Investigation of experimental cell lines and non-invasive online sensor technologies in a 3D bioreactor system for extracorporeal liver support therapy

Liver transplantation is currently the only successful treatment to cure acute or acute-on-chronic liver failure. However, the number of patients waiting for a donor organ is constantly rising due to scarcity of organ donations. To overcome this bottleneck different bio-artificial liver technologies are under investigation, which may take over functions of the diseased liver until a suitable donor organ can be transplanted or the diseased liver has recovered and regained its functions. A drawback so far is the lack of an effective cell source compensating human liver functions in vivo during extracorporeal liver support therapy. To be used in extracorporeal liver support therapy, cells need to be available in sufficient numbers, and they should be of human origin to minimize immunologic complications and safety risks for the patient. In this study, a novel cell source and sensor-based online monitoring methods were investigated using a multi-compartment hollow-fibre bioreactor technology developed at the Charité in Berlin for application in clinical extracorporeal liver support therapy. In the first part of the study, the rat pancreatic progenitor cell line (AR42J-B-13) was investigated as a model cell source for use in the bioreactor system. The ability of B-13 cells to trans-differentiate to liver-like cells (B-13/H cells) and the maintenance of hepatic functions were determined by means of metabolic parameters in addition to gene expression and histological analyses. Experiments revealed successful trans-differentiation in the bioreactor system. Bioreactor cultures showed increasing liver-specific functions, namely production of albumin and urea as well as cytochrome P450 activity. In contrast, secretion of amylase, typical for undifferentiated B-13 cells, declined over the culture period. Metabolic observations were confirmed by data from gene expression and protein analysis. Immune histochemical staining showed the expression of hepatic markers (CYP2E1, albumin, CK18, CEBP-ß and MRP2) in B-13/H cells after hepatic trans-differentiation in the bioreactor system. In the second part of the study, the integration of multi-parametric sensors into the bioreactor system was investigated to allow for cell culture surveillance in real-time. For this purpose oxygen and pH sensors (PreSens-Precision Sensing GmbH), as well as ammonia sensors and impedance sensors developed by the cooperation partners CEA-Leti-France and Fraunhofer IBMT, respectively, were integrated in an analytical-scale bioreactor for evaluation. In order to evaluate the ability of sensor-based methods to detect cell injury, the toxic drug methapyrilene was applied to B-13/H cells trans-differentiated in the bioreactor system. Online measurement of ammonia concentrations showed results comparable to offline values measured in samples from the culture medium. However, further optimisation concerning sterilisation, sensitivity, minimization of noise detection and sensor leakage is needed before using the technology in clinical applications. Impedance measurement enabled safe, sensitive and non-invasive detection of changes in the culture condition with a distinct response to toxic stress. To evaluate the bio-artificial liver system in a clinical setting, primary porcine liver cells (ppL) were investigated in the bioreactor system. As a model for toxic plasma exposure during clinical liver support sessions, the effect of the hepatotoxic drug acetaminophen (APAP) was evaluated. The response of the cells to toxic drug exposure was successfully monitored by sensor-based measurements, confirming the results from B-13/H cultures. Based on the results a procedure for culture prediction and decision-making conceived for extracorporeal liver support in a clinical setting was established. In the third part of the work, an up-scaled version of the bioreactor system was used in a pilot study to investigate the efficiency of cell culture and sensor-based monitoring in a clinical setting. As a cell source the H-14 cells developed by the cooperation partner Newcastle University were used as an experimental human equivalent of the B-13 cell line. The results of the pilot study indicate the feasibility of sensor-based monitoring during cell culture in the large-scale bioreactor. However, additional work has to be conducted to ensure sufficient cell numbers and to optimize sensor techniques for extracorporeal liver support in clinical application. In conclusion, the B-13 cell line represents a suitable model cell source in combination with the four-compartment bioreactor system for in vitro and clinical research. The integration of non-invasive online sensors enables sensitive culture surveillance and culture prediction. Finally, the genetically modified HPAC cell line H-14 might be a vital step towards the establishment of a human cell source in sufficient quality and quantity for extracorporeal liver support.

Author dc:creator

• Richter, Marco

Advisors dc:contributor.advisor

• Lauster, Roland
• Zeilinger, Katrin

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-5571

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/5984