Transcript Folie 1 - Forska Utan Djurförsök

Dynamic 3D Bioreactor Culture Model For Long-Term Maintenance
Of Human Hepatocyte Metabolic Function
Thomas Schreiter1, Malin Darnell3, Therese Söderdahl2, Nils Bohmer1, Daniel Knobeloch4, Birgitta Dillner2, Anna-Lena
Berg2, Andreas K.N. Nüssler5, Jörg C. Gerlach1,6, Katrin Zeilinger1, Tommy B. Andersson2,3
1Div.
of Experimental Surgery, BCRT and 4Department of Surgery, Charité Universitätsmedizin Berlin, Germany; 2AstraZeneca R&D, Sweden; 3Karolinska Institute,
Stockholm, Sweden; 5Department of Traumatology, TU Munich, MRI, Germany; 6McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA, USA
Introduction
Results
A major requirement in the development of new potential drug
candidates is adequate safety testing. The liver represents the
central target for human drug metabolism, and hepatic drug toxicity
is one of the main reasons for withdrawal of drugs from clinical use.
In vitro models based on human liver cells would be useful to
provide human-predictive information on the metabolism and
possible side-effects of drugs.
We focus on bioreactor technologies that address the cellular needs
of 3D tissue organisation in a highly physiological environment.
Primary human hepatocytes as well as hepatic cell lines survive and
can be maintained in the bioreactor over several weeks. In this
study the pattern and long-term stability of various cytochrome P
450 (CYP) activities was investigated in primary human hepatocytes
(pHHC) and two different liver cell lines (C3A, Huh7) and cultured in
a miniaturized bioreactor prototype.
Methods
Bioreactor system
The bioreactor (Fig. 1) consists of two independent bundles of
microfiltration capillaries for the transport of culture medium (red and
yellow), interwoven with one bundle of oxygenation capillaries
(blue). The capillaries provide a 3D scaffold for the cells residing in
the extra-capillary space (cell compartment). The bioreactor is
integrated into a perfusion system equipped with pump units for
medium supply, pressure and flow control, a gas mixing unit and an
electronically controlled heating unit (Fig. 2). Immunohistochemical
stainings demonstrate cell reorganisation and formation of tissue
like aggregates in the system, including biliary and vascular
structures as well as expression of hepatic transporters (Fig. 3).
Comparison of CYP activities in primary human hepatocytes, C3A and Huh7 cells
Primary human hepatocytes cultured in bioreactors show more than 10x higher CYP activities than the liver
cell lines for all three CYP isoforms tested (Fig. 4). Among the cell lines investigated, the Huh7 cell line
shows distinctly higher CYP activities, in particular regarding CYP1A1/2 and CYP2C9.
Fig. 4: Course of CYP metabolite formation from phenacetin (CYP1A1/1A2), diclofenac (CYP2C9) and
midazolam (CYP3A4) in human hepatocytes (blue), Huh7 cells (red) and C3A cells (orange)
cultured in bioreactors.
Evaluation of the long-term stability of CYP activities of primary human hepatocytes
cultured in bioreactors
All three CYP isoforms investigated exhibit distinct and reproducible activities in primary human hepatocyte
bioreactors after 3-5 days (Fig. 5, blue curves). In one bioreactor maintained over 23 days human
hepatocytes still show marked CYP activities at the end of the culture period (Fig. 5, red curves).
Fig. 5: Course of CYP1A1/1A2, CYP2C9 and CYP3A4 activities in primary human hepatocyte
bioreactors. The blue curves each represent the mean of five bioreactors cultured with human
hepatocytes from five donors for 3-5 days. The red curves show CYP activities of human
hepatocytes cultured in a bioreactor for 23 days.
CYP activity is correlated with urea production
Fig. 1: Bioreactor (schematic)
Fig. 2: Bioreactor perfusion system
We observed that as long as human hepatocytes produce urea above detection limit they exhibit CYP
activity. The heat map (Fig. 6) was created to compare urea production and CYP activities for several
bioreactors at different time points of bioreactor culture. The fact that urea and CYP activities show the
same color at most time points representing either low or high levels indicates a correlation. The
calculated correlation coefficient was highest for CYP1A1/1A2 (0.9) and less evident for CYP2C9 and
CYP3A4 (0.63 and 0.51, respectively).
Human Liver
Bioreactor
CK19
CD68
MDR1
Fig. 3: Immunohistochemical stainings of human liver tissue
and liver cell material from a bioreactor
Cell preparation / Bioreactor operation
A total of 6.0 to 8.0 x 107 cells of the human hepatoma cell line C3A
or Huh7 were inoculated in bioreactors with a cell compartment
volume of 2 ml, respectively. Cells were allowed to adapt to the 3D
environment and to reach a stable functional level before starting
experiments.
Primary human hepatocytes (pHHC) were prepared by collagenase
digestion of healthy tissue obtained from partial liver resections and
between 5.0 and 12.0 x 107 cells were inoculated into one bioreactor,
respectively.
During the entire run biochemical parameters for cell viability,
including glucose consumption and production of urea, lactate and
ammonia were measured daily. Cell damage was surveyed by
release of LDH, AST, ALT and GGT into the culture medium.
CYP activity analysis
CYP1A1/1A2, CYP2C9 and CYP3A4 activities were determined by
incubation of the cultures with suitable model substrates (phenacetin,
diclofenac and midazolam). Concentrations of parent substances
and their metabolites were analyzed at ten time points over 24 h by
liquid chromatography/ mass spectrometry (LC/MS).
Fig. 6: Heat map illustrating levels of urea production and degrees of CYP activity (low – yellow, high –
orange) for six human hepatocyte bioreactors at different time points of culture.
Conclusion
The results show that the analysed CYP activities are well expressed and can be maintained in human liver
cell bioreactors over several weeks. CYP dependent metabolism was markedly higher in primary
hepatocytes than in liver cell lines. CYP activity correlated with urea production in primary human
hepatocyte bioreactors.
The 3D four-compartment bioreactor technology allows long-term cell maintenance for repeated studies in
the same experimental setup enabling drug metabolism studies and long running drug exposure on human
liver cells in a highly controlled, dynamic environment.
11th European Regional Meeting of the International Society for the
Study of Xenobiotics (ISSX), 17 to 20 May 2009, Lisbon, Portugal