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.

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.

Removal of heavy metals from water effluents using supermacroporous metal chelating cryogels.

Applications of IDA in, for example, immobilized metal ion affinity chromatography for purification of His-tagged proteins are well recognized. The use of IDA as an efficient chelating adsorbent for environmental separations, that is, for the capture of heavy metals, is not studied. Adsorbents based on supermacroporous gels (cryogels) bearing metal chelating functionalities (IDA residues and ligand derived from derivatization of epoxy-cryogel with tris(2-aminoethyl)amine followed by the treatment with bromoacetic acid (defined as TBA ligand)) have been prepared and evaluated on capture of heavy metal ions.

Dried-reswollen immobilized biocatalysts for detoxification of organophosphorous compounds in the flow systems.

New immobilized biocatalysts based on polypeptides containing N- or C-terminal polyhistidine sequences and possessing organophosphorus hydrolase activity were investigated for detoxification of organophosphorous neurotoxic compounds in the flow systems. The biocatalysts were revealed to have a high catalytic activity within wide pH and temperature ranges 7.5-12.

Removal of endocrine-disrupting compounds from water using macroporous molecularly imprinted cryogels in a moving-bed reactor.

Highly efficient removal of endocrine-disrupting compounds (EDCs) such as 17beta-estradiol (E2), 4-nonylphenol (NP) and atrazine from water was achieved using a novel macroporous adsorption medium. The medium consisted of a macroporous poly(vinyl alcohol) (PVA) cryogel with molecularly imprinted polymer (MIP) particles embedded in it. The MIP was prepared using E2, NP and atrazine as templates.

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries.

Background: Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library.

Results: Both phages and E.

Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles.

Cell proliferation and long-term production of monoclonal antibody IgG(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 x 9 mm(2)). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer.

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.

Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model.

Biodegradable macroporous cryogels with highly open and interconnected pore structures were produced from dextran modified with oligo L-lactide bearing hydroxyethylmethacrylate (HEMA) end groups in moderately frozen solutions. Tissue responses to these novel scaffolds were evaluated in rats after dorsal subcutaneous implantation, iliac submuscular implantation, auricular implantation, or in calvarial defect model. In no case, either necrosis or foreign body reaction was observed during histological studies.

Bioresorbable and nonresorbable macroporous thermosensitive hydrogels prepared by cryopolymerization. Role of the cross-linking agent.

Macroporous poly( N-isopropylacrylamide) (pNIPA) gels (so-called cryogels), cross-linked with different bis-acrylic compounds, N,N'-methylenebisacrylamide (MBAAm) and dimethacrylate-tyrosine-lysine-tyrosine (DMTLT), were prepared through free-radical polymerization at subzero temperature in dioxane/water media. DMTLT is a hydrolytically degradable cross-linker with relatively hydrophobic character. The effects of different synthesis conditions, namely the concentration of monomers, the cross-linker, and the initiator in the reaction mixture, on the structure of the pNIPA-cryogels have been studied.

Monolithic macroporous albumin/chitosan cryogel structure: a new matrix for enzyme immobilization.

A novel monolithic macroporous material was developed by cross-linking hen egg albumin (HEA) and chitosan with glutaraldehyde at subzero temperatures. A macroporous cryogel structure allowed efficient mass transport of solutes within the material. In one application, albumin was partially replaced with active enzymes (glucose oxidase and horseradish peroxidase) resulting in the production of macroporous biocatalyst preparations suitable for flow-injection analysis of glucose in the low millimolar range.

Macroporous gel particles as robust macroporous matrices for cell immobilization.

A new design of robust matrices for cell immobilization is described. Macroporous gels (MGs) with immobilized microbial cells were prepared at subzero temperatures and were formed inside a plastic core (so-called, protective housing). Due to the protective housing the macroporous gel particles with immobilized cells can be used in well-stirred bioreactors.

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.

Macroporous monolithic gels, cryogels, with immobilized phages from phage-display library as a new platform for fast development of affinity adsorbent capable of target capture from crude feeds.

Selected phage clones expressing a peptide with high binding affinity for recombinant human lactoferrin or von Willebrand factor (vWF) were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column. The macroporous monolithic columns were successfully used for the direct affinity capture of target proteins from particulate containing feeds like milk containing casein micelles and fat globules (1-10 microm in size) or even whole blood containing blood cells (up to 20 microm in size).

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications.

Macroporous gels (MGs) with a broad variety of morphologies are prepared using the cryotropic gelation technique, i. e. gelation at subzero temperatures.

Chromatography of living cells using supermacroporous hydrogels, cryogels.

The preparative cell separation is an intrinsic requirement of various diagnostic, biotechnological and biomedical applications. Affinity chromatography is a promising technique for cell separation and is based on the interaction between a cell surface receptor and an immobilised ligand. Most of the currently available matrices have pore size smaller than the size of the cells and are not suitable for cell chromatography due to column clogging.

Macroporous molecularly imprinted polymer/cryogel composite systems for the removal of endocrine disrupting trace contaminants.

A new concept for the preparation of selective sorbents with high flow path properties is presented by embedding molecularly imprinted polymers (MIPs) into various macroporous gels (MGs). A MIP was first synthetized with 17beta-estradiol (E2) as template for the selective adsorption of this endocrine disrupter. The composite macroporous gel/MIP (MG/MIP) monoliths were then prepared at subzero temperatures.

Effect of matrix elasticity on affinity binding and release of bioparticles. Elution of bound cells by temperature-induced shrinkage of the smart macroporous hydrogel.

The first step of bacterial or viral invasion is affinity and presumably multisite binding of bioparticles to an elastic matrix like a living tissue. We have demonstrated that model bioparticles such as inclusion bodies (spheres of about 1 microm in size) Escherichia coli cells (rods 1 x 3 microm), yeast cells (8 microm spheres), and synthetic microgel particles (0.4 microm spheres) are binding via different affinity interactions (IgG antibody-protein A, sugar-lectin, and metal ion-chelate) to a macroporous hydrogel (MH) matrix bearing appropriate ligands.

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding.

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE.

Direct capture of plasmid DNA from non-clarified bacterial lysate using polycation-grafted monoliths.

Monolith columns from macroporous polyacrylamide gel were grafted with polycations, poly(N,N-dimethylaminoethyl methacrylate) (polyDMAEMA), (2-(methacryloyloxy)ethyl)-trimethyl ammonium chloride (polyMETA) and partially quaternized polyDMAEMA prepared via treating polyDMAEMA-grafted columns with propylbromide. The polymer grafting degrees varied between 34 and 110%. The polycation-grafted monolithic columns are able to capture plasmid DNA directly from alkaline lysate of Escherichia coli cells.

Immobilised peptide displaying phages as affinity ligands. Purification of lactoferrin from defatted milk.

An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column.

Pore structure in supermacroporous polyacrylamide based cryogels.

Pore size and thickness of pore walls in macroporous polyacrylamide gels, so-called cryogels (pAAm-cryogels), were controlled by varying the content of monomers in the initial reaction mixture and the cross-linker used. The thickness of pore walls in pAAm-cryogels increased with increasing concentration of monomers in the initial reaction mixture. Pore volume in the supermacroporous pAAm-cryogels was in the range of 70-93% and decreased with increasing concentration of monomers in the reaction feed.

Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format.

A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli.

Capture of bacterial endotoxins using a supermacroporous monolithic matrix with immobilized polyethyleneimine, lysozyme or polymyxin B.

Bacterial endotoxins (BEs) are integrated part of Escherichia coli, a microorganism widely used for the production of recombinant proteins. BEs should be eliminated in the course of down stream processing of target protein produced by these bacteria. Supermacroporous monolith (continuous bed) columns, so called cryogel columns, with immobilized polyethyleneimine (PEI), polymyxin B (PMB) and lysozyme were employed for BEs capture.

Cell chromatography: separation of different microbial cells using IMAC supermacroporous monolithic columns.

Supermacroporous monolithic columns with Cu(2+)-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 microm) 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.