchange transfusion. A novel approach using an extracorporeal blood filter containing immobilized bilirubin oxidase was recently proposed to detoxify jaundiced blood, and a prototype device markedly reduced serum bilirubin in genetically jaundiced Gunn rats. The primary toxicologic effect in that study was a 20% reduction in red blood cell count. Using a compartmental model for bilirubin metabolism, a mathematical simulation of the extracorporeal treatments ability to reduce serum bilirubin levels in jaundiced infants is presented. Using a 10-mL reactor volume containing immobilized bilirubin oxidase, the simulation predicts a 32 to 65% decrease in plasma bilirubin concentration over a 4-h treatment for a 2 kg preterm hyperbilirubinemic newborn. In addition, a new approach to altering support material has essentially eliminated red blood cell lysis in vivo using Gunn rats and in vitro using adult blood.