Purification of immunoglobulins from Serum Using Thiophilic Cellulose Beads

This study evaluates the chromatographic performance of a support obtained by the reaction of mercaptoethanol with cellulose beads activated with divinyl sulfone. Cellulose beads 500-800 microns (pm) in diameter and with a solids content of 3.5% were selected for this study. A two-step sequence of permeation and reaction was used to install thiophilic sites throughout the cross section of the bead. The distribution of thiophilic sites was visualized by immobilizing fluorescent antibodies. Human and porcine serum proteins were separated on the thiophilic support at different linear velocities. Thiophilic cellulose beads were observed to bind human and porcine immunoglobulins (IgG) selectively from serum. Overall total protein recoveries in the range of 85%-99% were obtained with human serum, porcine serum and cell supernatant. Human TgG yields of 75% and 50% were obtained at linear velocities of 1 cmlmin and 3 cmlmin, respectively. Thiophilic cellulose beads were observed to bind monoclonal antibodies from cell culture supematant but yields in the range of 40-508 were obtained. Purity of the products, obtained from a single chro~natographics tep, as judged by electrophoretic analysis was estimated to be greater than 80%

Chromatographic Purification of MAbs with Non-Affinity Supports

The introduction of new protein-based therapeutics such as monoclonal antibodies (MAbs), MAb-based vaccines, growth factors and plasma proteins implies the need to study, characterize, and purify. The separation step is likely to be a bottleneck and cost-effective technology will be needed to rectify it. The currently prevalent matrices for chromatographic separation of immunoglobulins (Igs) are based on Protein A or its recombinant versions (Protein G). They display excellent selectivity and specificity, but are expensive. A Protein A matrix costs $8,000 to 12,000 per L-resin. The typical production column volume is 100 L. The million-dollar matrix is far more expensive than the production hardware

Immunoaffinity Chomatography

Recent devclopments in recombinant DNA technology have enabled the synthesis of valuable therapeutic proteins in bacterial cells as well as in novel eucaryotic expression systems. However, the purification of proteins of interest from either the conventional sources, cell culture, or novel routes in a highly purified form necessitates the development of separation tcchniques capable of recovering proteins from these feed streams in a highly purified form (1,2). Purification of therapeutic proteins from biological sources is usually complicated by the presence of endogenous proteins (2). Purification methodologies based on ion exchange or adsorption serve as excellent prepurification steps, but they fail to resolve complex protein mixtures to yield a homogeneous protein tein product (l).P urification techniques based on affinity interactions between molecules (i.e., immunoaffinity chromatography, IAC) have rapidly evolved using a variety of biological and synthetic ligands (2)

Remodeling of chromatin under low intensity diffuse ultrasound

A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51- s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms

The Effect of Ultrasound Stimulation on the Cytoskeletal Organization of Chondrocytes Seeded In 3D Matrices

The impact of low intensity diffuse ultrasound (LIDUS) stimulation on the cytoskeletal organization of chondrocytes seeded in 3D scaffolds was evaluated. Chondrocytes seeded on 3D chitosan matrices were exposed to LIDUS at 5.0 MHz (~15kPa, 51-secs, 4-applications/day) in order to study the organization of actin, tubulin and vimentin. The results showed that actin presented a cytosolic punctuated distribution, tubulin presented a quasi parallel organization of microtubules whereas vimentin distribution was unaffected. Chondrocytes seeded on 3D scaffolds responded to US stimulation by the disruption of actin stress fibers and were sensitive to the presence of ROCK inhibitor (Y27632). The gene expression of ROCK-I, a key element in the formation of stress fibers and mDia1, was significantly up-regulated under the application of US. We conclude that the results of both the cytoskeletal analyses and gene expression support the argument that the presence of punctuated actin upon US stimulation was accompanied by the up-regulation of the RhoA/ROCK pathway

Remodeling of chromatin under low intensity diffuse ultrasound

A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51- s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves

Improved cellular infiltration into nanofibrous electrospun crosslinked gelatin scaffolds templated with micrometer sized polyethylene glycol fibers

Gelatin-based nanofibrous scaffolds with a mean fiber diameter of 300 nm were prepared with and without micrometer-sized polyethylene glycol (PEG) fibers that served as sacrificial templates. Upon fabrication of the scaffolds via electrospinning, the gelatin fibers were crosslinked with glutaraldehyde, and the PEG templates were removed using tert-butanol to yield nanofibrous scaffolds with pore diameters ranging from 10 to 100 μm, as estimated with mercury intrusion porosimetry. Non-templated gelatin-based nanofibrous matrices had an average pore size of 1 μm. Fibroblasts were seeded onto both types of the gelatin-based nanfibrous surfaces and cultured for 14 days. For comparative purposes, chitosan-based and polyurethane (PU)-based macroporous scaffolds with pore sizes of 100 μm and 170 μm, respectively, also were included. The number of cells as a function of the depth into the scaffold was judged and quantitatively assessed using nuclei staining. Cell penetration up to a depth of 250 μm and 90 μm was noted in gelatin scaffolds prepared with sacrificial templates and gelatin-only nanofibrous scaffolds. Noticeably, scaffold preparation protocol presented here allowed the structural integrity to be maintained even with high template content (95 %) and can be easily extended towards other classes of electrospun polymer matrices for tissue engineering

Theoretically proposed optimal frequency for ultrasound induced cartilage restoration

Background: Matching the frequency of the driving force to that of the system’s natural frequency of vibration results in greater amplitude response. Thus we hypothesize that applying ultrasound at the chondrocyte’s resonant frequency will result in greater deformation than applying similar ultrasound power at a frequency outside of the resonant bandwidth. Based on this resonant hypothesis, our group previously confirmed theoretically and experimentally that ultrasound stimulation of suspended chondrocytes at resonance (5 MHz) maximized gene expression of load inducible genes. However, this study was based on suspended chondrocytes. The resonant frequency of a chondrocyte does not only depend on the cell mass and intracellular stiffness, but also on the mechanical properties of the surrounding medium. An in vivo chondrocyte’s environment differs whether it be a blood clot (following microfracture), a hydrogel or the pericellular and extracellular matrices of the natural cartilage. All have distinct structures and compositions leading to different resonant frequencies. In this study, we present two theoretical models, the first model to understand the effects of the resonant frequency on the cellular deformation and the second to identify the optimal frequency range for clinical applications of ultrasound to enhance cartilage restoration. Results: We showed that applying low-intensity ultrasound at the resonant frequency induced deformation equivalent to that experimentally calculated in previous studies at higher intensities and a 1 MHz frequency. Additionally, the resonant frequency of an in vivo chondrocyte in healthy conditions, osteoarthritic conditions, embedded in a blood clot and embedded in fibrin ranges from 3.5 − 4.8 MHz. Conclusion: The main finding of this study is the theoretically proposed optimal frequency for clinical applications of therapeutic ultrasound induced cartilage restoration is 3.5 − 4.8 MHz (the resonant frequencies of in vivo chondrocytes). Application of ultrasound in this frequency range will maximize desired bioeffects