FAQs -- Frequently Asked Questions
Hepatocytes
Below are the answers to many frequently asked questions submitted
by our customers. If there are other questions which you would like to
have answered please email them to us at hepatocytes_gentest@bd.com.
- How do I
obtain human hepatocytes from BD Gentest?
- Is it possible to provide pooled
cryopreserved human hepatocytes?
- Is it possible to plate cryopreserved
human hepatocytes?
- What information is available regarding the human donors?
- How long can cryopreserved hepatocytes be stored?
- Can cryopreserved hepatocytes be thawed and refrozen?
- When are hepatocytes to be used in Drug Discovery?
- How do hepatocytes compare to HLM, Supersomes, etc.?
- How do hepatocytes compare to in vivo?
- Does Gentest use hepatocytes for in vitro metabolism
and/or induction studies?
- What is 7-HFC (7-hydoxy 4-trifluoromethyl-coumarin)
metabolic activity specific for; glucuronidation or sulfation?
- Is 7-MFC an isoform specific substrate?
- Is 7-Ethoxycoumarin (7-EC) an isoform specific substrate
for hepatocytes?
- Do I need to add cofactors or cytosol to Hepatocytes?
- What is the recommended density of (cryo)hepatocytes
in a metabolism assay?
- Which solvents should be used with test compounds?
- How are enzyme activities in Gentest Hepatocytes characterized?
(refer to batch data sheets)
- What is the density of hepatocytes on plated flasks
and multiwell plates?
- Do Gentest plated hepatocytes come with gel stabilizers
to prevent the media from sloshing during shipment?
- How are Gentest plated hepatocytes shipped to me? Do
you use standard overnight delivery like Federal Express or Airborne?
- Is it expensive to custom courier plated hepatocytes
to me? What sort of delivery charges can I expect?
- How are BD Gentest Cryopreserved Hepatocytes shipped
to me?
- Does shipment of cryopreserved hepatocytes in dry ice
reduce their activity?
- Will Gentest ever ship hepatocytes on Saturdays or Sundays?
Do I need to be prepared for unusual deliveries on weekends?
- What sort of safety precautions should I take when using
Gentest Hepatocyte products?
- Are Gentest human hepatocytes always available from
stock?
Q. How
do I obtain human hepatocytes from BD Gentest? <BACK
to top>
A. To start the process contact Gentest
Technical Support Personnel (1-800-334-5229 x2246) or hepatocytes@gentest.com
to obtain the necessary documentation.
Q.
Is it possible to provide pooled cryopreserved human hepatocytes? <BACK
to top>
A. No. The frequency of receipt of
fresh tissue and need to cryopreserve hepatocytes immediately after isolation
prevents preparation of hepatocyte pools. However, the end user can thaw
and pool hepatocytes from several different donors. This approach is most
cost effective for applications that require a large number of hepatocytes.
Q.
Is it possible to plate cryopreserved human hepatocytes? <BACK
to top>
A. No. While cryopreserved hepatocytes
retain substantial drug metabolism capability, they generally lose the
ability to attach to common substrates such as collagen. BD Gentest now
offers particular lots which have been specifically tested for the ability
to replate and to induce after cryopreservation. The Catalog Nos. are
454550 and 454551.
Q.
What information is available regarding the human donors? <BACK
to top>
A. The following donor information
is typically available: age, gender, race, social, over the counter and
prescription drug use, significant medical history and cause of death.
Q.
How long can cryopreserved hepatocytes be stored? <BACK
to top>
A. Cryopreserved hepatocytes should
always be stored at ultra-cold temperatures (vapor phase of liquid nitrogen:
< -150° C). Stability studies are ongoing. Previous studies have reported
Cryopreserved Hepatocytes to be stable for an excess of 1 year when stored
at liquid nitrogen temperatures (Chem.-Biol. Interactions, 1999, 121,
17-35).
Q.
Can cryopreserved hepatocytes be thawed and refrozen?
<BACK to top>
A. No, even a single additional freeze
and thaw cycle will result in a very low recovery of viable hepatocytes.
Q.
When are hepatocytes to be used in Drug Discovery? <BACK
to top>
A. They can be used at any stage of
drug discovery. They are the best in vitro model to determine toxicity,
metabolic stability, enzyme induction, drug-drug interaction and screening
drug candidates.
Q.
How do hepatocytes compare to HLM, Supersomes, etc.?
<BACK to top>
A. Hepatocytes are whole cells where
as microsomes are cellular fractions, therefore hepatocytes include both
Phase I and Phase II metabolism.
Q.
How do hepatocytes compare to in vivo? <BACK
to top>
A. Hepatocytes are the closest thing
to human trials, although there is no standard for comparison.
Q.
Does BD Gentest use hepatocytes for in vitro metabolism and/or
induction studies?
<BACK to top>
A. Yes. Plated hepatocytes are ideal
for studying induction of metabolic enzymes. Cryopreserved hepatocytes
are best for investigating metabolic enzyme activity.
Q.
What is 7-HFC (7-hydoxy 4-trifluoromethyl-coumarin) metabolic activity
specific for; glucuronidation or sulfation? <BACK
to top>
A. In human Hepatocytes 7-HFC is primarily
a substrate to measure UGT glucuronidation activity; there is some sulfation
activity occurring but it is minimal compared to glucuronidation.
Q.
Is 7-MFC an isoform specific substrate?
<BACK to top>
A. MFC is a non-specific substrate
for most CYP enzymes, including 2B6, 1A2, 2C19, 2D6, 2E1, and 2C9. MFC
is not a substrate for 3A4.
Q.
Is 7-Ethoxycoumarin (7-EC) an isoform specific substrate
for hepatocytes?
A. 7-EC is a non-specific substrate
for many CYP enzymes leading to the product 7-hydroxycoumarin. The 7-hydroxycoumarin
is then a subsequent substrate for the phase II reactions of sulfation
and glucuronidation. Generally, sulfation is the predominant activity
in rat hepatocytes whereas glucuronidation is predominant in humans.
Q.
Do I need to add cofactors or cytosol to Hepatocytes? <BACK
to top>
A. No, hepatocytes contain all the
necessary co-factors (e.g. NADPH and UDPGA) for both phase I and phase
II drug metabolism.
Q.
What is the recommended density of (cryo)hepatocytes in a metabolism assay?
<BACK to top>
A. 0.5-e6 - 1.0e6 cells/ml is the
recommended density. Refer to posted protocol.
Q.
Which solvents should be used with test compounds? <BACK
to top>
A. DMSO is the preferred solvent and
should be used in concentrations less than 0.2%. If DMSO is not appropriate
for a specific test compound ACN could also be used at concentrations
less than 1%. For induction experiments the final DMSO concentration in
the culture media should be less than 0.1%.
Q.
How are enzyme activities in BD Gentest Hepatocytes characterized?
(refer to batch data sheets) <BACK to top>
A. Enzyme activities in Cryopreserved
Human Hepatocytes are characterized with the following substrates:
Testosterone for CYP3A4
Diclofenac for CYP2C9
Bufuralol for CYP2D6
Phenacetin for CYP1A2
Umbelliferone (7-HC) for UGT's
*Respective metabolites are resolved using HPLC.
Enzyme activities of Plated Human Hepatocytes
are determined with the following substrates:
Testosterone; measuring CYP3A4 activity
7-MFC; measuring multiple CYP isoform activity
7-HFC; measuring multiple UGT glucuronidation isoforms
*Respective metabolites are resolved Fluorometrically.
Enzyme activities in Cryopreserved Rat Hepatocytes
are characterized with the following substrates:
7-Hydroxycoumarin for general P450 activity
7-Hydroxycoumarin sulfate for Phase 2 activity
7-Hydroxycoumarin glucuronide for Phase 2 activity
We have characterized general lots with the Testosterone, Phenacetin
and Chlorzoxazone.
*Respective metabolites are resolved using HPLC.
Q.
What is the density of human hepatocytes on plated flasks and multiwell
plates? <BACK to top>
A. Hepatocytes are seeded at 2.0 e5
cells/ cm2. We estimate that 90-100% adhere to the surface to form the
monolayer. We recommend protein quanitation of individual monolayers for
the purposes of calibrating data of induction/enzyme studies.
Q.
Do BD Gentest plated hepatocytes come with gel stabilizers to prevent
the media from sloshing during shipment? <BACK
to top>
A. No. Gentest plated hepatocytes
come with a special sealing gasket lid to minimize leakage of media during
shipment, therefore the use of gel stabilizers is not necessary. Once
the product is received, the cardboard lid sealer can be cut open and
the gasket lid can be replaced with a normal FALCON™ lid, enclosed with
each shipment. Gentest plated hepatocytes in T-flasks do not require any
special gaskets for shipment.
Q.
How are BD Gentest plated hepatocytes shipped to me? Do you use standard
overnight delivery like Federal Express or Airborne?
<BACK to top>
A. Each shipment of Gentest plated
hepatocytes are custom couriered to each account to ensure proper temperature
control and rapid delivery. Most shipments are delivered using Sterling
Couriers to ensure maximum viability upon receipt.
Q.
Is it expensive to custom courier plated hepatocytes to me? What sort
of delivery charges can I expect? <BACK to top>
A. All Gentest plated hepatocytes
are shipped FOB Woburn, Massachusetts. Shipment charges vary with distance.
To get a reasonable estimate for your location, contact hepatocytes@gentest.com
and be sure to give your site location. Typical courier charges vary from
$100-150/shipment.
Q.
How are BD Gentest Cryopreserved Hepatocytes shipped to me?
<BACK to top>
A. BD Gentest Cryopreserved Hepatocytes
are shipped to customers in dry ice using priority overnight delivery
from our warehouses in North America and Europe. US customers may also
request shipment in liquid nitrogen as a extra cost option. Ask your BD
Gentest customer service representative for more details.
Q.
Does shipment of cryopreserved hepatocytes in dry ice reduce their
activity? <BACK
to top>
A. Our studies have shown that there
is very little difference between cryopreserved hepatocytes shipped overnight
in dry ice vs. liquid nitrogen.
Data shown below represents the average of 5 different lots tested at
BD Gentest (+/- std. error of the mean):
| Type of Assay |
Value Relative to Liquid Nitrogen |
Mean Value (Liquid nitrogen) |
| Testosterone 6(beta)-hydroxylase (CYP3A4) |
1.07 +/- 0.18 |
266 pmole/min/million cells |
| Diclofenac 4'-hydroxylase (CYP2C9) |
1.00 +/- 0.16 |
265 pmole/min/million cells |
| Total viable cells recovered using BD Gentest Cryopreserved
Hepatocyte Purification Kit (Cat. No. 454500) |
| # Cells |
0.92 +/- 0.06 |
3.6 million cells |
| % Viability |
1.02 +/- 0.02 |
80% |
Q.
Will BD Gentest ever ship hepatocytes on Saturdays or Sundays? Do I need
to be prepared for unusual deliveries on weekends? <BACK
to top>
A. Gentest plated hepatocytes are
grown in Hepato-STIM™ media for maximum viability and longevity.
It is generally not necessary to ship plated hepatocytes over weekends.
Customers who register with Gentest (contact hepatocytes@gentest.com for
an application) will be notified of any impending shipments.
Q.
What sort of safety precautions should I take when using BD Gentest Hepatocyte
products? <BACK to top>
A. Gentest plated human hepatocytes
should always be handled using universal precautions, as mandated by the
U.S. Occupational Safety and Health Administration Bloodborne Pathogens
Final Standard. See your local Occupational Health and Safety Officer
for more information on any local procedures that you may need to follow
to use human derived products safely in your institution. Always dispose
of human hepatocytes in compliance with local, state and federal regulations.
Q.
Are BD Gentest human hepatocytes always available from stock? <BACK
to top>
A. Cryopreserved human hepatocytes
can be purchased at any time; however, fresh hepatocytes are only available
when we receive fresh tissue to process. On average, we receive a fresh
liver every 7-14 days, so any delays in receiving products would be minimal.
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