Comparative Analysis of Metabolism of Medium-and Plasma Perfused Primary Pig Hepatocytes Cultured around a 3-D Membrane Network

Abstract

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.

Kinetic Analysis of Lidocaine Elimination by Pig Liver Cells Cultured in 3D Multi-Compartment Hollow Fiber Membrane Network Perfusion Bioreactors

“…The model predicts that the kinetic constant of the MEGX further biotransformation (i.e., k 2, A , k 2, B ) decreases in time faster than that of MEGX formation from lidocaine (i.e., k 1, A , k 1, B ). This causes the shift towards longer t max and eventually causes the MEGX concentration profile to lose its typical bell shape with downward concavity as cells die off in adhesion culture, as shown in Figure 5 and Figure 8 [ 18 ]. Maintenance of a good liver cell state and competence in the bioreactor is possibly permitted by the favorable cell microenvironment produced by the controlled intermembrane spaces enabled by the orderly membrane arrangement and by the bleed/feed perfusion mode.…”

“…If deemed useful, lidocaine in the adsorbed phase could be predicted by the model for the best-fit parameter values. Experimental data previously reported [ 18 ] were included in the analysis. Lidocaine binding to serum proteins was accounted for with an unbound lidocaine fraction fu = 0.65 [ 19 ].…”

Cryopreservation and gel collagen culture of porcine hepatocytes

“…Matrix is an important part of affecting hepatocytes function. Many researches showed that collagenous matrix could provides an environment that more closely resembles that in vivo and allows to maintain the expression of certain liver-specific function of hepatocytes [44][45][46][47] . In this study, the hepatocytes keep their biological characters well after storage in liquid nitrogen for 4 mo.…”

Thermography as potential real-time technique to assess changes in flow distribution in hemofiltration

“…The filter was kept at room temperature under light exclusion until reuse and after priming the filter with normal saline that contained an in vitro dosage of antibiotics proven to be effective in maintaining sterile conditions and membrane functionality in hollow fiber bioreactors (penicillin 100 IU/ml and streptomycin 100 mg/ml). 18 Another 10 l of normal saline was used to extensively rinse the filter again just before priming for a second time with blood for CT measurement. Blood priming was performed in single-pass mode and the system was operated with blood flow and filtration rates similar to the first experiments.…”