A Self-Folding Hydrogel In Vitro Model for Ductal Carcinoma.
ABSTRACT: A significant challenge in oncology is the need to develop in vitro models that accurately mimic the complex microenvironment within and around normal and diseased tissues. Here, we describe a self-folding approach to create curved hydrogel microstructures that more accurately mimic the geometry of ducts and acini within the mammary glands, as compared to existing three-dimensional block-like models or flat dishes. The microstructures are composed of photopatterned bilayers of poly (ethylene glycol) diacrylate (PEGDA), a hydrogel widely used in tissue engineering. The PEGDA bilayers of dissimilar molecular weights spontaneously curve when released from the underlying substrate due to differential swelling ratios. The photopatterns can be altered via AutoCAD-designed photomasks so that a variety of ductal and acinar mimetic structures can be mass-produced. In addition, by co-polymerizing methacrylated gelatin (methagel) with PEGDA, microstructures with increased cell adherence are synthesized. Biocompatibility and versatility of our approach is highlighted by culturing either SUM159 cells, which were seeded postfabrication, or MDA-MB-231 cells, which were encapsulated in hydrogels; cell viability is verified over 9 and 15 days, respectively. We believe that self-folding processes and associated tubular, curved, and folded constructs like the ones demonstrated here can facilitate the design of more accurate in vitro models for investigating ductal carcinoma.
Project description:The in situ fabrication of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microstructures within poly(dimethylsiloxane) (PDMS)-based microfluidic networks is a versatile technique that has enabled unique applications in biosensing, medical diagnostics, and the fundamental life sciences. Hydrogel structures have previously been patterned by the lithographic photopolymerization of PEGDA hydrogel forming solutions, a process that is confounded by oxygen-permeable PDMS. Here, we introduce an alternate PEG patterning technique that relies upon the optical sculpting of features by patterned light-induced erosion of photodegradable PEGDA deemed negative projection lithography. We quantitatively compared the hydrogel micropatterning fidelity of negative projection lithography to positive projection lithography, using traditional PEGDA photopolymerization, within PDMS devices. We found that the channel depth, the local oxygen atmosphere, and the UV exposure time dictated the size and resolution of hydrogel features formed using positive projection lithography. In contrast, negative projection lithography was observed to deliver high-resolution functional features with dimensions on the order of single micrometers enabled by its facilely controlled mechanism of feature formation that is insensitive to oxygen. Next, the utility of photodegradable PEGDA was further assessed by encapsulating or conjugating bioactive molecules within photodegradable PEG matrixes to provide a route to the formation of complex and dynamically reconfigurable chemical microenvironments. Finally, we demonstrated that negative projection lithography enabled photopatterning of multilayered microscale objects without the need for precise mask alignment. The described approach for photopatterning high-resolution photolabile hydrogel microstructures directly within PDMS microchannels could enable novel microsystems of increasing complexity and sophistication for a variety of clinical and biological applications.
Project description:A stereolithography-based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell-loaded) hydrogel bioinks, to achieve layer-by-layer multimaterial bioprinting. Compared to conventional stereolithography-based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell-laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high-fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms.
Project description:Poly(dimethylsiloxane) (PDMS) microstructures have been widely used in bio-microelectromechanical systems (bio-MEMS) for various types of analytical, diagnostic and therapeutic applications. However, PDMS-based soft lithographic techniques still use conventional microfabrication processes to generate a master mold, which requires access to clean room facilities and costly equipment. With the increasing use of these systems in various fields, the development of benchtop systems for fabricating microdevices is emerging as an important challenge in their widespread use. Here we demonstrate a simple, low-cost and rapid method to fabricate PDMS microstructures by using micropatterned poly(ethylene glycol) diacrylate (PEGDA) master molds. In this method, PEGDA microstructures were patterned on a glass substrate by photolithography under ambient conditions and by using simple tools. The resulting PEGDA structures were subsequently used to generate PDMS microstructures by standard molding in a reproducible and repeatable manner. The thickness of the PEGDA microstructures was controllable from 15 to 300 µm by using commonly available spacer materials. We also demonstrate the use of this method to fabricate microfluidic channels capable of generating concentration gradients. In addition, we fabricated PEGDA microstructures by photolithography from the light generated from commonly available laminar cell culture hood. These data suggest that this approach could be beneficial for fabricating low-cost PDMS-based microdevices in resource limited settings.
Project description:In this work, poly(ethylene glycol) diacrylate (PEGDA) molecules were grafted to silk fibroin (SF) molecules via a thiol-ene click reaction under 405 nm UV illumination for the fabrication of a PEGDA/SF composite hydrogel. The composite hydrogels could be prepared in a short and controllable gelation time without the use of a photoinitiator. Features relevant to the drug delivery of the PEGDA/SF hydrogels were assessed, and the hydrogels were characterized by various techniques. The results showed that the prepared PEGDA/SF hydrogels demonstrated a good sustained-release performance with limited swelling behavior. It was found that a prior cooling step can improve the compressive strength of the hydrogels effectively. Additionally, the MTT assay indicated the prepared PEGDA/SF hydrogel is non-cytotoxic. Subcutaneous implantation of the PEGDA/SF hydrogel in Kunming mice did not induce an obvious inflammation, which revealed that the prepared PEGDA/SF hydrogel possessed good biocompatibility. Furthermore, the mechanism of the gelation process was discussed.
Project description:Collagen hydrogels have been widely investigated as scaffolds for vascular tissue engineering due in part to the capacity of collagen to promote robust cell adhesion and elongation. However, collagen hydrogels display relatively low stiffness and strength, are thrombogenic, and are highly susceptible to cell-mediated contraction. In the current work, we develop and characterize a sequentially-formed interpenetrating network (IPN) that retains the benefits of collagen, but which displays enhanced mechanical stiffness and strength, improved thromboresistance, high physical stability and resistance to contraction. In this strategy, we first form a collagen hydrogel, infuse this hydrogel with poly(ethylene glycol) diacrylate (PEGDA), and subsequently crosslink the PEGDA by exposure to longwave UV light. These collagen-PEGDA IPNs allow for cell encapsulation during the fabrication process with greater than 90% cell viability via inclusion of cells within the collagen hydrogel precursor solution. Furthermore, the degree of cell spreading within the IPNs can be tuned from rounded to fully elongated by varying the time delay between the formation of the cell-laden collagen hydrogel and the formation of the PEGDA network. We also demonstrate that these collagen-PEGDA IPNs are able to support the initial stages of smooth muscle cell lineage progression by elongated human mesenchymal stems cells.
Project description:Hydrogels of 2-hydroxyethyl methacrylate/polyethylene glycol diacrylate (HEMA/PEGDA) have been extensively studied for their use in biomedical and pharmaceutical applications owing to their nontoxic and highly hydrophilic characteristics. Recently, cells immobilized by HEMA/PEGDA hydrogels have also been studied for enhanced production in fermentation. Hydrogel films of HEMA/PEGDA copolymer were generated by Ultraviolet (UV)-initiated photopolymerization. The hydrogel films were used to immobilize viable Lactobacillus brevis RK03 cells for the bioconversion of monosodium glutamate (MSG) to ?-aminobutyric acid (GABA). The mechanical properties and fermentation yields of the L. brevis RK03 cells immobilized on polyacrylate hydrogel films with different monomeric formulations were investigated. Fermentation was carried out in 75 mL de Man, Rogosa and Sharpe (MRS) medium containing various concentrations of MSG. We found that HEMA (93%)/PEGDA (3%) hydrogels (sample H) maximized GABA production. The conversion rate of MSG to GABA reached a maximum value of 98.4% after 240 h. Bioconversion activity gradually declined after 420 h to 83.8% after five cycles of semi-continuous fermentation. Our results suggest that HEMA (93%)/PEGDA (3%) hydrogels have great potential for use in GABA production via semi-continuous fermentation.
Project description:Microscale hydrogels have been shown to be beneficial for various applications such as tissue engineering and drug delivery. A key aspect in these applications is the spatial organization of biological entities or chemical compounds within hydrogel microstructures. For this purpose, sequentially patterned microgels can be used to spatially organize either living materials to mimic biological complexity or multiple chemicals to design functional microparticles for drug delivery. Photolithographic methods are the most common way to pattern microscale hydrogels but are limited to photocrosslinkable polymers. So far, conventional micromolding approaches use static molds to fabricate structures, limiting the resulting shapes that can be generated. Herein, we describe a dynamic micromolding technique to fabricate sequentially patterned hydrogel microstructures by exploiting the thermoresponsiveness of poly(N-isopropylacrylamide)-based micromolds. These responsive micromolds exhibited shape changes under temperature variations, facilitating the sequential molding of microgels at two different temperatures. We fabricated multicompartmental striped, cylindrical, and cubic microgels that encapsulated fluorescent polymer microspheres or different cell types. These responsive micromolds can be used to immobilize living materials or chemicals into sequentially patterned hydrogel microstructures which may potentially be useful for a range of applications at the interface of chemistry, materials science and engineering, and biology.
Project description:Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21?days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Voice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.
Project description:PEG-based hydrogels have become widely used as drug delivery and tissue scaffolding materials. Common among PEG hydrogel-forming polymers are photopolymerizable acrylates such as polyethylene glycol diacrylate (PEGDA). Microfluidics and microfabrication technologies have recently enabled the miniaturization of PEGDA structures, thus enabling many possible applications for nano- and micro- structured hydrogels. The presence of oxygen, however, dramatically inhibits the photopolymerization of PEGDA, which in turn frustrates hydrogel formation in environments of persistently high oxygen concentration. Using PEGDA that has been emulsified in fluorocarbon oil via microfluidic flow focusing within polydimethylsiloxane (PDMS) devices, we show that polymerization is completely inhibited below critical droplet diameters. By developing an integrated model incorporating reaction kinetics and oxygen diffusion, we demonstrate that the critical droplet diameter is largely determined by the oxygen transport rate, which is dictated by the oxygen saturation concentration of the continuous oil phase. To overcome this fundamental limitation, we present a nitrogen micro-jacketed microfluidic device to reduce oxygen within the droplet, enabling the continuous on-chip photopolymerization of microscale PEGDA particles.
Project description:The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a "photomask" (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and/or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches.