Engineering an endocrine Neo-Pancreas by repopulation of a decellularized rat pancreas with islets of Langerhans

Decellularization of pancreata and repopulation of these non-immunogenic matrices with islets and endothelial cells could provide transplantable, endocrine Neo- Pancreata. In this study, rat pancreata were perfusion decellularized and repopulated with intact islets, comparing three perfusion routes (Artery, Portal Vein, Pancreatic Duct). Decellularization effectively removed all cellular components but conserved the pancreas specific extracellular matrix. Digital subtraction angiography of the matrices showed a conserved integrity of the decellularized vascular system but a contrast emersion into the parenchyma via the decellularized pancreatic duct. Islets infused via the pancreatic duct leaked from the ductular system into the peri- ductular decellularized space despite their magnitude. TUNEL staining and Glucose stimulated insulin secretion revealed that islets were viable and functional after the process. We present the first available protocol for perfusion decellularization of rat pancreata via three different perfusion routes. Furthermore, we provide first proof-of-concept for the repopulation of the decellularized rat pancreata with functional islets of Langerhans. The presented technique can serve as a bioengineering platform to generate implantable and functional endocrine Neo-Pancreata

Impact of Percoll purification on isolation of primary human hepatocytes

Abstract Research and therapeutic applications create a high demand for primary human hepatocytes. The limiting factor for their utilization is the availability of metabolically active hepatocytes in large quantities. Centrifugation through Percoll, which is commonly performed during hepatocyte isolation, has so far not been systematically evaluated in the scientific literature. 27 hepatocyte isolations were performed using a two-step perfusion technique on tissue obtained from partial liver resections. Cells were seeded with or without having undergone the centrifugation step through 25% Percoll. Cell yield, function, purity, viability and rate of bacterial contamination were assessed over a period of 6 days. Viable yield without Percoll purification was 42.4 × 106 (SEM ± 4.6 × 106) cells/g tissue. An average of 59% of cells were recovered after Percoll treatment. There were neither significant differences in the functional performance of cells, nor regarding presence of non-parenchymal liver cells. In five cases with initial viability of <80%, viability was significantly increased by Percoll purification (71.6 to 87.7%, p = 0.03). Considering our data and the massive cell loss due to Percoll purification, we suggest that this step can be omitted if the initial viability is high, whereas low viabilities can be improved by Percoll centrifugation

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepatocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation