Impact of Percoll purification on isolation of primary human hepatocytes.

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.

[High donor age for liver transplantation : Tackling organ scarcity in Germany]. / Hohes Spenderalter bei Lebertransplantation : Wie begegnen wir dem Organmangel in Deutschland?

BACKGROUND: Liver transplantation is the only curative treatment option for patients with end-stage liver disease; however, the 40% decline of available organ donors in recent years in Germany necessitates the optimization of available resources and possibly extending the criteria to older donors. MATERIAL AND METHODS: All 2652 livers made available to the Charité Universitätsmedizin Berlin from 2010 to 2016 were retrospectively analyzed and the clinical outcome of 526 liver transplantations during this time frame were evaluated. RESULTS: The median age of donors of transplanted organs increased from 49.3 years in 2010 to 57.3 years in 2016 (p = 0.02). Organs from donors ≥65 years were more frequently discarded than organs from younger donors (n = 344, 18.4% vs. n = 220, 28.1%; p = 0.005). Moreover, the older donors had higher rates of diabetes mellitus and hepatic steatosis. Organs from older donors had a higher donor risk index (2.8 vs. 2.2; p < 0.001) and were transplanted more often in patients with preserved liver function and hepatocellular carcinoma and liver cirrhosis (n = 121, 74.7% of indications). The 3­year survival after liver transplantation from donors ≥65 and ≥80 years old was not significantly reduced in comparison to younger donors; however, there was an increased retransplantation rate (28.6%; p = 0.005) after transplantation of organs from donors ≥80 years old. CONCLUSION: Despite conservative organ acceptance there were higher rates of retransplantation after transplantation from very old donors. In the light of an increasing scarcity of suitable organs this mandates caution and highlights the need for adequate assessment instruments for marginal donor organs before transplantation.

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.

Computed tomography-based survey of the vascular anatomy of the juvenile Göttingen minipig.

Over the past 50 years, image-guided procedures have been established for a wide range of applications. The development and clinical translation of new treatment regimens necessitate the availability of suitable animal models. The juvenile Göttingen minipig presents a favourable profile as a model for human infants. However, no information can be found regarding the vascular system of juvenile minipigs in the literature. Such information is imperative for planning the accessibility of target structures by catheterization. We present here a complete mapping of the arterial system of the juvenile minipig based on contrast-enhanced computed tomography. Four female animals weighing 6.13 ± 0.72 kg were used for the analyses. Imaging was performed under anaesthesia, and the measurement of the vascular structures was performed independently by four investigators. Our dataset forms a basis for future interventional studies in juvenile minipigs, and enables planning and refinement of future experiments according to the 3R (replacement, reduction and refinement) principles of animal research.

Diluted and undiluted Mercox severely destroy unfixed endothelial cells. A light and electron microscopic study using cultured endothelial cells and tadpole tail fin vessels.

Mercox is a methylmethacrylate-based resin which is widely used for vascular corrosion casting with subsequent scanning electron microscopic analysis. In the present study the effect of undiluted and diluted Mercox (4 + 1; volume + volume; Mercox: monomeric methyl-methacrylate (MMA); 0.02 g catalyst MA/ml Mercox) and methyl-methacrylate with and without catalyst MA (0.625 g/10 ml MMA) on fixed and unfixed endothelial cells was studied. Light microscopy (LM) of cultured capillary endothelial cells (ECs), which were replicated with diluted or undiluted Mercox shows degranulation and membrane perturbation of ECs, while no morphological changes occur in glutaraldehyde-prefixed ECs. Scanning electron microscopy (SEM) of replicas (= resin blocks) polymerized on prefixed ECs reveals unchanged ECs and replicas show many details. Unfixed ECs are destroyed and replicas reveal aberrant features. Transmission electron microscopy (TEM) of prefixed and unfixed ECs (cultured endothelial cells, endothelial cells of perfusion prefixed and of unfixed tadpole tail fin vessels) substantiates LM and SEM findings. Prefixed ECs resist Mercox without fine structural changes, while unfixed cells undergo destruction. It is recommended to fix vessels prior to casting. Extravasations in micro-vessels are considered to be caused by focal chemical destruction of endothelial cells.

Idiopathic hypertrophy of the oesophagus in children. A case report and review of the literature.

Idiopathic hypertrophy of the oesophagus is a rare entity. Of approximately 50 cases reported in the literature, only 5 are in children. The case of an 8-year-old girl is presented and compared with those previously reported.