Fatima M. Plieva

Poly(vinyl alcohol) cryogels employed as matrices for cell immobilization. 3. Overview of recent research and developments

Poly(vinyl alcohol) cryogels, which are prepared by the freeze-thawing of concentrated aqueous solutions of the polymer, are promising gel carriers for cell immobilization. These carriers possess definite advantages when compared to other hydrogels commonly used for the same purposes. Similar benefits are as follows: (i) PVA cryogels have very high micro- and macroporosities which provide favored conditions for the nonhindered mass transfer of substrates and metabolites; (ii) the rheological characteristics of the nonbrittle matrix are excellent and allow the use of these carriers in most types of reactors; (iii) thermostability of PVA cryogels exceeds that of other commonly used thermoreversible gel carriers; (iv) the cryogels are highly resistant to biological degradation as well as being of a low sensitivity to culture media compositions; (v) PVA itself is a biologically compatible, nontoxic, and readily available low-cost polymer. By cell entrapment within PVA cryogel matrices, diverse immobilized biocatalysts were prepared which were capable of performing both a simple monoenzyme biotransformation of respective substrates and a complex biosynthesis of various high- and low-molecular-weight products, decompose pollutants, and operate in biosensors. Compilation data on these biocatalysts are presented as well as the potential of this immobilization technique.

The potential of polymeric cryogels in bioseparation

This is a review discussing the production and properties of cryogels (from the Greek κριoσ (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point.Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.

Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent

A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10-100 microm size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 degrees C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts.

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.

Cryogel applications in microbiology

There is a great demand for improved technologies with regard to rapid processing of nano- and microparticles. The handling of viruses in addition to microbial and mammalian cells requires the availability of appropriate adsorbents. Recent developments in macroporous gels produced at subzero temperatures (known as cryogels) have demonstrated an efficiency for processing cell and virus suspensions, cell separation and cell culture applications. Their unique combination of properties such as macroporosity, tissue-like elasticity and biocompatibility, physical and chemical stability and ease of preparation, renders these materials interesting candidates for a broad range of potential applications within microbiological research. This review describes current applications of macroporous cryogels in microbiology with a brief discussion of future perspectives.

The performance of laminin-containing cryogel scaffolds in neural tissue regeneration

Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin - the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds pore size for neural tissue engineering was 80-100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in niche-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be transplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of hosts neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.

Cell Chromatography: Separation of Different Microbial Cells Using IMAC Supermacroporous Monolithic Columns

Supermacroporous monolithic columns with Cu2+‐IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross‐linked with methylenebis(acrylamide) and has a network of large (10–100 μm) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild‐type Escherichia coli(w.t. E. coli) and recombinant E. coli cells displaying poly‐His peptides (His‐tagged E. coli) and of w.t. E. coli and Bacillus haloduranscells. Wild‐type E. coli and His‐tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8‐fold enriched with His‐tagged E. colicells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. colicells. Haloalkalophilic B. haloduranscells had low affinity to the Cu2+‐IDA cryogel column and could be efficiently separated from a mixture with w.t. E. colicells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.

Preparation of macroporous cryostructurated gel monoliths, their characterization and main applications.

Cryostructuration platform renders it possible to form macroporous materials (known as cryogels) with a broad range of porosity, from structures with combination of meso- and macropores to structures with 100-μm sized macropores. When these materials are formed in the shape of monoliths (monolithic cryogels), they present a unique monolithic stationary medium for specific applications. This review summarizes the recent research on the preparation and characterization of cryostructurated monolithic cryogels for (bio)separation and points to some future perspectives.

Cryogelation for preparation of novel biodegradable tissue-engineering scaffolds

2-Hydroxyethyl methacrylate-L-lactate (HEMA-LLA) and HEMA-L-lactate-dextran (HEMA-LLA-D) were synthesized. 1H-NMR confirmed the formation of these oligomers and macromers. Cryogels with different pore structures were prepared using different amounts of HEMA, HEMA-LLA and HEMA-LLA-D by a cryogelation technique. SEM micrographs exhibited pore morphologies. Cryogels were highly porous with interconnected pore structures, opaque, spongy and highly elastic. It was possible to compress them to remove the water in the pores and to return to their original form just by immersing them in water in few minutes, which was quite reproducible. Their swelling abilities, compressive strengths and degradation in buffer solutions were found to be related with their structural properties which was controlled by changing the cryogelation recipe.

Purification of His 6 –organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)–polyacrylamide cryogel (PAA) and Me2+-N,N,N’-tris (carboxymethyl) ethylendiamine (TED)–PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10xa0mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA–PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA–PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA–PAA and Ni-nitrilotriacetic acid–agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.