Co-culture of Hepatocytes and Kupffer
Cells as a Model for Liver Inflammation.
Rafal P. Witek, Kirsten B. Amaral, Manda A. Edwards, Adam M. Farmer, Kimberly M. Freeman, Erica Deibert, Tania Bembridge, Jonathan P. Jackson,
Cornelia Smith, Jasminder Sahi, Stephen S. Ferguson, Mark J. Powers.
ADMETox/ Life Technologies, Durham NC USA
ABSTRACTIntroduction: Kupffer cells are activated liver resident macrophages that play an important rolein liver immunity and in the modulation of xenobiotic metabolism during liver inflammation.
These cells suppress the expression of multiple cytochrome P450 enzymes through theexchange of soluble factors, following activation with either lipopolysaccharide (LPS) or otherpro-inflammatory cytokines. Since those interactions lead to severe pharmacological andtoxicological consequences, there is an evident need to develop a commercially available model
mimicking the liver inflammatory state that could be used for in vitro drug metabolism anddiscovery ADME/Tox applications. We use a liver co-culture system consisting of plated primary
hepatocytes and Kupffer cells in a physiologic 2:1 ratio, which is relevant to inflammation, andcompare these to monocultures of Kupffer cells or hepatocytes. Material and Methods: Primary
rat Kupffer cells and hepatocytes were isolated using a modified two step collagenase perfusion.
Purity of isolated Kupffer cells was determined by culturing cells for 2 days and analyzing themby immunostaining for ED1 and ED2 expression. To create inflammatory co-cultures, Kupffer
cells were seeded at a density approximating half that of hepatocytes and cultured for 48hr prior
to all experiments. Kupffer cell activation was induced by treating hepatocyte cultures, Kupffer
cell cultures, or co-cultures with LPS (1µg/mL) or IL2 (200ng/mL) for a durations of 24, 48 and72hrs. Following treatment, all cultures were analyzed for morphology, cytokine production byELISA, and P450 mRNA expression (CYP3A and CYP1A2). Results: Isolated Kupffer cells
expressed ED1 at 88.7% (±1.05) and ED2 at 75.5% (±5.4) after 2 days in culture. Following 6days in culture, expression of ED1 remained at 85.6% (±1.9) and ED2 expression significantly
increased to 93.5% (±0.4) suggesting Kupffer cell activation. As expected, following plating andactivation, primary Kupffer cells significantly increased production of pro-inflammatory cytokines
IL-6 and TNFa in co-cultures and Kupffer cell monocultures. However, at 72hr after treatment
with either LPS or IL2, only co-cultures showed upregulated expression of IL6 by nearly 2-foldcompared with monocultures of Kupffer cells. This suggests auto-assembly of cellularinteractions between both types of cells. After 72hr treatment, LPS and corresponding IL-6 upregulation correlated with approximately 20% down-regulation of CYP3A and CYP1A2expression. IL2 treatment correlated with approximately 80% down-regulation of both enzymes.
Conclusion: Our data suggests that Kupffer cell and hepatocyte co-cultures can self-assemble
within 72hr of treatment with pro-inflammatory cytokines or LPS, and can function by effectively
modulating P450 expression in co-cultured hepatocytes. As such, co-culture of primary
hepatocytes and Kupffer cells mimicking liver inflammatory state offer a powerful in vitro
ADME/Tox tool to assess the effects of xenobiotics on drug metabolizing enzymes and moreclosely model more complex hepatic cell biology in vitro.

RESULTS

Figure 5. IL6 production in Kupffer cells after LPS and IL2 stimulation for 24, 48 and72hr. As expected, following activation with LPS treatment, IL6 production is significantly upregulated at 24hr and then it progressively decreases suggesting desensitization of Kupffer
cells to chronic stimulation with LPS.

Figure 1. Morphological characterization of primary rat Kupffer cells. A. At 2hr after
plating, rat Kupffer cells display fried egg like morphology of immature macrophages; B. Within
24hr after plating, rat Kupffer cells expand and activate to their mature macrophage-likephenotype; C-D. After 48hr to 96hr after plating and thereafter, Kupffer cells become fully
activated to their stellate-like phenotype that is characteristic of liver resident macrophages andfurther validated by ED2 (CD163) immune stain.

Figure 6. IL6 production in Kupffer cells and hepatocyte co-cultures after LPS and IL2
stimulation for 24, 48 and 72hr. Note that IL6 is significantly up-regulated in co-cultures at
all time points of 24, 48 and 72hr. This suggests cellular self assembly between Kupffer cells
and hepatocytes that synergistically allows those cells to function together during resolution ofinflammation.

INTRODUCTION
The largest solid organ in the human body, the liver is responsible for diversity of function withmost important being metabolism, detoxification and protein synthesis. Most of those functions takeplace within hepatocytes which represent a major cell type of the hepatic parenchyma; however, liver
also contains a large proportion of non-parenchymal cells (NPCs) which include liver sinusoidalendothelial cells, hepatic stellate (ito) cells, cholangiocytes and Kupffer cells. In general, NPCs provide
physical and biochemical structure to the liver. Out of all NPCs, Kupffer cells, which are the liver resident
macrophages, play an important role in liver physiology and homeostasis by participating in the acuteand chronic responses of the liver to toxic compounds during liver inflammation.
In the liver, Kupffer cells are located on the sinusoidal side of hepatic parenchyma and use theirstellate like cytoplasmic extensions for direct cell-to-cell contact with hepatocytes. This contact isessential for proper modulation and the development of a fulminant hepatic inflammatory response. In
addition to cell contact, activation of Kupffer cells results in the release of a variety of inflammatory
cytokines and growth control mediators that suppress the expression of multiple cytochrome P450
enzymes. Two of those factors, Interleukin 6 (IL6) and Tumor Necrosis Factor-a (TNFa) are known toinduce the synthesis of acute phase proteins and suppress CYP1A2, CYP2C19 and CYP3A activities.
Those three P450 enzymes represent the main group of metabolic enzymes involved in detoxification ofxenobiotic substances.

Figure 2. Activation of primary rat Kupffer cells in culture. A1-A3. Immune characterizationof Kupffer cells at 48hr after plating for presence of ED1 (A1) and ED2 (A2) macrophage
markers reveals their expression at 88.7% and 75.5%, respectively (A3). B1-B3. Following
activation at 6 days after plating, Kupffer cells express ED1 (B1) at 85.6% and ED2 (B2) at
93.5% (B3).

Figure 7. Modulation of CYP3A2 and CYP1A2 enzyme activities in co-cultures after LPSand IL2 stimulation for 72hr. Note near 80% decrease of CYP3A23 and CYP1A2 after IL2treatment.

CONCLUSIONS
MATERIALS AND METHODSRat Kupffer cell isolation: Primary rat Kupffer cells were isolated using proprietary enzymatic digestion
method developed at Life Technologies. Using this technique, Kupffer cells can be isolated at viabilities
higher than 98% and purities of approximately 90% or higher as determined by immune stain for ED1(CD68) and ED2 (CD163) markers. Contaminating cells represent small populations of endothelial cells~3%, fibroblasts ~2%, hepatocytes 1%, and Kupffer cells that did not stain sufficiently to be counted).
Isolated Kupffer cells attach within 15-30min and display macrophage morphological characteristics
within 24hr. They become activated and ready for experiments within 48hr after plating. Kupffer cells arecultured in supplemented WEM.

Figure 3. Primary rat Kupffer cell co-cultures with hepatocytes. A. Representative image ofprimary rat monoculture of hepatocytes plated for 48hr displaying characteristics of epithelialmonolayer; B. Representative image of inflammatory co-culture representing the 1:2 ratio ofKupffer cells to hepatocytes. Note stellate like morphology of Kupffer cells and their proximity tohepatocytes.

Rat Hepatocyte isolation: Primary rat hepatocytes are isolated using two step collagenase perfusion.
Cells with viability of at least 85%, as determined by trypan blue exclusion, are used in these studies.
Supplemented WEM was used during culture and changed every 24 h.

REFERENCES
Hoebe KH, Witkamp RF, Fink-Gremmels J, Van Miert AS, Monshouwer M. Direct cell-to-cell
contact between Kupffer cells and hepatocytes augments endotoxin-induced hepatic injury. Am JPhysiol Gastrointest Liver Physiol. 2001 Apr;280(4):G720-8.

Co-Cultures: Hepatocytes and Kupffer cells were co-cultured in 24-well dishes at ratios of 1:2 of Kupffer
cells/hepatocytes to approximate physiological inflammatory liver state. Additional 1:4, 1:8 and 1:16
ratios were used to determine linearity of inflammatory response. Supplemented WEM was used in coculture and changed every 24 h.
Data Analysis: To activate inflammatory response in Kupffer cells, co-cultures were treated with either
LPS (1ug/ml) or IL2 (200ng/ml). Response was analyzed at 24, 48 or 72hr for IL6 and TNFa production
using ELISA assays. For those assays, media was collected from each well at predetermined time
points and stored at -80oC until analysis.
To perform qRT-PCR, at terminal time point, cells were washed with HBSS and total RNA was collected.
Following cDNA synthesis, TaqMan analysis was performed using probes for rat CYP1A2 and CYP3A23-Kupffer cells secrete potent mediators of the inflammatory response that control liverinflammation and hepatocyte metabolic rates through direct interactions with phase I and phase IIenzymes.
-Kupffer cells can be used to create co-culture liver inflammatory system when plated together
with hepatocytes-Isolated primary rat Kupffer cells are >90% pure as determined by immune stain for ED1 andED2.
-Plated Kupffer cells culture activate as determined by increase expression of resident
macrophage marker ED2.
-Kupffer cells and hepatocytes self-assembly in culture-IL2 or LPS activated Kupffer cells modulate CYP3A and CYP1A2 activitiesSunman JA, Hawke RL, LeCluyse EL, Kashuba AD. Kupffer cell-mediated IL-2 suppression ofCYP3A activity in human hepatocytes. Drug Metab Dispos. 2004 Mar;32(3):359-63.

Figure 4. Progressive media accumulation of IL6 and TNFa in co-cultures of Kupffer cells
and hepatocytes. Cells were plated at different ratios corresponding to those of normal (1:16)
and inflamed (1:8 – 1:2) liver at 24hr after treatment with 1ug/ml of LPS. .

Roberts RA, Ganey PE, Ju C, Kamendulis LM, Rusyn I, Klaunig JE. Role of the Kupffer cell in
mediating hepatic toxicity and carcinogenesis. Toxicol Sci. 2007 Mar;96(1):2-15. Epub 2006 Nov
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