Hydrogel-based Microfluidic Devices Research Articles

Abstract 4577: An innovative platform to mimic the tumoral vascular microenvironment (TME) of patients with metastatic colorectal cancer (mCRC) using bioprinted hydrogel microfluidics

Abstract Introduction: Metastasis involves a set of complex events that finally result in malignant cells from primary tumors invading distant tissues by the pass of circulant tumor cells (CTC) through the blood circulation. To authentically comprehend the complex chain of events and even enable new therapeutic approaches in mCRC patients, ex-vivo functional models are an unmet research need. Experimental Plan: PROMISE project is combining bioengineering solutions such as bioprinting biomaterials and microfluidics to mimic the tumor microenvironment (TME) of metastatic sites in mCRC. In this project, we are developing a bioprinted hydrogel-based microfluidic device that recapitulates this TME. Then, CTCs are isolated from mCRC patients, cultured, and characterized by microdroplet technology. Those CTCs are then infused on the biodegradable polymer that mimics vascular channels and surrounding stromal cells, through microfluidic connection, in order to mimic CTC-endothelial interactions, study the procedures of extravasation (the invasiveness potential of extravasated patient-derived CTCs into TME) and intravasation (the process of CTCs-derived and biopsy-derived tumor spheroids from the TME to the vascular channel). The photopolymerizable hydrogel mimics vascular structures, supporting the growth of HUVEC cells, and is printed in a cost-effective manner (30 minutes) using a semi-direct printing method with visible light. The CROSS chip is used for fast and efficient isolation of CTCs from unprocessed blood of mCRC patients. In parallel, multi-omics of mCRC are done in circulant tumor DNA (ctDNA) and tumor biopsy of mCRC, therefore correlating the phenotypic and genetic profile of the patient cohorts studied. A first pilot test has been carried out successfully obtaining blood from mCRc refractory patients, and excellent coordination between the multidisciplinary consortium has been achieved to draw blood from the patient, isolate CTCs and infuse them into the hydrogel. Extravasation and intravasation studies are ongoing. Conclusion: This multidisciplinary project, which brings together bioengineering, microfluidic, and oncology experts’ teams, is developing a successful ex-vivo, in vitro, and dynamic model that will permit a better study and understanding of the metastatic process in mCRC, from the process of intravasation to extravasation of CTCs, passing thought the better multi-omic characterization of CTCs and ctDNA. Citation Format: Nadia Saoudi González, Francesc Salvà Ballabrera, Ariadna García, Javier Gonzalo-Ruiz, Iosune Baraibar, Javier Ros, Marta Rodriguez, María García Díaz, Marta Falcó Fusté, Melika Parchehbaf Kashani, Carlos Honrado, Alar Ainla, Josep Tabernero, Lorena Diéguez, Elena Martínez Fraiz, Elena Élez. An innovative platform to mimic the tumoral vascular microenvironment (TME) of patients with metastatic colorectal cancer (mCRC) using bioprinted hydrogel microfluidics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4577.

Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose–response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

A new insight into a thermoplastic microfluidic device aimed at improvement of oxygenation process and avoidance of shear stress during cell culture

Keeping the oxygen concentration at the desired physiological limits is a challenging task in cellular microfluidic devices. A good knowledge of affecting parameters would be helpful to control the oxygen delivery to cells. This study aims to provide a fundamental understanding of oxygenation process within a hydrogel-based microfluidic device considering simultaneous mass transfer, medium flow, and cellular consumption. For this purpose, the role of geometrical and hydrodynamic properties was numerically investigated. The results are in good agreement with both numerical and experimental data in the literature. The obtained results reveal that increasing the microchannel height delays the oxygen depletion in the absence of media flow. We also observed that increasing the medium flow rate increases the oxygen concentration in the device; however, it leads to high maximum shear stress. A novel pulsatile medium flow injection pattern is introduced to reduce detrimental effect of the applied shear stress on the cells.

CO2 laser fabrication of hydrogel-based open-channel microfluidic devices.

This study proposed a rapid and low-cost fabrication method for open-channel hydrogel-based microfluidic devices using CO2 laser ablation. The agarose hydrogel substrate was prepared with agarose gelation in DI water upon microwave heating, then a commercial CO2 laser system was used for the direct laser ablation of microchannels on the surface of agarose hydrogel substrate, the hydrophilic nature of the microchannels fabricated on hydrogel substrate enables the self-driven of the liquid inside the microchannels with capillary force. The profiles of the laser ablated microchannels on agarose hydrogel substrate with various laser power and scan speed were studied in detail. Due to the loss of water when exposed to the atmosphere, significant deformation of the fabricated microchannels was observed, and the profile change was recorded for 48h for comparison. An easy-to-access storage method of hydrogel-based microfluidic device in DI water was also proposed in this study. Unlike compact silicon or polymer-based microfluidic devices, the hydrogel is formed by cross-linked polymer chains filled with water, for a better understanding of the diffusion of small molecules into the bulk hydrogel material during fluid propagation inside the microchannel, the Nile red fluorescent was added into the liquid and the diffusion across the hydrogel-based microchannels with time was measured and discussed in this study. Several open-channel agarose hydrogel-based microfluidic devices were fabricated in this study for the demonstration of the proposed fabrication method. The CO2 laser ablation approach for agarose hydrogel-based microfluidic devices has the advantages of rapid processing time, low-cost, highly biocompatible, and self-driven without pumps and could have wide application potentials in biological and medical fields.

Design and characterization of hydrogel-based microfluidic devices with biomimetic solute transport networks.

Hydrogel could serve as a matrix material of new classes of solar cells and photoreactors with embedded microfluidic networks. These devices mimic the structure and function of plant leaves, which are a natural soft matter based microfluidic system. These unusual microfluidic-hydrogel devices with fluid-penetrable medium operate on the basis of convective-diffusive mechanism, where the liquid is transported between the non-connected channels via molecular permeation through the hydrogel. We define three key designs of such hydrogel devices, having linear, T-shaped, and branched channels and report results of numerical simulation of the process of their infusion with solute carried by the incoming fluid. The computational procedure takes into account both pressure-driven convection and concentration gradient-driven diffusion in the permeable gel matrix. We define the criteria for evaluation of the fluid infusion rate, uniformity, solute loss by outflow and overall performance. The T-shaped channel network was identified as the most efficient one and was improved further by investigating the effect of the channel-end secondary branches. Our parallel experimental data on the pattern of solute infusions are in excellent agreement with the simulation. These network designs can be applied to a broad range of novel microfluidic materials and soft matter devices with distributed microchannel networks.

Study on pH-sensitive hydrogel micro-valves: A fluid–structure interaction approach

Hydrogels are categorized as soft materials that undergo large deformation when they are subjected to even minor external forces. In this work, the performance of a variety of micro-valves, based on pH-sensitive hydrogel jackets coated on rigid pillars, is studied considering the gel deformation under fluid flow, employing fluid–structure interaction simulations. In this regard, an analytical solution to plane-strain inhomogeneous swelling of a cylindrical jacket is proposed. This is used as a tool to validate the finite element model. Then, a micro-valve consisting of one hydrogel jacket is studied in various inlet pressure and pH values performing fluid–structure interaction simulations. Thereafter, a variety of jacket patterns are investigated in order to identify the effects of the pattern on the micro-valve performance for various fluid stream pressures and pH values. The leakage pressure of the valves is also computed for each of the patterns. Fluid–structure interaction simulation is found to be essential to accurate design of the hydrogel-based microfluidic devices.

Engineering a "Self-Healing" Hydrogel-Based Microvasculature-on-a-Chip for Investigating the Effects of Cellular and Biomolecular Interactions on Endothelial Permeability in Sickle Cell Disease

Background: Endothelial activation and dysfunction play critical roles in vaso-occlusive crises and vasculopathy in sickle cell disease (SCD). However, it remains unclear how the myriad of cellular and biomolecular interactions that occur in SCD directly affect endothelial cell activation and injury, due largely in part to the lack of robust in vitro models for studying these complex biophysical processes. To this end, we engineered the first perfusable hydrogel-based microfluidic device comprised of "endothelialized" channels at the microvascular sizescale. Unlike typical microfluidic devices, which are silicone(PDMS)-based, this device is comprised of a collagen-based hydrogel that is not only more physiologic but also enables real-time monitoring of the endothelium permeability while allowing the user to tightly control hemodynamic conditions and the cellular and molecular components of the perfusate. Interestingly, in this system, upon removal of injurious stimuli, the endothelial cells are able to "self-heal" after injury and fully establish their barrier function. Using this system, we first tested whether interaction of SCD patient RBCs with endothelium under flow can directly cause endothelial permeability. We then studied how shear stress promotes endothelial cell activation and injury caused by free hemin, a byproduct of hemolysis in SCD.Results and Discussions: After seeding into the hydrogel-based device, human endothelial cells formed a monolayer that covered the entire inner surface of the microchannels and can be maintained for >1 month under flow conditions. Establishing that cultured endothelial cells are functional in this system, the cells appropriately formed continuous adherens junctions under flow as indicated by VE-cadherin staining (Figure 1A) and also deposited their own subendothelial extracellular matrices, including collagen IV (Figure 1B) and laminin (Figure 1C). Finally, perfusion of fluorescently-tagged albumin (BSA) sufficient endothelial barrier function of our system as all fluorescence signal was contained within the "vascular" space (Figure 1D and 1E).RBCs isolated from SCD patients were perfused into the endothelialized channels for 4 hours. Strikingly, the direct interaction of the perfused SCD RBCs with the engineered endothelium, in and of itself, was sufficient to induce endothelial permeability (Figure 2A), a phenomenon that did not occur with perfusion of control RBCs isolated from healthy volunteers. Interestingly, impermeability was reestablished as the endothelium "healed" 1 day post-interaction with patient RBCs (Figure 2B). We then perfused hemin in the channels under two different flow rates and the flow velocity profile, wall shear stress, shear rate, and pressure were characterized using COMSOL (Figure 3A and 3B). Interestingly, 1 hour of hemin exposure (10 µM) at higher shear stress compared to lower shear stress not only caused increased endothelial permeability and loss of endothelial cells at 1 day post-treatment, but also dampened the "healing" of injured endothelial cells and reestablishment of impermeability after removal of hemin from the system (Figure 3C and D).Conclusions and on-going work: Our physiologic hydrogel-based microvasculature-on-a-chip system enables investigation of how cell/molecular interactions directly affect endothelial permeability in real time and represents a milestone in the use of microfluidic devices for SCD research. In addition, the "self-healing" capability mimics the in vivo microvasculature and is a unique capability of this system as compared to current in vitro models. With this novel system, we determined that RBC-endothelial interactions under physiologic flow are sufficient to induce endothelial barrier dysfunction. We also demonstrated the additive role of hemodynamics in promoting hemin-induced endothelial activation and dysfunction. Ongoing work investigating the mechanisms of our observations will unveil insight into SCD pathogenesis and our system can be applied to other vascular diseases as well. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Facile fabrication processes for hydrogel-based microfluidic devices made of natural biopolymers.

We present facile strategies for the fabrication of two types of microfluidic devices made of hydrogels using the natural biopolymers, alginate, and gelatin as substrates. The processes presented include the molding-based preparation of hydrogel plates and their chemical bonding. To prepare calcium-alginate hydrogel microdevices, we suppressed the volume shrinkage of the alginate solution during gelation using propylene glycol alginate in the precursor solution along with sodium alginate. In addition, a chemical bonding method was developed using a polyelectrolyte membrane of poly-L-lysine as the electrostatic glue. To prepare gelatin-based microdevices, we used microbial transglutaminase to bond hydrogel plates chemically and to cross-link and stabilize the hydrogel matrix. As an application, mammalian cells (fibroblasts and vascular endothelial cells) were cultivated on the microchannel surface to form three-dimensional capillary-embedding tissue models for biological research and tissue engineering.

Alginate-based microfluidic system for tumor spheroid formation and anticancer agent screening

We demonstrate a microfluidic system for long-term tumor cell culture and drug testing. Three-dimensional cell culture is critical in characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Breast tumor cells were encapsulated within alginate which was gelled in situ within the microchannels. Tumor spheroid formation was observed several days after cell seeding, and various concentrations of doxorubicin were applied to the encapsulated cell aggregates. Drug effects on cell viability and proliferation were measured. In future, hydrogel-based microfluidic devices can comprise part of systems which replace labor intensive screening platforms currently implemented in the laboratory, and they address a need for improving preclinical testing of cancer cell sensitivity to anti-cancer drugs.

A hydrogel-based microfluidic device for the studies of directed cell migration

We have developed a hydrogel-based microfluidic device that is capable of generating a steady and long term linear chemical concentration gradient with no through flow in a microfluidic channel. Using this device, we successfully monitored the chemotactic responses of wildtype Escherichia coli (suspension cells) to alpha-methyl-DL-aspartate (attractant) and differentiated HL-60 cells (a human neutrophil-like cell line that is adherent) to formyl-Met-Leu-Phe (f-MLP, attractant). This device advances the current state of the art in microchemotaxis devices in that (1) it demonstrates the validity of using hydrogels as the building material for a microchemotaxis device; (2) it demonstrates the potential of the hydrogel based microfluidic device in biological experiments since most of the proteins and nutrients essential for cell survival are readily diffusible in hydrogel; (3) it is capable of applying chemical stimuli independently of mechanical stimuli; (4) it is straightforward to make, and requires very basic tools that are commonly available in biological labs. This device will also be useful in controlling the chemical and mechanical environment during the formation of tissue engineered constructs.