ADME/Tox support
Protocols for ADME/Tox products• Cell counting of primary hepatocytes using trypan
blue exclusion analysis• Metabolic stability using suspension andplated hepatocytes• Induction potential in plated hepatocytes• Co-culture of hepatocytes and Kupffer cells
Need assistance selecting a product?
Our product specialists are available to help you with productand lot selection, usage protocols, and orders.

Americas and Asia Pacific—orders and technical support
Tel: +1 716 774 0538
Toll-Free Tel: +1 866 952 3559
Email: hepaticproducts@thermofisher.com
Europe—orders and technical support
Tel: +44 (0) 141 814 5900
Email: hepaticproducts@thermofisher.com
Japan—orders and technical support
Tel: +81 3 6832 6980
Email: JPCS@thermofisher.com

Rapid Alert notifications
For customers in North America, we offer Rapid Alert emails that
notify you right away about the impending availability of freshprimary human or animal hepatocytes. When you receive a
Rapid Alert email, you can then decide if you need hepatocytes
and contact us to place an order.
To sign up for Rapid Alert notifications, visit
thermofisher.com/rapidalert
For a complete listing of fresh hepatocytes and available formats,visit thermofisher.com/hepatocytes

Find out more at thermofisher.com/admetox
For Research Use Only. Not for use in diagnostic procedures. © 2015 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. HepaRG is a trademark of BioPredic International. CO019703 1115

ADME/Tox Sourcebook

Thermo Fisher Scientific ADME/Tox products

ADME/Tox products and services
At Thermo Fisher Scientific, we strive to offer industry-leading ADME/
Tox products that provide physiologically relevant results, so you canmake the right decisions related to your compounds. We are a team of
hepatic scientists helping fellow scientists—supporting IND and NDA
submissions with our products and services, and participating in theadvancement of research in enzyme induction, CYP inhibition, hepatictransport, and other metabolism-related fields. Our goal is to provide
the right tools and technical support for your needs.
ADME/Tox products for drug
metabolism and safety
Physiologically relevant in vitro
systems, such as primary hepatocytes and liver subcellular fractions,can be used to address a wide arrayof research questions related toin vivo applications, including thoserelated to xenobiotic metabolism,
drug–drug interactions, andcytotoxicity (Table 1). We offer
products for:
• CYP450 inhibition and enzyme
induction• Metabolic profiling and stability• Transporter applications

Complete selection of in vitro
hepatic cell products
Our comprehensive offering includes
hepatocytes and subcellular fractions
isolated from human, rat, mouse, dog,
rabbit, nonhuman primate, trout, andother species upon request. Our
products are prequalified for particularresearch applications, and most aresupplied as pooled or single donors,
often in large lot sizes to facilitate
multiple and multi-site experiments.
These products include:• Cryopreserved primary hepatocytes
and Kupffer cells

Gibco™ quality—every step of
the way
As hepatic scientists, we deeply
understand the importance of ahigh-quality, reliable supply chain andstringent characterization methods.
We offer:• A robust, extensive tissue
procurement network• Carefully honed isolation techniques• Rigorous quality control standards• Ongoing research and development• Hepatic product specialists toprovide technical support• Freshly isolated primary
hepatocytes (North America only)• HepaRG™ cells• Cell culture media

3

1

Table 1. ADME/Tox products from Thermo Fisher Scientific and their applications.
Application

PurposeFDA
guidance*

Plateable
hepatocytes

Suspension
hepatocytes

Liver
Liver S9
Other
microsomes fractions

Featured productCat. No.

Gibco Human Cryopreserved Plateable Hepatocytes,
Induction Qualified
Also available:• Fresh plateable human hepatocytes• Cryopreserved and fresh plateable animal
hepatocytes

HMCPIS

Gibco Human Pooled Microsomes
Also available:• Cryopreserved suspension hepatocytes

HMMCPL

Gibco Human Cryopreserved Plateable Hepatocytes,
Metabolism Qualified
Also available:• Fresh and cryopreserved human and animal
hepatocytes

HMCPMS

Gibco Human Pooled Microsomes
Also available:• Animal microsomes• Human and animal, fresh and cryopreserved
hepatocytes• Human and animal liver S9 fractions

HMMCPL

Gibco Human Microsomes, Single Donor
Also available:• BACULOSOMES Plus reagents and Vivid kits

HMMCSD

Gibco Human Cryopreserved Suspension Hepatocytes,
Transporter Qualified
Also available:• Human plateable hepatocytes, transporter qualified• Animal hepatocytes

HMCSTS

Gibco Human Cryopreserved Plateable Hepatocytes,
Transporter Qualified
Also available:• GeneMembrane ABC inside-out vesicles• Animal plateable hepatocytes

HMCPTS

Gibco Fish (rainbow trout) S9 Fractions
Also available:• Animal and human, fresh and cryopreserved
hepatocytes

TRS9PL

Gibco HEP10 Pooled Human Cryopresereved
Hepatocytes
Also available:• Animal and human, fresh and cryopreserved
hepatocytes• Animal and human microsomes

HMCS10Gibco Human Cryopreserved Plateable Hepatocytes,
Transporter Qualified
Also available:• Human fresh plateable hepatocytes• Animal cryopreserved plateable hepatocytes

HMCPTS

Drug development research (ADME, DMPK, toxicology)
Enzyme induction andinhibition

Determine if a compound has the potential to induce or inhibit
hepatic enzymes

Yes

+++

Hepatotoxicity

Determine if a compound and its metabolites have the potential tobe hepatotoxic

Yes

+++

++

Metabolic profiling

Determine which enzymes metabolize a compound

Yes

+++

+++

Metabolic stability

Measure the disappearance of a compound in the presence of
metabolizing enzymes

Yes

++

+++

Reaction phenotyping(metabolic identification)

Identify the metabolites formed from a compound when exposed tometabolizing enzymes

Yes

++

Transporter uptake

Determine if a compound has the potential to inhibit or induce livertransporter uptake

No

+++

Transporter efflux

Determine if a compound has the potential to inhibit or induce liver
basolateral transporter efflux

No

+++

+++

+++

+

+++

Recombinant
CYP450
enzymes+++

ABC vesicles

Other research applications (R&D, toxicology, environmental safety)
Environmental
bioaccumulation

Evaluate bioaccumulation of drugs or chemicals in humans and fish

No

+++

++

Liver disease research

Improve understanding of how the liver is involved in disease

No

+++

+++

siRNA, basic research

Identify the effects of gene suppression on disease

No

+++

+++

++

*Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling, Draft Guidance for Industry, USFDA, September 2006 and Guidance for Industry on Drug Interaction Studies,
2012.
+++ Product highly recommended for this application.
++ Product recommended for this application.
+ Our lowest recommendation, but product may still be useful for some assays.

2

3

Gibco™ Cryopreserved Human and Animal Hepatocytes• Viabilities routinely >80%
• Large and multiple lots—ideal for long-term studies
across sites• Characterization for phase I and phase II drugmetabolizing activities• Metabolic activity testing—ECOD, 7-HCG, 7-HCS;
human hepatocytes include testing for CYP1A2,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,
CYP2E1, CYP3A, and FMO• Application qualification tests—attachment efficiency,monolayer confluency (Figure 2), fold induction with
prototypical inducers, transporter uptake and efflux,
in situ intrinsic clearance• Additional characterization data—genotyping, optimal
seeding densities (Figures 3 and 4), viability stabilities,
donor demographic

Lack of monolayer integrity(“unhealthy” morphology)

Ideal monolayer integrity(“healthy” morphology)

Rifampicin

8,000

4,000

0
100%

50%

33%

25%

Figure 3. Seeding density effects. Induction response varies based on
the level of plating density. A better response to rifampicin is seen herewith an optimized plating density.

These plateable and suspension hepatocytes areassessed for transporter function using uptake transporter
assays. These cells:• Have stringent release specifications: ≥80% viability and≥80% confluency (plated cells)

Figure 1. Confluent, healthy, sandwich-cultured human hepatocytes
shown after five days of culture. The high level of confluency (>90%)
and observable canaliculi are signs that that this lot is suitable formetabolism, induction, and transporter uptake experiments.

- High confluency (>90%)- Limited cell–cell contact- Good cell–cell contact- Grainy cytoplasm- Clear cytoplasm- Visible cell stretching- 3D configuration- Severe cell flattening- Cuboidal cell structure- Absence of bile canaliculi- Bile canaliculi formation

Figure 2. Monolayer integrity of human hepatocytes. Representative
images that demonstrate lack of monolayer integrity and ideal monolayer
integrity from cryopreserved human hepatocytes isolated of separate
donor tissues and cultured for multiple days. Note: An unhealthymonolayer can demonstrate confluence but lack integrity due to cell
stretching and flattening.

4

*

50%

Plating density*0.6–0.9 x 106 cells/mL for 24-well plate

Figure 4. General relationship between plating density andmonolayer confluence. For human hepatocytes, the optimal plating
range to achieve maximal confluence is 0.6–0.9 x 106 cells/mL for a
24-well plate.

Gibco™ Human Cryopreserved Hepatocytes, Transporter Qualified• Facilitate effective transporter uptake and basolateral
efflux (plated) experiments- Low confluency (<70%)

100%

Optimal plating density• Contain functional membrane receptors and transporters

B

Monolayer confluency

Characterization and quality control• 10-point cell morphology check backed byphotomicrographs—cell membrane integrity, organellesize, presence of lipid droplets, nucleus size and shape,
cytosolic clarity, cell shape, level of cell debris, cellexcretion products, cell–cell contacts (plateable cellsonly), and reestablishment of bile canalicular networks(plateable cells only)

Testosterone 6β-hydroxylation(pmol/mg/min)Primary hepatocytes isolated from the liver are effective
tools for the in vitro evaluation of metabolism, drug–druginteractions, hepatotoxicity, and transporter assessment.
Our technicians are extensively trained in proper
techniques to help ensure optimal cell health. As a result,
Gibco hepatocytes have high viabilities, in vivo–like enzyme
expression levels, and if released as plateable cells,
excellent confluencies that contribute to polarization andfunctioning cell–cell contacts (Figure 1). Our primary
hepatocytes offer:

12,000Measurement of transporter uptake using
suspension hepatocytes
Transporter-qualified cryopreserved suspension
hepatocytes are suitable for hepatic uptake studies, which
typically measure the rate of appearance of substrate incells after a relatively short incubation period, in mostcases from 15 seconds to 3 minutes. Each of ourtransporter-qualified lots (suspension and plateable) has
been functionally tested for the activities of NTCP,OATP1B3, and OATP transporter pathways using thesubstrates taurocholate, digoxin, and estradiol 17β
glucuronide (E2-17G), as well as phase I and phase II
metabolic activities. Visualization of the bile canalicular
networks is attained by fluorescence microscopy using thecompound 5-(6)-carboxy-2´,7´-dichlorofluorescein diacetate(CDFDA). CDFDA is a substrate of the MRP2 transporter
protein and accumulates in the bile canaliculi as the cells
polarize and form bile canaliculi over 3–4 days in culture(Figure 5). Transporter uptake data have been compared
before and after cryopreservation with similar results,suggesting that transporter gene expression in suspension
hepatocytes is not affected by cryopreservation (Figure 6).

5

A
25

25

fresh
Digoxin accumulation (pmol/mg)

TC accumulation (pmol/mg)

Gibco™ Human Cryopreserved
Plateable Hepatocytes,
Metabolism Qualified

B
cryo

20
15
10
5

fresh
cryo

20
15

These plateable hepatocytes are suitable for use inapplications involving metabolism of compounds:

10
5
0

0
0.5

1

0.5

2

1

Figure 5. Visualization of functional
bile canaliculi networks showing theaccumulation of 5-(6)-carboxy-2’,7’dichlorofluorescein (CDF).

2

Time (min)Time (min)Figure 6. Comparsion of human suspension hepatocyte activities before and after
cryopreservation. A single lot of human hepatocytes was functionally tested for transporter
uptake after 2 hours post-isolation and subsequent cryopreservation. Average accumulation of two
substrates, (A) Na-taurocholate and (B) digoxin, was similar under both conditions.

Gibco™ Human Plateable Hepatocytes, Induction Qualified
These plateable hepatocytes are tested with clinically
relevant inducers and are suitable for use in experiments
monitoring in vitro enzyme induction:• Prequalified for CYP1A2, CYP2B6, and CYP3A induction• ≥80% viability and ≥80% confluency (plated cells)• Minimum specific activities:

Qualification of lots for use in experiments
monitoring in vitro enzyme induction
Our induction-qualified hepatocytes have passed ourquality tests for specific activity and mRNA levels in
response to prototypical inducers. We culture ourcryopreserved hepatocytes in 24-well collagen-coatedplates and dose in triplicate with vehicle (0.1% DMSO),
omeprazole, phenobarbital, and rifampicin for 72 hours.
Once monolayers are washed, they are incubated with
substrates phenacetin, bupropion, and testosterone todetermine CYP1A2, CYP2B6, and CYP3A activities,
respectively (Figure 7). Fold induction of specific activity isexpressed as the ratio of induced activity to vehicle activity.
mRNA content is also determined by TaqMan™ qRT-PCR
analysis after 48 hours of treatment.

6

1,200

2.6

1,000

• ≥80% viability and ≥75% attachment efficiency
Metabolic assay conditions for plated metabolismqualified human hepatocytes
Our plated metabolism-qualified hepatocytes have beentested for the enzymatic functions of CYP3A4, CYP2C9,and CYP2D6, using the prototypical CYP450 substrates
midazolam, tolbutamide, and dextromethorphan,respectively (Figure 8). Using 48-well collagen-coatedplates, hepatocytes are allowed to attach prior to
incubation in serum-free Williams Medium E, with reactions
stopped with ice-cold acetonitrile. Well contents are stored
at –70°C prior to analysis. The disappearance of the parent
compound is monitored by LC-MS/MS and intrinsic
clearance values determined by linear regression.

• Characterized for CYP1A2, CYP2B6, CYP2C8, CYP2C9,CYP2C19, CYP2D6, CYP3A, ECOD, 7-HCG, and
7-HCS activities• Pooled as high-quality lots with batch sizes of>500 vials• Available as specialty lots, including low
CYP2D6 metabolizers

11.1

800

25.3

600
400

21.2
59.6 67.3

200

18.2

6.2

0

61
Hu 7
7
Hu 07
40
Hu 19
40
Hu 25
40
Hu 29
40
Hu 37
40
Hu 49
40
Hu 52
41
Hu 00
80
40

–– ≥3-fold induction of CYP3A

Vehicle control
Rifampicin (10 μM)
8.2

17.6

1,400

• Prequalified according to CLint of midazolam,
tolbutamide, and dextromethorphan

Our HEP10 pooled and single-donor human cryopreserved
suspension hepatocytes are ideal for metabolism studies
such as metabolic profiling and metabolic stability. As an
industry leader in liver tissue sourcing and hepatocyte
isolations, we offer one of the largest selections of
single-donor and pooled lots (Figure 9). Our pooled lots areproduced from normal donors with average CYP450
values, simplifying your experiments that may require theuse of multiple donors. HEP10 hepatocytes are:Hu–– ≥5-fold induction of CYP2B6

1,600

Testosterone 6β-hydroxylase(pmol/min/million cells)–– ≥10-fold induction of CYP1A2• Useful for the assessment of intrinsic clearance (CLint) of
low-turnover compounds

Gibco™ HEP10™ Pooled andSingle-Donor Human
Cryopreserved Hepatocytes,
Metabolism Qualified

CYP3A enzymatic activity
Figure 7. Testing for CYP3A fold induction. Gibco human cryopreserved
hepatocytes are tested for response to prototypical inducers to determine
suitable applications. In this example, the fold induction of CYP3A (number shown above the bar line) is calculated, illustrating inherent variability
in individual lots of hepatocytes.

Figure 8. Cryopreserved human hepatocytes prequalified for plated
metabolism (intrinsic clearance). CL int (µL/1 x 106 cells/min) results
were midazolam, 14.6; toIbutamide, 134; dextromethorphan, 7.20.

Figure 9. Human hepatocytes qualified for suspension metabolism
applications.

7Gibco™ Animal Cryopreserved Plateable andSuspension Hepatocytes
Our animal cryopreserved hepatocytes are prepared using the same careful isolation and cryopreservation techniques as
our human lots. We routinely isolate hepatocytes from mouse, rat, dog, and nonhuman primate, with other major
toxicology research species available upon request. Viabilities for these cells are routinely >80%. Characterization
methods include ECOD, 7-HCG, and 7-HCS for phase I and phase II enzyme activities. We also closely monitor cellmorphology, attachment efficiency, monolayer confluency (plateable cells), and viability stability over time.

Gibco™ Cryopreserved Kupffer Cells

Co-cultured Kupffer cell and hepatocytes can self-assemble within 72 hours of treatment with pro-inflammatory
cytokines or lipopolysaccharides (LPS), and can function
by effectively modulating P450 expression, thus giving amore physiologically relevant result (Figures 10 and 11).
Studies indicate that co-cultures of hepatocytes with NPCs
better represent both normal liver physiology and diseasestates. Cryopreserved purified human and rat Kupffer cells
are a convenient way to produce hepatocyte and Kupffer
cell co-cultures for the study of various hepatic functions.
With Gibco Cryopreserved Kupffer Cells, you get:• High viability and purity, routinely >90%
• Response to activation with LPS• Minimum 1 million viable cells per vial
• Hepatocyte co-culture protocols

Kupffer cells

Hepatocytes

Co-culture: normal liver state

Co-culture: inflamed liver state

Figure 10. Co-cultures modeling normal and inflamed liver states.
These models enable researchers to study the interactions between
hepatocytes and Kupffer cells during liver inflammation.

Co-cultures

600
500
IL-6 (pg/mL)

Kupffer cells provide improved physiologicalmodeling
Growing evidence shows that under both normal andpathological conditions, many hepatocyte functions are
regulated by substances released from neighboring
nonparenchymal cells (NPCs). These cells, particularly
Kupffer cells, play an important role in the modulation ofxenobiotic metabolism in the liver. Kupffer cells secrete
potent mediators of the inflammatory response that
controls liver inflammation. These cytokine mediators
control hepatocyte metabolic rates through directinteractions with phase I and phase II enzymes.

400
300
200
100
0

Vehicle LPS

24 hr

IL-2

Vehicle LPS

48 hr

IL-2

Vehicle LPS

IL-2

72 hr

Figure 11. IL-6 production in co-cultures of Kupffer cells andhepatocytes after LPS and IL-2 stimulation for 24, 48, and 72 hours.
Note that IL-6 is significantly up-regulated in co-cultures at all time
points. This suggests cellular self-assembly between Kupffer cells andhepatocytes that synergistically allows those cells to function together
during resolution of inflammation.

89• Enabling—designed to provide maximum cell viability
and cell health when culturing primary plateable
hepatocytes• Extended cell survival—days in culture can be extended
to >10 days while maintaining normal cell morphology
and bile canaliculi• Obtain biologically relevant results from a metabolicallycomplete system• Assess the drug–drug interaction potential of yourcompound• Experience reproducible results from a single populationof cells

CYP2B6CYP2B6
activity
activity150 150

6

4

4

2

2

0

0

0 1 02 1 2

5
5
10 10
DaysDaysin culture
in culture14

140 1 02 1 2

0

20
15
10
5
0

14

CYP3A4CYP3A4activity
activity

60
60

1,000
1,000

40
40
20
20

00 11 22

55
10
10
Days
Daysininculture
culture

500
500

00

14
14

00 11 22

200
150
200
100
150

CYP1A2
CYP1A2

55
10
10
Days
Daysininculture
culture

14
14

50
100
4
50
4
2
2
0

0
Control

Control

Omeprazole

200

Omeprazole Omeprazole
Control

20

CYP3A420 CYP3A4
15

CYP3A4CYP2B6200

CYP1A24

20Fold Foldchangechangefrom fromcontrol
control
standard
standard
media
media

2

14

1,500
1,500

Fold change fromcontrol standard media

4

5
5
10 10
DaysDaysin culture
in culture

80

Figure 12. Cytochrome P450 specific activity over the course of a
14-day culture. HepExtend Supplement cultures enhance cytochrome
P450 activity compared to standard Williams E Maintenance Medium.

Fold Foldchangechangefrom fromcontrol
control
standard
standard
media
media

Fold change fromcontrol standard media

0

00

14
14

Consistent performance
HepaRG cells are essentially a single donor. This enables
users to obtain physiologically relevant results formetabolism-based drug–drug interactions without the200
concern of donor variability and limited lot sizes that come150
100
with relying on donor tissue. Note: The cells we provide are50
terminally differentiated.

Fold change fromcontrol standard media

06b-hydroxytestosterone
6b-hydroxytestosterone(pmol/mg protein/min)(pmol/mg protein/min)

Dextrorphan
Dextrorphan(pmol/mg protein/min)(pmol/mg protein/min)Predict
metabolism-based drug–druginteractions
100
100
44
HepaRG cells respond to prototypical P450 inducers and50
50
22
inhibitors to the same extent as primary hepatocytes,
0000
0 11 2255 cells to
10
10be used1414
00 11 2255
10
10
allowing 0HepaRG
to evaluate
potential
Days
Daysininculture
cultureDays
Daysininculture
culturedrug–drug interactions.

10

50

6

CYP2D6
CYP2D6activity
activity66

Gibco™ HepExtend™ Supplement (50X)
HepExtend Supplement (50X) has been
optimized for use with cryopreserved
primary hepatocytes to improve cell
viability, function, and number of days inculture. This enables researchers to
perform metabolic and toxicity experiments not achievable using standard
culture conditions (Figures 12 and 13).
Simply add HepExtend Supplement to

50

80
80

Fold Foldchangechangefrom fromcontrol
control
standard
standard
media
media

Hydroxybupropion
Hydroxybupropion(pmol/mg protein/min)(pmol/mg protein/min)

APAP
APAP
(pmol/mg protein/min)(pmol/mg protein/min)

100 100

CYP3A4CYP3A4
activity
activity
Gibco™ Hepatocyte
Plating
Supplement Pack
1,5001,500
The Hepatocyte Plating Supplement Pack contains
60 60
1,0001,000
prequalified fetal
bovine serum, dexamethasone, and a
40 40
cocktail solution of penicillin-streptomycin, bovine insulin,
500 500
20 20
GlutaMAX™ medium, and HEPES to supplement up to 500
mL
of Williams
E withoutphenolred, or a suitable
000Medium0
0 1 02 1 2 5
5
10 10
14 14
0 1 02 1 2 5
5
10 10
14 14
DaysDaysin culture
in culture
DaysDaysin culture
in culture
alternative basal medium, for the purpose of plating freshor cryopreserved hepatocytes.
80

Hydroxybupropion(pmol/mg protein/min)Hydroxybupropion(pmol/mg protein/min)

APAP(pmol/mg protein/min)
APAP(pmol/mg protein/min)

8

CYP2D6
CYP2D6
activity
activity

8

Dextrorphan(pmol/mg protein/min)
Dextrorphan(pmol/mg protein/min)

CYP1A2
CYP1A2
activity
activity
200 200

Biologically relevant
HepaRG cells exhibit many characteristics of primary
human hepatocytes, including morphology, expression of
key metabolic enzymes, expression of nuclear receptors,
and drug transporters. Unlike HepG2 and Fa2N-4 cells,
CYP1A2activity
activity
CYP2B6CYP2B6activity
activity
HepaRG
cells CYP1A2
have
high
P450 activity88 and complete
200
200
expression of all nuclear receptors.

Cell culture reagents

Gibco™ Williams Medium E
We recommend a phenol red–free Williams Medium E for
hepatocyte research involving LC-MS/MS analysis.

• Consistent—manufactured in a cGMP-compliant facility
using the highest-quality materials

200

Control

CYP2B6CYP2B6

4
4
2
2
0
0

Control

Phenobarbital Rifampicin

Phenobarbital
Rifampicin
Rifampicin
Control Phenobarbital

15
10
10
5

Control

500Control

Phenobarbital Rifampicin

Phenobarbital
Phenobarbital
Rifampicin
Rifampicin
Control

Figure 13. Human hepatocytes cultured in HepExtend Supplement
respond to inducers for Ah receptor, PXR, and CAR.

Gibco™ Hepatocyte Maintenance Supplement Pack
The Hepatocyte Maintenance Supplement Pack contains
dexamethasone and a cocktail solution of penicillin-streptomycin, ITS+ (insulin, transferrin, selenium complex, BSA,
and linoleic acid), GlutaMAX medium, and HEPES to
supplement up to 500 mL of Williams Medium E without
phenol red, or a suitable alternative basal medium, for the
purpose of incubating hepatocytes in suspension orplated cultures.
Gibco™ Collagen I, Rat Tail
Collagen is the most widely used extracellular matrix (ECM)
protein for cell culture, facilitating cell attachment anddifferentiation. In addition to our 5 mg/mL Collagen I
solution, we also offer Collagen I precoated 6-, 24-, and96-well plates for your hepatocyte experiments.

100%
75%
Cell viability

The HepaRG cell line is an immortalized hepatic cell linethat retains many characteristics of primary humanhepatocytes. HepaRG cells are terminally differentiatedand provided in a convenient cryopreserved format. For
scientists who need reproducible metabolism data,
HepaRG cells are an in vitro tool that provides results in a
metabolically complete and scalable system. These cells
enable you to:

150
150

Thawing media
Gibco™ Hepatocyte Thaw Medium (HTM) and Gibco™
Cryopreserved Hepatocyte Recovery Medium (CHRM) are
proprietary formulations designed to enhance the recoveryof viable hepatocytes while removing cryoprotectant aftercell cryopreservation. When used appropriately, they have
proven to result in healthier hepatocytes with consistently
higher viability (Figure 14). Both are easy to use: simply add
one vial of thawed hepatocytes to the conical tube,
centrifuge briefly, remove the medium, and gently
resuspend the cell pellet.

standard Williams E Hepatocyte Maintenance Medium anduse with your current hepatocyte culturing methods andinstrumentation. The supplement does not contain any
small molecules or fetal bovine serum, components thatare known to interfere with primary hepatocyte function.

6b-hydroxytestosterone(pmol/mg protein/min)
6b-hydroxytestosterone(pmol/mg protein/min)

Gibco™ HepaRG™ cells

50%
25%
0%

HTM or CHRM Medium

DMEM

Figure 14. Typical results of HTM or CHRM vs. DMEM in the ability toenhance hepatocyte viability post-cryopreservation.

Gibco™ Geltrex™ LDEV-Free Reduced Growth Factor
Basement Membrane Matrix
Geltrex matrix is a soluble form of reduced growth factor(RGF) basement membrane extract (BME) purified fromcontinuous sheets of specialized extracellular matrix thatform an interface between Engelbreth-Holm-Swarm (EHS)
tumor cells. The major components of Geltrex matrix
include laminin, collagen IV, entactin, and heparin sulfate
proteoglycan, which provide the foundation for three-dimensional (3D) culture studies.

11Gibco™ Fresh Hepatocytes (North America only)
Primary human hepatocytes, under appropriate culture
conditions, model metabolism, induction, and inhibition
similar to the in vivo liver. Fresh human hepatocytes areused to test potential therapeutic compounds for their
metabolic stability, metabolic profile, probability ofhepatotoxicity, and ability to inhibit or induce metabolic
enzymes and hepatic transporters. To achieve optimalin vitro results, primary hepatocytes must be carefully
isolated to preserve their drug-metabolizing enzymes,transporters, and cellular activities. Our technicians arehighly trained in the proper techniques to help ensure
optimal cell health. As a result, our fresh hepatocytes havehigh viabilities, excellent confluencies (Figure 15), and invivo–like enzyme expression levels. Our fresh hepatocytes:• Offer a reliable supply from one of the industry’s largest
sourcing networks• Have stringent release specifications: ≥80% viability and≥80% confluency (plated cells)• Are ready for use—plated to your specifications orprovided in suspension

Gibco™ Human Fresh Hepatocytes
We source liver tissue from a network of hospitals that
perform surgical resections on living donors, as well asfrom deceased donors whose organs were rejected fortransplant. Typically, we have access to one or moretissues per week. With such a large sourcing network, we
can supply healthy human hepatocytes on a consistentbasis, as well as keep you informed of tissues that meetyour study’s specific requirements.
• Weekly availability (typically)• Rapid Alert™ emails keep you informed
Gibco™ Animal Fresh Hepatocytes
We carry hepatocytes from the major toxicology research
species and accept many custom requests. Isolations are
conducted weekly to facilitate your research. To view ouranimal isolation calendar, visit our website atthermofisher.com/hepatocytes.
• Multiple isolations every week
• Published calendar to facilitate planning
your experiments
Species of animal fresh hepatocytes routinely available
include:• Rat (Sprague-Dawley)• Mouse (CD-1)

pool of donors. Human microsome
pools are fully characterized(Km and Vmax) according to GLP
standards for major cytochrome P450
activities and selected phase II
enzymes using FDA-recommended
substrates (Table 3). We have:

S9 fractions are available from thefollowing species:• Large pooled lots for reproducible,
long-term studies• Nonhuman primate (cynomolgus)• Multi-well plates (6-, 12-, 24-, 48-, and 96-well)• Cytochrome P450 inhibition studies
and phenotyping• Suspension culture• Metabolic stability• Specialty human pools based onage, BMI, high CYP3A4, or high
CYP2D6• Dog (beagle)• Nonhuman primate (cynomolgus)

Figure 15. Human fresh hepatocytes shown post-plating on day 2.
Good cellular morphology is indicated by the close cell–cell contacts,
clearly defined nuclei, and cytosolic clarity.

Gibco™ Liver Microsomes and S9 Fractions

Our formats are flexible—you can order hepatocytes insuspension or let us do the plating for you. We plate ourcells to maximum confluency and offer an optional Geltrex
matrix overlay to create a more in vivo–like environment.
Available in:Subcellular fractions derived from theendoplasmic reticulum of liver containa variety of metabolic enzymes forassessing the in vitro metabolism of
drug candidates (Table 2) and aresuitable for a variety of experiments:• Metabolite characterization

We provide liver microsomes from avariety of human and other toxicology
research species. Each productcontains an average representative• Human (single donor, specialty
pools, population pools)• Rat (Sprague-Dawley)• Mouse (CD-1)• Dog (beagle)• Fish (rainbow trout)• Other subcellular fractions available,
including S9 and cytosol fractions

For information on our Rapid Alert notifications, see the back cover.
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Table 2. Metabolic enzymes found in liver subcellular fractions.
Metabolic enzymes

Liver microsomes

Aldehyde oxidase

Liver S9 fractions Liver cytosol
X

Cytochromes P450 (CYP)

X

X

Flavin monooxygenases (FMO)X

X

Glutathione transferase (GST)X

Monamine oxidase (MAO)

X

Sulfurotransferases (SULT)X

Uridine glucuronide transferase (UGT)

X

Cytochrome P450 BACULOSOMES™ Plus Reagents and Vivid™
CYP450 Screening Kits

X

X

X

Table 3. Kinetic parameters for the current lot of Gibco™ Human Liver Microsomes, 50 Donor Pool.
Isoform

Metabolite

Km (µM)

Vmax (nmol/min/mg)

CYP1A2

Acetaminophen

78

0.73

CYP2A6

7-Hydroxycoumarin

1.1

0.53

CYP2B6

Hydroxybupropion

64

0.29

CYP2C86α-Hydroxypaclitaxel

5.5

0.15

CYP2C9Hydroxytolbutamide

220

0.19

CYP2C19

4´-Hydroxymephenytoin

34

0.031

CYP2D6

Dextrorphan

3.2

0.13

CYP2E16-Hydroxychlorzoxazone

70

1.4

CYP3A46β-Hydroxytestosterone

19

4.0

CYP3A4

1´-Hydroxymidazolam

1.6

1.1

CYP450 BACULOSOMES Plus Reagents are microsomes
prepared from insect cells infected with a recombinantbaculovirus containing a human CYP450 isozyme, as wellas human cytochrome P450 reductase. Vivid CYP450
Screening Kits are high-throughput, fluorescence-based
assays for detection of enzyme–drug interactions andCYP450 inhibition. CYP450 BACULOSOMES Plus
Reagents and Vivid CYP450 Screening Kits offer:• A single human P450 isozyme for detailed drug
metabolism studies• An easy three-step procedure, “mix and read” format;
reactions performed at room temperature or 37˚C
• High signal-to-background ratio, broad
dynamic range• Compatibility with multiple assay formats from 96-well to
1,536-wellUnique Vivid™ reagents for bright fluorescent signals
and low background
Vivid™ fluorogenic substrates are blocked dyes that yield
minimal fluorescence signal until cleaved or hydroxylated.
Oxidation at either of two potential sites releases the highly
fluorescent product. They have superior fluorescence,solubility, and kinetic properties compared to conventional
fluorogenic probes. This results in higher sensitivity, greater
signal-to-noise ratios, and better assay reproducibility. The
choice of substrate yields a product that emits blue, green,
red, or cyan fluorescence.
Flexible assay formats for optimized results
The sensitivity of the Vivid CYP450 assays allows detectionof weak inhibitors and miniaturization to as little as 2 µL per
reaction. Assays may be set up in kinetic mode or in
endpoint mode to facilitate multi-plate screening. Assays
may be performed at room temperature or 37˚C.

Single overexpress human CYP450 isozyme
CYP450 BACULOSOMES Plus Reagents offer a distinct
advantage over human liver microsomes in that only one
CYP450 isozyme is expressed, thereby preventing
metabolism by other CYP450 isozymes or other classes ofdrug-metabolizing enzymes.

For full listings of CYP450 enzymes and Vivid kits, visit thermofisher.com/admetox

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ABC Transporter Vesicles
ATP-binding cassette (ABC) transporter vesicles areeasy-to-use, efficient reagents for early assessment of adrug candidate’s substrate and drug interaction potential.
Prepared from Sf9 cells, which have been engineered tooverexpress specific ABC transporters, these “inside-out”
vesicles provide high levels of transporter activity with low
background, giving you a clear signal if your compound isa substrate or inhibitor of a specific efflux transporter.
ABC Transporter Vesicles are prepared from ABC
transporter membranes for use in vesicular transport
assays. While ABC transporters typically mediate the effluxof substrates from cells, transporters expressed on theseinside-out vesicles import substrates into the vesicles. It istherefore possible to quantitatively evaluate transport
activity for your compound by determining the amountincorporated into the vesicles.
These vesicles, manufactured by GenoMembrane, areprepared by advanced methodologies using plasma
membrane purified from an insect cell system (Sf9 cells
transfected with baculovirus) that overexpresses
ABC transporters.

For a full listing of ABC vesicles, visit thermofisher.com/admetox

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