Introduction
It is clear enough that a complete understanding of gradient effects
on cellular behaviour is not terminated and more technological platforms
are required to increase the knowledge of many cellular mechanisms. Thus
it is a big technological challenge the realization of systems that makes the
in vitro environment more similar to the in vivo one, including spatially and
temporally organized signals supplemented with sensors for monitoring
cellular parameters.
Furthermore, wide interest was also devoted to the investigation of
the physical stimulation of cells: several types of substrates were presented
in literature reproducing selected aspects of the extracellular environment,
confirming that the bare topography affects cell [7] adhesion, morphology,
orientation, differentiation and phagocytotic activity [8]. Moreover, the
phenomenon of cell alignment, called contact guidance, was found to depend
on different parameters of micro/nanofabricated structures (i.e. grooves
depth and width) and on cell types: one example is given by Recknor et al.
[9], who developed a permissive substrate for in vitro neural stem cell
differentiation, using a photolithographic technique to pattern periodical
physical barriers with adsorbed laminin on polystyrene dishes. This
combination of chemical (adsorbed protein) and physical (microgrooves)
cues succeeded in the directional guidance of Rat type-1 astrocytes.
A wide interest is thus devoted to the development of systems for
high resolution analysis and control of cellular physiology, where the cell
development can be controlled not only by the physical and chemical
properties of the substrate but also delivering drugs and chemical agents in
a precise manner.
This work is focused on the realization of a microfluidic chip for
controlled cell culture, integrating a gradient generator and a
micropatterned substrate. Exploiting the advantages of soft lithographic
techniques, a polymeric structure in polydimethylsiloxane (PDMS) is
conceived with two orthogonal sets of fluidic channels and active
7
Introduction
components, such as switching valves. The system is designed to be applied
on a substrate, previously patterned by non-conventional micro contact
printing of adhesion proteins. Finally, the developed system guarantees the
production of a complex user-defined gradient, with tailored spatial and
temporal profiles; secondly, it allows immobilizing and observing a selected
small population of adherent cells.
The remainder of this thesis is organised as follows. Chapter 1
presents a brief description of microfluidic gradient generators, highlighting
the limits of the published architectures. In Chapter 2 a novel microfluidic
gradient generator is proposed. This system is designed to allow generating
multiple concentration gradients. The microfluidic network is combined
with a glass substrate which presents cell-adhesive and cell-repellent areas.
Materials and methods for the fabrication of the chip and for the substrate
micropatterning are presented in Chapter 3. Specifically, Multilayer Soft
Lithography (MLSL), used for the microfluidic chip fabrication, and micro-
contact printing (μ-CP), required for the substrate micropatterning are
described in detail. In Chapter 4 all the specific fabrication protocols and an
extensive experimental characterization and validation of the system are
reported and discussed. In particular, three major results are presented: i)
demonstration of complex gradient generation, ii) micro-patterning of
fibronectin/PLL-g-PEG on glass substrates, iii) immobilization on the
microfabricated systems. Chapter 5 outlines obtained results and
contributions and suggests directions for the future work.
8
Chapter 1
MICROFLUIDIC GRADIENT GENERATION
As previously introduced, the importance of biomolecular gradients in
biological events such as directing the growth, differentiation and migration
of various cell types in vivo has motivated researchers to develop numerous
methods for generating chemical gradients in vitro. Among the simplest
methods, they should be mentioned the deposition of soluble biomolecules
on the surface of a hydrogel matrix [1] or the use of a glass micropipette
filled with a biomolecule solution and its tip positioned at a set distance
from cells using mechanical manipulators [2].
Although these methods led to some interesting experimental results,
they are still not appropriate to generate highly reproducible or controllable
gradients [3]. This limit can be overcome with microfluidic gradient generators
which provide a way to create predictable, reproducible, and easily-
quantified biomolecule gradients in vitro [3]. Microfluidic gradient
generators allow the creation of multiple biomolecule gradients each with
its own user-defined spatio-temporal distribution.
The ability to create complex, user-defined gradient environments
would enable quantitative elucidation of multi-gradient signal integration
and provide the specific recipes for engineering the growth, migration, and
differentiation of a variety of cell types [3].
Recently, several microfluidic devices have been developed, many of
them offering significant control over the shape and temporal
characteristics of the gradient, as it is going to be described [25-27, 34]. A
few of these devices were also coupled with patterned substrates in order to
provide a physical stimulation [30-33]. Surface patterning can be obtained
by standard photolithography or soft lithography techniques (microcontact
10
Microfluidic gradient generation
printing and fluidic patterning) or by photoreactive chemistry [4].
Microfabrication methods, combined with micropatterning techniques and
advanced surface chemistry, enable the reproducibility of cell micro-
environment at cellular resolution possible.
In this section a brief introduction to microfluidics is presented
covering the aspect of the fluid behaviour at the nanolitre scale. The rest of
chapter is focused on the state of the art regarding the gradient generation
methods in biology. Particular attention is paid to a Microfluidic Multi-
Injector generator.
1.1 INTRODUCTION TO MICROFLUIDIC SYSTEMS
Microfluidics refers to the science and technology of systems that
manipulate small amounts of fluids, generally on the nanoliter scale and
below [5]. It is a multidisciplinary research field aiming to the precise
control and manipulation of fluids that are geometrically constrained to
small, typically sub-millimetre, length scale [5]. The small dimensions of
microfluidic systems exploit the not obvious characteristic of fluids at small
scale: the laminar flow. At the same time, the reduced dimensions of the
microsystems enable a precise control of concentrations of molecules in
space and time that cannot be realized in common millifluidic applications
[6].
Microfluidics has emerged in the beginning of the 1980s and
tremendous advances have been realized, for example, in revolutionizing of
molecular biology procedures for enzymatic analysis (e.g., glucose and
lactate assays), DNA analysis (e.g., polymerase chain reaction and high-
throughput sequencing), and proteomics. The basic idea of microfluidic
devices for biological applications is to integrate assay operations such as
detection, sample pre-treatment and preparation on a single chip with the
advantages of cheap device production, little reagents consumption and
11
Microfluidic gradient generation
high-throughput analysis. As microfabricated integrated-circuits
revolutionized computation by dramatically reducing the space, labour, and
time required for calculations, so microfluidic systems hold similar promise
for the large-scale automation of chemistry and biology, suggesting the
possibility of numerous experiments performed rapidly and in parallel [7].
Recently, microfluidic devices have found increasing applications in
basic and applied biomedical research [8]. Novel designs that exploit the
advantages of miniaturization have been proposed in devices for cell
migration, drug screening and long-term culture of stem cells and neurons
[9]. Therefore, microfluidic devices are starting to offer new capabilities for
gaining biological insights owning to their ability to control and manipulate
cellular microenvironments that have not been available in conventional
macroscale methods [10].
1.2 FLUID PHYSICS AT THE NANOLITER SCALE
The mathematical description of the state of a moving fluid is
effected by means of functions which give the distribution of the fluid
velocity vector v=v(x,y,z,t) and of any two thermodynamic quantities
pertaining to the fluid, for instance the pressure p(x,y,z,t) and the density
ρ(x,y,z,t). At small scales (channel diameters of around 100 nanometers to
several hundred micrometers) some interesting fluids properties
appear [11]. Fluids flowing in micrometer-scale conduits, or microchannels,
are dominated by the viscous properties of the fluid at the expense of the
inertial forces generated by the fluid. This flow regime, called laminar
flow [12] allows the movement of momentum, heat, and chemical species
inside a microfluidic device to be calculated with great accuracy [13].
The newtonian fluid behaviour is described by the Navier-Stokes
equation (1.1) that, together with the equation of mass conservation (1.2) and
well formulated boundary conditions, accurately models fluid motion:
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Microfluidic gradient generation
fvpvv
t
v
+∇+⋅−∇=
⎟
⎠
⎞
⎜
⎝
⎛
∇⋅+
∂
∂
2
ηρ
(1.1)
0)( =⋅∇+
∂
∂
v
t
ρ
ρ
(1.2)
where ρ is the density, v the velocity, p the pressure, η the dynamic viscosity
and f the external applied body forces. As previously stated, in microfluidic
devices inertial forces are small compared to viscous ones thus the
nonlinear term can be neglected, leading the Stokes equation:
fvp
t
v
+∇+⋅−∇=
∂
∂
2
ηρ (1.3)
Considering a constant density ρ, equation (1.2) becomes
0=⋅∇ v (1.4)
The relative importance between inertial and viscous forces is expressed
with the dimensionless Reynolds number Re:
v
i
f
fLU
=≡
η
ρ
00
Re (1.5)
with f
i
and f
v
being inertial and viscous stress density respectively, U
0
velocity of the fluid and L
0
typical length scale. For common microfluidic
devices considering water as the working fluid, typical velocities in the range
of 1 μm/s – 1 cm/s and channel radii of 1−100 μm, Reynolds number
ranges between O(10
−6
) and O(10). Having these low Reynolds numbers
microfluidic systems exploit linear regular deterministic flows. However, in
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Microfluidic gradient generation
presence of particular physical processes, such as capillary effects at free
surfaces, viscoelasticity in polymer solution and electrokinetic effects, some
nonlinearities can rise and generate peculiar microfluidic phenomena.
Another important physical characteristic in low Reynolds number
flows is the absence of turbulent mixing. Laminar flows make mixing to
occur only by diffusion, thus increasing mixing times. The dimensionless
Peclet number expresses the ratio between convection and diffusion:
D
wU
Pe
0
≡ (1.6)
where D is the diffusivity, w the width of the channel [6].
Thus low Reynolds and Peclet numbers mean that the flow will remain
laminar, two joining fluids will not mix readily via turbulence, thus, finally,
the diffusion must cause the two fluids to mingle. In this way gradients of
concentration are produced only with diffusion. This phenomenon
becomes tangible observing the mass continuity equation which describes the
conservative transport of mass of a species a, in this case expressed in
molar quantities [12]
aaa
a
RJvcD
t
c
+∇−⋅−=
∂
∂
)()( (1.7)
where c
a
is the molar concentration defined as the number of moles of a per
unit volume of solution, J
a
is the molecular molar flux of species defined as
the number of moles of a flowing through a unit area per unit time, v is the
molar average velocity, R
a
is the molar rate of production of a per unit
volume. If the convective term is negligible and no chemical reactions
occur (this means all chemical production terms are zero) the equation is
governed only by the diffusion term. In these conditions the equation 2.1
becomes equal to
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