in vitro microscale systems for systematic drug toxicity study
TRANSCRIPT
MINI REVIEW
In vitro microscale systems for systematic drug toxicity study
Jong Hwan Sung Æ Michael L. Shuler
Received: 28 May 2009 / Accepted: 4 August 2009 / Published online: 23 August 2009
� Springer-Verlag 2009
Abstract After administration, drugs go through a
complex, dynamic process of absorption, distribution,
metabolism and excretion. The resulting time-dependent
concentration, termed pharmacokinetics (PK), is critical to
the pharmacological response from patients. An in vitro
system that can test the dynamics of drug effects in a more
systematic way would save time and costs in drug devel-
opment. Integration of microfabrication and cell culture
techniques has resulted in ‘cells-on-a-chip’ technology,
which is showing promise for high-throughput drug
screening in physiologically relevant manner. In this
review, we summarize current research efforts which ulti-
mately lead to in vitro systems for testing drug’s effect in
PK-based manner. In particular, we highlight the contri-
bution of microscale systems towards this goal. We envi-
sion that the ‘cells-on-a-chip’ technology will serve as a
valuable link between in vitro and in vivo studies, reducing
the demand for animal studies, and making clinical trials
more effective.
Keywords Microfluidics � Pharmacokinetics �ADME
Introduction
Drug development is an expensive, time consuming pro-
cess. Despite continuing effort to improve the productivity
of the drug development process, only one out of ten drug
candidates entering the clinical trial reaches the final
approval stage [1, 2]. The number of new molecular enti-
ties (NMEs) that are approved by US FDA has been
declining over the last decade, from 53 in 1996 to 21 in
2008 [3]. The main reasons for such a low success rate are
primarily unforeseen lack of efficacy (accounting for about
30% of failures) and toxicity (accounting for another 30%)
[1]. When the lack of efficacy or unexpectedly high toxi-
city is revealed in later stages of clinical trials, it increases
the total cost, since the cost of clinical testing is consid-
erably higher than the cost of preclinical testing, increasing
with successive phases [4]. Improving the ability of in vitro
assay systems to predict the efficacy and toxicity of drug
candidates earlier in the drug discovery process will greatly
enhance productivity. Consequently, there has been a sig-
nificant amount of effort geared towards improvement of in
vitro systems for assessing drug efficacy and toxicity [5, 6].
Typically, cell-based assessment of drug toxicity and
efficacy is evaluated in multi-well microtiter plate systems.
Cells are cultured in multi-well plates, incubated with
drugs for a specific amount of time, and the viability of the
cells are measured. A large number of human cell lines
intended for specific target organ toxicity are available [7].
Although this methodology has been well-established and
has been used for several decades as a standard method for
evaluation of drugs, many drugs move onto successive
stages only to fail, due to unforeseen toxicity or lack of
efficacy. The predictability of multi-well, cell-based drug
assay is not satisfactory because this system differs from
the human body in many aspects. First, in a cell-based
J. H. Sung � M. L. Shuler (&)
Department of Chemical and Biomolecular Engineering,
Cornell University, Ithaca, NY, USA
e-mail: [email protected]
J. H. Sung
e-mail: [email protected]
M. L. Shuler
Department of Biomedical Engineering,
Cornell University, Ithaca, USA
123
Bioprocess Biosyst Eng (2010) 33:5–19
DOI 10.1007/s00449-009-0369-y
assay, only a single cell type is cultured at a time, which
means that multi-organ interaction is absent. On the other
hand, in the human body, drugs go through a complex,
dynamic process of absorption, distribution, metabolism
and excretion (collectively known as ADME) [8]. Conse-
quently, the effective drug concentration at a target site is
often different from what is expected from the adminis-
tered dosage. Pharmacokinetics (PK), which describes the
concentration profile of a drug after administration, has a
profound effect on the observed efficacy and toxicity of a
drug [2], but current in vitro cell-based assays lack the
ability to reproduce the PK of drugs. The second issue is
that the cell culture in multi-well format is different from
cells in their native environment. Cells are typically cul-
tured in 2-D monolayer, immersed in an excess amount of
liquid medium. In their tissue environment, cells are sur-
rounded by extracellular matrix (ECM) and other sup-
porting cells, with nutrients supplied by diffusion from
blood vessels. The microenvironment surrounding the cells
in 2-D culture is drastically different from that of native
tissue in terms of nutrient transport, shear stress, chemical
and mechanical signaling. As a result, cell behavior is often
not ‘authentic’, which means that cells behave differently
from the manner they would behave in their native envi-
ronment. This could be the reason behind a drug candidate
seeming to have a promising efficacy/toxicity profile in the
preclinical stage, but showing a different profile in a human
clinical testing. Thirdly, often there are individual varia-
tions in patients’ responses to a drug, resulting in unac-
ceptably high toxicity or lack of efficacy in a group of
patents while the same dosage works well for others [5].
Such individual variations are often due to genotypic or
phenotypic variations, which cause alterations in the PK of
a drug. For example, the rate at which a drug is metabo-
lized can vary depending on the enzyme level in a patient,
resulting in a different PK and toxicity profile [9]. Current
in vitro assay systems are not suitable for tackling these
issues, since these systems cannot reproduce the PK of a
drug. A mathematical approach called PK–pharmacody-
namic (PK–PD) modeling can help determine the effect of
such genotypic and phenotypic differences on the observed
pharmacological effect of a drug [2]. However, it has
limitations in that accurate parameter estimation is neces-
sary for model development, and unknown mechanisms of
action cannot be predicted with a model.
Such shortcomings of conventional cell-based assay
systems demand a more realistic in vitro system, which can
test the effect of a drug in a more systematic, that is,
PK-based way. Recently, integration of conventional cell
culture techniques and microfabrication technology
resulted in a new area called ‘cells-on-a-chip’ technology
[10, 11]. The area of cells-on-a-chip has grown rapidly
during the past few years, with the expectation that it can
offer advantages that cannot be provided by conventional
cell culture techniques. Among the many advantages of
microscale systems, the ability to fabricate complex
microscale structures and then to culture multiple cell types
inside them allows researchers to create novel microscale
systems mimicking multi-organ interactions. Such a device
allows one to observe the systematic, whole-body response
to drugs rather than the response of a single cell population.
Furthermore, the recent advance in microfluidic systems
has made it possible to precisely reproduce multi-organ
interaction with blood circulation, which has been termed
as a ‘body-on-a-chip’ approach [12]. Together with three-
dimensional cell cultures, such recent advances in micro-
scale systems can contribute to developing a more realistic,
in vitro system that can reproduce the whole-body response
to drugs (Fig. 1).
This paper reviews current research efforts for devel-
opment of microscale cell culture systems to overcome the
aforementioned limitations of conventional cell-based
assay systems. First, we summarize the recent development
in the area of cells-on-a-chip with a focus on high-
throughput implementation. Here we describe research
efforts to combine microfabrication and conventional cell
culture techniques, to create novel systems with advantages
that cannot be offered by conventional cell cultures. In the
Fig. 1 A diagram showing the concept of in vitro research efforts
towards achieving a more realistic, whole-body response to drugs.
Microfluidics and 3-D cell culture technology can be combined with
conventional cell culture techniques to enable reproduction of a
whole-body response. Microfluidics can contribute to mimicking the
dynamics of drug exposure to human body, and 3-D cell cultures can
help cells behave in a more authentic manner. Furthermore, the
typical length scale in microfluidics helps to reproduce the native
cellular microenvironment better, as it becomes easier to control
parameters such as a flow rate, shear stress, and liquid-to-cell ratio
6 Bioprocess Biosyst Eng (2010) 33:5–19
123
following section, we summarize microscale systems for
metabolism-dependent toxicity assay, and microscale sys-
tems based on the concept of physiologically based phar-
macokinetic (PBPK) modeling. Among many multi-organ
interactions, liver metabolism is the most critical element
for the realization of PK-based drug assay, and microscale
systems for reproducing the liver metabolism in vitro will
be reviewed. We will conclude with a summary of issues
that need to be overcome to achieve the PK-based drug
toxicity study, such as stable operation of microfluidic
devices and integration of real-time detection.
Advantages of combining microfabrication and cell
cultures
One obvious advantage of miniaturization is the possibility
of high-throughput implementation, and there have been
numerous examples of microscale systems for high-
throughput drug screening [13]. High-throughput imple-
mentation is necessary to screen a large number of drug
candidates in a given time, but also because it is often
required to perform a large number of tests per drug, to
obtain statistically meaningful data for various doses and
under various conditions. In case of microbial cells, we
have seen a significant amount of progress in high-
throughput and large-scale implementation in bioreactor
cultures with medium perfusion [14, 15]. With mammalian
cells, a high-throughput implementation has been achieved
at a high level in conventional multi-well assay systems,
but the area of microbioreactor systems for high-through-
put screening has begun to gain wide interest only recently.
There are unique advantages that can be offered only by
combining microfabrication/microfluidics and cell cultures.
Tailoring the architecture of microenvironment for cells is
possible, owing to the precise control over microscale
structures. The ability to easily manipulate the geometry
and the flow pattern gives researchers a control over the
transport of growth factors, reagents and oxygen inside the
system, as well as hydrodynamic shear stress upon cells
[16]. As will be reviewed further in this paper, such factors
can have an enormous influence on cell behavior and help
researchers create a more in vivo-like environment for
cells. It would be of a tremendous advantage if such novel
systems can be operated in high-throughput manner to
obtain large data set more easily.
Wang et al. reported a high-density microfluidic arrays
for cell cytotoxic assays (Fig. 2a) [17]. The device con-
sisted of fluidic channels for cell seeding, and another array
of fluidic channels for toxin exposure in an orthogonal
direction. 24 by 24 (=576) arrays of cell culture chambers
were fabricated, which were used to screen three cell types
against five toxins. Fluorescence live/dead staining was
used to evaluate the cell viability, and the result obtained
from this microfluidic array was comparable to a similar
experiment in a microtiter plate assay. This is one of the
early examples where relatively high-throughput toxicity
screening of multiple drugs against multiple cell types was
achieved on a single chip. Wu et al. developed a device
with a similar concept of orthogonal channels for cell
seeding and toxin application, but with 3-D cell culture in
agarose [18]. The device consisted of 15 microbioreactors,
which were perfused with medium using the same number
of pneumatic micropumps. Due to the use of integrated,
pneumatic pump, a significant amount of automation was
achieved with the device. An 8 by 5 microchamber array
with pressure-driven medium perfusion was developed,
and used to test seven toxins at a time [19]. A unique
terrace structure surrounding the microchamber prevented
air bubbles from invading the chambers and protected cells
inside. Microfluidic gene expression array with 16 by 16
(=256) 10-nl chambers was developed with microvalves for
row-cell seeding and column-stimulation (Fig. 2b) [20].
Using this device together with cells transfected with
fluorescent reporter genes, expression dynamics of eight
inflammatory genes were monitored in response to eight
stimuli.
As demonstrated by these examples, there has been a
significant amount of effort in the cells-on-a-chip area
during the past few years directed to automation of oper-
ation, such as easier cell loading and toxin application,
medium perfusion and evaluation of cell viability [21–24].
However, to compete with conventional microtiter plate
systems equipped with robotic liquid handling for high-
throughput experimentation, such microfluidic systems
need to demonstrate advantages other than high-throughput
processing to justify all the ‘hassle’ of using a microfluidic
system. Perhaps one of the most important and useful
advantages of a microfluidic cell culture system is that it
enables formation of well-controlled concentration gradi-
ent of chemicals inside the system. The laminar flow inside
the device restricts the transport process to be dominantly
diffusive. A concentration gradient can be formed by
simply having two parallel channels, one channel working
as a source and the other as a sink. Several other types of
more sophisticated devices have also been developed for
the formation of a more complex concentration gradient
[25]. Formation of concentration gradient offers an
advantage over multi-well plate system, since a multi-
dosage study can be performed in one shot, as opposed to
testing multiple doses individually in separate wells. In
addition, many drugs are administered in combination to
achieve a synergistic effect, but finding the optimal com-
bination ratio can be challenging. The concentration gra-
dient maker can be useful for testing various combination
ratios in a more efficient manner, rather than mixing and
Bioprocess Biosyst Eng (2010) 33:5–19 7
123
testing them individually. Recently a microfluidic gradient
maker was utilized to test the effect of various concentra-
tions of anesthetics, bupivacaine and lidocaine on myo-
blasts [26]. In this device, a gradient maker was integrated
with a cell culture chamber, so that cells are exposed to a
concentration gradient of toxins (Fig. 2c). By performing
cell viability assay after the exposure and correlating the
width across the chamber with the concentration predicted
by mathematical simulation, a quantitative dose–response
relationship was achieved. This type of study illustrates the
advantage of utilizing a microfluidic device to increase the
efficiency of drug testing. A similar concept was demon-
strated in a study by Hung, where 10 by 10 cell culture
chamber arrays were fabricated and a gradient maker was
attached to the columns [27, 28]. By introducing a flow in
horizontal direction in addition to the concentration gra-
dient in vertical direction, a series of combination ratios
were easily generated across columns and rows. The
potential of a microfluidic system for drug screening pur-
pose was demonstrated by testing the effect of a serum
concentration on cells. While these devices created a
continuous concentration gradient of chemicals, a micro-
fluidic device which generates a linear dilution of chemi-
cals on chip has been developed [29]. Nine dilutions were
generated by mixing two input fluid streams and control-
ling the geometry of fluidic channel network. Such a
controlled-microfluidic delivery of reagents illustrates the
potential of cells-on-a-chip technology to achieve novel
experimentation not possible with conventional multi-well
based platforms.
Another advantage of integrating microfabrication
technology with cell culture techniques is that the micro-
environment can be easily tailored by controlling param-
eters such as the geometry of the device structure and the
flow rate. Devices with various chamber volumes ranging
from nanoliters to several milliliters have been demon-
strated [30–32]. Such a wide spectrum of culture volumes
can have a profound effect on cell behavior by dramatically
altering the transport phenomena. To characterize cell
culture microenvironment, the concept of ‘effective culture
volume’ has been introduced, but the understanding of
microscale cell culture environment is still at its infancy
[16]. Another interesting and important parameter in a
microfluidic cell culture system is the flow rate. The effect
of flow rate on the growth kinetics of human mammalian
cells has been studied [33]. Fibroblasts were grown for up
to 2 weeks in a microbioreactor in various flow conditions,
from static (no flow) condition to high flow rate. The cells
Fig. 2 (a) High-density microfluidic array for cytotoxic assays.
24 9 24 (576) chambers are used to screen three cell types against
five toxins. The figure reproduced from [17] with permission of RSC
Publishing (b) 16 9 16 Microfluidic living cell array (shown in
yellow, layer 1). Fluid is controlled by two sets of PDMS barriers,
shown in green and purple. Cells are drawn from left to right, and
stimuli are drawn from top to bottom. Reproduced from [20] with
permission of RSC Publishing (c) A mathematical simulation of
gradient generation, reproduced from [26] with permission from
Elsevier (d) A microfluidic device for 3-D hydrogel-cell culture.
Human hepato-cellular carcinoma cells (HepG2) were encapsulated in
collagen composite, OPLA, and Puramatrix scaffolds inside the
device made of PDMS. Cellular activity (albumin production) and
cytotoxicity assay (Triton X-100) were performed using the device.
The image taken from [45] with permission from Springer
8 Bioprocess Biosyst Eng (2010) 33:5–19
123
did not grow well in either the no flow or high flow rate
(0.3 ml/h) condition, but grew best in low flow rate
(\0.2 ml/h) conditions. In a study with a similar purpose, a
microfluidic device which can generate a logarithmic range
of flow rates was developed and used to study the effect of
flow rate on the proliferation of embryonic stem cells [34].
In this study, cells exposed to a low flow rate (1 9 10-3 ll/
min, residence time of 319 min) grew considerably slower
than the cells exposed to a high flow rate (1.1 ll/min,
residence time of 0.29 min). Theses preliminary studies
have demonstrated that microfluidic environment in an
important factor in determining the behavior of culture
cells. It is also pertinent to toxicity assays, since it implies
that cell response to toxins can vary widely depending on
the microenvironment they are exposed to. A more para-
metric study on underlying mechanism of how cells
interact with surrounding microenvironment and how
microenvironment affects cell behavior is likely to yield
valuable information for creating more in vivo like
microscale cell culture systems.
It is believed and has been demonstrated that a 3-D cell
culture can offer a more in vivo-like environment for cells
than 2-D monolayer culture by providing a more realistic
cell–cell interaction, chemical and mechanical signals [35,
36]. Encapsulation of cells in 3-D matrix has been utilized
for various purposes, for both mammalian and microbial
cells [37]. It has been demonstrated that offering cell
adhesion, growth and organization in 3-D space can induce
authentic response of cells, leading to in vivo like differ-
entiation, proliferation, metabolic activity, and metastasis
potential of tumor cells [38–41]. It is also notable that the
response of tumor cell lines to chemotherapeutic agents can
change drastically (more than 1,000-fold) when they were
cultured in 3-D environment compared to 2-D monolayer
[42].
Although the importance of 3-D cell culture has been
well-recognized, one limitation of 3-D cell culture is an
inefficient transport into the matrix. With diffusive trans-
port being dominant in 3-D matrix, often nutrients and
oxygen are not able to reach the inside area of 3-D cell
culture matrix at sufficiently high level, especially in case
where the size of matrix is greater than a millimeter range.
Given the ability of microscale systems to precisely control
transport of molecules, it is likely that combination of 3-D
cell cultures and microfabrication techniques will yield an
in vitro system with more efficient and controlled transport.
In a recent study, hydrogel-cell matrix modules of sub-mm
size were made using a PMDS mold [43]. A PDMS
membrane with sub-mm size holes was fabricated, which
was filled with cell-hydrogel mixture in liquid. After the
gel formation the gel modules were removed from the
PDMS membrane, creating cell-hydrogel modules with
sub-mm size. The feasibility of the method was tested with
collagen, MatrigelTM
and agarose, and various cell densities
(105–108 cells/cm3) were obtained. The size of the fabri-
cated modules was determined by the thickness of the
PDMS membrane (down to 40 lm) and the size of holes
(200, 500, and 1,000 lm).
Incorporation of 3-D cell cultures into a microfluidic
device has been gaining increasing attention recently. Kim
et al. reported a 3-D microfluidic platform for a cell-based
assay (Fig. 2d) [44, 45]. A hepatoma cell line was cultured
inside a peptide hydrogel in the device, and the cytotoxicity
assay was performed using a linear concentration gradient
of Triton X-100. A microfluidic system with immobilized
hydrogel-encapsulated mammalian cells was developed
[46]. Poly(ethylene) glycol (PEG) hydrogel was photoli-
thographically polymerized in a microfluidic device and
the cytotoxicity assay was performed. Torisawa et al.
developed a 3-D cell culture array using collagen as a
matrix [47]. Human breast cancer (MCF-7) cells were
grown inside the 4 by 5 array of collagen matrix and
proliferation of cells was monitored for 5 days. In addition
to the cytotoxicity assay result, an interesting result was
observed when the proliferation kinetics of the cells was
compared with that of the same cells grown in 2-D
monolayer. The cells in 3-D matrix grew significantly
slower than the cells in monolayer, which again demon-
strates that 3-D environment causes a significant change in
the cell behavior.
Microscale systems for metabolism-dependent toxicity
study
Testing metabolism-dependent toxicity requires at least
two components, that is, activating system (metabolism)
and target cells. A simple and straightforward way to
achieve this at macroscale level is to incubate both systems
in the same cell culture well. Such an early attempt was
made in 1980 by Spielberg et al., who incubated human
lymphocytes with a drug in the presence of liver micro-
somes [48]. The metabolizing microsomes were removed
after 2 h of exposure, and cells were further incubated for
16 h before cell viability was measured. This system was
used to study the metabolism-dependent toxicity of several
antiepileptic drugs. Tabatabaei et al. developed a system
with a similar concept, using an external metabolizing
system (rabbit microsomes) and lymphocytes as target cells
[49]. The major modification made by the authors was
implementation of a membrane-impermeable fluorescent
dye and multiwell plate scanner, which gave the advantage
of high-throughput cell viability assay over the trypan blue
exclusion assay used in the previous study. In a more recent
study by Vignati et al., metabolism-dependent toxicity
was assessed by combining CYP3A4 bioactivation with
Bioprocess Biosyst Eng (2010) 33:5–19 9
123
hepatoma cell line (HepG2) [50]. Cytochrome P450 (CYP)
is the major metabolizing enzyme family in the liver, and
CYP3A4 is an isozyme that is involved in about 50% of
drugs currently on the market [51]. The activating system
was either human CYP3A4 cDNA expressed microsomes,
or HepG2 cell line transiently transfected with CYP3A4.
The HepG2 cell line was used as a target cell, since it is
widely used for evaluating hepatotoxicity and it also con-
tains coenzyme NADPH-cytochrome reductase, which is
required for CYP-mediated drug metabolism. Ten drugs in
various categories were tested, and the metabolism-induced
alteration in the cytotoxic effect was observed. Primary
human hepatocytes have also been gaining attention as an
in vitro system for assessing metabolism-dependent toxic-
ity [52]. Primary hepatocytes are known to have metabolic
activity profile similar to the liver, at least immediately
after isolation. However, the poor availability, the high
instability of enzyme activity (the enzyme level drops
drastically with increasing culture time) has prevented a
wider application of primary hepatocytes. All of these
approaches can mimic some aspect of metabolism-depen-
dent toxicity, but macroscale devices have limitations that
make high-throughput implementation inherently difficult,
and reproduction of the liver function is incomplete.
During the last few years, there have been several
attempts to develop an in vitro microscale system for
assessing metabolism-dependent toxicity. Microscale
systems offer advantages of easier high-throughput
implementation, and physiologically realistic environment
for more complete liver function, as will be discussed in
more detail later. One prominent example was reported by
Lee et al., who developed a 15 9 35 array of sol–gel
encapsulated P450 enzymes (Fig. 3a, denoted by the
authors as MetaChip). A prodrug was added onto each spot,
which would generate the active drug, and cytotoxicity was
assessed by overlapping with a layer of target cells (breast
cancer cell line MCF-7). Two main cytochrome P450
isozymes CYP 3A4 and 2B6 were prepared, and three
prodrugs (cyclophophamide, Tegafur, and acetaminophen)
were tested. All three drugs showed toxicity profiles similar
to a solution control (CYP enzymes in solution rather than
sol-gel droplets), which verified that the nanoliter scale sol-
gel arrays could produce comparable toxicity assay results,
but in a more high-throughput manner. The system was
further improved with the development of a 20 by 54 array
of hydrogel-encapsulated cells (called DataChip by
authors), which can be superimposed on the MetaChip with
a CYP enzyme array [53]. Using this system, three CYP
isozymes or mixtures of the three against nine compounds
were tested. The IC50 values obtained from this system
were also comparable to the results from a conventional
96-well plate assay.
As mentioned earlier, microfluidics provides an ideal
platform for fabricating and connecting several components
Fig. 3 Microscale systems for
metabolism-dependent toxicity
assay. a CYP enzymes are
spotted in an array (MetaChip),
and target drugs are incubated
with the enzymes, which
generates metabolites. DataChip
is provided by spotting an array
of target cells. By
superimposing the two and
incubating, the effect of the
target drugs and their
metabolites can be tested. The
artwork was drawn as described
in [53]. b A three-layer
microfluidic device consists of
cell culture chambers, human
liver microsomes (HLM), and
fluidic channels for medium
perfusion and metabolites
detection, as described in [54].
Via diffusion, the drugs and
their metabolites are transported
to the cell culture chamber.
c The concept of ‘wells-within-
a-well’, which allows
interaction of multiple cell types
through overlying cell culture
medium [59]. The artwork was
drawn as described
10 Bioprocess Biosyst Eng (2010) 33:5–19
123
in a single device. Recently, a microfluidic device for testing
metabolism-dependent drug toxicity was developed. This
microfluidic integrates a sol-gel human liver microsomes
(HLM) bioreactor with a cell culture chamber array [54].
This device consists of three layers (PDMS-quartz-PDMS),
quartz layer in the middle with microwells for HLM and
bottom PDMS layer for cell culture chambers. A drug con-
taining solution is introduced continuously into the top
PDMS layer and diffuses into HLM in the middle layer, and a
drug together with metabolites generated from the HLM
diffuse further down to the cell culture chamber (Fig. 3b).
Acetaminophen (AP) and its UDP-glucuronosyltransferase
(UGT) metabolism were tested using HepG2 as a target cell
line for hepatotoxicity. AP undergoes detoxification by
competing phase II conjugation reactions, glucuronidation
and sulfation, which convert AP to nontoxic conjugates for
elimination in bile or urine [55]. As expected, upon metab-
olism of AP to nontoxic glucuronidation metabolite (APG),
its cytotoxic effect was observed to be reduced. When
overdosed AP saturates the UGT detoxification pathway, it is
metabolized by CYP enzymes to a toxic metabolite, quinine
imine. When authors blocked the UGT detoxification path-
way by omitting uridine 50-diphosphate glucuronic acid
(UDPGA, coenzyme for UGT detoxification reaction) from
the reaction mixture, AP was metabolized by CYP and the
cytotoxicity of AP was increased. Furthermore, a metabo-
lism-based drug–drug interaction was tested. Addition of
UGT inhibitor such as phenotin would result in increased
drug toxicity, which was again observed experimentally.
Another notable feature of this device is that the microfluidic
device was integrated with a UV-absorbance detection sys-
tem previously developed by the authors, which enabled
simultaneous detection of the drug and its metabolite con-
centrations [56].
Achievement of multi-organ interaction in vitro inevi-
tably requires seeding multiple cell types in a device with a
precise control. A novel method of direct 3-D cell writing
for in vitro PK model has been developed [57]. A 3-D cell
culture in alginate was created by the syringe-based, layer
by layer bioprinting method, which is to be enclosed by a
housing made from a soft-lithography micropatterning
technique. The proliferation and urea synthesis rate of a
hepatoma cell line (HepG2) encapsulated in alginate matrix
was monitored. This method has a direct implication for in
vitro PK-based drug testing, since it will allow free-form
fabrication of multiple cell types onto a device, which can
be used to reproduce multi-organ interaction. Nevertheless,
direct-writing of only one cell type was demonstrated in
this study, and a further study of integrating the cell matrix
with the microfluidic device is required to achieve this
goal.
An in vitro platform with multiple cell types has been
developed by having small, separate inner-wells inside a
larger well [58, 59]. Termed as ‘‘wells-within-a-well’’
concept by the authors, multiple cell types are cultured
separately in small wells (Fig. 3c). This provides a simple,
rudimentary method to reproduce multi-organ interaction,
without different cell types physically contacting one
another. Cell culture medium is shared by flooding the
inner wells and filling the large well. A chemotherapeutic
agent tamoxifen was tested against six cell types, liver
(hepatocytes), kidney (kidney cortical cells), lung (small
airway epithelial cells), central nervous system (astro-
cytes), blood vessels (aortic endothelial cells), and breast
cancer (MCF-7). Using this device, EC50, EC90, EC99
(concentration to cause 50, 90, and 99% decrease in sur-
vival, respectively) values were obtained for the six cell
types. Unfortunately, multi-organ interaction was not
characterized by the authors, nor any comparison with
separate cell culture tests was made. However, it is plau-
sible that such a system will be able to mimic at least some
aspect of multi-organ interaction.
In an effort to combine a conventional perfusion
bioreactor with a microscale cell culture device, a hepa-
tocyte-bioreactor coupled was developed to assess hepato-
activated transformation of substrates [60]. In this device,
primary pig hepatocytes were cultured in a recirculating
50 ml perfusion bioreactor, which was connected to
a commercially available cell culture chamber called
microphysiometer at a flow rate of 150 ll/min. Inside the
microphysiometer the target cells, ZR 751, a breast cancer
cell line was cultured at 1.5 9 105 cells/chamber. As a
model drug, cyclophophamide (CYCL) was fed into the
bioreactor with liver cells, and after seven hours the via-
bility of the target cells were measured. The viability of ZR
751 cells dropped only in the presence of the prodrug
(CYCL) and the bioreactor activation.
All of the in vitro systems described so far attempt to go
beyond testing the direct effect of a drug, and reproduce
metabolism-dependent actions of the drug. One potentially
important aspect which these systems lack is the ability to
mimic the dynamics of multi-organ interaction; that is,
cells are not simply exposed to the drug or its metabolites
in a bolus dose, but the circulation of blood and transport
inside tissue environment alter the dynamics of drug
exposure.
Microscale cell culture analog (lCCA) based
on a PBPK model
Pharmacokinetics refers to the time-dependent concentra-
tions of a substance in a living system [61]. It examines the
ADME processes. A PK model is a mathematical model
which attempts to predict the concentration profile of a
drug in the blood or at the target site from a given dose.
Bioprocess Biosyst Eng (2010) 33:5–19 11
123
PK models can be categorized into several subtypes,
depending on their complexity of structure [62]. The most
comprehensive model, a physiologically based PK
(PBPK) model, is based on physiological consideration.
They comprise of several compartments, each represent-
ing tissue and organs. These compartments are connected
to one another with hypothetical blood flow, in the same
organization as the blood circulation. The volume of each
compartment is same as actual physical volume of a
corresponding organ. Physiological blood flow rates into
and out of each organ are given as the flow rates into and
out of each compartment, respectively. A set of simple
mass-balance equations accounting for flux in, flux out,
absorption, and reactions is constructed for each com-
partment, and the ordinary differential equations (ODEs)
are solved using numerical methods. A large number of
commercial mathematical software packages are also
available [63]. The output result of PBPK model is the
time-dependent concentrations of a drug and its metabo-
lites in each compartment, which can be directly com-
pared with experimental results.
The microscale cell culture analog (lCCA), also known
as a ‘body-on-a-chip’ or an ‘animal-on-a-chip’, was con-
ceived as a physical realization of a PBPK model, as an in
vitro system to emulate body’s dynamic response to drugs
[12]. Just like a PBPK model, compartments representing
different organs are connected with fluidic channels mim-
icking blood circulation, and in each chamber the corre-
sponding cell type is cultured. Before the advance of
microfabrication technology, the prototype cell culture
analog device was constructed with two milk dilution
bottles and a spinner flask, connected with Teflon tubing
and a peristaltic pump [64]. Lung and liver-related cell
lines were cultured in the two bottles separately, and
toxicity of naphthalene and its metabolites generated from
the liver cell line was observed. Since the first develop-
ment, several variations of the device were developed,
along with a mathematical PBPK model that described the
system [65–68].
Following the integration of microfabrication technol-
ogy with cell culture techniques, a microscale version of
cell culture analog device was developed (lCCA) [69].
Incorporation of microfabrication technology offers several
advantages; (1) fluidic residence times, which determine
the dynamics of drug exposure to each organ, can be easily
controlled to mimic physiological values. (2) fluidic shear
stress can be controlled to be in a physiological range for
specific cell types (3) microscale devices allow incorpo-
ration of authentic tissue samples, for example tumor
biopsy samples. As a proof-of-concept study, naphthalene
toxicity was assessed using the lCCA. It was demonstrated
that naphthaquinone, rather than naphthalene epoxide,
was the reactive metabolite generated from the liver
compartment causing glutathione depletion and loss of
viability in the lung compartment [70].
Since the initial development, several improvements
have been made. The device assembly and operation have
been optimized for long-term experimentation (several
days, rather than several hours), and multi-drug resistance
(MDR) and combination chemotherapy have been tested
using the device [71]. In this four-chamber lCCA, cells
representing the liver (HepG2/C3A), bone marrow (MEG-
01), uterine cancer (MES-SA), and MDR variant of uterine
cancer (MES-SA/DX-5) were cultured in the device
(Fig. 4a and (c)), and treated with various combinations of
drugs for either 24-h acute toxicity or 72-h proliferation. In
both studies, it was demonstrated that combining a che-
motherapeutic, doxorubicin, with modulators (cyclosporine
and nicardipine) resulted in greater efficacy than doxoru-
bicin or modulators alone. Furthermore, the combination
was shown to be particularly effective against MDR variant
cell line (MES-SA/DX-5), which was not observed in
96-well plate assays. This cell-specific synergy is believed
to be due to the pharmacokinetic interaction between
compartments. PBPK models for human and the lCCA
device were constructed with the predicted pharmacokinetic
profiles of a drug and its metabolites.
It is believed that 3-D hydrogel-cell cultures can elicit
more authentic behavior from culture cells [36]. The
lCCA device has been modified to accommodate 3-D
hydrogel-cell construct, and the metabolism-dependent
toxicity of Tegafur–uracil combination has been observed
[72]. In this three-chamber device, cells representing the
liver (HepG2/C3A), colon cancer (HCT-116), and bone
marrow (Kasumi-1) were cultured (Fig. 4b and d). Unlike
previous lCCA studies where cells were cultured in
monolayer, cells were encapsulated inside hydrogel
(alginate or MatrigelTM
). Tegafur, a drug widely used to
treat colon cancer, is a prodrug which needs activation in
the liver to become an active (toxic) metabolite 5-fluo-
rouracil (5-FU). Tegafur showed a cytotoxic effect in a
lCCA due to the metabolism by the liver cells, whereas it
had a negligible effect in a 96-well plate assay with colon
cancer cells only. The metabolism-dependent toxicity was
further verified by treating cells with Tegafur in a lCCA
with the liver chamber empty, in which case the cytotoxic
effect was significantly reduced. Uracil acts as an inhib-
itor of the liver enzyme dihydropyrimidine dehydrogenase
(DPD), which is responsible for the catabolism of 5-FU,
the active metabolite of Tegafur. Uracil is often added to
Tegafur as a modulator, since inhibition of 5-FU metab-
olism results in increased efficacy of the drug [73]. In a
lCCA, addition of uracil also enhanced the cytotoxic
effect of Tegafur on tumor cells. These results demon-
strate the strength of lCCA systems in testing the com-
bination of drugs in pharmacokinetic-based way, which
12 Bioprocess Biosyst Eng (2010) 33:5–19
123
would have been possible only in animal or human
studies.
Microscale systems for reproducing biotransformation
in vitro
Perhaps the most important prerequisite for the realization
of in vitro PK-based drug toxicity study is to reproduce the
liver metabolism in vitro. Biotransformation, especially in
the liver, has a significant effect on the pharmacokinetic
profile and the pharmacological effect of a drug [74].
Metabolism in the liver is responsible for not only detox-
ification, but also activation of a drug and drug–drug
interactions. Drug biotransformation is divided into two
types, phase I (hydrolysis, oxidation and reduction), and
phase II (conjugation) [75]. The cytochrome P450 (CYP)
enzymes are the major enzymes in the phase I reactions,
and are divided into several isoforms. Among many known
isoforms, the majority of metabolic reactions are carried
out by CYP 1A2, 2C9, 2D6, and 3A4 [74]. Drug–drug
interactions occur when one drug either induces or inhibits
the activity of an enzyme that is responsible for the
metabolism or absorption of another drug. Alternatively,
drugs can interact due to their pharmacodynamic proper-
ties, for example compete for the same receptor or
enzymes.
Prediction of drug metabolism and potential drug–drug
interactions is critical for successful drug development, and
there have been enormous amount of efforts directed
toward reproducing the liver metabolism in vitro [75].
Currently, there is a whole range of in vitro systems
available to researchers, from cDNA-expression systems to
perfused liver tissues. Unfortunately, each system has pros
and cons, and yet there is no single system that can
reproduce the liver function at a satisfactory level.
Advantages and disadvantages of various in vitro systems
are well documented in several review articles [75–77].
Fig. 4 Examples of microscale cell culture analog (lCCA) devices.
a A four-chamber lCCA for testing MDR cancer. Cell lines
representing the liver (HepG2/C3A), MDR-variant uterine cancer
(MES-SA/DX-5), normal uterine cancer (MES-SA), and bone marrow
(MEG-01) are cultured in chamber 1, 2, 3, and 4, respectively. The
image was reproduced from [71] with permission. Copyright 2009,
Wiley Periodicals, Inc. b A lCCA for testing colon cancer drugs with
three chambers. Cell lines cultured are 1: HCT-116 (colon cancer), 2:
HepG2/C3A (liver), and 3: Kasumi-1 (marrow) [72]c. A PBPK model
corresponding to the four-chamber lCCA for MDR cancer. d A
PBPK model corresponding to the three-chamber lCCA for colon
cancer. Note that the chambers are interconnected differently in the
two cases, to mimic the physiological blood circulation. The
chambers in a lCCA and the corresponding compartments in a
PBPK model are marked with numbers. The medium reservoir, which
corresponds to the plasma/other tissues, is not shown in the figure
Bioprocess Biosyst Eng (2010) 33:5–19 13
123
Brief descriptions, pros and cons of conventional in vitro
systems are summarized in Table 1.
Microscale systems offer unique advantages in control-
ling the transport of nutrients, mechanical stimulation,
surface interaction, and organizing cells in a defined
geometry. These advantages have been exploited by
researchers to develop an in vitro system with an improved
liver function. In a typical length scale of a microfluidic
system, a laminar flow is generated and diffusion plays a
dominating role in the transport of nutrient and oxygen.
With a combination of a mathematical modeling, concen-
tration of oxygen and nutrients can be predicted and con-
trolled [78, 79]. A microfluidic flat-bed bioreactor was
created, in which rat hepatocytes and non-parenchymal
cells were co-cultured (Fig. 5a) [80]. By controlling the
flow rate, it was possible to create a gradient of oxygen
concentration across the bioreactor, so that cells are
exposed to a heterogeneous oxygen environment. The
oxygen gradient in the bioreactor mimics the physiological
condition of the actual liver, where the localized function
of the liver is thought be controlled by the local oxygen
concentration [81]. For example, a gradient of oxygen
concentration is formed from the inlet at the periportal side
(high oxygen region), where glucose, albumin, urea bio-
synthesis and glutathione conjugation take places, to the
perivenous outlet (low oxygen region), where glucuroni-
dation and oxidation by CYP enzymes mainly occur. This
has an implication that a low oxygen tension might be
beneficial to achieve high CYP enzyme activity of cultured
hepatocytes. Indeed, the authors found that a differential
CYP activity was measured across the length of the bio-
reactor, and perfusion of acetaminophen, which is known
to have liver toxicity, caused the most damage to the cells
in the area of low oxygen concentration. This example
demonstrates the power of microfluidic system in control-
ling the transport process for more authentic cell function.
PDMS (Polydimethylsiloxane) has been the most com-
monly used material for microfluidic devices, owing its
popularity to several advantages it can offer, such as bio-
compatibility, gas permeability, and optical transparency.
One of the earliest attempts to culture liver-related cells in
a PDMS microfluidic device was demonstrated by a series
of papers by Leclerc et al., who developed a microfluidic
device for culturing HepG2 cells in 3-D by stacking two
layers of PDMS. Cells were maintained in the device for
several days, and cells were observed to attach and grow
successfully on the surface of PDMS. The measurement of
glucose consumption and albumin production confirmed
that the metabolic activity of the cells was maintained
during the culture period [82]. In a subsequent paper, the
authors attempted a high-density cell culture by stacking
10 layers of PDMS, and achieved the cell density of up to
4 9 107 cells/cm2, which is close to the density in a
macroscale bioreactor [83]. In addition to the HepG2 cell
line, authors also demonstrated culturing fetal human
hepatocytes (FHH) in the same device, and successfully
Table 1 In vitro systems for reproducing liver metabolism
System Description Advantages Disadvantages
cDNA-expression system
(supersomes or
baculosomes)
Human CYP and UGT can be
transfected into insect cells
and microsomes are
separated
Useful for characterizing
single enzyme reaction in
high-throughput manner
May lack in vivo relevance
since whole liver enzyme
repertoire is lacking
Human liver microsomes
(HLM)
Separated from liver tissues
by centrifugation, and
commercially available
Whole array of CYP and
UGT enzymes present
Cytosolic and phase II
enzymes are lacking
Relatively simple to use high-
throughput implementation
Can be recovered from frozen
tissue samples
Liver cell line Immortalized cell lines
originated from
hepatocellular carcinoma
Unlimited availability and
easy handling
Only a subset of in vivo liver
functions is retained
Hepatocytes Primary hepatocytes isolated
from the liver
Good reproduction of liver
metabolic profile
Limited culture time and
rapid loss of enzyme
activity (4 weeks in
maximum)
Relatively high-throughput No 3-D structure
Liver slices or perfused
liver
Best representation of in vivo
liver function
3-D cell–cell and cell–matrix
interactions are intact
Limited availability of human
models
Function is preserved for
only a few hours
14 Bioprocess Biosyst Eng (2010) 33:5–19
123
demonstrated that the perfusion culture in a microfluidic
device enhanced the metabolic function of cells in both
FHH and HepG2 cells [84].
One way to improve the liver-specific function of
hepatocytes is to co-culture them with various supporting
cell types, such as epithelial, Kupffer, fibroblasts [85]. In
conventional methods, those cells would be cultured in a
random mixture, with the only available control on the
ratio of mixing the cell populations. With the advent of
micropatterning techniques, it has become possible to
organize multiple cell types in a defined geometry [86].
One of the techniques has been used to create a micro-
patterned co-culture of rat hepatocytes and fibroblasts [87].
A PDMS stencil with through-holes was used to selectively
seed hepatocytes in a defined area of a polystyrene well,
where rest of the area was filled with fibroblasts. The
maximum liver function was observed at 500 lm size of
hepatocytes ‘islands’, with 1,200 lm spacing. Gene
expression profiles, metabolizing enzyme activity, secre-
tion of liver-specific proteins and response to hepatotoxins
were measured and the liver-specific functions were found
to be maintained for several weeks. The simplified proce-
dure of the cell patterning is shown in Fig. 5b.
Mechanical stimulation, a shear stress in particular, is
also thought play an important role in modulation of the
tissue-specific function [88, 89]. In addition, organization
of cells in 3-D, rather than 2-D monolayer culture, is
thought to provide essential chemical/mechanical stimula-
tions necessary for authentic cell behavior [36]. Microflu-
idic systems are ideal for providing both aspects with a
precise control. A microfluidic bioreactor with perfused
3-D liver cell culture was developed (Fig. 5c) [90]. Rat
primary hepatocytes were cultured in 3-dimensional con-
figuration made by through-holes in a silicon wafer, while
providing hydrodynamic shear at a physiological level
(\2 dyne/cm2). After up to 2 weeks of culture, cells rear-
ranged to form tissue-like structures. Preaggregation of
cells into spheroid-like form prior to seeding into the
device further improved the liver-specific cell behavior,
such as albumin secretion and urea synthesis [91]. This
concept was further developed into a scalable, multi-unit
structure and it was demonstrated from gene expression
profiles and biochemical activity measurements that the rat
primary hepatocytes cultured in this system was closer to
the human liver than 2-D collagen sandwich culture or 3-D
MatrigelTM
culture [92].
Recently, Vozzi et al. described a multicompartment
bioreactor (MCB), where multiple cell types are cultured in
separate chambers of a single device, connected with flu-
idic conduits (Fig. 5d) [93]. This is similar to the concept
Fig. 5 Microscale systems for
reproducing the liver function in
vitro. a A concentration
gradient of oxygen is formed
inside a flat-bed microreactor
with hepatocytes. The oxygen
concentration gradient mimics
the physiological situation of
the liver, in which different liver
functions are active depending
on the position and the
local oxygen concentration.
The microreactor is drawn
as described in [80].
b Micropatterning of
hepatocytes with supporting cell
types was shown to enhance the
liver-specific function.
A simplified procedures for the
cell patterning is shown, as
described in [87]. c Microfluidic
perfusion cell culture system,
where hepatocytes were
cultured in 3-dimensional
organization in a silicon
scaffold, as described in [90–
92]. d A simplified schematic of
a multi-compartmental
bioreactor (MCB) with
hepatocytes and human
umbilical vein endothelial cells
(HUVEC), as described in [93]
Bioprocess Biosyst Eng (2010) 33:5–19 15
123
of a lCCA, as a downscaled in vitro ‘human body’. One
thing notable in this study was that an attempt was made to
scale down the human body by using an allometric scaling
law, to conserve the ratio relationships of kinetic, meta-
bolic, volumetric parameters between cells. In this study,
the authors investigated the crosstalk between hepatocytes
and endothelia cells by culturing murine hepatocytes and
human umbilical veins endothelial cells (HUVEC) in the
device and measuring the rate of glucose consumption,
albumin and urea synthesis. An enhancement in the albu-
min and urea synthesis was observed in case when the
hepatocytes were co-cultured with HUVEC in the device,
even without a direct physical contact.
Outlook and conclusion
Cells-on-a-chip technology is a promising field, and it is
likely that in the next few years we will be seeing a
plethora of microscale devices for toxicity studies. The
concept of pharmacokinetic-based testing has been dem-
onstrated at a proof-of-concept level [68, 70–72]. In par-
ticular, combination of a mathematical modeling approach
such as a PBPK modeling and an in vitro platform such as a
lCCA can provide a valuable insight into the mechanisms
for drug toxicity [66]. A mathematical modeling has been
used to test various dosing strategies such as drug combi-
nation ratios and dosing schedule [94], and a microfluidic
system such as a lCCA can work as an in vitro platform for
verifying the mathematical simulation. However, there are
several obstacles that need to be overcome before it
becomes widely accepted outside the research community.
The first, and the most important obstacle is the lack of
realistic in vitro liver models, which has been discussed in
detail in the previous section. The second issue is the
problem of detection and analysis in microscale systems.
Miniaturization offers many advantages but also renders
detection and analysis difficult. Smaller sample size, for
example liquid medium in nanoliter scale, makes it difficult
to analyze the sample with many conventional analytical
methods. The device in microscale, especially when con-
nected with an external pump for medium perfusion, is
difficult to visually examine under a microscope in real-
time. The problem is further aggravated when multiple
microscale devices need to be analyzed for high-through-
put implementation. Consequently, there have been active
research efforts in improving or modifying current ana-
lytical techniques to adapt to the new microscale devices
[95–99]. While these systems have relied on fluorescent
signal, which is the most widely used method for non-
invasive detection, there are other approaches using elec-
trical signal [100, 101] or cellular impedance to monitor
cell viability [102], which may be advantageous for high-
throughput screening. The third challenge is the straight-
forward and stable operation of microscale systems,
especially microfluidic systems with cell cultures. The
Table 2 Practical issues in microfluidic systems and solutions
Issue Description Possible solution References
Bubble formation Microfluidic systems are susceptible to air
bubble formation, due to surface tension
in a micrometer length scale. Bubbles
can block channels, distort flow pattern,
and damage cells.
Use in-line or separate bubble traps [44, 104–107]
Place medium reservoir in elevated
position to increase pressure
Extensive rinsing with liquid with low
surface tension (ethanol or surfactant)
prior to the experiment to flush out air
completely
Bacterial contamination Bacterial contamination should be
avoided for mammalian cell culture.
Microfluidic devices require several
components from different various
materials, which may require different
sterilization methods.
Autoclaving [69, 71, 72, 104]
Washing with (acidified) ethanol
Air or oxygen plasma cleaning
Ethylene oxide sterilization
Effect of shear Shear stress from the flow can affect the
physiology and the viability of cells
Groove structure to protect cell layer from
flow
[90, 109]
Encapsulation of cells in 3-D matrix
Authenticity of cell function Cells cultured in vitro often display cell
functions different from their native
environment
Co-culture with supporting cells [87, 92]
3-D cell culture
High-throughput implementation Complex fluidic connections and flow
control required in microfluidic systems
present challenge in high-throughput
experimentation and automation
Microfluidic array [19, 27, 110]
Automated flow control
16 Bioprocess Biosyst Eng (2010) 33:5–19
123
utility of microscale systems have already been proven, but
much progress has to be made to make the systems simple,
but still versatile at the same time before they are adopted
by researchers in medicine or life sciences [21, 103]. In line
with this effort, several critical issues with microfluidic cell
culture systems have been discussed [104], and some of the
issues and their suggested solutions are summarized in
Table 2. In particular, formation of air bubbles in micro-
fluidic channels have been one of the most serious prob-
lems in microfluidic perfusion cell cultures, and several
researchers have developed microfluidic bubble traps with
different designs [105–107]. The achievements made by
many research labs in developing various microfluidic
components to enable more high-throughput, automated
multiplexing and screening will foster the development of
user-friendly microscale devices [108]. As these challenges
are overcome with novel solutions, we foresee that the
concept of PK-based drug testing will become more widely
accepted and will be able to complement the animal and
human studies, ultimately reducing the costs and time of
drug discovery process.
Acknowledgments This work was supported in part by Nanobio-
technology center (NBTC, project CM-2 (Nanotechnological
Assessment of Drug Toxicity)), NSF (National Science Foundation),
and CNF (Cornell Nanoscale Science and Technology Facility), and
by the Army Corp of Engineers (CERL) W9132T-07.
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