W3P.093
PLATE READER COMPATIBLE MEMBRANE-INTEGRATED MICROFLUIDIC
PLATFORM FOR HIGH-THROUGHPUT CELLULAR ASSAYS
E. Vereshchagina1, D. Mc Glade1, M. Glynn2, J. Ducrée1,2
School of Physical Sciences, 2Biomedical Diagnostics Institute
National Centre for Sensor Research, Dublin City University, IRELAND
1
ABSTRACT
This work reports a novel, versatile, and membraneintegrated 3-dimensional microfluidic platform (MP) for
high-throughput biodiagnostics, with an aim towards
applications in clinical cancer studies. The hybrid MP is
designed to be compatible with standard equipment used
in high throughput screening, featuring membrane
supported micro-wells and gradient generating
microstructures to enable exposure of cell samples to
various concentrations of reagents of interest, suitable to
run both fluorometric and colorimetric assays. This paper
discusses design and fabrication of the MP, and also
describes preliminary testing of colorimetric and
fluorometric assays carried out on the device.
KEYWORDS
Microfluidic platform, polycarbonate membrane,
polymer rapid prototyping, cell, high-throughput assay
INTRODUCTION
Overview of high throughput platforms
There is an increased need for high-throughput, low
cost, and automated microfluidic systems for cell culture
and analysis. Potentially, these systems allow for a more
controlled experimental manipulation of the cellular
microenvironment, better approximation of in vivo
conditions, compatibility to a wide range of cell assays
and, thus, a better understanding of the underlying
biology [1]-[3]. The overall objective is to make devices
for cell-based assays cheaper and feasible for highthroughput screening to be applied on the early stage of
drug testing [4]. Despite this evident trend, many highthroughput screening platforms are designed to support
only a single target bioassay [5], and often do not meet
design standards of commonly used lab analysis
equipment [6].
One approach to increase throughput of cellular
assays is to introduce a matrix of (optionally valved)
micro channels connecting culture wells in a 96 well
microtiter plate (TP) compatible format. A number of
examples of such systems can be found in the literature
[7]-[11]. However, most of the systems are PDMS-based
[8], [12] (PDMS has high adsorptive properties towards
cells or bio-compounds and is incompatible with a large
range of chemicals), comprise of a limited number of cell
culture chambers per device, and require sophisticated
loading - all hindering its wide-spread implementation as
a research tool. Additionally, a number of systems using
pumpless methods of liquid transport were demonstrated,
i.e. that make use of capillarity forces and absorbing
materials [13], gravity and surface tension [3] and droplet
dispending [14].
978-1-4673-5983-2/13/$31.00 ©2013 IEEE
Figure 1: Schematic of the device (A), close-up on the
wells (B), cross-sectional view of the gradient generating
layer (layer 4 on E) (C) a photograph of the
manufactured prototype (D) and the exploded view (E).
The micro-wells can be designed to fill either individual
chambers or several wells with the same reagent
composition (elongated wells); individual micro-wells
may serve (optionally) as a calibration standard, controls
or pre-storage of dry reagents.
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Despite the undisputed advantage of pumpless
sample transport, these systems require longer time per
step in assay and can fill only a limited number of wells
(up to three, as demonstrated). Also, due to the passive
nature of flow control over the delivered reagents, it may
not be suitable to a wide range of cell assays.
Membrane-integrated plate reader compatible
microfluidic platform
The 3D microfluidic platform described here is the
follow-up development of the previously demonstrated
membrane-based microsystem for convective and
diffusive perfusion of supported cell cultures [15]. The
system comprises of micro-wells interconnected via a
common microfluidic network that generates a
concentration gradient [16] (Figure 1). This channel
network is used to fill each of the biomimetric membranesupported micro-wells with culture media that may
include a chemical agent under investigation. This
multiplexed format allows several distinct cell samples to
be exposed to the generated concentrations of chemotoxic
agents being screened. Typically, TPs are loaded by
dedicated robotic systems designed for high-throughput
screenings. Yet, the availability of these expensive
machines is restricted to a rather small number of hightech lab infrastructures as they are notably expensive.
Moreover, these automated liquid handling systems
display a limited speed of loading and precision to dose
small liquid volumes [2]. Therefore the here presented
development
with
integrated
microfluidics
is
advantageous.
Figure 2: SEM image of the milled microchannels (A)
with the close up (B).
solution towards personalized medical prescriptions.
Compared to conventional TPs, that require at least 50 100 µL of sample consumed on each step of an assay, in
the presented system, reagent loading is accurately
achieved by pressure-driven flow and reagent
consumption is reduced, thereby reducing the costs per
device to run an assay. Moreover, a TP is a static system
whilst here the flow control can be used to study
important flow-based mechanisms imposed on suspended
cell culture. Monitoring the viability of the HL60 cell line
following exposure to DNA-damaging mitomycin C
(MMC) was chosen as a model system because MMC is a
common reagent used in anticancer drug treatments [17].
In this work we are aiming at the development of a
platform suitable for both colorimetric and fluorometric
biodiagnostic studies, and compatible with the format of
standard 96 well plate readers, thus proving a higher
degree of flexibility in streamlining and up-scaling preclinical tests on demand. The total sample-to-answer
times can be considerably reduced when introducing these
devices in the clinical environment. Additionally, the
system eliminates the risk of user and environment
contamination by dangerous reagents such as toxic MMC,
as well as the risk of cross-contamination between the
wells occasionally occurring in standard TPs. The MP
requires only one standard pump to fill all wells, thus the
need for a complex fluidic interfacing or multiple pumps
as in microfluidic valve arrays is eliminated [18]. Also,
such important aspects as automation of readout, e.g.
compatibility
with
conventional
high-throughput
screening equipment (multi well plate readers) and ease of
handling are addressed in this design. This approach
reduces costs and complexity of integrated assays.
Figure 4. A photograph of the assembled prototype being
tested on the 96 well micro-plate reader (Tecan Safire 2).
MATERIALS AND METHODS
Design and manufacturing of prototype
Figure 3: SEM image of the PC membrane featuring
homogeneous distribution of pore diameter.
The here described MP is suitable for studying the
impact of single drug concentration as well as
concentration ratios in drug mixtures essential in early,
off-lab diagnosis assessment in patients with the risk of
cancer and, therefore, represents a convenient engineering
The 3D multi-layer system is based on low cost
heterogeneous integration technology and thermoplastic
materials, e.g. micromachined poly (methyl methacrylate)
(PMMA) sheets with embedded thin WHATMAN®
polycarbonate (PC) membranes. The MP was designed
using AutoCAD 2012 and Solid Works 2011, and
manufactured by polymer rapid prototyping technologies:
CO2 laser ablation system for structuring 1.5 mm thick
PMMA sheets, a knife plotter for cutting 56 µm thin
pressure sensitive adhesives (PSA) subsequently used to
irreversibly seal all individual layers, and CNC precision
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Bioassay
The toxicity effect of MMC on HL60 cell was
determined using the XTT Cell Viability Kit (Cell
Signaling Technology Inc., Boston, MA, USA). The
signal from the black membranes was recorded with
fluorescein sodium salt (Sigma Aldrich, Germany). The
color- and fluorescent intensities were compared with the
same assay performed in transparent and black titer plates
(TP), respectively.
5
Fluorescence signal [a.u.]
10
TP
MP
4
10
3
10
2
10
1
10
-6
-4
10
-2
10
0
10
10
-1
Fluorescein Na-salt concentration [mol·L ]
Figure 6. Measurements of fluorescein sodium salt for
black titer plate (TP) and membrane platform (MP) at
491 nm (excitation) / 521 nm (emission).
100
Normalized cell viability [%]
milling machine for defining micro patterns in PMMA
(Figure 2). WHATMAN® polycarbonate membranes
shown in Figure 3 with porosities in the range of 0.015 –
0.4 µm, both transparent and black, for colorimetric or
fluorometric target analysis, respectively, were leak-tight
bonded between the PMMA layers.
Figure 1A - 1D show the top and cross sectional
views of the 3D CAD design and the prototype (Figure
1D). The platform is designed in a 96 well compatible
architecture, i.e. suitable for readout by standard plate
readers used in high throughput screening of assays. The
system secures generation of a stable reagent gradient in
the micro channels and its delivery to the cell culture
allocated inside the micro wells through the dead-volume,
25 µm thick micro-porous membranes. The built-in
microfluidic gradient [16] enables analysis of samples
being exposed to multiple chemotoxic reagents of up to
eight progressive dilutions. Two gradient designs were
used: 2 inlets with 8 outlets, and 3 inlets with 8 outlets.
Thus, the system can be used either for exposure of cells
to various concentrations of a single drug or to a mixture
of two drugs at different ratios. The integrated membranes
mimic in vivo environment for cell growth up to some
extent, support sterile conditions and act as a physical
mass transfer barrier for the range of drug exposures.
Each gradient generating network (two inputs and eight
outputs) of meander microchannels is connected with the
culture micro-wells via through holes (Figure 1C).
Cell viability
Model:
80
G-(cMMC)
n
Viability=Viabilitystart·e
60
40
20
0
0
10
20
30
40
50
-1
MMC concentration [μmol·L ]
Figure 7. Results of the XTT viability test on HL60
cancer cells after 24 hr incubation with MMC in TP. The
assay is used as a model bio-system.
Figure 5: Gradient generator (A): concentration
profile (B), pressure profile (C) and schematic cross
section of a single well.
Simulations in Comsol 4.2a were run to determine
the design parameters of a splitter unit. The goal of the
simulations was to identify initial design parameters
(width and length of distributing channels) that allow for
stable gradient formation inside the micro-wells. The
modules that were used were: laminar flow, Darcy flow
through porous media (polycarbonate membrane) and
transport of diluted species. Example for part of the
gradient is shown in Figure 5A, where a unit splitter
ensures homogeneous mixing inside the micro wells
(Figure 5B, for reference the diameter of wells is taken as
10 mm) at uniform pressure distribution across the wells
(Figure 5C).
RESULTS AND DISCUSSION
Performance of the micro-platform
The manufactured membrane-integrated prototype
was tested on a Tecan® micro-plate reader (Figure 4) for
colorimetric and fluorometric assays whilst commercial
TPs served as a reference. The calibration of intensities
has been performed on the prototype for the blank
(material background), blank and cell media and blank
with water containing food dye for which the data is
summarized in Table 1. As it follows from analysis of
these data, the materials comprising the prototype gave
higher intensity and this offset factor is to be taken into
consideration during the experiments. In fact, there is
always a calibration chamber available to eliminate effect
of the absorptive properties of materials. Figure 6 shows
the analysis of intensities obtained from a black TP and
integrated black membranes on the MP. The XTT
colorimetric cell viability assay on HL60 leukemia cells
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following MMC is used as a model system to study
metabolic activity of cells under the drug treatment
(Figure 7). We continue to develop the technology with
further experiments to transfer the assay to the MP.
Table 1: Absorption intensities [a.u.] for a standard TP
and the MP at 450 nm.
Parameter
TP
MP
Blank
(3.4±1.5)×10-3
(50±5)×10-3
Blank + cell media
(65±8)×10-3
(77±3)×10-3
Blank + dye
1.59±0.18
0.65±0.1
CONCLUSIONS AND FUTURE WORK
Future work will be directed towards further assay
integration as well as towards advancements of fluidic
design and device assembly. We envision that the
developed low-cost, multi-well, membrane-integrated
system will simplify, speed up and give a good foundation
for the high-throughput pre-clinical toxicology studies
required in assessing cancer risks in patients at an early
stage and ultimately may increase successful treatment
outcomes.
ACKNOWLEDGEMENTS
This research has been partially supported by the FP7
ENIAC project CAJAL4EU, the ERDF and Enterprise
Ireland under Grant No IR/2010/0002. The authors would
like to acknowledge Barry O’Connell (NCSR, Dublin
City University) for his help with high-resolution SEM
analysis of the membranes and Prof. Richard O’Kennedy
for giving access to the cell culture facilities.
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CONTACT
Prof. Jens Ducrée, +35317005377, [email protected]
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