Agarose Concentration Research Articles

ENDOTHELIAL PROGENITOR CELLS ENCAPSULATED IN MATRIX-SUPPLEMENTED MICROGEL IMPROVES CELL RETENTION AND THERAPEUTIC EFFICACY IN PULMONARY ARTERIAL HYPERTENSION

Late outgrowth (L) endothelial progenitor cells (EPC) represent a uniform, highly endothelial-like progenitor cell population; however, their therapeutic benefits in models of pulmonary arterial hypertension (PAH) are limited by poor cell persistence, due to rapid cell loss by apoptosis (anoikis) and redistribution to non-target organs. Temporary single-cell microencapsulation (i.e. cocooning) provides a portable stem cell niche, that can promote cell survival and retention in animal models of organ lung and heart injury. We hypothesize that microencapsulation of L-EPC with an agarose hydrogel supplemented with integrin-binding proteins will increase survival and retention of L-EPCs injected into the jugular vein and result in greater therapeutic benefits compared to non-cocooned cells in a rat monocrotaline (MCT) model of PAH. L-EPCs were encapsulated by vortex-emulsion using various concentrations of agarose, together with fibronectin and fibrinogen, and capsule size and initial cell viability were assessed. Encapsulated and non-encapsulated L-EPCs were transduced with luciferase and administered to SD rats three days after injection of MCT. L-EPCs were tracked in vivo by bioluminescence imaging (BLI) to assess cell persistence and bio-distribution for up to three weeks post cell injection. At end-study, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were assessed for therapeutic efficacy. The initial BLI signals at 15 minutes after delivery were similar in non-cocooned and cocooned L-EPCs; however, only cocooned cells could be detected by BLI after four and 24 hours (28±12% and 12±8% of baseline signal, respectively; p< 0.0001 and 0.05, n=11). Microencapsulation of L-EPCs led to significant improvement in RVSP three weeks after delivery compared to MCT alone (56±24 vs. 80±7 mmHg, respectively; p<0.05), whereas no improvement in pulmonary hemodynamics was seen with delivery of non-encapsulated cells. These results demonstrate that single-cell cocooning can significantly increase retention of L-EPCs within the lungs. Furthermore, even a modest increase in L-EPC persistence over 24 hours can provide an important therapeutic benefit, not seen with non-encapsulated L-EPCs in the rat MCT model of PAH.

Incorporating 4D into Bioprinting: Real-Time Magnetically Directed Collagen Fiber Alignment for Generating Complex Multilayered Tissues.

In vitro multilayered tissues with mimetic architectures resembling native tissues are valuable tools for application in medical research. In this study, an advanced bioprinting strategy is presented for aligning collagen fibers contained in functional bioinks. Streptavidin-coated iron nanoparticles are embedded in printable bioinks with varying concentrations of low gelling temperature agarose and type I collagen. By applying a straightforward magnetic-based mechanism in hydrogels during bioprinting, it is possible to align collagen fibers in less concentrated hydrogel blends with a maximum agarose concentration of 0.5 w/v%. Conversely, more elevated concentrations of agarose in printable blends show random collagen fiber distribution. Interestingly, hydrogel blends with unidirectionally aligned collagen fibers show significantly higher compression moduli compared to hydrogel blends including random fibers. Considering its application in the field of cartilage tissue engineering, bioprinted constructs with alternating layers of aligned and random fibers are fabricated. After 21 days of culture, cell-loaded constructs with alternating layers of aligned and random fibers express markedly more collagen II in comparison to solely randomly oriented fiber constructs. These encouraging results translate the importance of the structure and architecture of bioinks used in bioprinting in light of their use for tissue engineering and personalized medical applications.

Nanosilicate embedded agarose hydrogels with improved bioactivity

This paper reports the synthesis of nanocomposite agarose hydrogels with improved bioactivity with the incorporation of anisotropic 2D nanosilicates (Laponite) to promote cell binding, growth and proliferation. Rheological measurements showed that the incorporation of nanosilicates slightly increased the gelation temperature (Tgel). The use of higher nanosilicate content at the constant agarose concentration improved the mechanical properties of the gels. Due to the non-swelling nature of agarose, the addition of nanosilicates did not result in any remarkable change in the swelling properties of the agarose gels, while collapsed agarose nanofibers were observed with the incorporation of nanosilicates. EDX analysis confirmed the presence of the embedded nanosilicates in the gel matrix. The existence of physical interactions between nanosilicate and agarose was demonstrated by FTIR over the shifting of SiO stretching band to a lower frequency. The encapsulated NIH/3T3 fibroblast cells showed enhanced proliferation and spreading in the presence of nanosilicates.

Agarose Hydrogel Beads: An Effective Approach to Improve the Catalytic Activity, Stability and Reusability of Fungal Amyloglucosidase of GH15 Family

This study deals with the immobilization of amyloglucosidase within agarose using method of entrapment. Enzyme was produced from Aspergillus fumigatus KIBGE-IB33 and then partially purified using 40% ammonium sulphate saturation. Using 40 gl−1 concentration of agarose and adjusting 3.0 mm size of hydrogels, maximum entrapment yield (78%) was obtained. The kinetic behavior was slightly changed after immobilization as reaction time and reaction temperature increases from 5.0 min (soluble) to 10.0 min (immobilized) and 60 °C (soluble) to 65 °C (immobilized), respectively while, pH optima remained same (pH 5.0). Substrate saturation kinetics revealed that Km was increased from 1.47 to 4.215 mg ml−1 while, the value of Vmax decreased from 947 to 611 kU mg−1 for soluble and entrapped amyloglucosidase, respectively. The stability profile of amyloglucosidase also significantly improved after entrapment in agarose hydrogels at 50, 60 and 70 °C for 120 min with retention of 77, 59 and 25% residual activity, respectively. Furthermore, the t1/2 of soluble and immobilized amyloglucosidase at 60 °C was 167 and 375 min respectively. Due to increase in reusability for various subsequent cycles of entrapped amyloglucosidase, about 8.73 mg ml−1 increase in glucose production was observed as compared to soluble enzyme.

Porosity effect on microstructure, mechanical, and fluid dynamic properties of Ti2AlC by direct foaming and gel‐casting

AbstractIn this study, Ti2AlC foams were fabricated by direct foaming and gel‐casting using agarose as gelling agent. Slurry viscosity, determined by the agarose content (at a fixed solids loading), as well as surfactant concentration and foaming time were the key parameters employed for controlling the foaming yield, and hence the foam porosity after sintering process. Fabricated foams having total porosity in the 62.5‐84.4 vol% range were systematically characterized to determine their pore size and morphology. The effect of the foam porosity on the room‐temperature compression strength and elastic modulus was also determined. Depending on the amount of porosity, the compression strength and Young's modulus were found to be in the range of 9‐91 MPa and 7‐52 GPa, respectively. Permeability to air flow at temperatures up to 700°C was investigated. Darcian (k1) and non‐Darcian (k2) permeability coefficients displayed values in the range 0.30‐93.44 × 10−11 m2 and 0.39‐345.54 × 10−7 m, respectively. The amount of porosity is therefore a very useful microstructural parameter for tuning the mechanical and fluid dynamic properties of Ti2AlC foams.

Photonic force optical coherence elastography for three-dimensional mechanical microscopy

Optical tweezers are an invaluable tool for non-contact trapping and micro-manipulation, but their ability to facilitate high-throughput volumetric microrheology of biological samples for mechanobiology research is limited by the precise alignment associated with the excitation and detection of individual bead oscillations. In contrast, radiation pressure from a low-numerical aperture optical beam can apply transversely localized force over an extended depth range. Here we present photonic force optical coherence elastography (PF-OCE), leveraging phase-sensitive interferometric detection to track sub-nanometer oscillations of beads, embedded in viscoelastic hydrogels, induced by modulated radiation pressure. Since the displacements caused by ultra-low radiation-pressure force are typically obscured by absorption-mediated thermal effects, mechanical responses of the beads were isolated after independent measurement and decoupling of the photothermal response of the hydrogels. Volumetric imaging of bead mechanical responses in hydrogels with different agarose concentrations by PF-OCE was consistent with bulk mechanical characterization of the hydrogels by shear rheometry.

Uniform polysaccharide composite microspheres with controllable network by microporous membrane emulsification technique.

High resolution has been constantly pursued in both preparative and analytical chromatography. Chromatographic media are a key factor during the entire separation process. Tailor-made chromatographic media have gained more attention because of their adjustable structure appropriate for application. Uniform polysaccharide composite microspheres were prepared with a mixture of agarose and dextran solution by membrane emulsification technique for the first time. Their pore structure was deliberately regulated by adjusting boththe polysaccharide composition and the molecular weight of dextran. Compared with pure agarose microspheres, polysaccharide composite microspheres had a higher separation resolution and their separation range was controllable. By increasing agarose concentration and decreasing dextran concentration at the same time during the preparation of composite microspheres, the mean pore size increased first and then decreased later, and also the pore size distribution became narrower. By increasing the molecular weight of dextran, the pores became smaller with a narrower pore size distribution. Microspheres with a composition of 10% agarose/2% dextran T40 or 8% agarose/4% dextran T150 showed a higher separation resolution for proteins within range of low molecular weight. Furthermore, the mechanical strength of this composite microsphere was improved by adjusting its composition. Atomic force microscope (AFM) results showed that pores were distributed evenly on both the surface and the inner part of microspheres, beneficial for the passage of biomolecules. These novel uniform polysaccharide composite microspheres have great potential for high-resolution bioseparation. Graphical abstract ᅟ.

Mathematical Characterization of Thermo-reversible Phase Transitions of Agarose Gels

ABSTRACTThe thermal phase transition temperatures of high (HMP) and low melting point (LMP) agarose gels were investigated by using UV–vis spectroscopy techniques. Transmitted light intensities from the gel samples with different agarose concentrations were monitored during the heating (gel-sol) and cooling (sol–gel) processes. It was observed that the transition temperatures, Tm, defined as the location of the maximum of the first derivative of the sigmoidal transition paths obtained from the UV–vis technique, slightly increased by increasing the agarose concentration in both the HMP and LMP samples. Here, we express the phase transitions of the agar-water system, as a representative of reversible physical gels, in terms of a modified Susceptible-Infected-Susceptible epidemic model whose solutions are the well-known 5-point sigmoidal curves. The gel point is hard to determine experimentally and various computational techniques are used for its characterization. Based on previous work, we locate the gel point, T0, of sol-gel and gel-sol transitions in terms of the horizontal shift in the sigmoidal transition curve. For the gel-sol transition (heating), T0 is greater than Tm, i.e. later in time, and the difference between T0 and Tm is reduced as the agarose content increases. For the sol-gel transition (cooling), T0 is again greater than Tm, but it is earlier in time for all agarose contents and moves forward in time and gets closer to Tm as the agarose content increases.

The effect of different methods and image analyzers on the results of the in vivo comet assay

IntroductionThe in vivo comet assay is a widely used genotoxicity test that can detect DNA damage in a range of organs. It is included in the Organisation for Economic Co-operation and Development Guidelines for the Testing of Chemicals. However, various protocols are still used for this assay, and several different image analyzers are used routinely to evaluate the results. Here, we verified a protocol that largely contributes to the equivalence of results, and we assessed the effect on the results when slides made from the same sample were analyzed using two different image analyzers (Comet Assay IV vs Comet Analyzer).FindingsStandardizing the agarose concentrations and DNA unwinding and electrophoresis times had a large impact on the equivalence of the results between the different methods used for the in vivo comet assay. In addition, there was some variation in the sensitivity of the two different image analyzers tested; however this variation was considered to be minor and became negligible when the test conditions were standardized between the two different methods.ConclusionBy standardizing the concentrations of low melting agarose and DNA unwinding and electrophoresis times between both methods used in the current study, the sensitivity to detect the genotoxicity of a positive control substance in the in vivo comet assay became generally comparable, independently of the image analyzer used. However, there may still be the possibility that other conditions, except for the three described here, could affect the reproducibility of the in vivo comet assay.

Rheological Characterization of Agarose and Poloxamer 407 (P407) Based Hydrogels

ABSTRACTPoloxamer 407 (P407) is a biocompatible thermo-setting polymer, while agarose is a biocompatible thermo-softening material. It is interesting to mix them to examine any possible synergy in thermomechanical properties. In this study, rotational rheometer was adopted to study rheological properties of the mixtures of agarose/P407 gels with different concentrations at various frequencies, strain rates and temperatures. It has revealed that the addition of P407 decreased the gel stiffness by an order of magnitude. For the given combinations in this study, the increase in agarose concentration would increase both the storage modulus and loss modulus of the gel mixtures. The variation in P407 concentration (2.5%-10%) minimally changes the composite moduli. These agarose/P407 gel mixtures also exhibited shear thinning behavior. However, the addition of P407 (2.5%-10%) to agarose gel only has very small effect on thermomechanical properties of agarose gels. The overall transition temperature for these gel mixtures is governed by P407 melting point where the phase change starts around 55°C and the gels completely collapse at the melting temperature of agarose.

Characterization of agarose microparticles prepared by water-in-water emulsification

ABSTRACTMicroparticles have a wide range of applications in various areas. In this study, agarose microparticles (AGM) were successfully prepared by using a water-in-water (w/w) emulsification (WWEM) technique to eliminate the use of a surfactant. An aqueous agarose solution was employed as the dispersed phase, and polyethylene glycol (PEG) was used as the continuous phase. We evaluated how the characteristics of the microparticles were affected by different processing factors, including temperature, agitation speed, stirring time, concentration of agarose, and proportions of the two phases. The agarose microparticles obtained were nearly perfect spheres, and their particle sizes decreased with an increase in the agitation speed. Physicochemical characterization suggested that agarose microparticles, due to their ubiquitous stability, could be applied in most mild environments.

Agarose as an Efficient Inhibitor for Aluminium Corrosion in Acidic Medium: An Experimental and Theoretical Study

The inhibition efficiency and mechanism of agarose on aluminium corrosion in 1 M HCl, 0.5 M H2SO4 and 0.5 M H3PO4 solutions were studied by means of conventional weight loss method, Tafel polarisation electrochemical impedance spectroscopy and scanning electron microscope. The inhibition efficiency (%IE) was observed to increase with addition of agarose concentration, and it adversely decreases with increasing temperature. Moreover, the maximum inhibition efficiency of ~81% is achieved for agarose concentration of 700 ppm in 1 M HCl. Potentiodynamic polarisation curves showed that agarose acts as a mixed-type inhibitor. The absorption isotherm calculations revealed that interactions of agarose on aluminium surface are physisorption and typically obey the Temkin adsorption isotherm. The quantum chemical calculations reveal that active sites promote the agarose to anchor on aluminium surface due to mostly presence of hydroxyl group.

Pengembangan Metode Identifikasi Kerusakan DNA Spermatozoa Ternak

The success of artificial insemination is very much determined by the quality of spermatozoa. The detection or identification of damaged chromatin of spermatozoa DNA is very important to forsee the adverse clinical outcome. However, the method of identification is still depended on expensive imported kits. Therefore, the objective of this research was to developed an identification kit to determine the quality of livestock spermatozoa DNA chromatin.This study consist of three step. Step 1) Determination of low melting point agarose (LMP-agarose) concentration which is 0,6%, 0,7% and 0,8%. 2) Comparison of three lysis solution (LS) which is LS I (0.4M Tris, 0.8M DTT, 1% SDS, pH 7.5), LS II (0.4M Tris,2 M NaCl, 1% SDS , pH 7.5), and LS III (0.4M Tris-HCl, 2M NaCl, 1% SDS 0,05 M EDTA, pH 7.5). 3) Comparison different staining which is Eosin yellow and Methylene blue. The results showed that 0.6% LMP-agarose demonstrated the best concentration to “trapped the spermatozoa” compared for sheep and goats. whereas the three concentration of spermatozoa cows can not be used to trap spermatozoa cow. The best formulation to lysis the membrane was LS III (0.4M Tris -HCl, 2M NaCl, 1% SDS 0,05 M EDTA). The best staining was eosin yellow and methylene blue with 2:1 ratio.

Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a hydrogel-based subcutaneous tissue surrogate and UV–vis imaging

Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using a novel UV–vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively, were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of the systems was followed by the study of changes in light transmission and absorbance as a function of time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation was determined and found to decrease with increasing the PLGA concentration from 20% to 40% (w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site environment. The resulting implant morphology depended on the stiffness of hydrogel matrix, indicating that the matrix in which implants are formed is of importance. Overall, the work showed that the UV–vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue holds potential in providing bio-relevant and mechanistic information on the phase separation processes of in situ forming implants.

Bio-catalytic performance and dye-based industrial pollutants degradation potential of agarose-immobilized MnP using a Packed Bed Reactor System

In this study, the matrix-entrapment technique was adopted to immobilize a novel manganese peroxidase (MnP). Agarose beads developed from 3.0% agarose concentration furnished the preeminent immobilization yield (92.76%). The immobilized MnP exhibited better resistance to changes in the pH and temperature as compared to the free counterpart, with optimal conditions being pH 6.0 and 45°C. Thermal and storage stability characteristics were significantly improved after immobilization, and the immobilized-MnP displayed higher tolerance against different temperatures than free MnP state. After 72h, the insolubilized MnP retained its activity up to 41.2±1.7% and 33.6±1.4% at 55°C and 60°C, respectively, and 34.3±1.9% and 22.0±1.1% activities at 65°C and 70°C, respectively, after 48h of the incubation period. A considerable reusability profile was recorded with ten consecutive cycles. Moreover, to explore the industrial applicability, the agarose-immobilized-MnP was tested for bioremediation of textile industry effluent purposes. After six consecutive cycles, the tested effluents were decolorized to different extents (with a maximum of 98.4% decolorization). In conclusion, the remarkable bioremediation potential along with catalytic, thermo-stability, reusability, as well as storage stability features of the agarose-immobilized-MnP reflect its prospects as a biocatalyst for bioremediation and other industrial applications.

Systematic Study of Functionalizable, Non-Biofouling Agarose Films with Protein and Cellular Patterns on Glass Slides.

Herein we demonstrate a systematic investigation of chemically functionalizable, non-biofouling agarose films over large-area glass surfaces. Agarose films, prepared with various concentrations of aqueous agarose, were activated by using periodate oxidation to generate aldehyde groups at the termini of the agarose chains. The non-biofouling efficacy and binding capabilities of the activated films were evaluated by using protein and cellular patterning, performed by using a microarrayer, microcontact printing, and micromolding in capillaries. Characterization by using a fluorescence slide scanner and a scanning-probe microscope revealed that the pore sizes of the agarose films played an important role in achieving desirable film performance; the 0.2 wt % agarose film exhibited the optimum efficacy in this work.

Nonmonotonic swelling of agarose‐carbopol hybrid hydrogel: Experimental and theoretical analysis

ABSTRACTA hybrid gel is synthesized by physical mixing of agarose and carbopol and an interpenetrating network of Agarose‐Carbopol (AC) hybrid gel is observed by atomic force microscopy. This hybrid gel exhibits pH‐responsiveness and mechanical stability as well as tunable swelling. These hydrogels depict a nonmonotonic swelling behavior as a function of pH. In particular, the equilibrium degree of swelling increases with pH of bath solution until its maximum value around pH = , followed by a decrease at higher pH values. The degree of swelling is increased by the concentration of carbopol when the concentration of agarose is low [ ], or decreased by the concentration of agarose when is high [ ]. A physics‐based model is also adapted to characterize the swelling‐shrinking behavior of different compositions of AC gels. This swelling‐shrinking behavior of AC hydrogels will have potential applications in smart hydrogel‐based devices. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2017, 55, 444–454

High-frequency linear rheology of hydrogels probed by ultrasound-driven microbubble dynamics.

Ultrasound-driven microbubble dynamics are central to biomedical applications, from diagnostic imaging to drug delivery and therapy. In therapeutic applications, the bubbles are typically embedded in tissue, and their dynamics are strongly affected by the viscoelastic properties of the soft solid medium. While the behaviour of bubbles in Newtonian fluids is well characterised, a fundamental understanding of the effect on ultrasound-driven bubble dynamics of a soft viscoelastic medium is still being developed. We characterised the resonant behaviour in ultrasound of isolated microbubbles embedded in agarose gels, commonly used as tissue-mimicking phantoms. Gels with different viscoelastic properties were obtained by tuning agarose concentration, and were characterised by standard rheological tests. Isolated bubbles (100-200 μm) were excited by ultrasound (10-50 kHz) at small pressure amplitudes (<1 kPa), to ensure that the deformation of the material and the bubble dynamics remained in the linear regime. The radial dynamics of the bubbles were recorded by high-speed video microscopy. Resonance curves were measured experimentally and fitted to a model combining the Rayleigh-Plesset equation governing bubble dynamics, with the Kelvin-Voigt model for the viscoelastic medium. The resonance frequency of the bubbles was found to increase with increasing shear modulus of the medium, with implications for optimisation of imaging and therapeutic ultrasound protocols. In addition, the viscoelastic properties inferred from ultrasound-driven bubble dynamics differ significantly from those measured at low frequency with the rheometer. Hence, rheological characterisation of biomaterials for medical ultrasound applications requires particular attention to the strain rate applied.

Photo-Modulated Therapeutic Protein Release from a Hydrogel Depot Using Visible Light.

The use of biomacromolecular therapeutics has revolutionized disease treatment, but frequent injections are required owing to their short half-life in vivo. Thus there is a need for a drug delivery system that acts as a reservoir and releases the drug remotely "on demand". Here we demonstrate a simple light-triggered local drug delivery system through photo-thermal interactions of polymer-coated gold nanoparticles (AuNPs) inside an agarose hydrogel as therapeutic depot. Localized temperature increase induced by the visible light exposure caused reversible softening of the hydrogel matrix to release the pre-loaded therapeutics. The release profile can be adjusted by AuNPs and agarose concentrations, light intensity and exposure time. Importantly, the biological activity of the released bevacizumab was highly retained. In this study we demonstrate the potential application of this facile AuNPs/hydrogel system for ocular therapeutics delivery through its versatility to release multiple biologics, compatibility to ocular cells and spatiotemporal control using visible light.

Enhancing purification efficiency of affinity functionalized composite agarose micro beads using Fe3O4 nanoparticles.

In this work, a series of magnetic and nonmagnetic agarose matrices were fabricated for protein purification. Certain amounts of Fe3O4 nanoparticles were encapsulated in agarose beads to form composite magnetic matrices with enhanced purification efficiency. Structure and morphology of prepared matrices were studied by optical and scanning electron microscopes, FT-IR, and BET-BJH analysis. The prepared matrices had regular spherical shape, followed by a uniform size distribution. By nanoparticles addition, the number of mesopores decreased while population of pores with radius ≤10nm increased; thus, higher specific area achieved. According to VSM results, magnetization degree was one of the characteristics affected by agarose content of the beads. A dye ligand, Cibacron Blue F3GA (CB), was covalently bound to beads to adsorb Bovine serum albumin. CB concentration was determined by elemental analysis. It was shown that magnetic beads hold higher CB concentrations than nonmagnetic ones due to higher specific area. As a result, magnetic 8%-agarose beads had the highest affinity adsorption capacity in static experiments. Moreover, breakthrough curves were monitored to calculate dynamic binding capacity. And, it was shown that magnetic 4%-agarose had the highest adsorbing amount (6.00mg/mL). It was implied that pore diffusion in magnetic 4%-agarose may be the reason for higher dynamic capacity. Plus, column efficiency was evaluated. It was revealed that all magnetic beads had lower HETP (0.11, 0.12 and 0.11cm for magnetic 4, 6, and 8%-agarose beads) than nonmagnetic ones (P-value<0.05).