Julian B. Chaudhuri

Bioreactors for tissue engineering

Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.

Biocatalysis in organic media using enzymes from extremophiles

Abstract Enzymes from extremophiles (extremozymes) show activity and stability at extremes of temperature, low water activity, and high hydrostatic pressure. Aqueous/organic and nonaqueous media allow the modification of reaction equilibria and enzyme specificity, creating pathways for synthesizing novel compounds. Used in combination with such media, extremozymes show great potential as shown by their unique properties in aqueous media. This review introduces organic media biocatalysis before addressing the state of the art of recent fundamental and applied aspects of extremozyme biocatalysis. The aim is to encourage further exploitation of this technology, drawing on the limited work published in this field and important methods developed using mesophilic enzymes. Enzymes from three classes of extremophile will be considered: psychrophiles, halophiles, and thermophiles. Low temperature processes using psychrophilic (cold-active) enzymes may enhance yields of heat-sensitive products and reduce energy consumption. Halophilic enzymes require KCl/NaCl from 1 M to saturation, i.e. low water activity media, a feature in common with organic solvent systems. Thermophilic enzymes can be active and stable at up to 130°C and are highly resistant to proteases, detergents, and chaotropic agents. These features may afford resistance to the effects of organic solvents. Enhancing extremozyme performance via chemical modification, complexation, immobilization, and protein engineering is also discussed.

Protein refolding at high concentration using size‐exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size‐exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel‐filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S‐100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step.

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.

Electrical characterization of hydroxyapatite-based bioceramics.

This paper studies the AC conductivity and permittivity of hydroxyapatite (HA)-based ceramics from 0.1 Hz-1 MHz at temperatures from room temperature to 1000 degrees C. HA-based ceramics were prepared either as dense ceramics or in porous form with interconnected porosity and were sintered in either air or water vapour. Samples were thermally cycled to examine the influence of water desorption on AC conductivity and permittivity. Surface-bound water was thought to contribute to conductivity for both dense and porous materials at temperatures below 200 degrees C. At temperatures below 700 degrees C the permittivity and AC conductivity of HA was also influenced by the degree of dehydration and thermal history. At higher temperatures (700-1000 degrees C), bulk ionic conduction was dominant and activation energies were of the order of approximately 2 eV, indicating that hydroxyl ions are responsible for conductivity.

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.

Studies of the hydrodynamic volume changes that occur during refolding of lysozyme using size-exclusion chromatography

A size-exclusion chromatography-based refolding process (SEPROS) has successfully been used to renature lysozyme at high concentrations. This process is based on the different hydrodynamic characteristics of folded and unfolded proteins and their interaction with gel filtration media. In this paper we have quantified the changes in Stokes radius, hydrodynamic volume and partition coefficient that occur when lysozyme is refolded from urea in a size-exclusion column. In 8 M urea partially folded and unfolded lysozyme were resolved using Superdex 75 HR. These two species were present at approximately the same concentration. As the urea concentration was decreased the unfolded species gradually decreased until at 4 M urea only partially folded lysozyme remained, which continued to fold on further reduction of the urea concentration. Using these results the initial mechanism for size exclusion chromatography protein refolding has been confirmed.

Three-Dimensional Culture Systems for the Expansion of Pluripotent Embryonic Stem Cells

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two‐dimensional (2D) static culturing techniques are inadequate for large‐scale production. The culture of mammalian cells in three‐dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher‐S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher‐S out‐performed the Solohill microcarriers. When cultured for successive passages on Cultispher‐S microcarriers, mESCs maintained their pluripotency, demonstrated by self‐renewal, expression of pluripotency markers and the ability to undergo multi‐lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher‐S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra‐1–60, NANOG, and OCT‐4. Our study highlights the importance of optimization of initial seeding parameters and provides proof‐of‐concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683–695.

Emulsion liquid membrane extraction of organic acids. I: A theoretical model for lactic acid extraction with emulsion swelling

Abstract A new quantitative model is developed which describes the transport mechanisms and kinetics of organic acid extraction using emulsion liquid membranes. The model includes the contributions from facilitated transport of the acid by a tertiary amine carrier, and convective acid transport resulting from water transport to the emulsions. The facilitated transport model considers mass transfer in the external phase, the solute-carrier chemical reaction, diffusion in the globule as described by a shrinking-core model, and the stripping reaction. Convective transport is accounted for in terms of solute dissolved in the transported water. The effects of swelling on globule size and diffusion distance are also considered.

MagicWand: a single, designed peptide that assembles to stable, ordered α-helical fibers

We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric alpha-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg , with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesisthat is, as opposed to blunt-ended discrete speciesthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to alpha-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 degrees C at 100 muM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled alpha-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptide i.e., at the b and f positionscombined with stability and microscopy measurements, highlights the role of electrostatic and cation-pi interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems.