Corning
®
Matrigel
®
Matrix Thin-Coat
and Corning Matrigel Matrix Overlay
Improved CYP450 Activities in Human
Cryopreserved Hepatocytes
Application Note 476
1
Haiyan Xia,
2
Charles L. Crespi,
2
Chris J. Patten, and
1
Rongjun Zuo
1
BD Biosciences – Woburn, MA
2
Corning Life Sciences, Tewksbury, MA
Abstract
The purpose of this study was to evaluate the effect of Corning
Matrigel Matrix Thin-Coat and Corning Matrigel Matrix Overlay
on the attachment, morphology, and CYP enzyme activities of
human cryopreserved hepatocytes.
Corning Gentest™ Human CryoHepatocytes were tested for
basal and induced CYP3A4 and CYP1A2 activities on Collagen I
and Corning Matrigel Matrix thin-coat surface with or without
a Corning Matrigel Matrix overlay. Changes in attachment and
morphology were also examined. The results showed that the
Corning Matrigel Matrix thin-coat surface signicantly improved
basal activities for both CYP3A4 and CYP1A2 compared to a
conventional collagen I-coated surface; the Corning Matrigel
Matrix thin-coat surface showed a lot-dependent effect towards
induced activities for both CYP3A4 and CYP1A2 for tested lots.
The Corning Matrigel Matrix overlay, in addition to the Corning
Matrigel Matrix thin-coat plate, further improved basal CYP450
activities, however, the Corning Matrigel Matrix overlay did not
change cell attachment. The Corning Matrigel Matrix thin-coat
can maintain typical hepatocyte morphology for a longer time
than the collagen I-coated surface. In conclusion, a culture
condition combining Corning Matrigel Matrix thin-coat surface
and Corning Matrigel Matrix overlay has a potential of maintain-
ing stable long-term basal metabolic activities for human cryopre-
served hepatocytes, facilitating their application in areas such as
in vitro chronic toxicity assays.
Introduction
Primary human hepatocytes are considered the “Gold Standard”
for drug metabolism studies. Induction in drug metabolizing
enzymes in hepatocytes by test compounds can provide infor-
mation for drug-drug interactions (DDI). Enzyme induction in
hepatocytes takes a few days and both fresh and cryopreserved
hepatocytes can be used for this purpose if they are both platable
and inducible. Human cryopreserved hepatocytes often have
limited application due to impaired cell attachment and enzyme
activities which is caused by the cryopreservation process. Some
cryopreserved lots have very low basal CYP450 activities, result-
ing in a very high fold of induction which could provide false DDI
information. As cryopreserved hepatocytes have the benets of
being convenient, readily available, and able to provide multiple
lots for comparison. It is however important to develop culture
conditions that support cryopreserved hepatocyte recovery,
hepatic morphology, and metabolizing activities, especially
when long-term cell treatment is needed for certain applications
such as in vitro chronic hepatotoxicity assays. It has been shown
that extracellular matrix (ECM)-based growth substrata provide
a physiological environment that maintains differentiation
character and supports key cellular functions. Collagen I has tra-
ditionally been the substratum of choice for hepatocyte attach-
ment and induction assays. Corning evaluated cell attachment,
cell morphology and enzyme activities (especially basal activity
which is an indicator of hepatocyte health) of cryopreserved
hepatocytes using a Corning Matrigel Matrix thin-coat surface in
the form of an overlay.
Methods
Culturing of Corning Gentest Human CryoHepatocytes
Corning Gentest Human CryoHepatocytes (Cat. Nos. 454550
and 454551) were thawed and puried using the Corning
CryoHepatocyte Purication Kit (Cat. No. 454500). Puried
hepatocytes were resuspended in ISOM’s media containing
10% FBS at a concentration of 1.0x10
6
cells/mL and seeded on
24 well plates (Corning BioCoat™ Collagen I-coated plate,
Cat. No. 354408, Corning Matrigel Matrix Thin-Coat Plate, Cat.
No. 354605) at a density of 400,000/well and incubated at 37°C
with 5% CO
2
. Corning Matrigel Matrix solution (0.25 mg/mL
in Corning HepatoSTIM™ Medium) was added 6 hours later at
500 mL/well to form an overlay. In one set of experiments,
Corning Matrigel Matrix overlay was added only on day 1; while
in another set of experiments, Corning Matrigel Matrix overlay
was added daily from day 1 to day 4.
The following graph illustrates the experimental set up for the
ECM coating/overlay affect on hepatocyte application.
2
Hepatocyte CYP450 Induction Assay
Induction was conducted by adding inducers at 400 µL/well
(20 mM Rifampicin for CYP3A4 and 20 mM ß-Naphthoavone
(ß-NF) for CYP1A2) daily from day 2 to day 4. Same amount of
DMSO was added as vehicle control to obtain basal activities.
On day 5, probe substrates (200 mM testosterone for CYP3A4 and
100 mM phenacetin for CYP1A2) were added and incubated with
hepatocytes for 30 minutes (for 3A4) or 60 minutes (for 1A2).
Supernatants were then collected into tubes containing stop
solution and protein samples were collected by incubating cells
with 1% SDS solution for 15 minutes. Metabolite samples were
analyzed by HPLC and protein concentration was performed by
Lowry assay.
Data analysis
The enzyme activity was expressed as pmol/mg protein/min.
Each data point represents mean of 3 wells.
Hepatocyte Collagen/Corning® Matrigel® Matrix Sandwich Culture
Hepatocyte
Corning Matrigel Matrix
Collagen I
Corning Matrigel Matrix overlay added only on day 1 Corning Matrigel Matrix overlay added daily from day 1-4
Corning Matrigel Matrix
added on Day 1
Corning Matrigel Matrix
added on Day 1
Inducer added
on Day 2-4
Enzyme assay
on Day 5
Enzyme assay
on Day 5
Corning Matrigel Matrix
and Inducer added
on Day 2-4
Results
90
80
70
60
50
40
30
20
10
0
Lot #
Cell Confluency +/- Corning Matrigel Matrix After Plating at 24 and 96 hours
Confluency %
117 122
24h, without Corning Matrigel Matrix
24h, with Corning Matrigel Matrix
96h, without Corning Matrigel Matrix
96h, with Corning Matrigel Matrix
124 128 134 137 141 147 153 HH238
Figure 1. Effect of Corning Matrigel Matrix Overlay
on Hepatocyte Attachment
Attachment of cryopreserved hepatocytes is not
changed by the Corning Matrigel Matrix overlay.
Corning Matrigel Matrix overlay concentration (mg/mL)
Corning Matrigel Matrix added on Day 1
Corning Matrigel Matrix added daily from day 1-4
Enzyme activity (pmol/mg protein/min)
2500
2000
1500
1000
500
0
0 0.0625 0.125 0.25 0 0.0625 0.125 0.25
4
3.5
3
2.5
2
1.5
1
0.5
0
Induced activity
Basal activity Fold of Induction
390
414
1077
2.8
1395
3.4
1958
2.7
714
682
455
1352
2.8
1940
3
2003
2144
2003
1036
2.1
1034
2
985
1.9
Figure 2. Optimization of Corning Matrigel Matrix
Overlay for CYP3A4 Activities
Corning Matrigel Matrix was added at different
concentrations and different frequencies to deter-
mine the optimum overlay format. It shows that
the Corning Matrigel Matrix overlay signicantly
improved CYP3A4 basal activities in a concentration-
dependent style up to 0.125 mg/mL. Induced activity
was also increased at a lesser degree. No signicant
difference was observed with the frequency of adding
Corning Matrigel Matrix.