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.