Photo and Soft Lithography for Organ-on-Chip Applications.

Organs-on-Chip devices are generally fabricated by means of photo- and soft lithographic techniques. Photolithography is a process that involves the transfer of a pattern onto a substrate by a selective exposure to light. In particular, in this chapter two different photolithography methods will be described: liquid and dry photolithography. In liquid photolithography, a silicon wafer is spin-coated with liquid photoresist and exposed to UV light in order to be patterned. In dry photolithography, the silicon wafer is laminated with resist dry film before being patterned through UV light. In both cases, the UV light can be collimated on top of the wafer either through photomasks or by direct laser exposure. The obtained patterned wafer is then used as a mold for the soft lithographic process (i.e., replica molding) to produce polymer-based microdevices.

A new microfluidic platform for the highly reproducible preparation of non-viral gene delivery complexes.

Transfection describes the delivery of exogenous nucleic acids (NAs) to cells utilizing non-viral means. In the last few decades, scientists have been doing their utmost to design ever more effective transfection reagents. These are eventually mixed with NAs to give rise to gene delivery complexes, which must undergo characterization, testing, and further refinement through the sequential reiteration of these steps. Unfortunately, although microfluidics offers distinct advantages over the canonical approaches to preparing particles, the systems available do not address the most frequent and practical quest for the simultaneous generation of multiple polymer-to-NA ratios (N/Ps). Herein, we developed a user-friendly microfluidic cartridge to repeatably prepare non-viral gene delivery particles and screen across a range of seven N/Ps at once or significant volumes of polyplexes at a given N/P. The microchip is equipped with a chaotic serial dilution generator for the automatic linear dilution of the polymer to the downstream area, which encompasses the NA divider to dispense equal amounts of DNA to the mixing area, enabling the formation of particles at seven N/Ps eventually collected in individual built-in tanks. This is the first example of a stand-alone microfluidic cartridge for the fast and repeatable preparation of non-viral gene delivery complexes at different N/Ps and their storage.

Plasma-enhanced protein patterning in a microfluidic compartmentalized platform for multi-organs-on-chip: a liver-tumor model.

A microfluidic technique is presented for micropatterning protein domains and cell cultures within permanently bonded organs-on-chip devices. This method is based on the use of polydimethylsiloxane layers coupled with the plasma ablation technique for selective protein removal. We show how this technique can be employed to generate a multi-organin vitromodel directly within a microscale platform suitable for pharmacokinetic-based drug screening. We miniaturized a liver model based on micropatterned co-cultures in dual-compartment microfluidic devices. The cytotoxic effect of liver-metabolized Tegafur on colon cancer cell line was assessed using two microfluidic devices where microgrooves and valves systems are used to model drug diffusion between culture compartments. The platforms can reproduce the metabolism of Tegafur in the liver, thus killing colon cancer cells. The proposed plasma-enhanced microfluidic protein patterning method thus successfully combines the ability to generate precise cell micropatterning with the intrinsic advantages of microfluidics in cell biology.

A three-dimensional in vitro dynamic micro-tissue model of cardiac scar formation.

In vitro cardiac models able to mimic the fibrotic process are paramount to develop an effective anti-fibrosis therapy that can regulate fibroblast behaviour upon myocardial injury. In previously developed in vitro models, typical fibrosis features were induced by using scar-like stiffness substrates and/or potent morphogen supplementation in monolayer cultures. In our model, we aimed to mimic in vitro a fibrosis-like environment by applying cyclic stretching of cardiac fibroblasts embedded in three-dimensional fibrin-hydrogels alone. Using a microfluidic device capable of delivering controlled cyclic mechanical stretching (10% strain at 1 Hz), some of the main fibrosis hallmarks were successfully reproduced in 7 days. Cyclic strain indeed increased cell proliferation, extracellular matrix (ECM) deposition (e.g. type-I-collagen, fibronectin) and its stiffness, forming a scar-like tissue with superior quality compared to the supplementation of TGFß1 alone. Taken together, the observed findings resemble some of the key steps in the formation of a scar: (i) early fibroblast proliferation, (ii) later phenotype switch into myofibroblasts, (iii) ECM deposition and (iv) stiffening. This in vitro scar-on-a-chip model represents a big step forward to investigate the early mechanisms possibly leading later to fibrosis without any possible confounding supplementation of exogenous potent morphogens.

High-Throughput Microfluidic Platform for 3D Cultures of Mesenchymal Stem Cells.

The design of innovative tools for generating physiologically relevant three-dimensional (3D) in vitro models has been recently recognized as a fundamental step to study cell responses and long-term tissue functionalities thanks to its ability to recapitulate the complexity and the dimensional scale of the cellular microenvironment, while directly integrating high-throughput and automatic screening capabilities.This chapter addresses the development of a poly(dimethylsiloxane)-based microfluidic platform to (1) generate and culture 3D cellular microaggregates under continuous flow perfusion while (2) conditioning them with different combinations/concentrations of soluble factors (i.e., growth factors, morphogens or drug molecules), in a high-throughput fashion. The proposed microfluidic system thus represents a promising tool for establishing innovative high-throughput models for drug screening, investigation of tissues morphogenesis, and optimization of tissue engineering protocols.

Generating Multicompartmental 3D Biological Constructs Interfaced through Sequential Injections in Microfluidic Devices.

A novel technique is presented for molding and culturing composite 3D cellular constructs within microfluidic channels. The method is based on the use of removable molding polydimethylsiloxane (PDMS) inserts, which allow to selectively and incrementally generate composite 3D constructs featuring different cell types and/or biomaterials, with a high spatial control. The authors generate constructs made of either stacked hydrogels, with uniform horizontal interfaces, or flanked hydrogels with vertical interfaces. The authors also show how this technique can be employed to create custom-shaped endothelial barriers and monolayers directly interfaced with 3D cellular constructs. This method dramatically improves the significance of in vitro 3D biological models, enhancing mimicry and enabling for controlled studies of complex biological districts.

Design of a microfluidic strategy for trapping and screening single cells.

Traditionally, in vitro investigations on biology and physiology of cells rely on averaging the responses eliciting from heterogeneous cell populations, thus being unsuitable for assessing individual cell behaviors in response to external stimulations. In the last years, great interest has thus been focused on single cell analysis and screening, which represents a promising tool aiming at pursuing the direct and deterministic control over cause-effect relationships guiding cell behavior. In this regard, a high-throughput microfluidic platform for trapping and culturing adherent single cells was presented. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. A round-shaped culture chamber (Φ = 250 µm, h = 25 µm) was conceived presenting two connections with a main fluidic path: (i) an upper wide opening, and (ii) a bottom trapping junction which modulates the hydraulic resistance. Starting from eight different layouts, the chamber geometry was computationally optimized for maximizing the single cell trapping efficacy and then integrated in a polydimethylsiloxane (PDMS) microfluidic device. The final platform consists in (i) 288 chambers for trapping single cells organized in six culture units, independently addressable through the lines of (ii) a chaotic-mixer based serial dilution generator (SDG), designed for creating spatio-temporally controlled patterns of both soluble factors and non-diffusive particles. The device was experimentally validated by trapping polystyrene microspheres, featuring diameters comparable to cell size (Φ = 10 µm).

VA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding.

The ability to replicate in vitro the native extracellular matrix (ECM) features and to control the three-dimensional (3D) cell organization plays a fundamental role in obtaining functional engineered bioconstructs. In tissue engineering (TE) applications, hydrogels have been successfully implied as biomatrices for 3D cell embedding, exhibiting high similarities to the natural ECM and holding easily tunable mechanical properties. In the present study, we characterized a promising photocrosslinking process to generate cell-laden methacrylate gelatin (GelMA) hydrogels in the presence of VA-086 photoinitiator using a ultraviolet LED source. We investigated the influence of prepolymer concentration and light irradiance on mechanical and biomimetic properties of resulting hydrogels. In details, the increasing of gelatin concentration resulted in enhanced rheological properties and shorter polymerization time. We then defined and validated a reliable photopolymerization protocol for cell embedding (1.5% VA-086, LED 2 mW/cm2) within GelMA hydrogels, which demonstrated to support bone marrow stromal cells viability when cultured up to 7 days. Moreover, we showed how different mechanical properties, derived from different crosslinking parameters, strongly influence cell behavior. In conclusion, this protocol can be considered a versatile tool to obtain biocompatible cell-laden hydrogels with properties easily adaptable for different TE applications.

Multi-gradient hydrogels produced layer by layer with capillary flow and crosslinking in open microchannels.

This technical note describes a new bench-top method for producing anisotropic hydrogels composed of gradient layers of soluble factors, particles, polymer concentrations or material properties. Each gradient layer was produced by a previous gradient method in which a droplet of one precursor solution was added to a thin layer of a second solution. The ensuing rapid capillary flow along the open channel generated a gradient precursor solution, which was then crosslinked to form a gradient gel. Repeating these steps allowed a layered gel to be iteratively constructed with as many gradient layers as desired. This technique renders the synthesis of multi-layered gradient gels accessible to virtually any researcher and should help simplify the production of more biologically relevant cellular microenvironments.

Microfabricated polyester conical microwells for cell culture applications.

Over the past few years there has been a great deal of interest in reducing experimental systems to a lab-on-a-chip scale. There has been particular interest in conducting high-throughput screening studies using microscale devices, for example in stem cell research. Microwells have emerged as the structure of choice for such tests. Most manufacturing approaches for microwell fabrication are based on photolithography, soft lithography, and etching. However, some of these approaches require extensive equipment, lengthy fabrication process, and modifications to the existing microwell patterns are costly. Here we show a convenient, fast, and low-cost method for fabricating microwells for cell culture applications by laser ablation of a polyester film coated with silicone glue. Microwell diameter was controlled by adjusting the laser power and speed, and the well depth by stacking several layers of film. By using this setup, a device containing hundreds of microwells can be fabricated in a few minutes to analyze cell behavior. Murine embryonic stem cells and human hepatoblastoma cells were seeded in polyester microwells of different sizes and showed that after 9 days in culture cell aggregates were formed without a noticeable deleterious effect of the polyester film and glue. These results show that the polyester microwell platform may be useful for cell culture applications. The ease of fabrication adds to the appeal of this device as minimal technological skill and equipment is required.

Anisotropic material synthesis by capillary flow in a fluid stripe.

We present a simple bench-top technique to produce centimeter long concentration gradients in biomaterials incorporating soluble, material, and particle gradients. By patterning hydrophilic regions on a substrate, a stripe of prepolymer solution is held in place on a glass slide by a hydrophobic boundary. Adding a droplet to one end of this "pre-wet" stripe causes a rapid capillary flow that spreads the droplet along the stripe to generate a gradient in the relative concentrations of the droplet and pre-wet solutions. The gradient length and shape are controlled by the pre-wet and droplet volumes, stripe thickness, fluid viscosity and surface tension. Gradient biomaterials are produced by crosslinking gradients of prepolymer solutions. Demonstrated examples include a concentration gradient of cells encapsulated in three dimensions (3D) within a homogeneous biopolymer and a constant concentration of cells encapsulated in 3D within a biomaterial gradient exhibiting a gradient in cell spreading. The technique employs coated glass slides that may be purchased or custom made from tape and hydrophobic spray. The approach is accessible to virtually any researcher or student and should dramatically reduce the time required to synthesize a wide range of gradient biomaterials. Moreover, since the technique employs passive mechanisms it is ideal for remote or resource poor settings.

Realization and efficiency evaluation of a micro-photocatalytic cell prototype for real-time blood oxygenation.

A novel, miniaturized, high-efficiency photocatalytic cell, able to work in dynamic conditions, has been designed and validated in this study. Microfluidic channels were molded out of polydimethylsiloxane (PDMS) by means of standard soft lithography techniques, so as to work as photocatalytic cells, where the coupling of anatase titanium dioxide thin films and platinum electrodes, allows an electrically assisted photocatalytic reaction to produce dissolved oxygen gas from the water content of flowing fluid (e.g. blood). The thin films were deposited onto quartz glass substrates at room temperature (300 K) using reactive radio-frequency sputtering with a titanium metal target. The photocatalytic activity was evaluated through reduction rate of methylene blue solution. The results of the current study, as a proof of concept, have shown that the device can generate oxygen at a rate of 4.06 µM O(2)/(cm(2)min), thus extending its possible application range to the full oxygenation of flowing venous blood.

How to embed three-dimensional flexible electrodes in microfluidic devices for cell culture applications.

This communication describes a simple, rapid and cost effective method of embedding a conductive and flexible material within microfluidic devices as a means to realize uniform electric fields within cellular microenvironments. Fluidic channels and electrodes are fabricated by traditional soft-lithography in conjunction with chemical etching of PDMS. Devices can be deformable (thus allowing for a combination of electro-mechanical stimulation), they are made from inexpensive materials and easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets.

Computational and functional evaluation of a microfluidic blood flow device.

The development of microfluidic devices supporting physiological blood flow has the potential to yield biomedical technologies emulating human organ function. However, advances in this area have been constrained by the fact that artificial microchannels constructed for such devices need to achieve maximum chemical diffusion as well as hemocompatibility. To address this issue, we designed an elastomeric microfluidic flow device composed of poly (dimethylsiloxane) to emulate the geometry and flow properties of the pulmonary microcirculation. Our chip design is characterized by high aspect ratio (width > height) channels in an orthogonally interconnected configuration. Finite element simulations of blood flow through the network design chip demonstrated that the apparent pressure drop varied in a linear manner with flow rate. For simulated flow rates <250 mul min, the simulated pressure drop was <2000 Pa, the flow was laminar, and hemolysis was minimal. Hemolysis rate, assayed in terms of [total plasma hemoglobin (TPH) (sample - control)/(TPH control)] during 6 and 12 hour perfusions at 250 mul/min, was <5.0% through the entire period of device perfusion. There was no evidence of microscopic thrombus at any channel segment or junction under these perfusion conditions. We conclude that a microfluidic blood flow device possessing asymmetric and interconnected microchannels exhibits uniform flow properties and preliminary hemocompatibility. Such technology should foster the development of miniature oxygenators and similar biomedical devices requiring both a microscale reaction volume and physiological blood flow.