Co-culture of Cryopreserved Primary Rat and Human Kupffer Cells andHepatocytes to Study APAP Cytotoxicity
Gemma L. Mendel, Todd W. Stewart, Kirsten B. Amaral, Adam M. Farmer, Rachel M. Witek, Manda Edwards, Kimberley Freeman,
Erica Deibert, Brian J. Paszkiet, Jonathan P. Jackson, Stephen S. Ferguson, Rafal P. Witek.
Life Technologies, Durham, NC, 27703
3Kupffer cell isolation: Primary rat and human Kupffer cells were isolated using aproprietary enzymatic digestion method developed at Life Technologies. Using thistechnique, Kupffer cells can be isolated at viabilities higher than 98% and purities ofapproximately 90% (Rat) and 60% (Human) as determined by immune stain for ED1(CD68) and ED2 (CD163) markers. Contaminating cells represent populations ofendothelial cells, fibroblasts, hepatocytes, 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 forexperiments within 48hr after plating.
Co-Cultures: Hepatocytes (obtained from Life Technologies, CA) and Kupffer cells wereco-cultured in 24-well plates 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 todetermine linearity of inflammatory response. DMEM supplemented with NEAA, ITS+, 2%(for maintenance) and 10% (for plating) FBS, P/S was used in co-culture and changed
every 24 h. No corticosteroids (dexamethasone or hydrocortisone) were used.
Data Analysis: To activate inflammatory response in Kupffer cells, co-cultures were treatedwith either LPS (1ug/ml), IL2 (200ng/ml) and APAP (10mM). Data analysis was performed
using ELISA assays, IHC, qRT-PCR .

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Kupffer cells (KCs) directly interact with hepatocytes during liver
inflammation by modifying immunity and xenobiotic metabolism. KCs are known tosuppress the expression of multiple cytochrome P450 enzymes following activation withlipopolysaccharide (LPS). Since KCs are involved in liver diseases that arecharacterized with inflammation including viral and autoimmune hepatitis and nonalcoholic steatohepatitis (NASH), there is a need for a KCs and hepatocyte co-culture
model for drug discovery and ADME/Tox applications. This study hypothesizes that theuse of cryopreserved rat and human KCs in co-culture with hepatocytes will recreate
liver inflammation and model acetaminophen (APAP) cytotoxicity observed during liver
inflammation. Methods: Fresh and cryopreserved Kupffer cells were characterized bymorphology, plateability, phagocytosis, immunohistochemistry staining for CD68 andCD163, and cytokine release for IL-6 and TNFα by ELISA in response to 24hrs
treatment with 1µg/mL LPS. KCs and hepatic co-cultures were cultured in DMEM
medium supplemented with non-essential amino acids (NEAA), Insulin-TransferringSelenium (ITS+) and Penicillin/Streptomycin. For plating and maintenance media,
additional 10% or 2% fetal bovine serum (FBS) was used, respectively. Since
corticosteroids inhibit inflammation, dexamethasone or hydrocortisone were not usedwith KCs. For experiments with APAP cytotoxicity, inflammatory ratios of approximately
1:2-1:4 KCs to hepatocytes were used. Co-cultures were cultured for 24 hours and thenpre-treated with 1µg/mL LPS for 24, 4 and 0 hours prior to addition of 10mM APAP for
additional 24hr in the same media containing LPS. Cells were evaluated for viability
using PrestoBlueTM reagent, morphology and cytokine release (e.g. IL-6, TNFα) by
ELISA. Real time qRT-PCR was performed to establish gene expression changes inmetabolism, inflammation and viability. Results: Fresh and cryopreserved rat KCs werefound to be similar after comparison of morphology, plateability, culture purity via
immunofluorescent staining. The purity of KCs was found to be at 85.6% (±1.9 SE) for
CD68 and at 93.5% (±0.4 SE) for CD163, commonly recognized cell surface markers ofKCs. Using cytokine panel, activated fresh and cryopreserved co-cultures revealed
comparable upregulated expression of IL-1α, IL-1β and IL-2 by at least two fold, and IL6 IL-10 and TNFα by at least four fold; conversely, they revealed down regulated
expression in IL-4 and IL-12 by at least two fold. For the cytotoxicity experiments,viability and morphology data analysis revealed time-dependent hepato-protection in cocultures at 4 hours pre-treatment with LPS, but not at 0 and 24 hours. A strong
correlation was observed with cytokine release of IL-6 and TNFα, and expression ofCYP1A2 and CYP2E1. Conclusions: Cryopreserved KCs can maintain known
characteristics and functions as compared to freshly isolated cells. APAP cytotoxicity
data show that these cells can be effectively used for modeling liver inflammation andreveal hepato-protective role of KCs as shown at 4 hour pre-treatment with LPS.
Cryopreserved KCs when co-cultured with hepatocytes, provide a powerful in vitro
ADME/Tox tool to evaluate the effects of xenobiotic metabolism during inflammation.

2.5

Inflammatory
co-culture

Normal control

Inflammatory
co-culture

Figure 4. Experimental design for using co-cultures of cryopreserved Kupffer cells
and hepatocytes to study APAP cytotoxicity. Following culture of Kupffer cells for 24hr,
hepatocytes were plated and allowed to attach for 24hr prior to experiment. At that point,
three experimental groups were created. 24hr pretreatment with LPS (1ug/ml) before APAP
addition was to simulate cytotoxicity observed during full liver inflammation. 4hr LPS
pretreatment was mimicking cytotoxicity in early stages of inflammation and 0hr wassimulating cytotoxicity at the start of inflammatory response. Following pretreatment, APAP(10mM) was added in the media also containing LPS (1ug/ml) for additional 24hr of culture.
At that point, samples were collected for various analysis.

B24hr

48hr
1.4

No LPS- <5% of control+ 5-25% of control++ 25-75% of control+++ >75% of control

Figure 2. Culture of cryopreserved primary Kupffer cells. A Characterization of fresh rat Kupffer cells
plated on CellBIND shows morphological similarities with cryopreserved Kupffer cells plated on either
CellBIND or Collagen type I; B. Fresh and cryopreserved Kupffer cells can be cultured in modified
hepatocyte growth medium without dexamethasone. In presence of dexamethasone Kupffer cells detachand die. C. Cytokine panel showing similar inflammatory response of fresh and cryopreserved Kupffer cells
following 24hr treatment with LPS. Note high level expression of IL6, TNFa and IL10, major cytokines
involved in P450 modulation during liver inflammation.

ED

1.2
10.8

1.4

10.80.60.40.4

1.4

Hep

4hr LPS

Control

Hep+KC

**

1.2F

10.8

1.4

*

0.8

0.4

0.4

Hep+KC

Hep+KC24hr LPS

10.6

Hep

Hep

1.20.6

Control0hr LPS

1.20.6

Control

Legend:**Fold Change

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C

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In the liver, Kupffer cells are located on the sinusoidal side of hepaticparenchyma and use their stellate like cytoplasmic extensions for direct cell-tocell contact with hepatocytes. This contact is essential for proper modulation andthe development of a fulminant hepatic inflammatory response. In addition to cellcontact, activation of Kupffer cells results in the release of a variety ofinflammatory cytokines and growth control mediators that suppress theexpression of multiple cytochrome P450 enzymes. Two of those factors,Interleukin 6 (IL6) and Tumor Necrosis Factor-a (TNFa) are known to induce thesynthesis of acute phase proteins and suppress CYP1A2, CYP2C19 and CYP3A
activities. In addition, it has been shown that IL10, an anti-inflammatory cytokine
produced during inflammation by Kupffer cells, suppresses CYP2E1, a major
P450 involved in APAP metabolism.

ACHep Hep+KC

Hep Hep+KC

Hep Hep+KC

No LPS

0hr LPS

4hr LPS

24hr LPS

7Hep Hep+KC

Hep Hep+KC

Hep Hep+KC

Hep Hep+KC

No LPS

0hr LPS

4hr LPS

24hr LPSCYP2E16543210Control

Figure 6. Preliminary real time qRT-PCR assessment of CYP1A2 and CYP2E1
in hepatocytes and co-cultures following pre-treatment with LPS and 24hr
incubation with APAP.

TLR4Fold Change

The largest solid organ in the human body, the liver is responsible fordiversity of function with the most important being metabolism, detoxification andprotein synthesis. Most of those functions take place within hepatocytes whichrepresent a major cell type of the hepatic parenchyma; however, the liver also
contains a large proportion of non-parenchymal cells (NPCs), which include liver
sinusoidal endothelial cells, hepatic stellate (ito) cells, cholangiocytes andKupffer cells. In general, NPCs provide physical and biochemical structure to theliver. Out of all NPCs, Kupffer cells, which are the liver resident macrophages,
play an important role in liver physiology and homeostasis by participating in theacute and chronic responses of the liver to toxic compounds during liver
inflammation.

B

Cryopreserved

Hep Hep+KC

Normal control

Figure 3. Simulating inflammatory response using progressive production of IL6and TNFa in co-cultures of Kupffer cells and hepatocytes at different ratios
following LPS treatment. Cells were plated at different ratios and treated with LPS(1ug/ml) for 24hr. Progressive increase of IL6 and TNFa indicates that 1:2-1:4 ratios ofKupffer cells to hepatocytes mimics high levels of cytokine production typical to that ofliver inflammation. Based on this data, 1:2-1:4 ratios were used to mimic liver
inflammatory state, and culture of hepatocytes only was used as control mimicking
normal non-inflamed liver.

INTRODUCTION
Fresh1Control

RESULTSA

1.50

APAP (10mM)

Figure 1. Morphological andimmuno-histologicalcharacterization of primary rat
Kupffer cells. A At 2hr after plating,rat Kupffer cells display fried egg likemorphology of immature
macrophages; B Within 24hr after
plating, rat Kupffer cells expand andactivate to their mature
macrophage-like phenotype; C-D.
Immune characterization of Kupffer
cells at 48hr after plating reveal
presence of CD68, a classicalmarker of macrophages (C) andCD163, marker of activatedmacrophages (D).

20.5

Fold Change

Introduction:

MATERIALS AND METHODS

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ABSTRACTCYP1A2Control

Hep

Hep+KC* p<0.05, ** p<0.01

For reach use only. Not intended for human or animal therapeutic or diagnostic use.
© 2011 Life Technologies Corporation. All rights reserved.

Figure 5. Morphological andfunctional assessment of
hepatocytes and co-cultures
following pre-treatment withLPS and 24hr incubation with
APAP. A-B Representative
images of hepatocytes only andco-cultures pretreated with LPS
for 4hr followed by 24hr
incubations with APAP,
respectively; C-F PrestoBlueTM
analysis of each experimentalgroup (no LPS pretreatment, 0,4, and 24hr LPS pre-treatment)
following 24hr incubation withAPAP. Each sample iscompared to control that used
the same pre-treatment
condition but was not treatedwith APAP.
Note, similar observation wasobtained using
cryopreserved human
Kupffer cells when cultured
with human hepatocytes.

Figure 7. Real time qRT-PCR assessment ofTLR-4 in co-cultures following pre-treatment
with LPS and 24hr incubation with APAP.
Note significant increase of TLR-4 mRNA in 4
and 24hr LPS pre-treatment groups suggestingthat protective effect in those groups could be
modulated through TLR-4 signaling, whereas inco-cultures without LPS or with 0hr pretreatment
the protective effect is independent of TLR-4.
* p<0.05, ** p<0.01

CONCLUSIONS- Cryopreserved Kupffer cells can maintain known characteristics and functions
comparable to freshly isolated cells.
- APAP cytotoxicity data show that Kupffer cells can be effectively used for modelingliver inflammation and reveal a hepato-protective role of KCs as shown at 4 hourpre-treatment with LPS- Current data indicates that there could be two independent hepato-protective
mechanisms that Kupffer cells use to decrease APAP cytotoxicity: TLR-4 dependentthat acts during inflammation and TLR-4 independent that functions in normal liver
without inflammatory stimuli.

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 J Physiol
Gastrointest Liver Physiol. 2001 Apr;280(4):G720-8.
Bourdi M, Masubuchi Y, Reilly TP, Amouzadeh HR, Martin JL, George JW, Shah AG, Pohl LR.
Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role ofinducible nitric oxide synthase. Hepatology. 2002 Feb;35(2):289-98.
Ju C, Reilly TP, Bourdi M, Radonovich MF, Brady JN, George JW, Pohl LR. Protective role of Kupffer
cells in acetaminophen-induced hepatic injury in mice. Chem Res Toxicol. 2002 Dec;15(12):150413.
Sunman JA, Hawke RL, LeCluyse EL, Kashuba AD. Kupffer cell-mediated IL-2 suppression of
CYP3A activity in human hepatocytes. Drug Metab Dispos. 2004 Mar;32(3):359-63.

ACKNOWLEDGEMENTS
I would like to acknowledge all members of Durham Life Technologies for helping withmultiple aspects of this project. Without their co-operation and help, this work would
not exist.
Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com