Dissection of Bitcoin's multiscale bubble history from January 2012 to February 2018.

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.

Culture of subconfluent human fibroblasts and keratinocytes using biodegradable transfer membranes.

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.

Comparison of four methods for mass hepatocyte isolation from pig and human livers.

Using the pig liver, parameters for large scale hepatocyte isolation were studied in order to develop a technique suitable for human organs. These investigations led to a 5-step modification of the original 2-step method. Four groups were compared. A nonenzymatic EDTA perfusion technique has been shown to be inconvenient for mass cell isolation. The enzymatic 2-step perfusion, using 0.08% collagenase and 20-kg pigs, resulted in a mean hepatocyte viability of 61 +/- 1.9%, with a mean yield of 67 +/- 6.5% wet weight of the organ. The enzymatic 5-step method resulted in a mean hepatocyte viability of 74 +/- 1.7% with a mean yield of 80 +/- 1.8% wet weight. Five-step portal venous perfusion in combination with arterial perfusion resulted in 76 +/- 2.6% viability with a yield of 82 +/- 6.1%. The results were dependent on collagenase concentration and weight of the donors, improving with decreasing body weight. The 5-step method with combined arterial and portal vein perfusion developed for pig liver was used for mass human liver cell isolation with a minimum viability of 57% and a minimum yield of 58% wet weight.

Bioreactor for a larger scale hepatocyte in vitro perfusion.

A bioreactor construction for hepatocytes and liver sinusoidal endothelial cells is described. The reactor is based on capillaries for hepatocyte immobilization. Four discrete capillary membrane systems, each serving different purposes, are woven to create a three-dimensional framework for decentralized cell perfusion with low metabolite gradients and decentralized oxygenation and CO2 removal. The biochemical performance of reactors initially seeded with 2.5 x 10(9) hepatocytes were evaluated over 3 weeks. On day 21, pig albumin synthesis was 4.7 mg/day, lidocaine metabolism was 813.7 +/- 23 micrograms/hr, galactose elimination was 210.1 +/- 3 mg/hr, and midazolam metabolism was 37.1 +/- 2 micrograms/hr. The specific construction of the reactor enables scale-up to hybrid liver support systems as extracorporeal bridging devices for liver transplantation.

Visualization of liver sinusoidal endothelial cell repair behavior after preservation by in vitro time-lapse video microscopy.

Sinusoidal endothelial cells are significantly more vulnerable to cold storage and reperfusion than hepatocytes. In this study, a method for assessing the repair behavior of sinusoidal endothelial cells in vitro, after preservation, was investigated. Time-lapse video microscopy analysis was performed and migration rates, division rates, and cell detachment rates were determined. Preservation intervals between 3 and 24 hr and reoxygenation times between 4 and 24 hr were compared. A comparison between sinusoidal endothelial cultures that were stored for 6 hr in University of Wisconsin solution and nonpreserved control cultures was performed. This method allows the investigation of the repair capability of individual cells in vitro. Indications of the kind of preservation/reoxygenation injury that occurs after treatment with several preservation solutions and the resultant repair behavior can be obtained.

Cell detachment during sinusoidal reperfusion after liver preservation: an in vitro model.

BACKGROUND: Sinusoidal endothelial cells (SEC) are significantly more vulnerable to cold storage and reperfusion than hepatocytes. Swelling and disruption of the sinusoidal lining induce the microcirculatory disturbances seen after reperfusion. In this article, the investigation of a method to assess the adhesion and morphology of SEC in vitro during reperfusion after preservation is described. METHODS: Time-lapse video microscopy analysis was performed and cell detachment rates and cell lengths were determined. Preservation intervals between 6 and 24 hr and flow rates ranging from 3 L/min to 9 L/min (resulting in shear stresses between 5.1 and 15.3 dynes/cm2 on the monolayer surface) during reperfusion period were compared. SEC that were stored for 6 hr in University of Wisconsin solution and nonpreserved control cultures were compared. RESULTS: Varying the preservation intervals from 6 hr to 24 hr during reperfusion at a flow rate of 3 L/min led to increased cell erosion rates (6 hr, 35.5+/-15.2%; 12 hr, 38.0+/-7.6%; 18 hr, 54.3+/-5.7%; 24 hr, 76.7+/-6.7%; nonpreserved cells, 3.4+/-3.4%). Storage periods from 12 hr to 24 hr led to significantly higher cell detachment rates than occurred in nonpreserved cells. CONCLUSIONS: This method allows the investigation of the adhesion capability and morphology of individual cells in vitro. Indications of the kind of preservation/reperfusion injury that occurs after treatment with several preservation solutions and the resultant repair behavior can be obtained.

Is a clinical application of hybrid liver support systems limited by an initial disorder in cellular amino acid and alpha-keto acid metabolism, rather than by later gradual loss of primary hepatocyte function?

The in-vitro amino acid (AA) and alpha-keto acid (KA) metabolism of bioreactors initially seeded with 2.5 x 10(9) pig hepatocytes was investigated with a perfusion technique. Considerable changes in the culture medium concentrations of AA and KA were measured during the first days in culture. This is indicative of dynamic cellular metabolism in the initial phase. While the concentration of pyruvate decreased starting on the first day, alpha-ketoglutarate, alpha-ketoisocaproate, alpha-ketoisovalerate, and alpha-keto-beta-methyl-n-valerate were synthesized. The long term use of hepatocyte cultures in bioreactors and thus a desirable clinical hybrid liver support therapy appears to be possible since the hepatocytes switched, after 15 days in culture, to steady-state conditions with a stable amino acid turnover featuring general AA uptake accompanied by KA release. The release of branched chain KA, in particular that of alpha-ketoisocaproate, reflects an effective transamination activity in the bioreactor system. Primary pig hepatocytes cultivated in hybrid liver support systems for therapy of acute liver failure or as devices for bridging to liver transplantation can sustain amino acid metabolism for at least 30 days in vitro. However, an initial disorder following the cell isolation that is demonstrated may limit immediate utilization of the systems prior to the reorganisation of the cells to tissue-like structures in bioreactors.

Adsorption of xenobiotics to plastic tubing incorporated into dynamic in vitro systems used in pharmacological research--limits and progress.

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.

Bioreactors for hybrid liver support: historical aspects and novel designs.

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.

Development of a hybrid liver support system.

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.

Use of hepatocyte cultures for liver support bioreactors.

Hybrid artificial liver systems are being developed as extracorporeal temporary liver support therapy. Here, an overview is given with emphasis on hepatocyte culture models for bioreactors, in vitro studies, animal studies and the clinical application of hybrid liver support systems. In vitro studies show long term external metabolic functions of primary isolated hepatocytes in bioreactors. These systems are capable of supporting essential liver functions. Animal experiments show the possibility of upscaling the bioreactors for clinical treatment. Since there is no reliable animal model for investigations on the treatment of acute liver failure, the promising results of these studies have limited relevance. The small number of clinical studies are not sufficient to give statements about a clinical improvement of therapy of acute liver failure. Although important progress has been made in the development of the systems, multiple different hepatocyte culture models and bioreactor constructions are discussed in the literature, indicating competition in this field of medical research.

Development of a hybrid liver support system: a review.

Hybrid liver support systems (LSS) for the use of the detoxifying, metabolic synthetic and regulatory capabilities of liver cells are under development for extracorporeal therapy of acute liver failure and for bridging to liver transplantation. A summary of our development is discussed. A five-step technique for primary liver cell isolation has been introduced in order to address larger scale procurement of hepatocytes. Immobilisation of the cells after isolation appears to be one of the main factors in maintaining hepatocyte function in vitro. Different techniques have been investigated. Using the cell-cell adhesion technique, a culture model was developed for the immobilisation of hepatocytes between capillary membranes. Four separate capillary membrane systems, each forming independent compartments are woven in order to create a three dimensional network. A bioreactor design has been developed. The construction provides different functions, including decentralised cell perfusion. The bioreactor enables 3 dimensional reorganisation of cells, integral oxygenation and decentralised metabolite exchange. The bioreactor has been scaled-up to allow hepatocytes and sinusoidal endothelial cells to be cultured in quantities sufficient for therapeutic application. In a healthy pig model, possible limiting side effects of therapy with this device were excluded. The efficacy of the system has been demonstrated in a hepatectomised pig model. Subsequently, a complete hybrid liver support system for human studies was introduced and applied clinically.

Consideration of potential immunological problems in the application of xenogenic hybrid liver support.

Hybrid liver support systems (LSS) for the use of pig liver cells are under development for extracorporeal therapy of acute liver failure and for bridging to liver transplantation. A literature overview about possible immunological side effects of a clinical application is given. The data summarised from experimental studies and those clinical applications of porcine cells reported so far, suggest that clinical use of LSS utilising porcine cells and an immuno-isolation membrane should not be compromised by severe immunological complications. The reported data suggest that clinical application should be conducted in conjunction with carefully planned immunological monitoring. Only after such applications of LSS have been carried out and further data have been evaluated, might one be able to judge the immunological consequences of broader application of hybrid liver support.

Nonenzymatic versus enzymatic hepatocyte isolation from pig livers for larger scale investigations of liver cell perfusion systems.

A comparison of nonenzymatic and enzymatic hepatocyte isolation was performed on pig livers. The collagenase perfusion showed superior results: mean viability 72 +/- 10% versus a maximum viability of 21% using EDTA-perfusion. A five-step collagenase perfusion technique, developed for pig livers enables larger scale investigations, in order to develop methods for hepatocyte cultures in therapeutical liver cell perfusion systems.

Comparison of hollow fibre membranes for hepatocyte immobilisation in bioreactors.

Various hollow fibre membranes of polyamide, cellulose and polypropylene were investigated as potential substrata for hepatocyte immobilisation in bioreactors for hybrid liver support systems. Membranes were subjected to a cytocompatibility test in which the attachment and morphology of primary hepatocytes were evaluated. The effect of coating with collagen and fibronectin was also studied. Adequate cell immobilisation was possible on polypropylene and polyamide membranes even without coating. The flattening process of the cells was dependent on the material and the coating. The incorporation of porous polypropylene hollow fibres in hybrid liver cell bioreactors and their specific permeability properties could also offer means for cell oxygenation, metabolite distribution and immuno-isolation purposes.

Development of a special catheterbag to enable artificial organ evaluation in conscious, unrestrained pigs: technical note.

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.

Hepatocyte aggregate culture technique for bioreactors in hybrid liver support systems.

Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.

Comparative analysis of metabolism of medium- and plasma perfused primary pig hepatocytes cultured around a 3-D membrane network.

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.

Immunoisolation of hybrid liver support systems by semipermeable membranes.

Immunoisolation of hybrid liver support systems (LSS) utilizing suitable semipermeable membranes as an immune barrier enables neither immunocompetent cytotoxic factors to cause damage to the hepatocytes in the bioreactor nor xenogenic hepatocyte products to cause immunological side effects in patients. To determine the capability of membranes as an immune barrier, 6 flat membranes were investigated: Cuprophan (C-100), cut-off MW 1000, Cuprophan (C-240), cut-off MW 10,000, Polypropylen hydrophilic and hydrophobic (PPhi, PPho), cut-off MW 500,000-1,000,000, Polysulfon (PS), cut-off MW 1,000,000, Polyamid (PA), cut-off beyond MW 1,000,000. The permeability of the membranes to plasma factors and liver protein fractions (LP) was studied by routine biochemical methods and gel electrophoresis. In a second study, pigs (n=7) were immunised by LP after membrane passage. The results showed PA, PS, and PPhi to be completely permeable for plasma factors and LP C-100 and C-240 for urophanic substances, and C-240 again for LP under MW 14.000. All 7 pig sera studied by Western blot discovered pre-formed xenoreactive natural IgG-antibodies (NAB) against human liver antigen (AG) with MW 26.000. AB de-novo-synthesis was demonstrated for AG with MW 45.000. No AB-synthesis was induced for epitopes under MW 26,000. These results suggest that limiting the cut-off of bioreactor outflow membranes to MW < 26,000 could avoid immunological side effects to patients.

Extracorporeal liver support: porcine or human cell based systems?

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.