Microfluidics for cell culture

1. Instituto Superior Técnico
Microfluidics and Cell Culture
David Conceição* & Johannes Österreicher**, May 2010
* Master in Bioengineering and Nanosystems, nº64405
** Master in Chemical Engineering, nº68694
Abstract:
Microfluidic devices (MFD) handling sub-micrometer-volumes of liquids open new
perspectives to cell culture, as they can mimic the in vivo-environment of the cells.
Furthermore, different laboratory operations can be joined on a MFD, creating a “lab
on a chip”. However, the behaviour of fluids on the micro-scale is very different than
on the macro-scale, creating new challenges. In this paper, the advantages and
disadvantages of this new technology for cell culture are discussed, micro-fabrication
methods are presented and the state of the art is expatiated on two case studies: a MFD
for depositing two cell cultures with intercellular communication capability and a
MFD facilitating integrated cell culture and lysis on a chip.
1. Introduction
Microfluidics is the science and technology of handling small volumes (typically
10⁻ ⁹ to 10⁻ ¹⁸ litres) in channels with dimensions in the range of micrometres. Due to
the small size, microfluidics present laminar flow of liquids and therefore offer
fundamental new possibilities for the control of concentration of molecules. This type of
technology is thereby able to show the fundamental differences between the physical
properties of fluids moving in large channels and those travelling through micrometre-
scale channels [1]. The biggest difference regards the phenomenon of turbulence and is
related to the concept of laminar flow. On large scales, fluids mix convectively, but at
micrometre-scales, fluids normally only mix by diffusion, unless a turbulent flow is
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achieved. The reason for this behaviour is that at these scales, viscosity assumes a more
important role than the fluids inertia. To control the fluids behaviour, devices that can
accomplish mixing might be needed and also a very deep analysis of the parameters that
characterizes the fluids flow regime must be taken into account. Such parameters
concern Reynolds number, the Navier-Stokes equations, Knudsen number and Peclet
number, for example.
The first applications of microfluidics were developed in the '80s in the field of
microanalytical chemistry (gas-phase chromatography, high pressure liquid
chromatography and capillary electrophoresis) because the technology allows the use of
very small amounts of reagents and presents short time of analysis, low cost, high
sensitivity and resolution amongst others[1]. Later, applications such as detectors for
chemical and biological threats, devices for high throughput DNA sequencing and
platforms in microelectronics were also developed. Following the first pioneer works in
microfluidics, new microfluidic components for fluid transport, fluid mixing and
valving were created. All these concepts suggested new and better ways to control the
fluids behaviour and also its targeting for several specific applications (e.g.: the use of
micropumps in the separation of molecules within miniaturized quantities of fluids)
[1,3].
Cell culture is a key step in cell biology, tissue engineering, biomedical
engineering, and pharmacokinetics for drug development. Although the in vitro cell
culture technique is widely used in conventional laboratory experiments, cells grown in
vitro often show different behaviour than in vivo as in vitro methods provide a static and
macroscale environment that is entirely different from the environments of biological
systems [2,4]. So, when it comes to cell culture, microfluidics present even more
possibilities besides the obvious; they can provide complex structures that mimic the in
vivo environment of cells [5]. Living organisms have complex systems of channels,
tissues, membranes etc. Cells growing within this systems can adhere to this structures
and communicate with other cell types. In traditional in vitro-cell culture systems, there
are no such structures nor other cell types to interact with. These inconsistencies result
in different growth rates, morphology and intracellular metabolism. Microfluidics, on
the other hand, can for example provide a lab-on-a-chip platform that resembles the
human circulatory system and in the way it supplies media to cells and removes their
waste products[2].
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2. Micro-Fabrication Methods
The technologies involved in the production methods for microfluidic devices
come from the microelectronics industry. Typically, micro-fabricated devices are made
on silicon wafers, glass, plastic, or with other types of substrate materials. The protocol
usually implied in the fabrication of such micro-devices includes several processes,
including photolithography, etching, thin-film deposition, thermal oxidation, and wafer
cleaning. However, techniques such conventional photolithography require clean room
facilities, a large photolithographic equipment and besides that, it requires the use of
several chemicals that are toxic to cells and that are not biocompatible. Taking that into
account, for biological applications, techniques of soft lithography are usually used.
These techniques use elastomeric stamps fabricated from patterned silicon wafers to
mold materials and are used to create several unit operations components and structures
such as micropumps and microchannels [3]. Such soft lithographic techniques include
replica molding, microcontact printing , microtransfer molding and micromolding. The
elastomer typically used is poly(dimethylsiloxane) (PDMS) because it is biocompatible,
optically transparent, permeable to gases, and durable [6]. It is also inexpensive
compared to other material used in conventional photolithography. Figure 1 shows the
procedure adopted for the fabrication of microfluidic devices using soft lithography and
PDMS (in this case the protocol used is for the fabrication of microchannels). It starts
with the exposure of the photoresist to UV light, using a photomask with the adequate
design. Then, having the patterned mold (as mentioned before, usually a silicon wafer is
used for the mold), PDMS is poured on the patterned substrate and undergoes through a
temperature change to solidify and gain the respective design. Finally, a layer of glass is
usually affixed to the patterned PDMS layer to 'close' the structure and to create the
microchannels.
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Figure 1. Schematic diagram of microfabrication
procedures of a microfluidic device. Image taken
from Yeon et al (2007).
3. State of the art
Microfluidic technology can be used to supply and transfer media, buffers, and
even air while the waste products by cellular activities are drained in a way resembling
the human circulatory system. In addition, many studies have focused on analytical
microsystems that are integrated into a microfluidic platform that carries out sample
mixing, buffer exchange, as well as cell seeding, transferring and separation in a
microchannel network. Therefore, microfluidic systems can provide an in vivo-like
environment for a cell culture as well as a reaction environment for a cell-based assay.
In the last years, simple two-dimensional microstructures were widely used to construct
a microfluidic cell culture system. However, as microfluidic devices have become
sophisticated in an effort to realize a perfect in vivo-environment on a chip, they have
been adapted for use with three-dimensional microstructures and polymer scaffolds,
ensuring multiple layers for co-cultures or three-dimensional cell cultivation.
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Other works have recently been developed: Khademhosseini et al. [7], proposed
the creation of microwells on a substrate to capture and immobilize cells within low
shear stress regions inside channels. By using an array of channels, it was possible to
deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells,
on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold
could be removed, generating a multiphenotype array of cells. This application is
illustrated in figure 2:
Figure 2. Schematic diagram of
reversible sealing of microfluidic
arrays onto microwell patterned
substrates to fabricate
multiphenotype cell arrays. Image
taken from Khademhosseini et al
(2005).
4. Case Studies
In this section, two different case studies will be analysed. One is regarding the
concept of fluid transport within a network of two distinct cell types and the other one is
relative to cell culture and cellular lysis on a chip.
4.1 A microfluidic device for depositing and addressing two cell
populations with intercellular population communication
capability
Lovchik et al.[8] have produced a PDMS microfluidic network for the
deposition of two different cell types in two chambers with flow across the chambers.
The device is composed of two cell chambers with a volume of 0.49µl, 6 microchannels
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for servicing the chambers, and one channel linking both chambers. The molded PDMS
is sealed with a Si lid having six vias and ports that can be linked to high precision
pumps, allowing flow rates of 0.1 to 10µl min⁻ ¹. The device can be observed using an
inverted microscope. Figure 3 shows the device.
Figure 3. Design and assembly of the microfluidic network with two cell chambers. (a) The
molded PDMS is aligned to a Si lid with vias. (b) Each via is connected to a port on the
back side of the lid. (c) The ports are connected to pumps or valves. Image taken from
Lovchik et al (2010).
The usage of the device was shown using murine N9 microglia (macrophages of
the brain and spinal chord) and human SH-S5Y5 neuroblastoma (can differentiate
neurites, and can thus mimic neuronal response in vitro). It is proposed that different
brain diseases result from changes in interaction between neurons, astrocytes and
microglia. Microfluidic devices able to study these interactions could thus result in
better understanding of diseases and new therapeutic strategies.
The device was first tested with colored water, where it was shown that pulling
the liquids is advantageous over pushing to avoid delamination of the PDMS from the
Si chip. Flushing of the chambers sequentially or independently, the formation of
different gradients in the chambers and other features were shown. Furthermore,
polystyrene beads with a size similar to that of cells (10 μm) were deposited in the
chamber; a homogeneous distribution of the beads was achieved at flow rates between
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0.5 and 5 μL min⁻ ¹. For experiments with living cells, the microfluidic device was
coated with fibronectin and the cells were deposited by sedimentation at low flow rates
in the two chambers. The chambers were then rinsed to remove cells in the channels
before and after the cell chambers; the cells were then stained with different dyes. The
microglia were then stimulated by flushing their chamber for 20min with ATP.
Stimulation with ATP is known to induce the release of microvesicles from microglia.
This process is known to be a pathway of intracellular communication during neuro-
inflammatory events. The microvesicles were then transferred to the chamber
containing neuroblastoma. A part of the supernatant was analysed with labelled
antibodies to verify that the transport of vesicles has in fact occurred.
It has been concluded that this microfluidic device may help to dissect the flux
of information that occurs between different brain cell types and that may contribute to
neuroinflammation. The device could easily be scaled to create a cascade of cell
chambers to study communication between three or more different cell types.
4.2 Integrated microfluidic cell culture and lysis on a chip
Nevill et al [9] created a platform which allowed them to culture several cell
types (HeLa, MCF-7, Jurkat, and CHO-K1 cells) for up to five days and also to do their
lysis without the addition of lysis buffers and subsequent washing steps, which increase
the complexity of such devices and reduce their ease of use. In this device, lysis is
accomplished by applying a DC voltage to electrochemically generate hydroxide inside
it.
The device was designed to have six chambers, individually addressable via
polymer tubing connections. In each chamber, four fluid-permeable cell holding
structures with 11 nl of volume each were created ('traps' in figure 4). They also
contained other structures, like filters, to help keeping debris from entering the trapping
region and to break up cell clumps into individual cells during loading; a high resistance
region, to reduce the pressure in the region where metal lines entered the fluidic
channels; and also spacers, to prevent the traps from bonding to the glass, as well as to
reduce the risk of releasing trapped cells due to fluctuations in pressure.
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Figure 5 shows a scheme of cell loading into this device, using simultaneously
two syringes: one for the media or buffer and another one for cells. Complete cell
loading usually occurred within five minutes, and they observed that cells remained
viable for up to five days of culturing under constant perfusion of media.
Electrochemical lysis was accomplished by applying a DC voltage across the
electrodes on either side of the trapping region. This generated hydroxide ions in the
cathode upstream of the cell chamber, which cleaved fatty acids groups of cell
membrane phospholipids.
Therefore, this work presented a new microfluidic cell analysis platform for
culturing and electrochemically lysing cells on demand. Fluorescence analysis
confirmed that genetic material and membrane bound proteins were present in the
lysate, assuring that it is possible to lyse cells with electrochemically generated
hydroxide without compromising downstream lysate analysis.
Figure 4. Integrated microfluidic cell culture and lysis on a chip. (a) A top view of the
chip with six separate devices (filled with dye for visualization).(b) A magnified image
of one chamber showing the trapping region structure which consists of an array of four
cell traps separated by spacers. Electrodes are on either side of the trapping region,
which is preceded by a high resistance, or pinched, section. Scale bars are 1.3 mm.
Image taken from Nevill et al (2007).
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Figure 5. Cell loading. (a) Schematic of how the chip
is loaded using a four way valve connected to a
syringe with cells and a syringe with media or buffer.
(b) Images taken from a movie of cells loading into
the traps. Flow rate was 80 µl min-1. Image taken
from Nevill et al (2007).
5. Conclusions
Microfluidics offer a complete set of fluidic unit operations (fluid transport, fluid
valving, fluid mixing, separation, detection, etc.) within a platform and assures an easy
way to have a set of elements ideally connected in an integrated way, with a well
defined (and low cost) fabrication technology. Devices are especially suitable for
biological applications, particularly on the cellular level, because the scale of channels
corresponds with that of cells and the scale of the devices allows important factors to
accumulate locally, forming a stable microenvironment for cell cultures [2].
Compared with traditional culture tools, microfluidic platforms provide much
greater control over the cell microenvironment and a rapid optimization of media
composition using relatively small numbers of cells. Given that a group of cells can
more easily maintain a local microenvironment within a microchannel than in a
macroscale culture flasks, cells in microchannels grow significantly slower than they
would in a traditional culture flask [5].
However, some problems arise on the micro-scale that are unknown on the
macroscale, particularly difficulties to control the fluids behaviour on the micro-scale;
furthermore, the high surface to volume-ratio can lead to a lower effective concentration
of reagents due to adsorption.[10] Clotting can also be a problem.
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6. References
[1] Whitesides, G. The origins and the future of microfluidics, Nature, Nature
Publishing Group, 2006, Vol. 442(7101), pp. 368-373;
[2] Yeon, J.H. & Park, J.K., Microfluidic cell culture systems for cellular analysis,
Biochip J , 2007, Vol. 1, pp. 17-27;
[3] Haeberle, S. & Zengerle, R., Microfluidic platforms for lab-on-a-chip applications,
Lab on a Chip, Royal Society of Chemistry, 2007, Vol. 7(9), pp. 1081-1220.
[4] Bruzewicz, D., McGuigan, A. & Whitesides, G. Fabrication of a modular tissue
construct in a microfluidic chip Lab on a Chip, Royal Society of Chemistry, 2008, Vol.
8(5), pp. 663-671;
[5] Young, E. & Beebe, D. Fundamentals of microfluidic cell culture in controlled
microenvironments Chemical Society Reviews, Royal Society of Chemistry, 2010, Vol.
39(3), pp. 1036-1048;
[6] Wheeler, A., Throndset, W., Whelan, R., Leach, A., Zare, R., Liao, Y., Farrell, K.,
Manger, I., Daridon, A. & others Microfluidic device for single-cell analysis Analytical
Chemistry, ACS Publications, 2003, Vol. 75(14), pp. 3581-3586;
[7] Khademhosseini, A., Yeh, J., Eng, G., Karp, J., Kaji, H., Borenstein, J., Farokhzad,
O. & Langer, R. Cell docking inside microwells within reversibly sealed microfluidic
channels for fabricating multiphenotype cell arrays Lab on a Chip, Royal Society of
Chemistry, 2005, Vol. 5(12), pp. 1380-1386;
[8] Lovchik, R., Tonna, N., Bianco, F., Matteoli, M. & Delamarche, E. A microfluidic
device for depositing and addressing two cell populations with intercellular population
communication capability Biomedical Microdevices, Springer, 2010, Vol. 12, pp. 275–-
282;
[9] Nevill, J., Cooper, R., Dueck, M., Breslauer, D. & Lee, L. Integrated microfluidic
cell culture and lysis on a chip Lab on a Chip, Royal Society of Chemistry, 2007, Vol.
7(12), pp. 1689-1695;
[10] Barbulovic-Nadi, I. & Wheeler, A.R., Cell Assays in Microfluidics, Encyclopedia
of Micro- and Nanofluidics; Li, D. Q., Ed.;Germany, 2008; Vol. 1, pp 209-216.
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