Pre-gel Solution Research Articles

Influence of gel preparation concentration on statistical mechanics of poly(dialkylaminoethyl methacrylate) gels on the basis of scaling concept: Toward tunable elasticity and thermomechanical parameters

ABSTRACTIn order to evaluate the scaling parameters of weakly cationic crosslinked network structures, poly(dialkylaminoethyl methacrylate)‐based hydrogels were synthesized via free‐radical crosslinking in aqueous solution varying systematically concentration of pregel solution. Based on the gel‐preparation concentration, variation in structural properties, effective crosslinking density, average molecular weight of polymer chains, and thermodynamic parameters from combined swelling and elasticity results were discussed using the scaling theory to predict various exponent identities. The concentration dependence of compressive elastic modulus as‐prepared state was described by a power‐law relationship with the exponent of m = 3.55 indicating the importance of the trapped entanglements. Two structural characteristics, the network chain length N and the average molecular weight of polymer chains have inverse dependence on the gel‐preparation concentration in the matrix, while the compressive moduli and effective crosslinking density show completely direct dependence. Experimentally determined N values of PDMAEMA hydrogels first decrease with increasing up to 0.2972 and the dependence of N on the gel‐preparation concentration gives the relation with a scaling parameter n = −1.80, which coincides with the prediction of scaling theory. Acceptable agreement was found between the estimate of crosslink density fluctuations deduced from mechanical measurements and the results derived from independent swelling observations. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48350.

4D Printing of Multi-Hydrogels Using Direct Ink Writing in a Supporting Viscous Liquid.

We propose a method to print four-dimensional (4D) stimuli-responsive hydrogel structures with internal gaps. Our 4D structures are fabricated by printing an N-isopropylacrylamide-based stimuli-responsive pre-gel solution (NIPAM-based ink) and an acrylamide-based non-responsive pre-gel solution (AAM-based ink) in a supporting viscous liquid (carboxymethyl cellulose solution) and by polymerizing the printed structures using ultraviolet (UV) light irradiation. First, the printed ink position and width were investigated by varying various parameters. The position of the printed ink changed according to physical characteristics of the ink and supporting liquid and printing conditions including the flow rates of the ink and the nozzle diameter, position, and speed. The width of the printed ink was mainly influenced by the ink flow rate and the nozzle speed. Next, we confirmed the polymerization of the printed ink in the supporting viscous liquid, as well as its responsivity to thermal stimulation. The degree of polymerization became smaller, as the interval time was longer after printing. The polymerized ink shrunk or swelled repeatedly according to thermal stimulation. In addition, printing multi-hydrogels was demonstrated by using a nozzle attached to a Y shape connector, and the responsivity of the multi-hydrogels to thermal-stimulation was investigated. The pattern of the multi-hydrogels structure and its responsivity to thermal-stimulation were controlled by the flow ratio of the inks. Finally, various 4D structures including a rounded pattern, a spiral shape pattern, a cross point, and a multi-hydrogel pattern were fabricated, and their deformations in response to the stimuli were demonstrated.

Sucrose Acetate Isobutyrate as an In situ Forming Implant for Sustained Release of Local Anesthetics.

In this study, an injectable Sucrose Acetate Isobutyrate (SAIB) drug delivery system (SADS) was designed and fabricated for the sustained release of Ropivacaine (RP) to prolong the duration of local anesthesia. By mixing SAIB, RP, and N-methyl-2-pyrrolidone, the SADS was prepared in a sol state with low viscosity before injection. After subcutaneous injection, the pre-gel solution underwent gelation in situ to form a drug-released depot. The in vitro release profiles and in vivo pharmacokinetic analysis indicated that RP-SADS had suitable controlled release properties. Particularly, the RP-SADS significantly reduced the initial burst release after subcutaneous injection in rats. In a pharmacodynamic analysis of rats, the duration of nerve blockade was prolonged by over 3-fold for the RP-SADS formulation compared to RP solution. Additionally, RP-SADS showed good biocompatibility in vitro and in vivo. Thus, the SADS-based depot technology is a safe drug delivery strategy for the sustained release of local anesthetics with long-term analgesia effects.

Conductive Polymer Hydrogel Microfibers from Multiflow Microfluidics.

Conductive hydrogels are receiving increasing attention for their utility in electronic area applications requiring flexible conductors. Here, it is presented novel conductive hydrogel microfibers with alginate shells and poly (3, 4-ethylenedioxythiophene): poly (4-styrenesulfonate) (PEDOT: PSS) cores fabricated using a multiflow capillary microfluidic spinning approach. Based on multiflow microfluidics, alginate shells are formed immediately from the fast gelation reaction between sodium alginate (Na-Alg) and sheath laminar calcium chloride flows, while PEDOT: PSS cores are solidified slowly in the hollow alginate hydrogel shell microreactors after their precursor solutions are injected in situ as the center fluids. The resultant PEDOT: PSS-containing microfibers are with features of designed morphology and highly controllable package, because material compositions or the sizes of their shell hydrogels can be tailored by using different concentrations or flow rates of pregel solutions. Moreover, the practical values of these microfibers in stretch sensitivity and bending stability are explored based on various electrical characterizations of the compound materials. Thus, it is believed that these microfluidic spinning PEDOT: PSS conductive microfibers will find important utility in electronic applications requiring flexible electronic systems.

Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model

In the last decades, the decellularization (DC) of organs has become an established technique in the field of regenerative medicine to yield complex and vascularized bioscaffolds. Furthermore, it has been demonstrated in vitro that these decellularized scaffolds retain their native tissue-specificity. This is also the case when this tissue-specific extracellular matrix (ECM) is solubilized and used as hydrogels or coatings to create a biomimetic environment. In this study we investigated if this specificity not only remains when applied to distinct tissues but even more, that these differences can be distinguished within the same tissue at different stages of proliferation. To address this question, a sensitive in vitro animal model was used: rabbit embryos at the third day of development were cultured on coatings made from acellular endometrium that was non-proliferating (non-synchronous, NS) and proliferating (synchronous with the embryo, S) and their development was compared.For this, we obtained whole NS and S rabbit uteri and subjected them to an adapted decellularization protocol. The acellular endometrium was carefully separated by microdissection and converted into a pre-gel solution to be used as hydrogels and coatings for in vitro assays. First, the characteristics of these NS and S hydrogels were investigated by proteomic analysis, electron microscopy and gelling kinetics. When used as substrata for day 3 embryos culture, it became apparent that only the acellular ECM from synchronous endometrial coating achieved similar results to the gold standard culture protocols and conditions, possibly because of the slow release of growth factors present in the synchronous/proliferating endometrium. Statement of SignificanceIt has been shown by in vitro culture of stem cells, progenitor cells and primary culture cells that decellularized tissues retain their specific functions and biochemical and structural compositions. The present work demonstrates that using a mild SDS and perfusion based decellularization (DC) protocol not only effectively decellularize whole rabbit uteri, adding to the growing field of reproductive tissue engineering, but more importantly that the differences in the proliferating endometrium are translated after DC. This implies that DC not only retains the interspecificity of tissues but also the intraspecificity of a developing hormonally stimulated tissue. For the first time, we demonstrate that the coating from decellularized synchronous endometrium acts as a biological support for in vitro embryo development, achieving comparable results with the current gold standard that only uses serum-containing media.

Hertzian elasticity and triggered swelling kinetics of poly(amino ester)-based gel beads with controlled hydrophilicity and functionality: A mild and convenient synthesis via dropwise freezing into cryogenic liquid

Poly(amino ester)-based homo- and co-polymeric gel beads were prepared via “dropwise freezing into cryogenic liquid” method and multi-responsive properties of the prepared spherical beads were confirmed by the pulsatile swelling kinetics and the stress-strain relationships based on Hertzian elasticity. Dual pH- and ionic strength-responsive homopolymeric PDMAEMA as well as copolymeric poly(hydroxyethyl methacrylate-co-dimethylaminoethyl methacrylate) P(HEMA-co-DMAEMA) gel beads crosslinked with diethyleneglycol dimethacrylate (DEGDMA) were prepared by aqueous phase crosslinking copolymerization at subzero temperature within the millimeter-sized frozen droplets in the paraffin oil as continuous phase. With this method, spherical ionic gel beads of sizes 2–5 mm could be synthesized without using a stabilizer. The successful synthesis of PDMAEMA and P(HEMA-co-DMAEMA) gel beads was confirmed by ATR-FTIR and SEM. Modulus of elasticity of cross-linked poly(amino ester)-based gel beads in swollen state was measured as a function of the bead diameter. The relationship between the compressive elastic force and corresponding deformation was obtained to compare the experimental results with theoretical-analytical formulations using the constitutive relations from Hertzian elasticity. The effective crosslink density of gel beads produced in the same synthesis batch increased as a function of increasing bead diameter, which was attributed to the formation conditions of the beads via frozen droplets of the pre-gel solution. The synthesis pathway used for the production of gel beads from methacrylate-based monomer with a polar tertiary amino group offered one-way control over material geometry to design soft and smart materials with controlled size and porosity.

Post-resection treatment of glioblastoma with an injectable nanomedicine-loaded photopolymerizable hydrogel induces long-term survival

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor. Despite available therapeutic options, the prognosis for patients with GBM remains very poor. We hypothesized that the intra-operative injection of a photopolymerizable hydrogel into the tumor resection cavity could sustain the release of the anti-cancer drug paclitaxel (PTX) encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles and prevent GBM recurrence. The tumor was resected 13 days after implantation and a pre-gel solution composed of polyethylene glycol dimethacrylate (PEG-DMA) polymer, a photoinitiator and PTX-loaded PLGA nanoparticles (PTX PLGA-NPs) was injected into the tumor resection cavity. A solid gel filling the whole cavity was formed immediately by photopolymerization using a 400 nm light. PTX in vitro release study showed a burst release (11%) in the first 8 h and a sustained release of 29% over a week. In vitro, U87 MG cells were sensitive to PTX PLGA-NPs with IC50 level of approximately 0.010 μg/mL. The hydrogel was well-tolerated when implanted in the brain of healthy mice for 2 and 4 months. Administration of PTX PLGA-NPs-loaded hydrogel into the resection cavity of GBM orthotopic model lead to more than 50% long-term survival mice (150 days) compared to the control groups (mean survival time 52 days). This significant delay of recurrence is very promising for the post-resection treatment of GBM.

Preparation of Multiple Parallel Protein-polysaccharide Hybrid Gel Filaments Under Shear

Fabrication of anisotropic gel with characteristic structures has been reported using a flow system with three dimensional microfluidic devices. Such gel structures are mainly achieved by design and preparation of the microchannel for flowing pre-gel solution. In this research, we propose a method for preparing multiple parallel gel filaments by controlling viscoelastic properties of polymer assembly as the fluid of microfluidic devices. To promote the formation of heterogeneous assembly of different types of macromolecules, an aqueous two-phase separation (ATPS) technique was utilized. Two layers of aqueous solutions appear when the ATPS conditions including ionic strength, temperature, and solute concentration in certain combinations of two components are satisfied. In respective phase, some solute molecules form heterogeneous assemblies because the solutes in respective phase are highly concentrated. We previously reported that the alginate (Alg) could form the assembly with polyethylene glycol (PEG) in the induced phase of Alg/PEG/NaCl system. In aqueous media, the Alg assembly has the rigid structure because diaxial linkages in a successive region of guluronic acid are stabilized by hydrogen bonds. Destruction of hydrogen bonds of Alg molecules by interaction with PEG enables deformation of Alg assembly by shear. Gel filaments were prepared with a co-axial flow device as double glass tubes, where an inner tube (inner diameter 0.7 mm) was fixed at the insert position of the outer tube. Pre-gel solution and crosslinker solution respectively were flown to inner and outer tube using syringe pumps. The morphology of the gel filaments was observed by a scanning electron microscope. A single thick gel filament was obtained from aqueous sodium alginate solution, whereas multiple thin gel filaments were obtained when a mixed solution of Alg and PEG under the ATPS condition. Appearance of shear-thinning behavior for the Alg-rich phase suggests that the complexation of Alg with PEG was responsible for destruction of the rigid assembly due to breaking hydrogen bonds. In this poster, the method will be applied to the protein containing system to fabricate the protein-polysaccharide hybrid filaments.

(Invited) Micro-Manipulation of Hydrogel Sheet through Photoresponsive Polymer Dissolution

Various functional hydrogels have been developed, and their applications to the fields of bioengineering have been examined actively. In these situations, we developed novel technologies to manipulate hydrogel sheet and living cells through photoresponsive polymer dissolution. Firstly, we proposed the fabrication of unique hydrogel microstructures through photolithography. As a photo-acid-generation (PAG) polymer, we synthesized a poly(methylmethacrylate) functionalized with visible- light-responsive PAG group (pPAGMMA), and spin-coated polystyrene surface with the polymer solution. Then we overcoated pregel solution containing hydrophilic polymer (hydroxypropyl cellulose) (HPC) and 1,3,4,6-Tetrakis(methoxymethyl)glycoluril, and carried out micro-patterned light projection. After post-bake and flushing, we obtained crosslinked hydrophilic polymer sheet only in the irradiated area. Due to another property of pPAGMMA getting dissolved in ethanol/water mixture by further irradiation, the cross-linked hydrophilic polymer sheet was lifted from the substrate surface and became swollen in the desired areas by the second micro-patterned projection in ethanol/water mixture. As a result, we obtained various microstructures which were composed of patterned swollen hydrogel sheet partially attached to the substrate in the patterned areas. Secondly, we developed a novel photoresponsive polymer materials by functionalizing poly(N-isopropylacrylamide) with o-nitrobenzaldehyde (pNBANIPAAm) to implement in situ photocontrol in aqueous systems containing bio-compatible environment. We observed that the polymer exhibited sharp aqueous dissolution upon light irradiation in wide pH range including biological conditions. We prepared closs-linked HPC (cHPC) layer on pNBANIPAAm thin layer and carried out micropatterned light projection in PBS. As a result, we observed that the cHPC layer lifted off of the substrate and swelled while nothing happened in unirradiated areas. Also living cells were manipulated by light in a similar manner.

(Invited) Shear-Induced Fabrication of Multiple Parallel Gel Filaments Using Aqueous Two-Phase Separation

Macroscopic structures of gel fibers have been prepared by control of the polymer solution properties and by design of the nozzle and flow path of the microfluidic device. In this research, we investigated a method to simultaneously fabricate hundreds to thousands of gel filaments simply by flowing the polymer solution inside the capillary without spinneret. An aqueous two-phase separation (ATPS) can be induced by adjusting their concentration in certain combinations of two polymers. The respective polymer concentration in each phase can be determined from the phase diagram, whereas an assembly of heterogeneous polymers generate in each phase. Hydrodynamic properties of pre-gel solution can be controlled by inducing such heterogeneous polymer assemblies. A co-axial flow double capillary was used to fabricate gel filaments, where an inner capillary (inner diameter 0.7 mm) was fixed at the insert position of an outer capillary. To induce ATPS, an ionic strength for the aqueous solution of sodium alginate (Alg) / polyethylene glycol (PEG) mixture was controlled by NaCl addition. The pre-gel Alg/PEG solution was flown as an inner flow of glass double capillaries, where a crosslinker solution (Ca2+ soln.) was flown as a sheath flow. The obtained gel was composed of multiple parallel gel filaments with a diameter of several micrometers, while a single gel without filament structure was obtained from the homogeneous solution without NaCl addition. In aqueous solution, the Alg chain has a structure with few entanglements because of its rigidity. The rigid assembly of alginate is mainly caused by guluronic acid block, that is stabilized by diaxial linkages via intramolecular hydrogen bonds. The shear stress generated by flowing the aqueous Alg solution inside the capillary cannot induce deformation of its rigid assembly, results in the single thick gel filament. By complexation with PEG, destruction of regular hydrogen bond was responsible for breaking the rigid structure of Alg. Thus, deformation of Alg/PEG assembly was enabled to elongate as the fibrous structure by the shear stress, followed by gelation in contact with Ca2+ ions at the outlet of the inner capillary. Such shear-induced deformation of the Alg/PEG assembly was studied by measuring viscoelastic properties with a spindle viscometer. Besides, Alg/PEG assembly generation in each phase was determined by size-exclusion chromatography with a multi angle light scattering detector. This presentation will discuss a simple technique for preparing multiple gel filaments simply by passing a polymer solution through a capillary.

Spring-shaped stimuli-responsive hydrogel actuator with large deformation

This study describes a novel microfluidics-based method for compressive/expanding actuation of stimuli-responsive hydrogel microsprings with large deformations. A continuous flow of mixed alginate and poly(N-isopropylacrylamide-co-acrylic acid) pre-gel solution can spontaneously form a hydrogel microspring with a wide range of gradient pitches via buoyancy force. This technique enables fabrication of hydrogel microsprings using only simple capillaries and syringe pumps. The resulting microsprings can be patterned via laminar flow inside the capillary, which can contribute to large deformation. Single-layered hydrogel microsprings shrunk isotropically while maintaining the shape of the spring. Compressing stimuli-responsive microsprings can be done by patterning the shrinking part of the spring. Here, the degree of compression in the double-layered spring depends on the initial pitch. Furthermore, large axial expansion of microsprings can be achieved by shrinking part of a microspring. Our large compression/expansion stimuli-responsive hydrogel microsprings have immense potential to be applied in various microengineering products including soft actuators, chemical sensors, and medical applications.

Responsive graphene oxide hydrogel microcarriers for controllable cell capture and release

Cell microcarriers have emerged as a powerful cell culture platform in biomedical areas, but their functions are usually limited to simply capturing and proliferating cells, because of the simplicity of their components. Thus, in this study, we developed a new near-infrared (NIR) light-responsive graphene oxide (GO) hydrogel microcarrier system for controllable cell culture. The microcarriers were generated by using capillary microfluidics to emulsify the GO dispersed poly( N -isopropylacrylamide) (pNIPAM) and gelatin methacrylate (GelMA) pre-gel solution. The composite GO hydrogel microcarriers exhibited photothermally responsive cell capture, as well as the capacity for proliferation and release due to the NIR absorption of GO, the thermally responsive shape transition of pNIPAM, and the high biocompatibility of GelMA. It was found that the NIR-responsive GO hydrogel microcarriers could prevent the cultured cells from being attacked by the immune system and promote the formation of tumor models in immunocompetent mice, which is desired for tumor and drug research. These features make the NIR-responsive GO hydrogel microcarriers excellent functional materials for different biomedical applications.

Structure–property relations for temperature-responsive gels

A model is developed for equilibrium unconstrained swelling of thermo-responsive gels that takes into account formation of hydrophobic aggregates above the volume phase transition temperature (VPTT). The solid phase of a gel is treated as a polymer network with two types of junctions: covalent bonds that are formed under cross-linking polymerization of a pre-gel solution, and physical junctions that are developed due to aggregation of hydrophobic segments above VPTT. Adjustable parameters are found by fitting experimental data on poly(N-isopropylacrylamide) gels with various molar fractions of monomers and cross-linker and various routes of cross-linking (chemical reaction versus irradiation), furnished with sacrificial chains in pre-gel solutions, and synthesized under phase separation condition in mixed solvents. The ability of the model to describe equilibrium swelling diagrams is confirmed by numerical simulation.

Tailoring the physico-chemical properties and elasticity of poly(hydroxy-functional methacrylate)-based cationically charged gel beads: Combined hydrophobicity and mechanical durability through frozen droplets

This study describes the experimental methodology to obtain hydroxy-functional methacrylate-based gel beads in millimeter-sized under production conditions by addition of the frozen pre-gel solution into the silicon oil as continuous phase using tetraethyleneglycol dimethacrylate (TEGDMA) as the crosslinking agent. Weakly basic cationic comonomer N,N-dimethylaminoethyl methacrylate (DMAEMA) was included into the polymerization procedure for the production of uniform polycationic moities exhibiting a drastic response against pH. The aim of this work is to determine the actual conditions for optimum bead production and to provide poly(hydroxypropyl methacrylate-co-N,N-dimethylaminoethyl methacrylate)-based P(HPMA-co-DMAEMA) gel beads with simple tools for the physical characterization. By the proposed experimental method, the physico-chemical properties of P(HPMA-co-DMAEMA) gel beads can be controlled by adjusting the pH, temperature of solution, ionic strength as well as the structure of the gel network. From the uniaxial compression tests, the swelling dependent stress–strain characteristics of resulting gel beads was evaluated. The obtained results were consistent with Hertzian elasticity, and were analyzed in terms of the compression modulus of the bead samples. It was found that force acting on the gel beads is the power-law function of the deformation F≈ΔD1.48 which is very close to the theoretical exponent 1.5 predicted by the Hertz equation. The water absorption capacities of P(HPMA-co-DMAEMA) gel beads with basic functional groups were repeatedly measured to obtain the optimal preparation conditions according to the variations of the bead diameters. Its maximal water absorption capacity appeared as 48 times among from 2.3mm to 11.0mm of swollen bead diameters. The swelling equilibria in various aqueous solutions were used to estimate the physico-chemical parameters asa function of gel bead size and swelling equilibrium capacity by correlating with the Flory-Rehner theory.

Microfluidic generation of Buddha beads-like microcarriers for cell culture

The fabrication of functional microcarriers capable of achieving in vivo-like three-dimensional cell culture is important for many tissue engineering applications. Here, inspired by the structure of Buddha beads, which are generally composed of moveable beads strung on a rope, we present novel cell microcarriers with controllable macropores and heterogeneous microstructures by using a capillary array microfluidic technology. Microfibers with a string of moveable and releasable microcarriers could be achieved by an immediate gelation reaction of sodium alginate spinning and subsequent polymerization of cell-dispersed gelatin methacrylate emulsification. The sizes of the microcarriers and their inner macropores could be well tailored by adjusting the flow rates of the microfluidic phases; this was of great importance in guaranteeing a sufficient supply of nutrients during cell culture. In addition, by infusing multiple cell-dispersed pregel solutions into the capillaries, the microcarriers with spatially heterogeneous cell encapsulations for mimicking physiological structures and functions could also be achieved.

Ultrastretchable and Self-Healing Double-Network Hydrogel for 3D Printing and Strain Sensor.

On the basis of the thermoreversible sol-gel transition behavior of κ-carrageenan in water, a double-network (DN) hydrogel has been fabricated by combining an ionically cross-linked κ-carrageenan network with a covalently cross-linked polyacrylamide (PAAm) network. The κ-carrageenan/PAAm DN hydrogel demonstrated an excellent recoverability and significant self-healing capability (even when notched). More importantly, the warm pregel solution of κ-carrageenan/AAm can be used as an ink of a three-dimensional (3D) printer to print complex 3D structures with remarkable mechanical strength after UV exposure. Furthermore, the κ-carrageenan/PAAm DN hydrogel exhibited a great strain sensitivity with a gauge factor of 0.63 at the strain of 1000%, and thus, the hydrogel can be used as sensitive strain sensors for applications in robotics and human motion detection.

Abstract 5788: Development of cellular morphology-based separation system for three-dimensional culture

Abstract 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 changes depending on its malignancy in 3D culture environment. The cell separation system in 3D culture environment should need to obtain the cells according to its morphology, which includes cell phenotypes. Recently, we developed gelatin-based photodegradable hydrogels, and applied this hydrogels to optical cell separation. The target cells in the photodegradable hydrogels were successfully separated by the optical cell separation, the separated cells was growth on another dish. On the other hand, we recently developed the predication model of stem cell differentiation by image analysis. The image analysis technique and the photodegradable gelatin hydrogels are included in the automated morphology-based cell separation system in the 3D culture environment. For forming cell encapsulated-photodegradable hydrogels, suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels. After the culture in 3D environment, 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. The hydrogels around the target area is irradiated with light (365nm). The cells in the irradiated area are collected by automated pipetting system. We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection. We demonstrated automated optical cell separation using the model culture system. Normal gastric mucosal cells were cultured in the photodegradable hydrogels. After cultivation for 1 week, the cells were irradiated the light for 5 to 20 min. The cells in the irradiated area were collected by automated pipetting and transferred into a collection dish. The collected cells were viable and attached in the collection dish after collection. 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. Citation Format: Hirofumi Matsui, Shinji Sugiura, Masato Tamura, Toshiyuki Kanamori, Toshiyuki Takagi, Taku Satou, Ryuji Kato, Kei Kanie, Mayu Shibuta. Development of cellular morphology-based separation system for three-dimensional culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5788. doi:10.1158/1538-7445.AM2017-5788

Combining ATRP and FRP Gels: Soft Gluing of Polymeric Materials for the Fabrication of Stackable Gels.

Stackable gels comprised of layers of dissimilar polymers were synthesized by combining conventional free radical polymerization (FRP) and atom transfer radical polymerization (ATRP) using two approaches: (i) polymerization of a pre-gel solution containing a monomer and cross-linker introduced on top of a previously prepared gel, and (ii) simultaneous polymerization of two immiscible pre-gel solutions remaining in contact. All permutations of FRP and ATRP yielded single-piece, connected, amphiphilic gels regardless of the order of polymerization. Furthermore, multi-layer ATRP gels combining different polymers were synthesized with the FRP layer as a gluing agent. A 10-layer amphiphilic stackable gel combining n-butyl methacrylate (BMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a 10-layer stackable gel combining BMA, DMAEMA and di(ethylene glycol) methyl ether methacrylate (PEO2MA) were synthesized. This patching method, combining conventional FRP gels with ATRP ones, offers an efficient path to the formation of complex stackable gel architectures.

Functionalized core-shell hydrogel microsprings by anisotropic gelation with bevel-tip capillary

This study describes a novel microfluidic-based method for the synthesis of hydrogel microsprings that are capable of encapsulating various functional materials. A continuous flow of alginate pre-gel solution can spontaneously form a hydrogel microspring by anisotropic gelation around the bevel-tip of the capillary. This technique allows fabrication of hydrogel microsprings using only simple capillaries and syringe pumps, while their complex compartmentalization characterized by a laminar flow inside the capillary can contribute to the optimization of the microspring internal structure and functionality. Encapsulation of several functional materials including magnetic-responsive nanoparticles or cell dispersed collagen for tissue scaffold was demonstrated to functionalize the microsprings. Our core-shell hydrogel microsprings have immense potential for application in a number of fields, including biological/chemical microsensors, biocompatible soft robots/microactuators, drug release, self-assembly of 3D structures and tissue engineering.

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 °C and can be used immediately following neutralization or stored at 4 °C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.