Biomedical subjects

Gesine Pless

Publications and source records attributed to Gesine Pless.

9 recordsLinked to original sources

PubMed2007

Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent.

Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 +/- 13 vs. 46.9 +/- 11%, P < 0.01) and plating efficiency (41.5 +/- 18 vs. 17.6 +/- 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation.

Albumins↗

PubMed2006

Evaluation of primary human liver cells in bioreactor cultures for extracorporeal liver support on the basis of urea production.

Primary human liver cells from donor organs unsuitable for transplantation were cultivated in bioreactors developed for extracorporeal liver support. Because each system contains cells originating from an individual organ, each bioreactor culture must be individually characterized. The objective of this study was to identify suitable decisive parameters for the evaluation of cell culture performance. We analyzed the data from 47 bioreactor cultures containing 437 +/- 110 g of cells. Choosing urea production as the decisive parameter, the bioreactor cultures were divided into high-performance (daily urea production > or = 110 mg per bioreactor between culture days 3 and 14) and low-performance cultures. Comparing the mean courses of the groups revealed a significant distinction in most other investigated biochemical parameters. In conclusion, urea production seems to be an appropriate parameter for evaluating the performance of liver cell cultures in bioreactors because it corresponds to all other evaluated parameters of cell function.

Adult↗

PubMed2005

"Blogs" and "wikis" are valuable software tools for communication within research groups.

Appropriate software tools may improve communication and ease access to knowledge for research groups. A weblog is a website which contains periodic, chronologically ordered posts on a common webpage, whereas a wiki is hypertext-based collaborative software that enables documents to be authored collectively using a web browser. Although not primarily intended for use as an intranet-based collaborative knowledge warehouse, both blogs and wikis have the potential to offer all the features of complex and expensive IT solutions. These tools enable the team members to share knowledge simply and quickly-the collective knowledge base of the group can be efficiently managed and navigated.

Communication↗

PubMed2005

In vitro evaluation of the transportability of viable primary human liver cells originating from discarded donor organs in bioreactors.

BACKGROUND: The use of primary human liver cells obtained from discarded donor organs is increasingly favored for cell-based extracorporeal liver support systems. However, as cryopreservation of primary human hepatocytes causes a significant loss of metabolic activity, the transport of bioreactors with viable liver cells is required. The aim of this study was to evaluate the impact of two major potential threats to viable cells during transport: temperature changes and mechanical stress. METHODS: In each experiment three hollow fiber-based bioreactors were charged with primary human liver cells originating from the same discarded donor organ and were simultaneously kept under culture conditions for 8 days. In total, 18 bioreactors were evaluated. On the fifth day the bioreactors were exposed to hypothermia (4 degrees C, n = 3), to hyperthermia (42 degrees C, n = 3), or served as normothermic controls (37 degrees C, n = 3). In a second test series bioreactors were exposed to vibration (21 Hz for 20 min, thereafter 7 Hz for 160 min, n = 3), or were operated as control cultures (n = 6). The release of hepatocyte-specific enzymes was determined as an indicator for cell damage. RESULTS: Hypothermic stress resulted in a significant release of transaminases and led to disturbances of the histological integrity, all indicating a high degree of cell damage. When compared with the control cultures, hyperthermia and mechanical stress in terms of vibration had no significant effect on the cells. CONCLUSION: The transport of hollow fiber bioreactors charged with viable primary human liver cells appears to be feasible in transport monitors for perfusion and temperature control.

Bioreactors↗

PubMed2005

The SlideReactor--a simple hollow fiber based bioreactor suitable for light microscopy.

Most bioartificial liver support systems are based on hollow fiber capillaries within modified dialysis cartridges or more sophisticated bioreactor constructions. Due to their design microscopic follow-up of reorganization and growth of tissue between the hollow fibers is not possible. The SlideReactor is a simple hollow fiber based bioreactor construction suitable for light microscopy and time-lapse video observation. The SlideReactor offers a cell compartment separated from a medium inflow and outflow compartment. Cell compartment access ports enable easy filling of the cell compartment with cell suspension, as well as fixation of the tissue. For more complex procedures or full access to all the cells, the bioreactor can be opened easily by cutting the silicone seal with a scalpel. Due to its simple design and the utilization of standard materials, it could serve as a suitable, cost-efficient tool to evaluate the behavior of cells cultured between hollow fiber capillaries. The paper describes the production process: similar to open source projects in software engineering, we would like to propose the concept as an open platform to anyone interested in hollow fiber based cell culture.

Bioreactors↗

PubMed2003

Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study.

INTRODUCTION: The development of a bioreactor providing a three-dimensional network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange enables the culture of primary human liver cells from discarded donor organs for extracorporeal liver support. METHODS: Primary liver cells were isolated from 54 discarded organs (donor age 56.7+/-13.2 years). Between 2.8x10(10) and 6.4x10(10) parenchymal cells (PC) were cocultured with nonparenchymal cells (NPC) of the same organ in bioreactors (n=36). The metabolic activity of the cells was regularly determined during culture. The cell morphology and ultrastructure were investigated after culture periods of 1 to 5 weeks. RESULTS: Cell metabolism was maintained over at least 3 weeks after a phase of adaptation lasting 2 to 3 days. Through the use of transmission electron microscopy and immunohistochemistry, it was demonstrated that PC and NPC spontaneously formed tissue-like structures. Vascular cavities (CD 31 immunoreactivity [IR]) and bile duct-like channels (CK 19 IR), both exhibiting proliferation activity (Ki-67 IR), were regularly distributed. Some of the bile duct-like channels showed similarities to the Canals of Hering found in the natural liver. Cells expressing morphologic and antigenic characteristics of adult liver stem cells (CD 34 IR and c-kit IR) and areas with cells that showed both hepatocyte and biliary characteristics were detected. CONCLUSION: The results show that primary human liver cells obtained from discarded donor organs recover and can be maintained in bioreactors for clinical liver support therapy. In addition, initial observations on adult liver stem-cell culture in bioreactors are presented.

Aged↗

PubMed2003

Extracorporeal liver support based on primary human liver cells and albumin dialysis--treatment of a patient with primary graft non-function.

METHODS: Following liver transplantation, a 26-year old female suffered from primary non-function of the transplant. The patient was subsequently treated with a modular extracorporeal liver support concept until a suitable organ became available. A bioreactor was charged with human liver cells, obtained from a discarded cadaveric graft (470 g, viability: 60%). The bioreactor was integrated into an extracorporeal circuit with continuous single pass albumin dialysis and continuous veno-venuous hemodiafiltration for detoxification and fluid reduction. RESULTS: Over the total system application time of 79 h, a significant reduction of the plasma levels of total bilirubin (21.1 mg/dl at start, 10.1 mg/dl at end of therapy) and ammonia (100 versus 22.7 micromol/l) was achieved. During treatment the patient's neurological status significantly improved from coma stage IV to I permitting extubation. Recovery of kidney function with a urine output of 1325 ml/24 h compared to 45 ml/24 h prior to system application, was noted. Over the treatment period, an improvement of coagulation status was observed. Adverse events were absent. CONCLUSIONS: This first successful clinical treatment of a patient with liver failure suggests that a modular approach combining both primary human liver cell bioreactor technology and detoxification methods is promising.

Adult↗

PubMedDate not supplied

Recovery of preservation-injured primary human hepatocytes and nonparenchymal cells to tissuelike structures in large-scale bioreactors for liver support: an initial transmission electron microscopy study.

This study investigated large-scale regeneration and tissue reorganization of adult human liver cells from preservation injured transplant organs. The use of basement membrane protein gels and growth factor enriched culture medium in standard culture flasks promotes liver tissue formation in isolated rat and pig hepatocytes, resulting in prolongation of phenotypic stability and metabolic competence of primary cells in vitro. A special bioreactor construction for high-density three-dimensional cell recovery was developed and isolation of cells from discarded human donor livers was enabled. In vitro regeneration of adult human liver cells isolated from preservation-injured organs took place over a period of 2 weeks in a purpose-built bioreactor. Basement membrane protein and growth factors were avoided. Reorganization of tissue structures was studied using transmission electron microscopy (TEM). This showed regeneration and tissue reorganization of adult human cells from preservation-injured organs by coculture with nonparenchymal cells in the bioreactor. The majority of the aggregated hepatocytes in the bioreactors showed morphological similarities to those in vivo (although not re-formed to hepatocyte plates), exhibiting cell-cell junctions and reconstituted bile canaliculi-like spaces between neighboring hepatocytes. Perfusion channels appeared regularly between cell aggregates. The arrangement of nonparenchymal cells between the hepatocyte aggregates exhibited similarities to liver sinusoids. Endothelial cells often covered the aggregates and formed a borderline to the perfusion channels between the capillaries. Similar to the space of Disse, further nonparenchymal cells were located between the endothelial cells and the parenchymal aggregates. Deposits of biomatrix fibers occurred spontaneously. The regenerated cell mass was close to that of a single liver lobe. In conclusion, the further optimization of bioreactors that enable cell recovery from preservation injury may lead to the utilization of cells from discarded whole or split transplants for extracorporeal temporary liver support therapy or hepatocyte transplantation.

Adult↗

PubMedDate not supplied

Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions.

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (CYP450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.

Albumins↗