Photodegradable Hydrogels Research Articles

Photodegradable hydrogels for external manipulation of cellular microenvironments with real-time monitoring

A designed hydrogel whose stiffness could not only be controlled but also monitored in situ by fluorescence.

Direct Gradient Photolithography of Photodegradable Hydrogels with Patterned Stiffness Control with Submicrometer Resolution.

Cell response to matrix mechanics is well-known; however, the ability to spatially pattern matrix stiffness to a high degree of control has been difficult to attain. This study describes the use of maskless photolithography as a flexible process for direct, noncontact gradient patterning of photodegradable hydrogels with custom graphics. Any input gray scale image can be used to directly chart hydrogel cross-link density as a function of spatial position. Hydrogels can be patterned with submicron resolution, with length scales within a single substrate spanning several orders of magnitude. A quantitative relationship between input grayscale image pixel intensity and output gel stiffness is validated, allowing for direct gradient patterning. Such physical gradient hydrogel constructs are rapidly produced in a highly controlled fashion with measured stiffness ranges and length scales that are physiologically relevant. Mesenchymal stem cells cultured on these physical gradients matrices congregate and align orthogonal to the gradient direction along iso-degraded lines. This approach results in a robust and high-throughput platform to answer key questions about cell response in heterogeneous physical environments.

Shape-Changing Photodegradable Hydrogels for Dynamic 3D Cell Culture.

Inspired by natural examples of swelling-actuated self-folding, we utilize photodegradable hydrogels as dynamically tunable, shape-changing scaffolds for culturing cells. Poly(ethylene glycol) diacrylate-based thin films incorporating ortho-nitrobenzyl (o-NB) moieties are transformed from flat 2D sheets to folded 3D structures by exposure to 365 nm UV light. As the UV light is attenuated through the thickness of the gel, a cross-link density gradient is formed. This gradient gives rise to differential swelling and a bending moment, resulting in gel folding. By tuning the UV light dose and the molar ratio of photodegradable to nondegradable species, both the initial degree of folding and the relaxation of tubular structures can be accurately controlled. These self-folding photodegradable gels were further functionalized with a cell-adhesive RGD peptide for both seeding and encapsulation of C2C12 mouse myoblasts. Light-induced folding of RGD functionalized hydrogels from flat sheets to tubular structures was demonstrated 1 or 3 days after C2C12 seeding. The C2C12s remained adhered on the inner walls of folded tubes for up to 6 days after folding. The minimum measured diameter of a tubular structure containing C2C12s was 1 mm, which is similar to the size of muscle fascicles. Furthermore, the viability of encapsulated C2C12s was not adversely affected by the UV light-induced folding. This is the first account of a self-folding material system that allows 2D-3D shape change in the presence of both seeded and encapsulated cells at a user-directed time point of choice.

Light-triggered RNA release and induction of hMSC osteogenesis via photodegradable, dual-crosslinked hydrogels.

To engineer a photodegradable hydrogel system for actively controlled release of bioactive unmodified RNA at designated time points to induce hMSC osteogenesis. RNA/polyethylenimine complexes were loaded into dual-crosslinked photodegradable hydrogels to examine the capacity of UV light application to trigger their release. The ability of released RNA to drive hMSC osteogenic differentiation was also investigated. RNA release from photodegradable hydrogels was accelerated upon UV application, which was not observed in non-photodegradable hydrogels. Regardless of the presence of UV light, released siGFP exhibited high bioactivity by silencing GFP expression in HeLa cells. Importantly, siNoggin or miRNA-20a released from the hydrogels induced hMSC osteogenesis. This system provides a potentially valuable physician/patient-controlled 'on-demand' RNA delivery platform for biomedical applications.

Cell biology and the "real world".

Cell biology and the "real world".

Development of photodegradable gelatin hydrogels and image analysis technique for automatic optical cell separation system based on the cellular morphology in embedding culture

Event Abstract Back to Event Development of photodegradable gelatin hydrogels and image analysis technique for automatic optical cell separation system based on the cellular morphology in embedding culture Masato Tamura1, Shinji Sugiura1, Taku Satoh1, Toshiyuki Kanamori1, Shibuta Mayu2, Kei Kanie2, Ryuji Kato2, Hirofumi Matsui3 and Masumi Yanagisawa4 1 National Institute of Advanced Industrial Science and Technology (AIST), Department of Life Science and Biotechnology, Japan 2 Nagoya University, Graduate School of Pharmaceutical Sciences, Japan 3 University of Tsukuba, Faculty of Medicine, Japan 4 Engineering System Co., Ltd., Japan This paper reports novel cell separation strategy and automated cell separation system based on the cellular morphology under 3D culture environment in the photodegradable hydrogel. Recently, we developed gelatin-based photodegradable hydrogels by NHS-activated-ester reaction [1], and applied this hydrogels to optical cell separation [2]. More recently, we developed “click-crosslinkable and photodegradable gelatin hydrogels” [3]. The present study applied this hydrogels to the most promising application, cell separation based on the cellular morphology. The present study includes proof concept of morphology-based cell separation and development of automatic cell separation system. 3D culture environment with a specific extracellular matrix regulates cellular function and phenotype. In addition, cancer cell morphology is alternated depending on its malignancy in 3D culture environment [4]. Recently we developed the predication model of stem cell differentiation by image analysis [5]. In this paper, we introduced this image analysis technique and the features of click-crosslinkable and photodegradable gelatin hydrogels to the automated morphology-based cell separation system in 3D culture environment in the above mentioned photodegradable gelatin hydrogels. Figure 1 shows the schematic procedure for optical cell separation based on the cellular morphology under 3D culture environment in the photodegradable hydrogel. Suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels (Figure 1a). After the culture in 3D environment (Figure 1b), microscopic images of the cells are captured. The captured images are analyzed to distinguish the target cells from the other cells by using the image analysis algorithm, which we previously developed for analyzing stem cells (Figure 2) [5]. The hydrogels around the target area is irradiated with UV light (Figure 1c). The cells in the irradiated area are collected by automated pipetting system (Figure 1d). We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection (Figure 3). We are currently developing an automated image analysis algorithm to distinguish cancer cells from normal cells under 3D environment. The automated optical cell separation system with image analysis algorithm will be applied to the establishment of novel cancer-cell lines from clinical samples such as biopsy tissue. KAKENHI (14J07186); NEDO (1009004)

Preparation of photodegradable polyacrylamide hydrogels via micellar copolymerization and determination of their phototunable elasticity and swelling behaviors

A photo-decomposable hydrophobic crosslinker was synthesized and utilized to obtain photo-tunable hydrogelsviafree radical micellar copolymerization.

User-programmable vasculature with 3D micron-scale resolution through hydrogel photodegradation

Event Abstract Back to Event User-programmable vasculature with 3D micron-scale resolution through hydrogel photodegradation Christopher K. Arakawa1 and Cole Deforest1, 2, 3, 4 1 University of Washington, Bioengineering, United States 2 University of Washington, Chemical Engineering, United States 3 University of Washington, Institute for Stem Cell and Regenerative Medicine, United States 4 University of Washington, Molecular Engineering & Sciences Institute, United States Introduction: The high metabolic demands of living tissue are satisfied by complex interconnected vascular networks capable of efficiently delivering oxygen and nutrients to, as well as removing waste from, all organs of the body. Though significant progress has been made over the last several decades in the isolation, maintenance, and expansion of cultured cells from a wide variety of sources, success towards the generation of 3D synthetic constructs containing physiologic cell densities and vascularized connectivity has been somewhat limited[1],[2]. While early strategies involving layer-by-layer assembly and bioprinting enable simplistic tissue architectures to be formed, robust strategies to engineer perfusable vasculature with biomimetic size scales and dimensionality would be of significant interest for both fundamental and translational biological studies. In this work, we outline a method based on hydrogel material photodegradation to enable for the first-time the generation of user-defined microvasculature with single-micron resolution able to support native cell densities in 3D. Materials: Photodegradable precursors were synthesized, purified by flash chromatography, and characterized by 1H and 13C NMR. Peptides were synthesized via microwave-assisted Fmoc peptide synthesis, purified by HPLC, and characterized by MALDI-ToF mass spectrometry. Human umbilical vein endothelial cells (HUVECs) were maintained at 37 °C and 5% CO2. Methods: Photodegradable polymer-peptide hydrogels[3]-[5] were formed upon reaction of a PEG tetra(cyclooctyne), a photodegradable bis(azide) polypeptide, and an azide-modified RGDS peptide. Multiphoton laser-scanning lithography was utilized to etch 3D vasculature of varying sizes within cell-laden biomaterials using pulsed focused laser light (λ = 740 nm). Networks were endothelialized with HUVECs. HUVEC viability and function was compared for networks of different geometries and vasculature sizes, under both static and perfusion culture conditions. Results and Discussion: Microvasculature networks with 10 – 250 μm diameter features were synthesized and demonstrated as perfusable, representing the first user-directed approach able to obtain capillary-sized vasculature (Fig. 1). Moreover, vascular features could be generated with full 3D structural control. HUVEC attachment readily occurred within 24 hours of cell seeding and complete endothelilization occurred by day 4 (Fig. 2). Vascular patterning and endothelization was performed in the presence of encapsulated cardiomyocytes, opening up the door to create dense endothelialized cardiac tissue. Fig. 1. Perfusability of photodegraded microchannels. A) 50 μm channels generated with full 3D control. B) 250, 100, 75, 50, 25, 10 μm diameter channels were generated. Fig. 2. Endothelialized channels. Complete vasculature endothelilization was observed 4 days after HUVEC seeding, imaged by phase-contrast light microscopy. HUVECs appear black, lining the vessel wall. Conclusion: We have developed a synthetic approach enabling user-defined 3D endothelialized microvasculature to be generated with single-micron resolution. We expect that these materials will prove useful in endothelial co-culture experiments, as well as in the eventual engineering of large-scale functional tissues.

Photodegradable gelatin-based hydrogels for 3D perfusion culture

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Click-crosslinkable and photodegradable gelatin hydrogels for cytocompatible optical cell manipulation in natural environment.

This paper describes the generation of “click-crosslinkable“ and “photodegaradable“ gelatin hydrogels from the reaction between dibenzocycloctyl-terminated photoclevable tetra-arm polyethylene glycol and azide-modified gelatin. The hydrogels were formed in 30 min through the click-crosslinking reaction. The micropatterned features in the hydrogels were created by micropatterned light irradiation; the minimum resolution of micropatterning was 10-μm widths for line patterns and 20-μm diameters for circle patterns. Cells were successfully encapsulated in the hydrogels without any loss of viability across a wide concentration range of crosslinker. In contrast, an activated-ester-type photocleavable crosslinker, which we previously used to prepare photodegradable gelatin hydrogels, induced a decrease in cell viability at crosslinker concentrations greater than 1.8 mM. We also observed morphology alteration and better growth of cancer cells in the click-crosslinked photodegradable gelatin hydrogels that included matrigel than in the absence of matrigel. We also demonstrated micropatterning of the hydrogels encapsulating cells and optical cell separation. Both of the cells that remained in the non-irradiated area and the cells collected from the irradiated area maintained their viability.

Bioactive Photodegradable Hydrogel for Cultivation and Retrieval of Embryonic Stem Cells

The development of a novel photodegradable heparin‐based hydrogel for cultivation and retrieval of embryonic stem cells is described. Mouse embryonic stem cells cultured atop the gel with encapsulated growth factors (GFs) express higher levels of differentiation markers compared to a standard protocol employing soluble GFs. Beyond improving differentiation of stem cells, the novel hydrogels can be used to release specific stem cell colonies without disturbing neighboring cells. This way, stem cell colonies can be retrieved at different time points and from different locations of the culture surface for polymerase chain reaction (PCR) analysis without the loss of the microenvironment context. The ability to retrieve some stem cell colonies without disturbing neighboring colonies will open possibilities for characterizing in‐dish heterogeneity of stem cell phenotype and will also allow to conserve cells/reagents. Overall, the bioactive photodegradable hydrogel developed in this study may offer new possibilities for cultivation and analysis of stem cells as well as other cell types.

A microsystem integrating photodegradable hydrogel microstructures and reconfigurable microfluidics for single-cell analysis and retrieval.

We developed a micropatterned photodegradable hydrogel array integrated with reconfigurable microfluidics to enable cell secretion analysis and cell retrieval at the single-cell level. The activity of protease molecules secreted from single cells was monitored using FRET peptides entrapped inside microfabricated compartments. Antibody-modified gel islands tethering cells to the surface could be degraded by UV exposure to release specific single cells of interest.

Facile One-step Micropatterning Using Photodegradable Methacrylated Gelatin Hydrogels for Improved Cardiomyocyte Organization and Alignment.

Hydrogels are often employed as temporary platforms for cell proliferation and tissue organization in vitro. Researchers have incorporated photodegradable moieties into synthetic polymeric hydrogels as a means of achieving spatiotemporal control over material properties. In this study protein-based photodegradable hydrogels composed of methacrylated gelatin (GelMA) and a crosslinker containing o-nitrobenzyl ester groups have been developed. The hydrogels are able to degrade rapidly and specifically in response to UV light and can be photopatterned to a variety of shapes and dimensions in a one-step process. Micropatterned photodegradable hydrogels are shown to improve cell distribution, alignment and beating regularity of cultured neonatal rat cardiomyocytes. Overall this work introduces a new class of photodegradable hydrogel based on natural and biofunctional polymers as cell culture substrates for improving cellular organization and function.

Partially photodegradable hybrid hydrogels with elasticity tunable by light irradiation

This paper reports a simple technique to synthesize elasticity tunable hybrid hydrogels using photocleavable (N-hydroxysuccinimide terminated photocleavable tetra-arm poly(ethylene glycol); NHS-PC-4armPEG) and non-photocleavable (N-hydroxysuccinimide terminated tetra-arm poly(ethylene glycol); NHS-4armPEG) activated-ester type crosslinkers. Partially photodegradable hybrid hydrogels were synthesized by reacting the crosslinker mixture with amino-terminated tetra-arm poly(ethylene glycol) (amino-4armPEG). The photocleavable crosslinks are cleaved by irradiating light while the non-photocleavable crosslinks remain intact, resulting in decreased elasticity. We demonstrate that hydrogel elasticity can be controlled by adjusting the ratio of photocleavable NHS-PC-4armPEG and non-photocleavable NHS-4armPEG, and by varying the light exposure energy. We also show how micropatterned elasticity can be obtained in the hydrogels by irradiating with micropatterned light. These techniques could provide a novel platform to tailor the elasticity of hydrogels with microscale precision for biological studies in the near future.

Activated-ester-type photocleavable crosslinker for preparation of photodegradable hydrogels using a two-component mixing reaction.

Photodegradable hydrogels have emerged as powerful platforms for studying and directing cellular behavior in a spatiotemporally controlled manner. Photodegradable hydrogels have previously been formed by free radical polymerizations, Michael-type addition reactions, and orthogonal click reactions. Here, an ester-activated photocleavable crosslinker is presented for preparing photodegradable hydrogels by means of a one-step mixing reaction between the crosslinker and a biocompatible polymer containing amino moieties (amino-terminated tetra-arm poly(ethylene glycol) or gelatin). It is demonstrated that photodegradable hydrogels micropatterned by photolithography can be used to culture cells with high viability and proliferation rates. The resulting micropatterned cell-laden structures can potentially be used to create 3D biomaterials for various tissue-engineering applications.

Design and characterization of a synthetically accessible, photodegradable hydrogel for user-directed formation of neural networks.

Hydrogels with photocleavable units incorporated into the cross-links have provided researchers with the ability to control mechanical properties temporally and study the role of matrix signaling on stem cell function and fate. With a growing interest in dynamically tunable cell culture systems, methods to synthesize photolabile hydrogels from simple precursors would facilitate broader accessibility. Here, a step-growth photodegradable poly(ethylene glycol) (PEG) hydrogel system cross-linked through a strain promoted alkyne-azide cycloaddition (SPAAC) reaction and degraded through the cleavage of a nitrobenzyl ether moiety integrated into the cross-links is developed from commercially available precursors in three straightforward synthetic steps with high yields (>95%). The network evolution and degradation properties are characterized in response to one- and two-photon irradiation. The PEG hydrogel is employed to encapsulate embryonic stem cell-derived motor neurons (ESMNs), and in situ degradation is exploited to gain three-dimensional control over the extension of motor axons using two-photon infrared light. Finally, ESMNs and their in vivo synaptic partners, myotubes, are coencapsulated, and the formation of user-directed neural networks is demonstrated.

Photodegradable hydrogels for capture, detection, and release of live cells.

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Photodegradable Hydrogels for Capture, Detection, and Release of Live Cells

AbstractCells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T‐cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV‐induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95 % purity of CD4 and CD8 T‐cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti‐CD4 and anti‐TNF‐α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence “halo” after immunofluorescent staining and could be retrieved by site‐specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Stem cell differentiation: sticky mechanical memory.

Physical cues from the extracellular environment influence the lineage commitment of stem cells. Now, experiments on human mesenchymal stem cells cultured on photodegradable hydrogels show that the cells' fate can also be determined by past physical environments.

Coumarin-Based Photodegradable Hydrogel: Design, Synthesis, Gelation, and Degradation Kinetics

The design, synthesis, and characterization of a new class of coumarin-based photodegradable hydrogels are reported. Hydrogel formation was achieved rapidly and efficiently under aqueous conditions using copper-catalyzed click chemistry, which afforded excellent control over the rate of network formation. Rapid photodegradation, to the point of reverse gelation, was observed using both 365 and 405 nm light, and micrometer-scale features were eroded using two-photon irradiation at wavelengths as long as 860 nm.