Photoregulation of cytochrome P450 activity by using caged compound

Article abstract of DOI:10.1021/ac202189h

Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP⁺) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP⁺ resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 μm; height, 50 μm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.

Full text of DOI:10.1021/ac202189h

ARTICLE
pubs.acs.org/ac
Photoregulation of Cytochrome P450 Activity by Using Caged
Compound
Kenichi Morigaki,*,†,‡ Kazuyuki Mizutani,†,‡ Emi Kanemura,†,‡ Yoshiro Tatsu,*,† Noboru Yumoto,† and
Hiromasa Imaishi*,‡
†National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda 563-8577, Japan
‡Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501 Japan
S Supporting Information
b
ABSTRACT: Cytochrome P450 (P450) species play an important role in
the metabolism of xenobiotics, and assaying the activities of P450 is
important for evaluating the toxicity of chemicals in drugs and food.
However, the lag time caused by the introduction and mixing of sample
solutions can become sources of error as the throughput is heightened by
increasing the sample number and decreasing the sample volume. To
amend this technological obstacle, we developed a methodology to
photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide
adenine dinucleotide phosphate (NADP+) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH
(cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP+
resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use
of caged-G6P enabled assays in isolated microchambers (width, 50 μm; height, 50 μm) by encapsulating necessary ingredients in
advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single
microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of
obtained kinetic constants.
e report on a methodology to regulate the activity of
cytochrome P450 (P450) by photoillumination. P450
To this end, we synthesized caged molecules of nicotinamide
adenine dinucleotide phosphate (NADP+) and glucose 6-phos-
phate (G6P). NADP+ can be converted into NADPH by glucose-
6-phosphate dehydrogenase, consuming G6P (NADPH regen-
eration reaction, Figure 1). (Direct photoprotection of NADPH
was not possible because of its chemical instability.) The synthesis of
caged-G6P and NADP+ was previously reported.10,11 Caged-G6P
and NADP+ were synthesized using 2-nitrophenyl-acetophenone
as the protecting group, and their abilities to photoregulate the
activity of P450 were evaluated. Upon establishing the con-
ditions for photoregulating enzymatic reactions, we conducted
assays in isolated microchambers. We encapsulated all necessary
ingredients, including P450 and substrate molecules, into each
chamber and initiated the reaction by UV illumination. By tem-
porally separating the mixing process and the reaction, we could
observe the initial velocity of the reaction, resulting in the deter-
mination of kinetic constants with significantly smaller errors
compared with the conventional methods.
W
species play an important role in the metabolism of xenobiotics
in organisms, and their metabolic activities in human liver are
closely related with the toxicity of chemicals in drugs and food.1,2
Methods to assay P450 activities in high throughput are sought
for drug development and toxicological inspections.3 One tech-
nological challenge in high-throughput screening is the lag time
caused by the introduction and mixing of sample solutions
(enzymes, substrates, cofactors, etc.), which can become sources
of error in quantitative evaluation of enzymatic activities. This
problem becomes critical as the volumes of solutions become
smaller and the number of samples increases. One feasible ap-
proach to amend this technological obstacle is to photoregulate
the enzymatic activities by using photoprotected (caged) com-
pounds. By using a caged cofactor of the enzyme, one can mix the
necessary ingredients in advance, initiate the reaction by illumi-
nating UV light in a temporally and spatially controlled manner,
and observe the reaction in real time. Although caged compounds
have been extensively used in vivo and in time-resolved studies,4ꢀ8
to the best of our knowledge, its application to bioassays in
microchambers has not been fully exploited.
’ MATERIALS AND METHODS
Materials. Nicotinamide adenine dinucleotide phosphate
(NADP+), reduced nicotinamide adenine dinucleotide phosphate
P450 requires electrons provided by P450 reductase for the
enzymatic activity. P450 reductase requires, in turn, the reduced
form of nicotinamide adenine dinucleotide phosphate (NADPH) as
the electron source.9 Therefore, it should be possible to control
the enzymatic reaction by regulating the supply of NADPH.
Received: August 19, 2011
Accepted: November 15, 2011
Published: November 15, 2011
r
2011 American Chemical Society
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2-nitrophenyl-acetophenone hydrazone (225 mg, 1.26 mmol) in
1.0 mL of dichloromethane was added to manganese oxide
(369.9 mg, 4.25 mmol). After stirring for 30 min at room tem-
perature, the solution was centrifuged. The supernatant was
filtered by PTFE filter (pore diameter 0.75 μm, Millipore), the
filtrate was added to 87.3 mg (0.31 mmol) of glucose-6-phos-
phate (G6P) sodium salt dissolved in 1 mL of deionized water
and stirred overnight at room temperature. The aqueous phase
was washed twice with dichloromethane and freeze-dried to give
116 mg of white powder. The solid was purified by C18 reverse
phase HPLC and freeze-dried twice to give the target caged-
G6P (Figure 2). EIMS calculated for C14H21NO11P, 409.07;
[M + Na+]m/z, 432.3.
Preparation of Membrane Fraction of Human P450 and
P450 Reductase from E. coli. A pCW1A1 vector for expressing
CYP1A1 in E. coli was constructed. The cDNA fragment encod-
ing human CYP1A1 was obtained from human liver-cDNA
libraries by using PCR. In order to modify N-terminal regions
of cDNA fragments of CYP1A1 and add a digestion site by
NdeI and SalI, a primer was prepared as follows: h1A1-F, 50-
GGAATTCCATATGGCTTTTCCAATTTCAATGTCAGC-
AACG-30, and h1A1-R, 50-AAGTCGACCTAAGAGCGCAG-
CTGCATTTGGAAGTGCT-30. The plasmid containing the
cDNAs of CYP1A1 was digested with NdeI and SalI, and the
fragments obtained were subcloned into the SalI and NdeI sites of
the pCW vector, which contains a cDNA of human NADPH-
cytochrome P450 reductase.13 E. coli JM109 cells were trans-
formed with the pCW1A1 vector. The transformed cells were
cultured in 5 mL of Luria broth (LB) at 37 °C for 1 day, and then
3 mL of this culture was added to 300 mL of LB and incubated at
37 °C to an OD600 of 0.2ꢀ0.3. All liquid cultures were shook in a
shaking incubator at 150 rpm. After the addition of IPTG (1 mM)
and delta-amino levulinic acid (0.5 mM), the cultures were grown at
25 °C for 24 h. The cells were centrifuged and suspended in 4 mL
of 100 mM potassium phosphate buffer (pH 7.5) containing 20%
glycerol. E. coli cells were homogenized by sonication. The
homogenate was centrifuged at 10000g for 15 min to obtain a
supernatant fraction, and then this supernatant was centrifuged
at 100 000g for 60 min. Pellets (1 mL) were then resuspended in
2 mL of 100 mM potassium phosphate buffer (pH 7.5) contain-
ing 20% glycerol and then stored at ꢀ80 °C until use.
Figure 1. (A) Schematic of the photoregulation of P450 catalysis by
using caged compound. (B) Chemical structure and reaction scheme of
caged-NADP+ and caged-G6P. The protecting groups are indicated with
dotted circles.
(NADPH), dithiothreitol (DTT), magnesium chloride, and
glucose-6-phosphate dipotassium (G6P) were purchased
from Nacalai Tesque (Kyoto, Japan). Isopropyl-1-thio-β-D-
galactopyranoside (IPTG), delta-amino levulinic acid, etha-
nol, methanol, and concentrated hydrochloric acid were
obtained from Wako Pure Chemical Industries (Osaka, Japan).
Glucose-6-phosphate dehydrogenase was purchased from Toyobo
(Osaka, Japan). 7-Ethoxyresorufin (7-ER) was purchased
from Toronto Research Chemicals Inc. (Toronto, Canada).
Milli-Q water with the resistivity more than 18 MΩ cm was
used to prepare aqueous solutions.
P450 Assays in Test Tubes. For measuring the enzymatic
activity of P450 using caged-G6P or caged-NADP+ in a test tube,
we used a fluorogenic substrate (7-ER). 7-ER can be dealkylated
by CYP1A1 and forms a fluorescent product (resorufin). The
reaction solution contained 0.1 M potassium phosphate buffer
(KPB), 0.4 U/mL G6P dehydrogenase, 3 mM magnesium chloride,
1 mM DTT, 10.25 μL of P450 membrane fragments, 0.3 mM G6P
(or 0.3 mM caged-G6P), and 0.01 mM NADP+ (or 0.01 mM caged
NADP+) (total volume, 500 μL). UV illumination was conducted
by using a UV lamp (Ushio, SP-7) and a bandpass filter (Schott,
UG1). The applied energy was 12 mW/cm2. FollowingtheUVlight
irradiation, 1.5 μM 7-ER was added and the sample was incubated
for 30 min at room temperature. The reaction was terminated by
adding 25 μL of 30% trichloroacetic acid. A volume of 500 μL of
chloroform was added to the reaction solution and vortexed for
1 min to extract resorufin generated by the enzymatic reaction.
The organic and aqueous phases were separated by centrifugation
(2 000g for 1 min), and 250 μL of chloroform was transferred to
another tube. A volume of 500 μL of 5 mM NaOH/50 mM NaCl
solution was added, and the mixture was vortexed for 1 min. The
organic and aqueous phases were separated by centrifugation,
Synthesis of Caged-NADP+ and Caged-G6P. Synthesis of
Caged-NADP+. Caged-NADP+ was prepared according to the
reported method with some modifications.10 A solution of
2-nitrophenyl-acetophenone hydrazone (26.9 mg, 0.15 mmol) in
0.3 mL of dichloromethane was added to manganese oxide (65.2 mg,
0.75 mmol). After stirring for 5 min at room temperature, the solution
was centrifuged. The supernatant was filtered with a PTFE filter
(porediameter 0.75μm, Millipore). The filtrate was added to 77 mg
(0.1 mmol) of NADP+ dissolved in 0.3 mL of deionized water and
stirred for 2 h at room temperature. The aqueous phase was washed
twice with dichloromethane and freeze-dried to give 116 mg
of white powder. The solid was purified by C18 reverse phase
high-performance liquid chromatography (HPLC) and
freeze-dried to give the target caged NADP+ (Figure 1).
Electrospray ionization mass spectrometry (EIMS) calcu-
lated for C29H37N8O19P3, 892.4; [M + H+]m/z, 893.1.
Synthesis of Caged-G6P. Caged-G6P was prepared according
to the reported method with some modifications.12 A solution of
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Figure 2. The enzymatic activity of h-CYP1A1 toward 7-ER, estimated from the fluorescence intensity of the product (resorufin), was plotted versus the
UV dose applied to decage either caged-NADP+ or caged-G6P. The measured fluorescence increase was normalized to the enzymatic reaction using
normal NADP+ and G6P to obtain the relative activity. The assays were conducted in a test tube by mixing the necessary ingredients (including
h-CYP1A1 and a caged compound) and illuminating UV light for a defined duration. (A) caged-NADP+ and normal G6P were used and (B) caged-G6P
and normal NADP+ were used. 7-ER was added after the UV illumination.
and 300 μL of the aqueous phase was transferred to a quartz
cuvette for the fluorescencemeasurement (F20-SQ-5, GL Sciences,
Japan). The concentration of resorufin was determined by a
fluorescence spectrophotometer (F4500, Hitachi, Tokyo, Japan)
with the excitation and emission wavelength of 530 and 583 nm,
respectively (Supporting Information, Figure S1). We have also
confirmed by HPLC that the metabolic reaction product was
resorufin (Supporting Information, Figure S2).
P450 Assays in Microwells. For P450 assays in microcom-
partments, a PDMS (polydimethylsiloxane) chip having micro-
wells (diameter, 50 μm; depth, 50 μm) was used. P450 reaction
solution contained 0.1 M KPB, 7-ER (varied concentrations),
0.3 mM caged-G6P, 0.4 U/mL G6P dehydrogenase, 3 mM mag-
nesium chloride, 0.01 mM NADP+, 1 mM DTT, and 10.25 μL of
P450 membrane fragments (total volume, 500 μL). The solution
was put on top of the PDMS chip and introduced into microwells by
applying a gentle vacuum. Then the tops of the wells were sealed
with a glass slide. The P450 reaction was observed by fluorescence
microscopy. An upright microscope (BX51WI, Olympus, Tokyo,
Japan) equipped with a xenon lamp (UXL-75XB, Olympus), a
20ꢁ water-immersion objective (NA 0.95), and a CCD camera
(DP30BW, Olympus) were used. Photoactivation of caged-G6P
was done by UV illumination with a bandpass filter (330ꢀ385 nm).
The conversion of 7-ER into resorufin was observed with a different
set of bandpass filters (excitation 545ꢀ580 nm, emission >610 nm).
background reaction. It is presumably because a small amount of
NADP+ present can be used repeatedly by the NADPH regenera-
tion reaction (Figure 1). For both caged-G6P and caged-NADP+,
the enzymatic activity increased swiftly with the UV illumination and
reached maximum values, which were more than 90% of the native
enzymatic activity (i.e., the enzymatic activity measured by using
normal NADP+ and G6P). For a longer UV illumination, the
enzymatic activity gradually decreased. Since the wavelength of UV
illumination overlaps the absorption band of heme in P450, a
prolonged illumination may cause photodeactivation of P450
(Supporting Information, Figures S4ꢀS6). If we used both caged-
NADP+ and caged-G6P for the assay, the onset of enzymatic activity
occurred much more slowly, and the maximum enzymatic reaction
reached was also significantly lower (Supporting Information,
Figure S7). Since the use of caged-G6P could avoid the background
reaction (unlike caged-NADP+) and resulted in an effective switch-
ing of P450 activity, we employed it in the following experiments.
Photoregulation of P450 Activity in Microchambers. To
demonstrate that initiation of enzymatic reactions can be con-
trolled spatially and temporally, we conducted the enzymatic
reaction in closed microchambers made of polydimethylsiloxane
(PDMS). A reaction solution containing h-CYP1A1, substrate
(7-ER), NADP+, caged-G6P, and other necessary ingredients
was encapsulated into PDMS microwells (diameter, 50 μm;
depth, 50 μm) by putting the solution on top of the PDMS slab
and evacuating gas from the microwells in vacuum. Subsequently,
the wells were sealed with a glass slide. After these procedures,
which took roughly 15 min, we started the assay. We could
induce the enzymatic reaction in a single well by a localized UV
illumination (Figure 3A). By applying an automated program
comprising UV illumination and observation, we could measure
the initial enzymatic reaction rate in this particular well (Figure 3B,C).
No enzymatic reaction occurred in the neighboring wells
(Figure 3A,B). In a different set of experiment, we measured
the fluorescence increase with different 7-ER concentrations
(Figure 4). From the concentration dependency of the initial
velocity, we could estimate the enzymatic kinetic parameters
according to the MichaelisꢀMenten model (Figure 5). The results
are compiled in Table 1. As a comparison, we estimated the
enzymatic parameters in test tubes (volume, 500 μL) using either
normal G6P or caged-G6P. (It is important to note that assays in
’ RESULTS AND DISCUSSION
Photoregulation of P450 Activity Using Caged-NADP+
and Caged-G6P. We first studied the photoactivation (decaging)
of caged-NADP+ and caged-G6P to regulate the activity of P450
enzymes. To this end, reaction solutions containing human CYP1A1
(h-CYP1A1), a caged compound (either caged-G6P or caged-
NADP+), and other necessary ingredients were subjected to UV
illumination (Figure 2). (We have chosen h-CYP1A1 as a model
P450, partially because its metabolic activity toward 7-ethoxyresor-
ufin (7-ER) is well established.14 It should be noted that we added
7-ER after the UV illumination in this experiment to avoid the
complication caused by the photobleaching.) In the case of caged-
G6P, no enzymatic reaction was observed without UV illumination.
On the other hand, the use of caged-NADP+ resulted in a little
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Figure 5. Initial reaction rates of h-CYP1A1 enzymatic activities were
plotted versus the concentrations of 7ER to estimate the Km and Vmax
values from the MichaelisꢀMenten’s equation. Black square, using
caged-G6P in microwells; red diamond, using caged-G6P in test tubes;
green triangle, using normal G6P in test tubes.
Table 1. Enzymatic Kinetic Constants Determined by Using
Normal and Caged-G6Pa
Km (μM) Vmax (pmol/min/pmol P450)
n
caged-G6P (microwells) 0.44 ( 0.04
caged-G6P (test tubes) 0.65 ( 0.04
4.78 ( 0.35
3.88 ( 0.03
5.65 ( 0.45
5
3
3
normal-G6P (test tubes) 1.11 ( 0.38
a Metabolic activity of h-CYP1A1 toward 7-ER was measured in test
tubes and microwells at room temperature (24 °C).
Figure 3. Spatially and temporally controlled activation of P450 in a
PDMS microwell: Solutions containing h-CYP1A1, 7-ER, caged-G6P,
and other necessary ingredients were encapsulated in PDMS microwells
(diameter, 50 μm; height, 50 μm) and sealed with a glass slide. The
enzymatic reaction in a single well was initiated by localized UV
illumination using a pinhole. (A) Bright field and fluorescence images
of the microwells after the reaction: Fluorescence from the reaction
product (resorufin) was observed only in the illuminated microwell. (B)
Time course of fluorescence increase after the illumination. Illuminated
microwell showed a distinctive fluorescence increase (black squares in
part B) whereas fluorescence in a neighbor chamber did not change (red
circles in part B). (C) Selected fluorescence images in the time course in
(B) (indicated as a, b, and c).
microwells using normal G6P were not possible because the
enzymatic reaction started upon mixing the solutions before
incorporating them into each well.) The obtained values for Km
were comparable to the literature values.15 On the other hand,
Vmax may be slightly underestimated in the case of caged-G6P
due to the effects of UV illumination. Importantly, errors of the
estimated Km and Vmax values were significantly lower for caged-
G6P compared with normal G6P (the results in test tubes). The
reduced uncertainty should stem from the fact that we could
measure the initial velocity of the enzymatic reaction by using
caged-G6P without the effects of the mixing process.
One of the most commonly applied modes of high-throughput
screening involving P450 is the competitive assay between a
fluorogenic substrate and a drug candidate (or potentially toxic
substance). The decrease of P450 activity toward a fluorogenic
substrate in the presence of an additional substance implies that
the substance is either an inhibitor or a substrate of this particular
P450 species.3 As a demonstration that we can apply caged-G6P
to P450 assays in a manner relevant to the high-throughput
screening setting, we conducted competitive assays in PDMS
microwells. Benzo[a]pyrene, a carcinogenic substance, was en-
capsulated into each well together with h-CYP1A1 and 7-ER. As
shown in Figure 6, a decrease in the metabolic reaction of
h-CYP1A1 toward 7-ER was observed as the concentration of
benzo[a]pyrene was increased, which is consistent with the
report that benzo[a]pyrene acts as a noncompetitive inhibitor for
the metabolic reaction of 7-ER by h-CYP1A1.14 This result clearly
demonstrates the feasibility to assay P450 activities using caged-G6P.
Advantages of Using Caged Compounds in Bioassays.
The use of caged-G6P has two distinct advantages. First, it enables
one to assay using isolated microchambers in a parallel fashion.
Currently, bioassays are typically conducted using microwell
Figure 4. Assaying P450 activity in PDMS microwells: Solutions
containing different concentrations of 7ER were incubated in PDMS
microwells together with h-CYP1A1, caged G6P, and other necessary
ingredients. The enzymatic reaction was started by UV illumination.
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major advantages. First, we could assay P450 in isolated micro-
chambers, drastically reducing the amount of solution necessary
and increasing the number of assays that can be conducted in
parallel. Second, the observation of initial velocity can minimize
uncertainties caused by the introduction and mixing process of
solutions, leading to significantly reduced errors of obtained
assay data (kinetic constants). These features should facilitate the
evaluation of P450 activities, which are important for a wide
range of pharmaceutical and diagnostic applications. We have
chosen h-CYP1A1 as a model P450, but we can certainly apply
the same methodology to other P450 species, including h-CYP3A4,
and their genetic variants. Genetic variations of P450 play im-
portant roles in the pharmacological effects of drugs to indivi-
duals. The use of caged cofactors opens a new avenue to assay a
large number of P450 variants in a parallel fashion using isolated
microchambers, minimizing the use of P450 enzymes and sub-
strate materials, and improving the accuracy of assay at the same
time. Furthermore, these merits of using caged cofactors can also
be readily transferred to other types of clinically relevant bioassays.
Figure 6. Competitive assay in PDMS microwells: Solutions containing
7ER and benzopyrene were incubated in PDMS microwells together
with h-CYP1A1, caged G6P, and other ingredients. The enzymatic reaction
was started by UV illumination at time zero. Concentration of benzopyrene:
black triangles, 0 μM; red circles, 0.1 μM; green diamonds, 0.2 μM.
plates, in which each microwell has a volume of ∼10 μL.
Microchambers such as demonstrated in the present study require
a much smaller volume of assay solution (∼100 pL). However,
bioassays in such small chambers have not been feasible because
of the fact that reactions already start (and possibly finish) during
the mixing and introduction of the solutions. Although femtoliter
chambers have been used to study single molecule enzymatic
activities,16 the difficulty to introduce the substrate solutions
into each well without starting the reaction prevented the use of
isolated microchambers for high-throughput assays. The use of
caged cofactors opens new possibilities to the parallel assay of
enzymatic activities in isolated microchambers. One can possibly
immobilize different CYPs in microwells on a chip by applying
the printing technology and assay their activities by introducing
the substrate solution and sealing the wells.
’ ASSOCIATED CONTENT
S
Supporting Information. Additional information as noted
b
in text. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Kenichi Morigaki: e-mail, morigaki@port.kobe-u.ac.jp; fax, +81-
78-803-5941. Yoshiro Tatsu: e-mail, y-tatsu@aist.go.jp; fax, +81-
29-862-6130. Hiromasa Imaishi: e-mail, himaish@kobe-u.ac.jp;
fax, +81-78-803-5940.
The second important advantage of using caged-G6P is the
fact that one can measure the initial velocity of the enzymatic
reaction. Kinetic models of enzymatic reactions are based on the
initial velocities, but experimentally obtained initial velocities are
often compromised by the lag time due to the sample introduc-
tion and mixing processes. This problem can become critical as
the volume of each sample decreases and the number of samples
increases. Our results suggested that we could obtain enzymatic
kinetic data with a lower data fluctuation by premixing the ingre-
dients and measuring the onset of the reaction. One potential
drawback of the present approach is the possibility that enzy-
matic activity is affected by the UV illumination. However, we
should be able to obtain accurate estimates of relative P450
activities and inhibition by applying the same decaging condi-
tions. (Obtaining accurate values for the relative activities is often
sufficient in the high-throughput screening setting because an
absolute value of enzymatic activities are very difficult to attain
due to many technical obstacles such as the homogeneity of P450
sources.) Furthermore, we should also be able to minimize the
effects of light illumination by designing more efficient photopro-
tective groups (having longer absorption wavelength and higher
quantum yield) and optimizing the illumination conditions.
’ ACKNOWLEDGMENT
This work was financially supported by Program for Promo-
tion of Basic Research Activities for Innovative Biosciences
(PROBRAIN). We thank Ms. Saori Mori, Mr. Takashi Irie
(AIST), and Mr. Yoshinao Mori (Kobe Univ.) for their assistance
in the experiments. We thank Dr. Hidenori Nagai and Dr.
Shinich Wakida (AIST) for supporting us with the fabrication
of PDMS microwells. Discussions with Dr. Kunio Isshiki and
Dr. Akira Arisawa (Mercian Corp.) were appreciated.
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