Photo‐crosslinkable hydrogel‐based 3D microfluidic culture device

We developed the photo‐crosslinkable hydrogel‐based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo‐crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular‐shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel‐based 3D microfluidic device, showing that 53–75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo‐crosslinkable hydrogel‐based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.     

Characterizing the impact of thermal gels on isotachophoresis in microfluidic devices

Thermally reversible Pluronic gels have been employed as separation matrices in microfluidic devices in the analysis of biological macromolecules. The phase of these gels can be tuned between liquid and solid states using temperature to vary fluidic resistance and alter peak resolution. Although separations in thermal gels have been characterized, their effect on isotachophoresis has not. This study used fluorescein as a model analyte to evaluate isotachophoretic preconcentration as a function of thermal polymer concentration and temperature. Results demonstrated that increasing polymer concentration in microfluidic channels increased the apparent analyte concentration. A critical minimum of 10% (w/v) Pluronic was required to achieve efficient preconcentration with maximum focusing occurring in 20 and 25% polymer gels. Temperature of the thermal gel also impacted analyte focusing. Most efficient focusing was achieved at 25°C with diminishing analyte accumulation at higher and lower temperatures. Under optimal conditions, isotachophoretic preconcentration increased an additional threefold simply by including thermal gels in the system. This approach can be readily implemented in other applications to increase detection sensitivity and measure low-concentration analytes within simple microfluidic devices.     

Development of analytic microdevices for the detection of phenol using polymer hydrogel particles containing enzyme-QD conjugates

We present the fabrication of a microdevice for the detection of phenol by combining microfluidic channels and poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel microparticles containing tyrosinase-quantum dot conjugates. PHEMA hydrogel microparticles containing conjugates of enzyme (tyrosinase) and quantum dot (QD) were prepared by dispersion photopolymerization and entrapped within a microfilter-incorporated reaction chamber in a microfluidic channel. The fluorescence change, due to the fluorescence quenching effect caused by the enzyme reaction between phenol and tyrosinase, was used to detect phenol. The fluorescence intensity of PHEMA hydrogel microparticles containing tyrosinase-QD conjugates at 585 nm decreased with phenol concentration. In conclusion, the microfluidic channels fabricated in this study entrapping PHEMA hydrogel microparticles containing enzyme-QD conjugates show the potential to be used as an analytic microdevice for the detection of phenol.     

Rapid purification of cell encapsulated hydrogel beads from oil phase to aqueous phase in a microfluidic device

In this paper, we demonstrate a new type of microfluidic chip that can realize continuous-flow purification of hydrogel beads from a carrier oil into aqueous solution by using a laminar-like oil/water interface. The microfluidic chip is composed by two functional components: (1) a flow-focusing bead generation module that can control size and shape of beads, (2) a bead extraction module capable of purifying hydrogel beads from oil into aqueous solution. This module is featured with large branch channels on one side and small ones on the opposite side. Water is continuously infused into the bead extraction module through the large branch channels, resulting in a laminar-like oil/water interface between the branch junctions. Simulation and experimental data show that the efficiency of oil depletion is determined by the relative flow rates between infused water and carrier oil. By using such a microfluidic device, viable cells (HCT116, colon cancer cell line) can be encapsulated in the hydrogel beads and purified into a cell culture media. Significantly improved cell viability was achieved compared to that observed by conventional bead purification approaches. This facile microfluidic chip could be a promising candidate for sample treatment in lab-on-a-chip applications.     

A novel single-step fabrication technique to create heterogeneous poly(ethylene glycol) hydrogel microstructures containing multiple phenotypes of mammalian cells

In this study, a novel method for the one-step fabrication of stacked hydrogel microstructures using a microfluidic mold is presented. The fabrication of these structures takes advantage of the laminar flow regime in microfluidic devices, limiting the mixing of polymer precursor solutions. To create multilayered hydrogel structures, microfluidic devices were rotated 90 degrees from the traditional xy axes and sealed with a cover slip. Two discreet fluidic regions form in the channels, resulting in the multilayered hydrogel upon UV polymerization. Multilayered patterned poly(ethylene glycol) hydrogel arrays (60 mum tall, 250 mum wide) containing fluorescent dyes, fluorescein isothiocyanate, and tetramethylrhodamine isothiocyanate were created for imaging purposes. Additionally, this method was used to generate hydrogel layers containing murine fibroblasts and macrophages. The cell adhesion promoter, RGD, was added to hydrogel precursor solution to enhance fibroblast cell spreading within the hydrogel matrix in one layer, but not the other. We were able to successfully generate patterns of hydrogels containing multiple phenotypes by using this technique.     

Solvent bonding of poly(methyl methacrylate) microfluidic chip using phase-changing agar hydrogel as a sacrificial layer

In this report, a solvent bonding method based on phase-changing agar hydrogel has been developed for the fabrication of poly(methyl methacrylate) (PMMA) microfluidic chips. Prior to bonding, the channels and the reservoir ports on PMMA channel plates were filled with molten agar hydrogel that could gelate to form solid sacrificial layers at room temperature. Subsequently, PMMA cover sheets were covered on the channeled plates and 1,2-dichlororethane was applied to the interspaces between them. The agar hydrogel in the channels could prevent the bonding solvent and the softened surface of the PMMA cover sheets from filling in the channels. After solvent bonding, the agar hydrogel in the channels and the reservoir ports was melted and removed under pressure. The sealed channels in the complete microchips had been examined by an optical microscope and a scanning electron microscope. The results indicated that high-quality bonding was achieved at room temperature. The prepared microfluidic microchips have been successfully employed in the electrophoresis separation and detection of three cations in combination with contactless conductivity detection.     

Microfluidic hydrogel layers with multiple gradients to stimulate and perfuse three-dimensional neuronal cell cultures

We present a simple and easy to handle PDMS microfluidic device for neuronal cell culture studies in three-dimensional hydrogel scaffolds. The hydrogel is structured in parallel layers to reconstruct cell layers close to the natural environment. Dissociated cortical neurons of embryonic rats have been cultured in 0.5% w/v agarose including 0.2% w/v alginate. The cells formed neurite networks through neighboring cell free hydrogel layers. The cell culture showed neurite outgrowth in the microfluidic channel over more than seven days in vitro without perfusion. Culturing neurons in hydrogel layers surrounded by a liquid phase containing culture medium resulted in denser neuronal networks.     

Solid supports for micro analytical systems

The development of micro analytical systems requires that fluids are able to interact with the surface of the microfluidic chip in order to perform analysis such as chromatography, solid phase extraction, and enzymatic digestion. These types of analyses are more efficient if there are solid supports within the microfluidic channels. In addition, solid supports within microfluidic chips are useful in producing devices with multiple functionalities. In recent years there have been many approaches introduced for incorporating solid supports within chips. This review will explore several state of the art methods and applications of introducing solid supports into chips. These include packing chips with beads, incorporating membranes into chips, creating supports using microfabrication, and fabricating gels and polymer monoliths within microfluidic channels.     

Engineered alginate hydrogels for effective microfluidic capture and release of endothelial progenitor cells from whole blood

Microfluidic adhesion-based cell separation systems are of interest in clinical and biological applications where small sample volumes must be processed efficiently and rapidly. While the ability to capture rare cells from complex suspensions such as blood using microfluidic systems has been demonstrated, few methods exist for rapid and nondestructive release of the bound cells. Such detachment is critical for applications in tissue engineering and cell-based therapeutics in contrast with diagnostics wherein immunohistochemical, proteomic, and genomic analyses can be carried out by simply lysing captured cells. This paper demonstrates how the incorporation of four-arm amine-terminated poly(ethylene glycol) (PEG) molecules along with antibodies within alginate hydrogels can enhance the ability of the hydrogels to capture endothelial progenitor cells (EPCs) from whole human blood. The hydrogel coatings are applied conformally onto pillar structures within microfluidic channels and their dissolution with a chelator allows for effective recovery of EPCs following capture.     

In situ fabrication of macroporous polymer networks within microfluidic devices by living radical photopolymerization and leaching

Novel fabrication techniques and polymer systems are being explored to enable mass production of low cost microfluidic devices. In this contribution we discuss a new fabrication scheme for making microfluidic devices containing porous polymer components in situ. Contact lithography, a living radical photopolymer (LRPP) system and salt leaching were used to fabricate multilayer microfluidic devices rapidly with various channel geometries and covalently attached porous polymer plugs made of various photopolymerizable substrates. LRPP systems offer the advantages of covalent attachment of microfluidic device layers and facile surface modification via grafting. Several applications of the porous plugs are also explored, including a static mixer, a high surface area-to-volume reactor and a rapidly responding hydrogel valve. Quantitative and qualitative data show an increase in mixing of a fluorescein and a water stream for channels containing porous plugs relative to channels with no porous plugs. Confocal laser scanning microscopy images demonstrate the ability to graft a functional material onto porous plug surfaces. A reaction was carried out on the grafted pore surfaces, which resulted in fluorescent labelling of the grafted material throughout the pores of the plug. Homogenous fluorescence throughout the depth of the porous plug and along pore surfaces indicated that the porous plugs were surface modified by grafting and that reactions can be carried out on the pore surfaces. Finally, porous hydrogel valves were fabricated which swelled in response to contact with various pH solutions. Results indicate that a porous hydrogel valve will swell and close more rapidly than other valve geometries made with the same polymer formulation. The LRPP-salt leaching method provides a means for rapidly incorporating porous polymer components into microfluidic devices, which can be utilized for a variety of pertinent applications upon appropriate selection of porous plug materials and surface treatments.     

Fabrication of cell-containing hydrogel microstructures inside microfluidic devices that can be used as cell-based biosensors

This paper describes microfluidic systems containing immobilized hydrogel-encapsulated mammalian cells that can be used as cell-based biosensors. Mammalian cells were encapsulated in three-dimensional poly(ethylene glycol)(PEG) hydrogel microstructures which were photolithographically polymerized in microfluidic devices and grown under static culture conditions. The encapsulated cells remained viable for a week and were able to carry out enzymatic reactions inside the microfluidic devices. Cytotoxicity assays proved that small molecular weight toxins such as sodium azide could easily diffuse into the hydrogel microstructures and kill the encapsulated cells, which resulted in decreased viability. Furthermore, heterogeneous hydrogel microstructures encapsulating two different phenotypes in discrete spatial locations were also successfully fabricated inside microchannels.     

Development of Gelatin‐Alginate Hydrogels for Burn Wound Treatment

Hydrogels are interesting as wound dressing for burn wounds to maintain a moist environment. Especially gelatin and alginate based wound dressings show strong potential. Both polymers are modified by introducing photocrosslinkable functionalities and combined to hydrogel films (gel‐MA/alg‐MA). In one protocol gel‐MA films are incubated in alg‐MA solutions and crosslinked afterward into double networks. Another protocol involves blending both and subsequent photocrosslinking. The introduction of alginate into the gelatin matrix results in phase separation with polysaccharide microdomains in a protein matrix. Addition of alg(‐MA) to gel‐MA leads to an increased swelling compared to 100% gelatin and similar to the commercial Aquacel Ag dressing. In vitro tests show better cell adhesion for films which have a lower alginate content and also have superior mechanical properties. The hydrogel dressings exhibit good biocompatibility with adaptable cell attachment properties. An adequate gelatin‐alginate ratio should allow application of the materials as wound dressings for several days without tissue ingrowth.     

Poly(ethylene glycol) hydrogel microstructures encapsulating living cells

We present an easy and effective method for the encapsulation of cells inside PEG-based hydrogel microstructures fabricated using photolithography. High-density arrays of three-dimensional microstructures were created on substrates using this method. Mammalian cells were encapsulated in cylindrical hydrogel microstructures of 600 and 50 micrometers in diameter or in cubic hydrogel structures in microfluidic channels. Reducing lateral dimension of the individual hydrogel microstructure to 50 micrometers allowed us to isolate 1-3 cells per microstructure. Viability assays demonstrated that cells remained viable inside these hydrogels after encapsulation for up to 7 days.     

Lipase-catalysed stereoselective esterifications using gelatin-based hydrogels

A hydrogel stable in an organic solvent has been developed. This pseudo-solid aqueous gel (PAG) consists of only native gelatin and water, and has been used for immobilization of enzymes. A relatively high amount of gelatin is required in order to obtain stable gels. PAGs containing the enzyme Candida antarctica lipase (SP 525) were successfully used in catalysing the esterification of R/S-(±)-2-octanol and hexanoic acid in hexane. The conversions as well as the enantiomeric excess values of the product, R-(−)-2-octyl hexanoate, were high and comparable to those obtained with microemulsion-based gels. The PAGs containing immobilized lipase gave reproducible results and may be re-used several times. The gels are easy to prepare and use, non-toxic and biocompatible. The PAGs retain their integrity in organic solvents and may be used in preparative-scale synthesis of organic compounds.     

Engineered 3D tissue models for cell-laden microfluidic channels

Delivery of nutrients and oxygen within three-dimensional (3D) tissue constructs is important to maintain cell viability. We built 3D cell-laden hydrogels to validate a new tissue perfusion model that takes into account nutrition consumption. The model system was analyzed by simulating theoretical nutrient diffusion into cell-laden hydrogels. We carried out a parametric study considering different microchannel sizes and inter-channel separation in the hydrogel. We hypothesized that nutrient consumption needs to be taken into account when optimizing the perfusion channel size and separation. We validated the hypothesis by experiments. We fabricated circular microchannels (r = 400 μm) in 3D cell-laden hydrogel constructs (R = 7.5 mm, volume = 5 ml). These channels were positioned either individually or in parallel within hydrogels to increase nutrient and oxygen transport as a way to improve cell viability. We quantified the spatial distribution of viable cells within 3D hydrogel scaffolds without channels and with single- and dual-perfusion microfluidic channels. We investigated quantitatively the cell viability as a function of radial distance from the channels using experimental data and mathematical modeling of diffusion profiles. Our simulations show that a large-channel radius as well as a large channel to channel distance diffuse nutrients farther through a 3D hydrogel. This is important since our results reveal that there is a close correlation between nutrient profiles and cell viability across the hydrogel. Young Seok Song and Richard L. Lin have contributed equally to this contribution     

Reversible gelatin-based hydrogels: Finetuning of material properties

The present work reports on the synthesis and evaluation of a crosslinkable thiolated gelatin derivative. The effect of varying two parameters including the pH of the reaction buffer and the thiolating agent applied (i.e. N-acetylhomocysteine thiolactone versus Traut’s reagent) on the obtained modification degree was studied in a first part. The gelatin derivatives synthesized starting from N-acetylhomocysteine thiolactone and Traut’s reagent were characterized in depth using size exclusion chromatography and UV–VIS spectrophotometry. In a subsequent part of the present work, hydrogel films were prepared starting from the thiolated gelatin derivative developed using N-acetylhomocysteine thiolactone. The contributions of both the chemical and the physical crosslinking of the hydrogels developed were studied in depth using rheology, swelling experiments and texturometry. The results indicate that the physical structuring, inherent to gelatin, contributes to a large extent to the mechanical properties. However, the chemical crosslinking mostly determines the final hydrogel properties and can be controlled to a large extent. The gelatin-based gels are flexible, strong and transparent. A major advantage of disulfide-crosslinked hydrogels is the fact that the crosslinking is reversible. The latter could be interesting in view of future applications as cell carriers for tissue engineering.     

Synthesis and characterization of thermoresponsive poly(ethylene glycol)‐based hydrogels and their magnetic nanocomposites

Temperature‐responsive hydrogels are one of the most widely studied types of stimuli‐responsive hydrogel systems. Their ability to transition between their swollen and collapsed states makes them attractive for controlled drug delivery, microfluidic devices, and biosensor applications. Recent work has shown that poly(ethylene glycol) (PEG) methacrylate polymers are temperature‐responsive and exhibit a wide range of lower critical solution temperatures based on the length of ethylene glycol units in the macromer chain. The addition of iron oxide nanoparticles into the hydrogel matrix can provide the ability to remotely heat the gels upon exposure to an alternating magnetic field (AMF). In this work, diethylene glycol (n = 2) methyl ether methacrylate and PEG (n = 4.5) methyl ether methacrylate copolymers were polymerized into hydrogels with 5 mol % PEG 600 (n = 13.6) dimethacrylate as the crosslinker along with 5 wt % iron oxide nanoparticles. Volumetric swelling studies were completed from 22 to 80 °C and confirmed the temperature‐responsive nature of the hydrogel systems. The ability of the gels to collapse in response to rapid temperature changes when exposed to an AMF was demonstrated showing their potential use in biomedical applications such as controlled drug delivery and hyperthermia therapy. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3229–3235, 2010     

Harnessing Joule heating in microfluidic thermal gel electrophoresis to create reversible barriers for cell enrichment

Gel electrophoresis is a ubiquitous bioanalytical technique used to characterize the components of cell lysates. However, analyses of bulk lysates sacrifice detection sensitivity because intracellular biomolecules become diluted, and the liberation of proteases and nucleases can degrade target analytes. This report describes a method to enrich cells directly within a microfluidic gel as a first step toward online measurement of trace intracellular biomolecules with minimal dilution and degradation. Thermal gels were employed as the gel matrix because they can be reversibly converted between liquid and solid phases as a function of temperature. Rather than fabricate costly heating elements into devices to control temperature—and thus the phase of the gel—Joule heating was used instead. Adjoining regions of liquid-phase and solid-phase gel were formed within microfluidic channels by selectively inducing localized Joule heat. Cells migrated through the liquid gel but could not enter the solid gel—accumulating at the liquid–solid gel boundary—whereas small molecule contaminants passed through to waste. Barriers were then liquified on-demand by removing Joule heat to collect the purified, non-lysed cells for downstream analyses. Using voltage-controlled Joule heating to regulate the phase of thermal gels is an innovative approach to facilitate in-gel cell enrichment in low-cost microfluidic devices.     

Shape-controlled production of biodegradable calcium alginate gel microparticles using a novel microfluidic device

In this paper we describe a novel method of manufacturing shape-controlled calcium alginate gel microparticles in a microfluidic device. Both manufacturing shape-controlled microparticles and synthesizing hydrogel microparticles could be performed simultaneously in the microfluidic device. The novel microfluidic device comprised of two individual flow-focusing channels and a synthesizing channel was successfully applied as a continuous microfluidic reactor to synthesize gel microparticles with size and shape control. By passive control based on the microchannel geometric confinement and liquid-phase flow rates, we succeeded in producing monodisperse sodium alginate microparticles with diverse shapes (such as plugs, disks, microspheres, rods, and threads) in the flow-focusing channels of the microfluidic device. The shape and size of the sodium alginate microparticles could be tuned by adjusting the flow rates of the various streams. Further stages of the chemical reaction could be initiated by mixing sodium alginate microparticles and calcium chloride (CaCl2) solution in the synthesizing channel. The shapes of the sodium alginate microparticles could be permanently preserved by the synthesis of calcium alginate gel microparticles. The preparation conditions of size- and shape-controlled calcium alginate microparticles and influence factors were studied.