Abstract
Integrating biological material within soft microfluidic systems made of
hydrogels offers countless possibilities in biomedical research to
overcome the intrinsic limitations of traditional microfluidics based on
solid, non-biodegradable, and non-biocompatible materials.
Hydrogel-based microfluidic technologies have the potential to transformin vitro cell/tissue culture and modeling. However, most
hydrogel-based microfluidic platforms are associated with device
deformation, poor structural definition, reduced
stability/reproducibility due to swelling, and a limited range in
rigidity, which threatens their applicability. Herein, we describe a new
methodological approach for developing a soft cell-laden microfluidic
device based on enzymatically-crosslinked silk fibroin (eSF) hydrogels.
Its unique mechano-chemical properties and high structural fidelity,
make this platform especially suited for in vitro disease
modelling, as demonstrated by reproducing the native dynamic 3D
microenvironment of colorectal cancer and its response to
chemotherapeutics. Results show that 14 wt% enzymatically-crosslinked
silk fibroin microfluidic platform has outstanding structural stability
and the ability to perfuse fluid while displaying in vivo -like
biological responses. Overall, this work shows how the combination of
enzymatically-crosslinked silk fibroin and microfluidics can be employed
for developing soft lab-on-a-chip platforms with superior performance.
Introduction
Biomedical research has increasingly moved towards the design,
fabrication and implementation of microfluidic systems to precisely
manipulate fluids and biological material
(1, 2).
Poly(dimethyl siloxane) (PDMS) has typically been employed in the
development of hard microfluidic platforms due to its unique properties,
such as high cell compatibility, optical transparency and oxygen
permeability (3). However, PDMS displays
serious drawbacks, including limited mechanical and biochemical
properties and degradability that threaten the clinical applicability of
PDMS-based devices (Figure 1). To solve this, softer biodegradable
materials, such as natural hydrogels, are an excellent alternative to
building microfluidic systems (i.e ., soft microfluidics)
(4, 5).
Observing Figure 1, the improvement of soft microfluidics such as the
one developed herein present some important features over the hard
microfluidics (PDMS), e.g. physiological relevance, functional space,
flexibility, ECM interaction analysis, optical properties and easy
manipulation. Typical hydrogels include collagen, gelatin, gelatin
methacrylate, agarose and alginate, all of them cell-compatible,
enabling better interfaces and cell adhesive moieties
(6, 7).
Similarly, synthetic hydrogels have been employed to build microfluidic
chips (5,
8).
The integration of microfluidic technologies with tissue engineering
strategies, such as engineered functional biomaterials as ECM
surrogates, is on the rise (2). This
combination can efficiently improve the drug discovery pipeline,
diagnostics, ameliorate tissue (disease) models, and tissue regeneration
processes (9,
10). Nevertheless, the stiffness of
traditional hydrogels is limited when compared to the wide range of
human tissues (11), limiting their
utility in mimicking physiological condition. Additionally, the
degradability of most of these hydrogels is far from desired. Recently,
new biomaterials, such as silk fibroin (SF), have been employed in
tissue engineering (12). SF is an
FDA-approved material for certain medical applications, gaining a lot of
attention in the biomedical field due to its excellent mechanical and
biochemical properties (10,
13). Together with its unique
biocompatibility, flexibility, tunable degradability, mild processing
conditions, and the presence of accessible chemical groups for
functional modifications, SF is an ideal biomaterial for the development
of biocompatible microfluidic systems with implantable potential
(14, 15).
Notably, SF has already been used in soft microfluidics
(11). Bettinger et al .,
(16) reported the fabrication of
microfluidic devices made by laminating water-stable micro-molded SF
membranes in β-sheet, which were then modified with macroscopic fluidic
connections. However, SF hydrogels in β-sheet lack the transparency and
friendly cellular habitat needed for microfluidic applications
(17).
A novel class of enzymatic-crosslinked SF (eSF) hydrogels has recently
been reported. Still, being its exploitation limited to engineeringin vitro 3D static models of disease, bioinks, and as conduits
for peripheral nerve regeneration applications
(15, 18).
The horseradish peroxidase (HRP)/hydrogen peroxide
(H2O2) cross-linking approach was
proposed by Yan et al . (19), and
it is used in polymers containing phenol group, including tyrosine,
tyramine or aminophenol (20). An
advantage of this method is that eSF hydrogel proteins remain in an
amorphous state, presenting the opportunity to induce β-sheet
conformation if necessary or naturally shifting after 7 to 10 days. This
high degree of versatility and control makes it ideal for developing
soft microfluidic systems.
Herein, we describe the development of a biomimetic eSF hydrogel-based
microfluidic platform in the amorphous state with mechanical properties
not achievable with β-sheet conformation
(14, 16).
The eSF microfluidic chip remains in unstructured conformation for long
periods, demonstrating unprecedented structural stability that allows
fluid perfusion. As a proof of concept, we designed a microfluidic model
of colorectal cancer (CRC) to investigate and characterize distinctive
features of the tumor microenvironment. For this, human colonic
microvascular endothelial cells (HCoMECs) were injected within the
microfluidic channel displaying a serpentine morphology that aimed to
reproduce, in a reductionist manner, the abnormal (tortuous)
architecture of the tumor vasculature. Next, HCT-116 colorectal cancer
cells were encapsulated in the bulk of the microfluidic platform.
Finally, to evaluate the performance of this model, Gemcitabine (GEM), a
typical chemotherapeutic drug currently being tested to treat patients
with advanced colorectal cancer (21,
22), was perfused through the
microchannel, and cell viability was measured. Overall, this
enzymatic-crosslinked silk hydrogel microfluidic platform promises new
ways of engineering and mimicking tissues and pathological states and
culturing in vitro under dynamic culture conditions. Drug
screening and precision nanomedicine are also targets of this advanced
technology.