Jessica A. Bonzo, Stacy L. Jones, Rachel M. Witek, Lauren E. Sangenario, Shyam Kumar, and Rafal P. Witek

Cell Biology, Life Technologies, Thermo Fisher Scientific, Frederick, MD, USA 21704

RESULTS

Figure 1. Morphological assessment of 14 day cultures of cryopreserved primary human

hepatocytes

ABSTRACT

Primary human hepatocyte cultures are the gold standard for preclinical drug safety

assessment as well as required by the FDA for evaluation of drug induction potential.

Cryopreserved human hepatocytes have been validated as a comparable model to

fresh human hepatocytes. However, these cultures are limited by their relatively short

life-span of 5 to 7 days accompanied by a rapid drop in metabolic function which

precludes, among other experiments, evaluation of low metabolic turnover compounds,

multi-day toxicity studies, and long term viral infection studies. The need for longer

term hepatocyte cultures has increased with recent initiatives by European regulatory

bodies to limit animal testing. To address this need, we are evaluating a new cell

culture media supplement (HepExtend™) that maintains the function and viability of

cryopreserved human hepatocytes for at least 10 days. The new culture media has

been tested for the ability to prolong hepatocyte life as determined by morphological

assessment. In addition, to understand the effect of long term culture on metabolic

function, Phase I enzymatic activities have been assessed by LC/MS/MS. Hepatocytes

cultured in HepExtend™ supplemented media displayed CYP1A2, CYP2B6, CYP2D6,

and CYP3A4 activity levels significantly greater than those achieved using standard

culture media after 5 days. Cultures maintained in the new media survived for at least

10 days with metabolic activities comparable to day 5; this was not achievable with

standard culture media. In addition supporting data shows hepatic cultures in the new

media after 10 days maintained polarity and bile canaliculi formation. As our data

suggests, HepExtend™ supplemented media supports hepatocyte health and

metabolic function for an extended period of time. This enables researchers to perform

metabolic and toxicity experiments not achievable using currently available standard

culture conditions. This further allows for multi-day toxicity studies and more predictive

data interpretation.

INTRODUCTION

Current standard monolayer human hepatocyte cultures are typically functional in

culture for 5 to 7 days. While this is sufficient for induction studies and high rate of

metabolic turnover compounds, there is an increasing need for longer term cultures of

hepatocytes to study low turnover compounds as well as longer term toxicity studies.

Recently developed coculture and 3D models have significantly increased the

functional life span of hepatocytes in culture. However, many of these models rely on

coculturing of human hepatocytes with other species of cells, are not amenable to high

throughput processes that have been developed with standard culture practices, and

are very costly. To address these concerns and extend the functional life of

hepatocytes in culture, we have developed a new supplement, HepExtend™

Supplement (50X), that is used in conjunction with our standard William’s E

Maintenance media. To address the inherent variability of hepatocytes, HepExtend™

was developed using 13 different lots of plateable cryopreserved primary human

hepatocytes across all three SKUs currently offered by Life Technologies (Metabolism,

Induction, Transporter). Our data indicates that HepExtend™ enhances the viability

and P450 function of human hepatocytes for at least 10 days.

MATERIALS AND METHODS

Cell Culture Cryopreserved primary human hepatocytes (Life Technologies, HMCPIS,

HMCPTS, HMCPMS) were briefly thawed at 37°, transferred to a 50 mL conical tube

of Hepatocyte Thaw Media (Life Technologies, CM7500), and centrifuged for 10 min at

100 x g. Cells were resuspended with William’s E media (Life Technologies,

A1217601) supplemented with serum-containing Hepatocyte Thaw and Plating Media

(Life Technologies, CM3000) and plated at a density of 0.8 x106 cells/mL on 24-well

collagen I coated plates (Life Technologies, A11428-2). Media was replaced 4 hours

after plating with William’s E media supplemented with serum-free Hepatocyte

Maintenance Supplements (i.e. William’s E Maintenance Media; Life Technologies,

CM4000) or William’s E Maintenance Media supplemented with HepExtend™

Supplement. An overlay of Geltrex® Basement Membrane Matrix (Life Technologies,

A1413201) diluted in William’s E Maintenance media was applied 24 hours after

plating. Media was changed daily for the duration of cultures.

P450 Enzymatic Activity On indicated days, cultures were washed with fresh

William’s E Maintenance Media and incubated with the following probe substrates for

15 minutes: CYP1A2-phenacetin 1; CYP2B6-bupropion; CYP2D6-dextromethorphan;

CYP3A4-testosterone. After incubation, supernatants were collected and frozen until

analysis. Metabolites were detected using LC//MS/MS.

Gene Expression Analysis Hepatocytes were cultured for 5 days in indicated media.

For induction studies, protoypical inducers were added on day 3 of the culture for a

total exposure time of 48 hours. RNA was harvested using the Purelink® RNA Mini kit

(Life Technologies, 12183018A). Five hundred nanograms of RNA was reverse

transcribed using the High Capacity cDNA Reverse Transcription kit (Life

Technologies, 4368813). Gene expression was assessed using a custom designed

TaqMan® Array card containing 48 hepatocyte-specific genes of interest to ADME/Tox

studies.

CONCLUSIONS

• Our HepExtend™ Supplement (50X) allows for long term (>5 days) culturing of primary

human hepatocytes with maintenance of hepatocyte polarity and basal P450 enzymatic

activities.

• Future studies will examine the impact of HepExtend™ on analysis of low turnover

compounds as well as low dose long term toxicity studies.

ACKNOWLEDGEMENTS

The authors thank Mukesh Kumar, Rhonda Newman, Kevin Vedvik, Kristen Vaverchak, Wendy

Bray, Tim Hurley, Hanna Nguyen, and Chelsie Fritz for research and program support.

Long Term Hepatocyte Culture for Drug Safety Assessment Screening

Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com

Da

y 5

D

ay 1

0

Da

y 1

4

Standard HepExtend™

Hu1651 (Transporter) Hu1552 (Induction) Hu1604 (Metabolism)

Figure 2. HepExtend™ supplementation increases cytochrome P450 activity in plated

cryopreserved human hepatocytes

Standard HepExtend™ Standard HepExtend™

Hu17

17

(Tra

nspo

rte

r)

Hu82

00

A

(In

du

ctio

n)

Hu82

09

(Me

tab

olis

m)

CYP1A2 CYP2B6 CYP2D6 CYP3A4

Supplementation with HepExtend™ increases the enzymatic activity of all 4 cytochrome P450s monitored as early as five days in culture. Cryopreserved

primary human hepatocytes were plated and cultured in standard William’s E Maintenance Media (black lines) or William’s E Maintenance Media supplemented with

HepExtend™ (blue lines). On indicated culture days, cells were assessed for P450 activity as described in Materials and Methods. The increase in activity is

observed in all three SKUs of primary cryopreserved human hepatocytes offered by Life Technologies.

Culturing with HepExtend™ extends the viable culture life of primary human hepatocytes for 10-14 days compared to standard Maintenance Media.

Cryopreserved primary human hepatocytes were plated and cultured in standard William’s E Maintenance Media or William’s E Maintenance Media

supplemented with HepExtend™ for 14 days. Cells were assessed for retention of markers of hepatocyte viability including: bright nuclei, bile canaliculi

formation, cuboidal shape, integrity of monolayer, accumulation of dead cells.

Figure 3. Basal gene expression of Phase I and II

drug metabolism enzymes and transporters

Hu1552

Hu1595

HepExtend™ supplementation increases the basal gene expression of many key genes involved

in drug metabolism and transport. Cryopreserved primary human hepatocytes were cultured in

standard William’s E Maintenance Media or William’s E Maintenance Media supplemented with

HepExtend™ for 5 days. Gene expression profiling was performed using a TaqMan® Array card

containing 48 hepatocyte-specific genes relevant to ADME/Tox research.

Figure 4. HepExtend™ cultures retain inducibility

Nuclear receptor activation and gene inducibility is retained in cultures treated with HepExtend™.

Cryopreserved primary human hepatocytes (Hu1552) were cultured in standard William’s E Maintenance

Media or William’s E Maintenance Media supplemented with HepExtend™ for 5 days. Prototypical

inducers were applied for 48 hours starting on day 3 of culture (Omeprazole-AhR; Phenobarbital-CAR;

Rifampicin-PXR). Gene expression profiling was performed using a TaqMan® Array card containing 48

hepatocyte-specific genes relevant to ADME/Tox research.

© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific

and its subsidiaries unless otherwise specified. TaqMan® is a registered trademark of Roche Molecular Systems, Inc.,

used under permission and license. For research use only. Not for use in diagnostic purposes.