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At least 433 records · Page 24Linked to original sources
PubMed2004

Cell orientation influences the biaxial mechanical properties of fibroblast populated collagen vessels.

Bioartificial tissues, composed of cells in a collagen matrix, can be fabricated with preferred cell orientations to mimic the histologic arrangement of biologic tissues. The influence of preferred cell orientations on the biaxial mechanical behavior of bioartificial tissues is unknown. Characterizing the biaxial mechanical behavior is necessary for better predicting the in vivo behavior of bioartificial tissues. Fibroblast populated collagen vessels (FPCVs) were fabricated with two different cell orientations by controlling the mechanical constraints during incubation. The cell orientation was verified by confocal microscopy and the collagen fiber organization was examined by confocal reflection and scanning electron microscopy (SEM). Pressure-diameter, force-length tests were performed to determine the influence of cell orientation on the biaxial mechanical behavior. FPCVs were more extensible in the direction perpendicular to the preferred cell orientation, than in the direction parallel to the cell orientation. Biaxial tests were also performed in the presence of Cytochalasin D (Cyto D) to minimize the mechanical contribution of the cells. After Cyto D treatment, the FPCVs remained more extensible in the direction perpendicular to the cell orientation, even though a preferred collagen fiber orientation was not observed in the microscopy images.

Animals↗
PubMed2005

Development and perspectives of perfusion treatment for liver failure.

To treat patients with severe liver failure, liver transplantation and blood purification therapy, including plasmapheresis, hemodiafiltration, and bioartificial liver support, are available. The two mainstream systems developed for bioartificial liver support are extracorporeal whole liver perfusion (ECLP) and the bioreactor system (BIS). We developed a method of cross-plasma perfusion, in which plasma is exchanged between the blood circuit of the patient and that of a hepatic functioning unit, through which immunologically free whole human blood is perfused. From the aspects of efficacy and epidemic safety, the best system of bioartificial liver support for clinical use is considered to be ECLP in cross-plasma perfusion. In opposition, a social antagonist for zoonosis has consistently been raised, with controversy surrounding the use of xenogeneic organs for human treatment, which might be final obstacle. It is possible that the combination therapy of hemodiafiltration and the administration of human serum albumin and anticoagulant factors, which minimizes the economic and medical resource costs through the development of transgenic livestock that secrete human pharmaceuticals systemically, will become a more desirable and practical treatment for patients with severe liver failure.

Animals↗
PubMed1995

Functional activity of isolated pig hepatocytes attached to different extracellular matrix substrates. Implication for application of pig hepatocytes in a bioartificial liver.

For the manufacture of a bioartificial liver for human application, large amounts of viable and active hepatocytes are needed. Pig hepatocytes are considered to be the best alternative to scarce human hepatocytes. In vitro hepatocyte functions have so far been tested under different circumstances, mainly with rat hepatocytes. Pig hepatocytes were isolated with a single two-step isolation procedure, resulting in a high yield of viable hepatocytes. The hepatocytes were tested for their ability to synthesise urea, to metabolise 7-ethoxycoumarin (cytochrome P450 activity), and to synthesise and secrete proteins. These activities of hepatocytes while attached to tissue culture plastic were compared to the activity of the cells attached to several extracellular matrix constituents: collagen I and IV, laminin, fibronectin, Engelbreth-Holm-Swarm Natrix and in the presence of Matrigel. With the exception of Matrigel, neither of the extracellular matrix substrates enhanced pig hepatocyte function compared to tissue culture plastic. However, relatively large amounts of murine proteins leak out of the Matrigel. The advisability of using Matrigel or other extracellular matrix proteins in a bioartificial liver loaded with pig hepatocytes is discussed.

Animals↗
PubMed1998

The development of bioartificial nerve grafts for peripheral-nerve regeneration.

This article describes recent, significant scientific advances leading to the development of the bioartificial nerve graft. Schwann cells, which play an active role in the repair and function of peripheral nerves, are used to seed a synthetic, often resorbable conduit, which is then used to bridge and repair nerve gaps caused by injury or disease. By enhancing the rate and extent of regeneration, the bioartificial nerve graft holds great promise for improving recovery in the peripheral (and central) nervous system.

Animals↗
PubMed1993

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: a potential bioartificial liver.

Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.

Albumins↗
PubMed1997

Galactosamine-induced fulminant hepatic necrosis in unanesthetized canines.

A large animal model of fulminant hepatic necrosis is necessary to test the efficacy of artificial liver support systems. A recent model was developed using D-galactosamine in anesthetized dogs. Because of the difficulties encountered with prolonged anesthesia, a similar protocol was used in 10 unanesthetized dogs. Intravenous infusions of D-galactosamine (1.0 to 1.5 gm/kg) did not result in uniform death of all animals at 72 hours or development of hypoglycemia. Severe hepatic necrosis was observed in all animals, but residual hepatocyte viability was evident in some. All animals developed severe consumption coagulopathy with histological evidence of disseminated intravascular coagulation (DIC) in four. A clinical picture characteristic of endotoxic shock was observed in most animals as a terminal event. The presence of endotoxin was confirmed in all dogs tested after 12 hours (7/10). The differences observed between this model and the anesthetized model are probably because of the toxic synergism between halothane and D-galactosamine. Neither model seems satisfactory for the testing of artificial liver support systems. The halothane model requires extremely long periods of anesthesia and mechanical ventilation. The present model does not cause uniform or universal death of the animals within 3 days. Foremost, both models result in DIC and endotoxic shock, neither of which is likely to respond to bioartificial liver support or treatment with conventional dialysis membranes.

Anesthesia↗
PubMed1992

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.

Actins↗
PubMed2004

Inverse modeling using multi-block PLS to determine the environmental conditions that provide optimal cellular function.

MOTIVATIONS: Tissue engineering constitutes an important field with its potential of addressing the current shortage in organ availability. To successfully develop tissue-engineered organs, it is crucial to understand how to maintain the cells under conditions that maximize their ability to perform their physiological roles, regardless of the environment, whether the cells are part of an extracorporeal system, such as the bioartificial liver assist device, or an implantable tissue-engineered device. Our goals are to (1) provide insight into how cells will behave when confronted with changes in its environment and (2) determine the optimal environmental factors to achieve a desired level of cellular function. RESULTS: Diverse sets of environmental factors were used to systematically perturb the metabolic behavior associated with pre-conditioning and plasma supplementation. To probe metabolic state of hepatocytes, metabolic flux analysis was used to obtain the metabolic profile. We applied a multi-block partial least square (MPLS) model to relate environmental factors and fluxes to levels of intracellular lipids and urea synthesis. The MPLS model identified: (1) the most influential environmental factors and (2) how the metabolic pathways are altered by these factors. Finally, we inverted the MPLS model to determine the concentrations and types of environmental factors required to obtain the most economical solution for achieving optimal levels of cellular function for practical situations.

Amino Acids↗
PubMed2003

Bioartificial liver support devices: historical perspectives.

Fulminant hepatic failure (FHF) is an important cause of death worldwide. Despite significant improvements in critical care therapy there has been little impact on survival with mortality rates approaching 80%. In many patients the cause of the liver failure is reversible and if short-term hepatic support is provided, the liver may regenerate. Survivors recover full liver function and a normal life expectancy. For many years the only curative treatment for this condition has been liver transplantation, subjecting many patients to replacement of a potentially self-regenerating organ, with the lifetime danger of immunosuppression and its attendant complications, such as malignancy. Because of the shortage of livers available for transplantation, many patients die before a transplant can be performed, or are too ill for operation by the time a liver becomes available. Many patients with hepatic failure do not qualify for liver transplantation because of concomitant infection, metastatic cancer, active alcoholism or concurrent medical problems. The survival of patients excluded from liver transplantation or those with potentially reversible acute hepatitis might be improved with temporary artificial liver support. With a view to this, bioartificial liver support devices have been developed which replace the synthetic, metabolic and detoxification functions of the liver. Some such devices have been evaluated in clinical trials. During the last decade, improvements in bioengineering techniques have been used to refine the membranes and hepatocyte attachment systems used in these devices, in the hope of improving function. The present article reviews the history of liver support systems, the attendant problems encountered, and summarizes the main systems that are currently under evaluation.

Cells, Cultured↗
PubMed2005

A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation.

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Alginates↗
PubMed1992

Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: an in vitro system with application to the bioartificial liver. Rat hepatocytes cultured in cylindrical collagen gels.

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.

Albumins↗
PubMed2006

Kidney epithelial cells.

Kidney tubules are an essential component of an organism's blood clearance mechanism, recovering essential metabolites from glomerular filtration by active transport. Tubules are subject to injury, usually as the result of ischemia-reperfusion events that damage the polarized tubular cell layer that coats the tubule basement membrane, causing dysfunction and necrosis that is often associated with acute renal failure. However, tubules are capable of self-repair, forming new proximal tubular cells to replace failing or necrotic cells. The origin of the progenitor cells that give rise to new tubular cells is unknown. At one extreme, it is possible that all or a fraction of tubular cells can undergo a form of dedifferentiation and subsequent mitosis to form new tubular cells, or alternatively, it is possible that tubular regeneration follows the stem cell/transit-amplifying cell paradigm described for more rapidly regenerating organ systems. Regardless of the mechanism employed to generate new tubular cells, human tubular cells are readily grown in primary cultures and can recapitulate many of the metabolic, endocrine, and immunological properties attributable to endogenous renal proximal tubules when engrafted into bioartificial devices.

Adult Stem Cells↗
PubMed1994

Primary hepatocytes outperform Hep G2 cells as the source of biotransformation functions in a bioartificial liver.

OBJECTIVE: Metabolic activity of transformed human liver (Hep G2) cells and primary rat hepatocytes were compared during in vitro application of a gel entrapment bioartificial liver. BACKGROUND: Clinical trials of bioartificial liver devices containing either transformed liver cells or primary hepatocytes have been initiated. A study comparing transformed liver cells and primary hepatocytes in a bioartificial liver under similar conditions has not been reported previously. METHODS: Gel entrapment bioartificial liver devices were inoculated with 100 million cells, Hep G2 cell line (n = 4), or rat hepatocytes (n = 16), and studied for up to 60 days of in vitro cultivation. RESULTS: Hep G2 cells grew to confluence within the gel entrapment configuration with a doubling time of 20 +/- 3 hours. Rat hepatocytes significantly outperformed Hep G2 cells at confluence in all categories of biotransformation, including ureagenesis (3.5 +/- 0.7 vs. 0.3 +/- 0.1 mumol/hr, p < 0.05), glucuronidation (630 +/- 75 vs. 21 +/- 2 nmol/hr, p < 0.005), sulfation (59 +/- 13 vs. 5 +/- 2 nmol/hr, p < 0.05), and oxidation (233 +/- 38 vs. < 1 nmol/hr, p < 0.005). At the conclusion of one experiment, Hep G2 cells were found in the extracapillary compartment of the bioartificial liver, analogous to the patient's compartment during clinical application. CONCLUSIONS: Primary rat hepatocytes were superior to the Hep G2 cell line as the source of hepatic function in a bioartificial liver and avoided the potential risk of tumor transmigration from the bioartificial liver into the patient's circulation.

Albumins↗
PubMed1998

[Bioartificial liver].

Fulminant hepatic failure remains without satisfactory medical treatment and with a mortality rate of nearly 90%. Liver transplantation is the only chance to save these patients. However, there is a shortage of donors and many patients die before transplantation. It is thus necessary to develop a liver support system to help maintain patients alive, neurologically intact until an organ becomes available for transplantation, or ideally until hepatic regeneration. Early systems had only a depuration function, but now these systems include hepatocytes to replace most hepatic functions.

Cell Transplantation↗
PubMed1998

Commercially available media for flushing extracorporeal bioartificial liver systems prior to connection to the patient's circulation: an in vitro comparative study in two and three dimensional porcine hepatocyte cultures.

Extracorporeal bioartificial liver (BAL) systems based on hepatocytes need to be flushed before clinical application, as hepatocyte culture media are not approved for medical use. Commercially available 0.9% NaCl solution and hemofiltration solution (both supplemented with 10% human albumin) were investigated in vitro to test their potential to wash BAL systems with minimal stress for the cultured hepatocytes. After a 2 hour incubation, the lidocaine metabolising capacity and release of liver enzymes were assessed. As hepatocytes have been cultured in bioreactors in either two or three dimensional cell configurations, we tested the media in respectively hepatocyte monolayers cultures and in our newly developed bioreactor in which hepatocytes reorganise as small hepatocyte aggregates. The three dimensional hepatocyte cultures tolerated both media well, and no significant differences were seen compared with hepatocytes cultured in Williams' E (reference hepatocyte culture medium). The two dimensional hepatocyte cultures tolerated the supplemented hemofiltration solution and the reference medium equally well, but the condition of the porcine hepatocytes monolayer cultures was significantly impaired when incubated with the supplemented physiological saline solution. In conclusion, as a supplemented physiological saline solution may have detrimental effects on the condition of the hepatocytes, the more complex hemofiltration solution (bicarbonate buffered, glucose, essential minerals) was considered the better alternative for flushing bioartificial liver systems.

Animals↗
PubMed2007

Integration of technologies for hepatic tissue engineering.

The liver is the largest internal organ in the body, responsible for over 500 metabolic, regulatory, and immune functions. Loss of liver function leads to liver failure which causes over 25,000 deaths/year in the United States. Efforts in the field of hepatic tissue engineering include the design of bioartificial liver systems to prolong patient's lives during liver failure, for drug toxicity screening and for the study of liver regeneration, ischemia/reperfusion injury, fibrosis, viral infection, and inflammation. This chapter will overview the current state-of-the-art in hepatology including isolated perfused liver, culture of liver slices and tissue explants, hepatocyte culture on collagen "sandwich" and spheroids, coculture of hepatocytes with non-parenchymal cells, and the integration of these culture techniques with microfluidics and reactor design. This work will discuss the role of oxygen and medium composition in hepatocyte culture and present promising new technologies for hepatocyte proliferation and function. We will also discuss liver development, architecture, and function as they relate to these culture techniques. Finally, we will review current opportunities and major challenges in integrating cell culture, bioreactor design, and microtechnology to develop new systems for novel applications.

Animals↗