Dynamic culture of human liver equivalents inside a micro-bioreactor for long-term substance testing [original]

Eva-Maria Materne, Ilka Wagner, Caroline Frädrich, Ute Süßbier, Reyk Horland,

Silke Hoffmann, Sven Brincker, Alexandra Lorenz, Matthias Gruchow, Frank

Sonntag, Udo Klotzbach, Roland Lauster, Uwe Marx

Dynamic culture of human liver equivalents

inside a micro-bioreactor for long-term

substance testing

Conference paper, Published version

This version is available at http://nbn-resolving.de/urn:nbn:de:kobv:83-opus4-70187.

Suggested Citation

Materne, Eva-Maria et al.: Dynamic culture of human liver equivalents inside a micro-bioreactor for

longterm substance testing. - In: BMC Proceedings. - ISSN 1753-6561 (online). - 7 (2012), suppl. 6, art.

P72. - doi:10.1186/1753-6561-7-S6-P72.

This work is licensed under a CC BY 2.0 License (Creative

Commons Attribution 2.0 Generic). For more information see

http://creativecommons.org/licenses/by/2.0.

POSTER PRESENTATION Open Access

Dynamic culture of human liver equivalents

inside a micro-bioreactor for long-term

substance testing

Eva-Maria Materne

, Ilka Wagner

, Caroline Frädrich

, Ute Süßbier

, Reyk Horland

, Silke Hoffmann

Sven Brincker

, Alexandra Lorenz

, Matthias Gruchow

, Frank Sonntag

, Udo Klotzbach

, Roland Lauster

Uwe Marx

1,3

From 23rd European Society for Animal Cell Technology (ESACT) Meeting: Better Cells for Better Health

Lille, France. 23-26 June 2013

Background

Current in vitro and animal tests for drug development

are failing to emulate the organ complexity of the

human body and, therefore, to accurately predict drug

toxicity. In this study, we present a self-contained, bior-

eactor based human in vitro tissue culture test system

aiming to support predictive substance testing at rele-

vant throughput. We designed a microcirculation system

interconnecting several tissue culture spaces within a

PDMS-embedded microfluidic channel circuit. The bior-

eactor is reproducibly perfused by a peristaltic on-chip

micro-pump, providing a near physiologic fluid flow and

volume to liquid ratio.

Materials and methods

Liver microtissue aggregates containing 4.8 × 10

HepaRG cells and 0.2 × 10

human hepatic stellate cells

(HHSteC) were formed in Perfecta3D

384-Well Hang-

ing Drop Plates (3D Biomatrix, USA). After two days of

hanging drop culture, 20 aggregates were loaded into a

single tissue culture compartment of the micro-bioreac-

tor. Each circuit of the micro-bioreactor device con-

tained 700 μl medium in total. During the first 7 days, a

40% media exchange rate was applied at 12 h intervals.

From day 8 onwards, a 40% exchange rate was applied

at 24 h intervals. Daily samples were collected for

respective analyses. Experiments were stopped at day 14

and 28 and tissues were subjected to immunohisto-

chemical stainings and qRT-PCR analyses. Experiments

were conducted with four replicates. To expose the

chip-cultures to troglitazone, liver microtissues were

cultured for one day in normal medium and were, sub-

sequently, treated with 0 μM, 5 μMand50μMsub-

stance, respectively. Application of troglitazone was

repeated at 12 h or 24 h intervals simultaneously with

the medium change.

Results

Cultures of human artificial liver microtissues have suc-

cessfully been cultivated over 28 days in the novel micro-

fluidic bioreactor. Glucose consumption and lactate

production indicated an aerobic metabolism which

reached a steady state after 7 days. Immunohistochemical

staining revealed the expression of phase I metabolic

enzymes CYP450 3A4 and CYP450 7A1, extracellular

matrix component collagen I, apical transporter MRP2

and tight junction protein ZO-1 (Figure 1A-D). Cell viabi-

lity over 28 days was increased in the bioreactor culture

compared to static control (Figure 1E, F). Furthermore,

the cultures revealed a dose-dependent response to a

7-day exposure to the toxic substance troglitazone. Liver

microtissues showed sensitivity at different molecular

levels. Concentration of LDH released to the medium

increased with troglitazone concentration and gene

expression of selected marker genes varied. An induction

of CYP450 3A4 by troglitazone treatment was also

recorded on protein level by immunhistochemistry.

Conclusion

A promising tool for long term culture of human liver

equivalents has been developed. The simple MOC design

presented, assisted the culture of human liver equivalents

* Correspondence: eva-maria.materne@tu-berlin.de

TU Berlin, Institute for Biotechnology, Faculty of Process Science and

Engineering, Gustav-Meyer-Allee 25, 13355 Berlin, Germany

Full list of author information is available at the end of the article

Materne et al.BMC Proceedings 2013, 7(Suppl 6):P72

http://www.biomedcentral.com/1753-6561/7/S6/P72

Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver

(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

over a period of up to 28 days. The cultures, operated at

a total on-chip volume of 700 μl medium at recirculation

rates of 40 μl/min assisted by an on-chip micropump,

stabilize approximately within a week at a metabolic

steady state. The prediction of toxicology profiles of com-

pounds metabolised by the liver was demonstrated possi-

ble by exposing the cells to different concentrations of

troglitazone. This platform is designed to generate high-

quality in vitro data predictive of substance safety in

humans. Tissue cultures can be exposed to pharmaceuti-

cal substances at regimens relevant to respective guide-

lines, currently used for subsystemic substance testing in

animals.

Acknowledgements

The work has been funded by the German Federal Ministry for Education

and Research, GO-Bio Grand No. 0315569.

Authors’details

TU Berlin, Institute for Biotechnology, Faculty of Process Science and

Engineering, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.

Fraunhofer IWS

Dresden, Winterbergstraße 28, 01277 Dresden, Germany.

TissUse GmbH,

Markgrafenstraße 18, 15528 Spreenhagen, Germany.

Published: 4 December 2013

doi:10.1186/1753-6561-7-S6-P72

Cite this article as: Materne et al.: Dynamic culture of human liver

equivalents inside a micro-bioreactor for long-term substance testing.

BMC Proceedings 2013 7(Suppl 6):P72.

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Figure 1 14-day tissue performance of the micro-bioreactor culture compared to static control Cell functionality shown by

immunostaining of (A) phase I enzyme CYP450 3A4 (red) and CYP450 7A1 (green), (B) collagen I (red) and vimentin (green), (C)

MRP2, an ABC transporter located at the apical membrane, (green) and (D) tight junction protein ZO-1 (red). Cell viability shown by

TUNEL KI67 staining of (E) liver equivalents cultivated for 28 days in the micro-bioreactor and (F) liver equivalents cultivated for 28 days under

static conditions. Nuclei are stained with hoechst 33342. Scale bar: 100 μm.

Materne et al.BMC Proceedings 2013, 7(Suppl 6):P72

http://www.biomedcentral.com/1753-6561/7/S6/P72

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