Functionality integration in stereolithography 3D printed microfluidics using a "print-pause-print" strategy.

Stereolithography 3D printing, although an increasingly used fabrication method for microfluidic chips, has the main disadvantage of producing monolithic chips in a single material. We propose to incorporate during printing various objects using a "print-pause-print" strategy. Here, we demonstrate that this novel approach can be used to incorporate glass slides, hydrosoluble films, paper pads, steel balls, elastic or nanoporous membranes and silicon-based microdevices, in order to add microfluidic functionalities as diverse as valves, fluidic diodes, shallow chambers, imaging windows for bacteria tracking, storage of reagents, blue energy harvesting or filters for cell capture and culture.

A versatile micromodel technology to explore biofilm development in porous media flows.

Bacterial biofilms that grow in porous media are critical to ecosystem processes and applications ranging from soil bioremediation to bioreactors for treating wastewater or producing value-added products. However, understanding and engineering the complex phenomena that drive the development of biofilms in such systems remains a challenge. Here we present a novel micromodel technology to explore bacterial biofilm development in porous media flows. The technology consists of a set of modules that can be combined as required for any given experiment and conveniently tuned for specific requirements. The core module is a 3D-printed micromodel where biofilm is grown into a perfusable porous substrate. High-precision additive manufacturing, in particular stereolithography, is used to fabricate porous scaffolds with precisely controlled architectures integrating flow channels with diameters down to several hundreds of micrometers. The system is instrumented with: ultraviolet-C light-emitting diodes; on-line measurements of oxygen consumption and pressure drop across the porous medium; camera and spectrophotometric cells for the detection of biofilm detachment events at the outlet. We demonstrate how this technology can be used to study the development of Pseudomonas aeruginosa biofilm for several days within a network of flow channels. We find complex dynamics whereby oxygen consumption reaches a steady-state but not the pressure drop, which instead features a permanent regime with large fluctuations. We further use X-ray computed microtomography to image the spatial distribution of biofilms and computational fluid dynamics to link biofilm development with local flow properties. By combining the advantages of additive manufacturing for the creation of reproducible 3D porous microarchitectures with the flow control and instrumentation accuracy of microfluidics, our system provides a platform to study the dynamics of biofilm development in 3D porous media and to rapidly test new concepts in process engineering.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples.

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor ß (TGFß) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

A microfluidic mechano-chemostat for tissues and organisms reveals that confined growth is accompanied with increased macromolecular crowding.

Conventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellular assemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they often do not allow the control of compressive forces emerging when cells proliferate in a confined environment. Here, we present a generic microfluidic device to control both chemical and mechanical compressive forces. This device relies on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable closed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression.

Rapid prototyping of a polymer MEMS droplet dispenser by laser-assisted 3D printing.

In this work, we introduce a polymer version of a previously developed silicon MEMS drop deposition tool for surface functionalization that consists of a microcantilever integrating an open fluidic channel and a reservoir. The device is fabricated by laser stereolithography, which offers the advantages of low-cost and fast prototyping. Additionally, thanks to the ability to process multiple materials, a magnetic base is incorporated into the cantilever for convenient handling and attachment to the holder of a robotized stage used for spotting. Droplets with diameters ranging from ∼50 µm to ∼300 µm are printed upon direct contact of the cantilever tip with the surface to pattern. Liquid loading is achieved by fully immersing the cantilever into a reservoir drop, where a single load results in the deposition of more than 200 droplets. The influences of the size and shape of the cantilever tip and the reservoir on the printing outcome are studied. As a proof-of-concept of the biofunctionalization capability of this 3D printed droplet dispenser, microarrays of oligonucleotides and antibodies displaying high specificity and no cross-contamination are fabricated, and droplets are deposited at the tip of an optical fiber bundle.

Modular microfluidic system for on-chip extraction, preconcentration and detection of the cytokine biomarker IL-6 in biofluid.

The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples.

Cell Aggregate Assembly through Microengineering for Functional Tissue Emergence.

Compared to cell suspensions or monolayers, 3D cell aggregates provide cellular interactions organized in space and heterogeneity that better resume the real organization of native tissues. They represent powerful tools to narrow down the gap between in vitro and in vivo models, thanks to their self-evolving capabilities. Recent strategies have demonstrated their potential as building blocks to generate microtissues. Developing specific methodologies capable of organizing these cell aggregates into 3D architectures and environments has become essential to convert them into functional microtissues adapted for regenerative medicine or pharmaceutical screening purposes. Although the techniques for producing individual cell aggregates have been on the market for over a decade, the methodology for engineering functional tissues starting from them is still a young and quickly evolving field of research. In this review, we first present a panorama of emerging cell aggregates microfabrication and assembly technologies. We further discuss the perspectives opened in the establishment of functional tissues with a specific focus on controlled architecture and heterogeneity to favor cell differentiation and proliferation.

Ion chromatograph with three-dimensional printed absorbance detector for indirect ultraviolet absorbance detection of phosphate in effluent and natural waters.

An ion chromatography system employing a low-cost three-dimensional printed absorbance detector for indirect ultraviolet detection towards portable phosphate analysis of environmental and industrial waters has been developed. The optical detection cell was fabricated using stereolithography three-dimensional printing of nanocomposite material. Chromatographic analysis and detection of phosphate were carried out using a CS5A 4 × 250 mm analytical column with indirect ultraviolet detection using a 255 nm light-emitting diode. Isocratic elution using a 0.6 mM potassium phthalate eluent combined with 1.44 mM sodium bicarbonate was employed at a flow rate of 0.75 mL/min. A linear calibration range of 0.5 to 30 mg/L PO4 3- applicable to environmental and wastewater analysis was achieved. For retention time and peak area repeatability, relative standard deviation values were 0.68 and 4.09%, respectively. Environmental and wastewater samples were analyzed with the optimized ion chromatography platform and the results were compared to values obtained by an accredited ion chromatograph. For the analysis of environmental samples, relative errors of <14 % were achieved. Recovery analysis was also carried out on both freshwater and wastewater samples and recovery results were within the acceptable range for water analysis using standard ion chromatography methods.

Wet spinning of a library of carbohydrate low molecular weight gels.

HYPOTHESIS: Recently, a low molecular weight hydrogel based on a carbohydrate alkyl amide has been successfully used as biomaterial for neuron cell culture and for 3D printing. Varying the molecular structure should make it possible to extend the library of carbohydrate low molecular weight hydrogels available for these applications and to improve their performances. EXPERIMENTS: Thirteen molecules easy to synthetize and designed to be potentially biocompatible were prepared. They are based on gluconamide, glucoheptonamide, galactonamide, glucamide, aliphatic chains and glycine. Their gelation in water was investigated in thermal conditions and wet spinning conditions, namely by dimethylsulfoxide-water exchange under injection. FINDINGS: Nine molecules give hydrogels in thermal conditions. By wet spinning, six molecules self-assemble fast enough, within few seconds, to form continous hydrogel filaments. Therefore, the method enables to shape by injection these mechanically fragile hydrogels, notably in the perspective of 3D printing. Depending on the molecular structure, persistent or soluble gel filaments are obtained. The microstructures are varied, featuring entangled ribbons, platelets or particles. In thermal gelation, molecules with a symmetrical polar head (galacto, glucoheptono) give flat ribbons and molecules with an asymmetrical polar head (gluco) give helical ribbons. The introduction of an extra glycine linker disturbs this trend.

In vitro models of intestinal epithelium: Toward bioengineered systems.

The intestinal epithelium, the fastest renewing tissue in human, is a complex tissue hosting multiple cell types with a dynamic and multiparametric microenvironment, making it particularly challenging to recreate in vitro. Convergence of recent advances in cellular biology and microfabrication technologies have led to the development of various bioengineered systems to model and study the intestinal epithelium. Theses microfabricated in vitro models may constitute an alternative to current approaches for studying the fundamental mechanisms governing intestinal homeostasis and pathologies, as well as for in vitro drug screening and testing. Herein, we review the recent advances in bioengineered in vitro intestinal models.

Colon Fibroblasts and Inflammation: Sparring Partners in Colorectal Cancer Initiation?

Colorectal cancer (CRC) is the third most common cause of cancer-related death. Significant improvements in CRC treatment have been made for the last 20 years, on one hand thanks to a better detection, allowing surgical resection of the incriminated area, and on the other hand, thanks to a better knowledge of CRC's development allowing the improvement of drug strategies. Despite this crucial progress, CRC remains a public health issue. The current model for CRC initiation and progression is based on accumulation of sequential known genetic mutations in the colon epithelial cells' genome leading to a loss of control over proliferation and survival. However, increasing evidence reveals that CRC initiation is more complex. Indeed, chronic inflammatory contexts, such as inflammatory bowel diseases, have been shown to increase the risk for CRC development in mice and humans. In this manuscript, we review whether colon fibroblasts can go from the main regulators of the ISC homeostasis, regulating not only the renewal process but also the epithelial cells' differentiation occurring along the colon crypt, to the main player in the initiation of the colorectal cancer process due to chronic inflammation.

In vitro bone metastasis dwelling in a 3D bioengineered niche.

Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

Tuning the Nanotopography and Chemical Functionality of 3D Printed Scaffolds through Cellulose Nanocrystal Coatings.

In nature, cells exist in three-dimensional (3D) microenvironments with topography, stiffness, surface chemistry, and biological factors that strongly dictate their phenotype and behavior. The cellular microenvironment is an organized structure or scaffold that, together with the cells that live within it, make up living tissue. To mimic these systems and understand how the different properties of a scaffold, such as adhesion, proliferation, or function, influence cell behavior, we need to be able to fabricate cellular microenvironments with tunable properties. In this work, the nanotopography and functionality of scaffolds for cell culture were modified by coating 3D printed materials (DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose nanocrystals (CNCs). This general approach was demonstrated on a variety of structures designed to incorporate macro- and microscale features fabricated using photopolymerization and 3D printing techniques. Atomic force microscopy was used to characterize the CNC coatings and showed the ability to tune their density and in turn the surface nanoroughness from isolated nanoparticles to dense surface coverage. The ability to tune the density of CNCs on 3D printed structures could be leveraged to control the attachment and morphology of prostate cancer cells. It was also possible to introduce functionalization onto the surface of these scaffolds, either by directly coating them with CNCs grafted with the functionality of interest or with a more general approach of functionalizing the CNCs after coating using biotin-streptavidin coupling. The ability to carefully tune the nanostructure and functionalization of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of natural microenvironments, which are key to understanding their impact on cells and developing artificial tissues.

Extracellular Matrix Mechanical Properties and Regulation of the Intestinal Stem Cells: When Mechanics Control Fate.

Intestinal stem cells (ISC) are crucial players in colon epithelium physiology. The accurate control of their auto-renewal, proliferation and differentiation capacities provides a constant flow of regeneration, maintaining the epithelial intestinal barrier integrity. Under stress conditions, colon epithelium homeostasis in disrupted, evolving towards pathologies such as inflammatory bowel diseases or colorectal cancer. A specific environment, namely the ISC niche constituted by the surrounding mesenchymal stem cells, the factors they secrete and the extracellular matrix (ECM), tightly controls ISC homeostasis. Colon ECM exerts physical constraint on the enclosed stem cells through peculiar topography, stiffness and deformability. However, little is known on the molecular and cellular events involved in ECM regulation of the ISC phenotype and fate. To address this question, combining accurately reproduced colon ECM mechanical parameters to primary ISC cultures such as organoids is an appropriated approach. Here, we review colon ECM physical properties at physiological and pathological states and their bioengineered in vitro reproduction applications to ISC studies.

3D deterministic lateral displacement (3D-DLD) cartridge system for high throughput particle sorting.

A new 3D architecture for the deterministic lateral displacement (DLD) microfluidic devices based on ultra-high aspect ratio arch shaped pillars is presented. The proposed system addresses the major flow rate and shear rate limitations of standard planar devices.

Multiplexed Remote SPR Detection of Biological Interactions through Optical Fiber Bundles.

The development of sensitive methods for in situ detection of biomarkers is a real challenge to bring medical diagnosis a step forward. The proof-of-concept of a remote multiplexed biomolecular interaction detection through a plasmonic optical fiber bundle is demonstrated here. The strategy relies on a fiber optic biosensor designed from a 300 µm diameter bundle composed of 6000 individual optical fibers. When appropriately etched and metallized, each optical fiber exhibits specific plasmonic properties. The surface plasmon resonance phenomenon occurring at the surface of each fiber enables to measure biomolecular interactions, through the changes of the retro-reflected light intensity due to light/plasmon coupling variations. The functionalization of the microstructured bundle by multiple protein probes was performed using new polymeric 3D-printed microcantilevers. Such soft cantilevers allow for immobilizing the probes in micro spots, without damaging the optical microstructures nor the gold layer. We show here the potential of this device to perform the multiplexed detection of two different antibodies with limits of detection down to a few tenths of nanomoles per liter. This tool, adapted for multiparametric, real-time, and label free monitoring is minimally invasive and could then provide a useful platform for in vivo targeted molecular analysis.

Sliding walls: a new paradigm for fluidic actuation and protocol implementation in microfluidics.

Currently, fluidic control in microdevices is mainly achieved either by external pumps and valves, which are expensive and bulky, or by valves integrated in the chip. Numerous types of internal valves or actuation methods have been proposed, but they generally impose difficult compromises between performance and fabrication complexity. We propose here a new paradigm for actuation in microfluidic devices based on rigid or semi-rigid walls with transversal dimensions of hundreds of micrometres that are able to slide within a microfluidic chip and to intersect microchannels with hand-driven or translation stage-based actuation. With this new concept for reconfigurable microfluidics, the implementation of a wide range of functionalities was facilitated and allowed for no or limited dead volume, low cost and low footprint. We demonstrate here several fluidic operations, including on/off or switch valving, where channels are blocked or reconfigured depending on the sliding wall geometry. The valves sustain pressures up to 30 kPa. Pumping and reversible compartmentalisation of large microfluidic chambers were also demonstrated. This last possibility was applied to a "4D" migration assay of dendritic cells in a collagen gel. Finally, sliding walls containing a hydrogel-based membrane were developed and used to concentrate, purify and transport biomolecules from one channel to another, such functionality involving complex fluidic transport patterns not possible in earlier microfluidic devices. Overall, this toolbox is compatible with "soft lithography" technology, allowing easy implementation within usual fabrication workflows for polydimethylsiloxane chips. This new technology opens the route to a variety of microfluidic applications, with a focus on simple, hand-driven devices for point-of-care or biological laboratories with low or limited equipment and resources.

Building a microfluidic cell culture platform with stiffness control using Loctite 3525 glue.

The study of mechanotransduction signals and cell response to mechanical properties requires designing culture substrates that possess some, or ideally all, of the following characteristics: (1) biological compatibility and adhesive properties, (2) stiffness control or tunability in a dynamic mode, (3) patternability on the microscale and (4) integrability in microfluidic chips. The most common materials used to address cell mechanotransduction are hydrogels, due to their softness. However, they may be impractical when complex scaffolds are sought and they lack viscous dissipative properties that are very important in mechanobiology. In this work, we show that an off-the-shelf, biocompatible photosensitive glue, Loctite 3525, may be used readily in mechanobiology assays without any special treatment prior to fabrication of cell culture platforms. Despite a high (MPa) stiffness easily tunable by UV exposure time at a fixed dose, 3T3 fibroblasts showed a response to the mechanics of the material similar to that obtained on much softer (kPa) hydrogels. Loctite's viscous dissipation properties indeed seemed to be responsible for such cell mechanical response, as suggested by recent works where more complex two-phase hydrogels were employed. More interestingly, it was possible to stiffen soft Loctite substrates by post-exposing them during cell culture, to observe changes in cell spreading caused by a dynamic stiffness modification. Thanks to Loctite 3525's patternability, micropillars were also fabricated to demonstrate the compatibility with traction force microscopy studies. Finally, the glue was used as an excellent adhesion layer for hydrogels on glass or PDMS, without the need for additional treatment, enabling the easy fabrication of microfluidic chips integrating hydrogels.

Fabrication of 3D scaffolds reproducing intestinal epithelium topography by high-resolution 3D stereolithography.

The small intestine is a complex tissue with a crypt/villus architecture and high tissue polarity. Maintenance of tissue integrity and function is supported by a constant renewal of the epithelium, with proliferative cells located in the crypts and differentiated cells migrating upward to the top of villi. So far, most in vitro studies have been limited to 2D surfaces or 3D organoid cultures that do not fully recapitulate the tissue 3D architecture, microenvironment and cell compartmentalization found in vivo. Here, we report the development of a 3D model that reproduces more faithfully the architecture of the intestinal epithelium in vitro. We developed a new fabrication process combining a photopolymerizable hydrogel that supports the growth of intestinal cell lines with high-resolution stereolithography 3D printing. This approach offers the possibility to create artificial 3D scaffolds matching the dimensions and architecture of mouse intestinal crypts and villi. We demonstrate that these 3D culture models support the growth and differentiation of Caco-2 cells for 3 weeks. These models may constitute a complementary approach to organoid cultures to study intestinal homeostasis by allowing guided self-organization and controlled differentiation, as well as for in vitro drug screening and testing.