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We present a detailed bubble analysis of the Bitcoin to US Dollar price dynamics from January 2012 to February 2018. We introduce a robust automatic peak detection method that classifies price time series into periods of uninterrupted market growth (drawups) and regimes of uninterrupted market decrease (drawdowns). In combination with the Lagrange Regularization Method for detecting the beginning of a new market regime, we identify three major peaks and 10 additional smaller peaks, that have punctuated the dynamics of Bitcoin price during the analysed time period. We explain this classification of long and short bubbles by a number of quantitative metrics and graphs to understand the main socio-economic drivers behind the ascent of Bitcoin over this period. Then, a detailed analysis of the growing risks associated with the three long bubbles using the Log-Periodic Power-Law Singularity (LPPLS) model is based on the LPPLS Confidence Indicators, defined as the fraction of qualified fits of the LPPLS model over multiple time windows. Furthermore, for various fictitious 'present' times t 2 before the crashes, we employ a clustering method to group the predicted critical times t c of the LPPLS fits over different time scales, where t c is the most probable time for the ending of the bubble. Each cluster is proposed as a plausible scenario for the subsequent Bitcoin price evolution. We present these predictions for the three long bubbles and the four short bubbles that our time scale of analysis was able to resolve. Overall, our predictive scheme provides useful information to warn of an imminent crash risk.

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Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.

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The number of human embryonic stem cell (hESC) lines that are available and that are subsequently being used in numerous research projects is increasing steadily. However, there is little coordination of hESC line derivation, and comparative information on the characteristics and quality of these cells is sparse. Obtaining consistent information on hESCs is hampered further by legislative fragmentation, particularly in Europe. Recognizing these obstacles, the European Commission has set up a Human Embryonic Stem Cell Registry (hESCreg) to make hESCs and their characterizing information accessible and to ensure that the results of research become more quickly available to the public. The primary objectives of hESCreg are to provide freely accessible information on existing hESC lines, their derivation, molecular characteristics, use and quality. Successful research with listed hESC lines will be used to evaluate clinical potential and thus directly influence policy decisions. The developing integration with other initiatives, such as characterization projects, registries and cell banks, is expected to lead to a common and internationally accepted central reference. The hESCreg provides a first step in this direction and might grow into an internationally funded and administered project.

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This study aims to assess the suitability of biodegradable membranes as transfer matrix materials for the culture of subconfluent fibroblasts and keratinocytes. The materials investigated were based on collagen, chitosan and enzyme-digestible cellulose. The proliferation and growth behaviour of human keratinocytes and dermal fibroblasts were analysed and morphology and distribution determined. Cultured fibroblasts exhibited no significant differences in proliferation for the different membrane types, whereas keratinocytes revealed significantly higher proliferation on collagen membranes compared with membranes based on cellulose and chitosan. Co-cultured fibroblasts and keratinocytes from the same donor on collagen membranes showed more homogenous cell distribution, but they segregated in heterologous co-cultures; this effect must be further investigated. Thus, collagen and collagen-coated chitosan membranes are suitable for the subconfluent transfer of human fibroblasts and keratinocytes.

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The kinetics of 18 amino acids, ammonia (NH3) and urea (UREA) in 18 liver cell bioreactor runs were analyzed and simulated by a two-compartment model consisting of a system of 42 differential equations. The model parameters, most of them representing enzymatic activities, were identified and their values discussed with respect to the different liver cell bioreactor performance levels. The nitrogen balance based model was used as a tool to quantify the variability of runs and to describe different kinetic patterns of the amino acid metabolism, in particular with respect to glutamate (GLU) and aspartate (ASP).

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The objective of this study was to evaluate the feasibility and safety of a hybrid liver support system with extracorporeal plasma separation and bioreactor perfusion in patients with acute liver failure (ALF) who had already fulfilled the criteria for high urgency liver transplantation (LTx). Eight patients (one male, seven female) were treated in terms of bridging to transplantation. The mean age was 36.5 yr (range 20 to 58). Etiology of liver failure was drug-related in two patients, hepatitis B infection in three patients, and unknown for three patients. The bioreactors were charged with primary liver cells from specific pathogen-free pigs. Cell viability varied between 91 and 98%. Continuous liver support treatment over a period of 8 to 46 h (mean 27.3 h) was safely performed and well-tolerated by all patients. No complications associated with the therapy were observed during the follow-up period. Thrombocytopenia was considered to be an effect of the plasma separation. Subsequently, all patients were transplanted successfully and were observed over at least 3 yr with an organ and patient survival rate of 100%. Screening of patient's sera for antibodies specific for porcine endogenous retroviruses (PERVs) showed no reactivity--either prior to application of the system, or after extracorporeal treatment. The results encourage us to continue the development of the technology, and further studies appear to be justified. The bioreactor technology has been integrated into a modular extracorporeal liver support (MELS) system, combining biologic liver support with artificial detoxification technology.

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Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed. The use of porcine cells or tumor cell lines is controversial. Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation. The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient. A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion. A cell mass of 400 g - 600 g enables the clinical application of a liver lobe equivalent hybrid organ. A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous veno-venuous hemodiafiltration may be included if required. Cells from 54 human livers have been isolated (donor age: 56 +/- 13 years, liver weight: 1862 +/- 556 g resulting in a viability of 55.0 +/- 15.9%). These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%). Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure. The overall treatment time was 7-144 hours. No adverse events were observed. Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system.

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Initial results of the clinical use of primary porcine liver cells for extracorporeal liver support are being reviewed as the cell source is controversial. According to Eurotransplant data 20-25% of explanted donor livers are not transplanted, due to factors such as steatosis or cirrhosis. This number corresponds to the number of patients with acute liver failure who require bridging therapy to transplantation. Primary human liver cells from transplant discards can be isolated, purified and maintained in bioreactors and provide an alternative for cell-based extracorporeal liver support therapy. A four-compartment bioreactor enables recovery from preservation and isolation injury in a three-dimensional network of interwoven capillary membranes with integrated oxygenation, rendering the liver cells from these discarded donor organs viable for clinical utilization. Patient contact with additional animal-derived biomatrix and fetal calf serum can be avoided. The initiation of an in vitro cultivation phase allows cell stabilization, quality control, and immediate availability of a characterized system without cryopreservation. The hypothesis of this paper is that with appropriate logistics and four-compartment bioreactor technology, cells from human liver transplant discards can serve the demand for cell-based therapy, including extracorporeal liver support.

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Acute liver failure has a poor prognosis. The introduction of liver transplantation as a therapeutic option reduced mortality to 20-40%.With the growing disparity between the number of organ donations and the number of patients waiting for liver transplantation, efforts have been made to optimize the allocation of organs and to design extracorporeal methods to support the failing liver. The modular extracorporeal liver support is a concept for the treatment of hepatic failure. The CellModule is a multicompartment bioreactor for extracorporeal liver support therapy. The construction provides efficient integrated oxygenator functions and decentralized mass transfer is effected by a woven array of capillary systems. The bioreactor promotes primary human liver cells to spontaneous neo-formation of liver sinusoidal structures in vitro. Small capillary subunits, in which interwoven membrane links represent the liver lobuli, are simultaneously perfused. The used cell mass of 400-600 g enabled the clinical application of a liver lobe equivalent hybrid organ. The DetoxModule enables albumin-dialysis for removal of albumin-bound toxins; a DialysisModule for continuous veno-venous hemofiltration can be added to the system, in the case of hepato-renal failure.

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OBJECTIVE: Hepatic in vitro studies, like those on hypoxia/reperfusion injury in liver transplants, demand large numbers of cultivated sinusoidal endothelial cells (SECs). In this article, we present and evaluate a new method for the isolation of SECs from porcine and human livers. METHODS: SECs were isolated employing a four-step collagenase perfusion. The sinusoidal character of the cells was validated by transmission and scanning electron microscopy, exclusion of Weibel-Palade bodies and factor VIII-related antigen, expression of scavenger receptor, and incorporation of latex beads. RESULTS: In 23 pigs, an average of 9 x 10(4) SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multilayer cocultures of isolated SECs and hepatocytes in a "sandwich" configuration. Standard cultures showed an average of 90% SECs in primary cultures and 100% SECs after the first passage. The possibility of isolation of SECs from human livers was demonstrated in eight cases. CONCLUSION: With the four-step collagenase perfusion it is possible to easily isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ.

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Commonly used materials incorporated into dynamic culture systems typically show the feature of adsorption of lipophilic xenobiotics. Yet, this phenomenon is strongly limiting the use of dynamic culture models and ex vivo organ perfusions in pharmacological and toxicological research. The aim of the study was to characterize different materials with respect to their capacity for drug adsorption and to find methods or materials to reduce the loss of substrate by adsorption in order to improve the use of dynamic in vitro systems. The adsorption of different xenobiotics (lidocaine, midazolam, lormetazepam, phenobarbital, testosterone, ethoxyresoroufine) to tubes used in dynamic in vitro systems (polyvinyl-chloride, silicone) were investigated and compared to a new material (silicone-caoutchouc-mixture). In addition, the role of protein deposition onto the tubing was studied and it was investigated whether it was possible to reach saturation of the inner tube surface by pre-loading it with the test compound. We found that silicone tubes provided the highest comfort with respect to handling and reusability, but they also demonstrated the highest capacity for substrate adsorption. Polyvinyl-chloride was the second best in handling but also demonstrated a high complexity in its adsorption behavior. The silicone-caoutchouc-mixture reached acceptable experimental results with respect to its handling and demonstrated a very low capacity for substrate adsorption.

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Hybrid liver systems are being developed as temporary extracorporeal liver support therapy. The overview given here emphasizes the development of both hepatocyte culture models for bioreactors and of systems for clinical therapy. In vitro studies demonstrate long term external metabolic function in isolated primary hepatocytes within bioreactors. These systems are capable of supporting essential liver functions. Animal experiments verify the possibility of upscaling bioreactors for clinical treatment. However, since there is no reliable animal model for investigating the treatment of acute liver failure, the promising results obtained from these studies have limited relevance to human beings. The small number of clinical studies performed thus far are not sufficient to enable any conclusions concerning improvements in the therapy of acute liver failure. Although important progress has been made in the development of these systems, multiple hepatocyte culture models and bioreactor constructions are being discussed in the literature, indicating competition in this field of medical research. For the use of hepatocytes and sinusoidal endothelial cells in coculture, a bioreactor has been designed. The construction is based on capillaries for hepatocyte aggregate immobilization. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. The parallel use of identical units enables easy upscaling. Initial studies on the use of discarded organs that are unsuitable for transplantation as a source for primary human liver cells seem to be promising.

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Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy. Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery. Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells. The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density. Treatment started after complete recovery from anesthesia and was performed continuously. A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group. Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.

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Pigs are widely used as models for a variety of human diseases, because many of their physiological functions closely resemble those of humans. However, information on instrumentation techniques is still scarce. In particular, experiments in conscious pigs focused on extracorporeal circuits are connected to a variety of methodical problems with respect to the handling of the animals. Usually, pigs are placed in restraint-slings during the application of an extracorporeal system. However, this method of restraint may lead to excessive mental distress even in trained animals. The latter might influence the results and certainly affects principles of animal welfare. Our own experiences with instrumented, conscious, but unrestrained dogs encouraged us to modify methods used for the fixation of in-dwelling central venous catheters in dogs with special regard to the species specific behaviour and phenotype of pigs. A cord retractable leash (CRL) was used for maintaining a safe distance between the animal and the outer ends of the catheters. To prevent dehiscences of the required fixation sutures a new catheter bag (CB) was designed to counteract tension forces caused by the CRL's spring-mechanism. The combination of both the CRL and CB enabled us to conduct safe experiments with conscious, unrestrained pigs. We alleviated the mental distress these animals were exposed to in comparison to former methods based on restraint of the animals.

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Culture media are frequently used in the evaluation of metabolical functions of hepatocytes in hybrid liver support systems (hLSS). However, media compositions differ substantially from those of plasma. Therefore, our study was designed to investigate whether current in vitro studies with medium are suitable to assess the metabolical competence of hLSS-cultures during clinical application as well as to explore whether the cell nutrition with medium provides a suitable modus operandi for stand by cultivation. Paired bioreactor cultures were perfused with either Williams' Medium E (MPB) or human plasma (PPB). About 6x108 primary pig hepatocytes (>97% viability) were cultured in three laboratory scale bioreactors designed according to Gerlach's bioreactor-concept. Different perfusion protocols were initiated after a standardised period allowing for cell attachment and reorganisation in aggregates. Whereas patterns of enzyme release were similar in both protocols the metabolical behaviour was different between MPB (anabolic state) and PPB (catabolic state). Furthermore, compared to MPB the lidocaine-MEGX-tests for PPB demonstrated lower MEGX-concentrations and a different reaction pattern. We conclude that the nutrition of hepatocytes with medium during the stand by period itself might influence the cell function and subsequently the efficacy of the hLSS-treatment during clinical application.

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A novel bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells. The reactor is based on capillaries for hepatocyte aggregate immobilization. Three separate capillary membrane systems, each permitting a different function are woven in order to create a three dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients are possible. The use of identical parallel units to supply hepatocytes facilitates scale up. In vitro studies demonstrate long-term external metabolic function in primary isolated hepatocytes within bioreactors. These systems are capable of supporting essential liver functions. Animal experiments have verified the possibility of scaling-up the bioreactors for clinical treatment. However, since there is no reliable animal model for investigation of the treatment of acute liver failure, the promising results obtained from these studies have limited relevance. The small number of clinical studies performed so far is not sufficient to reach conclusions about improvements in the therapy of acute liver failure. Although important progress has been made in the development of these systems, various hepatocyte culture models and bioreactor constructions are being discussed in the literature, which indicates competition in this field of medical research. An overview, which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies, and clinical application, is also provided.

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Hybrid liver support systems, which combine living cells of the liver in an extracorporeal circuit, have entered the first clinical trials in patients with acute liver failure. The goal of the different developments is to provide within a few hours a biological support of the patient's failing liver. Different technical solutions can be found to provide a sufficient functional mass of liver cells to the patient. Within this article, the biotechnical and clinical backgrounds of these developments are discussed, initial clinical results are summarized and an evaluation of this method as experimental treatment is given.

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