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.

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.

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. The macroporous composite molecularly imprinted cryogels were formed inside the open-ended protective shells, known as Kaldnes carriers. These adsorbents (defined as Macroporous Gel Particles, MGPs) were evaluated on the removal of E2, NP and atrazine from water using different column configurations, namely column filled with the MGPs (packed-bed column) and in moving-bed reactors (defined here as moving-bed MGPs reactor). Complete binding (> 99%) of E2 from a spiked aqueous solution (1 mg/L) was achieved using E2-MIP/MGPs in a moving-bed MGPs reactor at the retention time in the reactor of 4 min, while only 77% was bound to the nonimprinted medium (NIP/MGPs). Similar results were also obtained for the adsorption medium imprinted with atrazine. All contaminants studied (E2, atrazine and NP) were effectively removed from water at low (environmentally relevant) concentrations by the respective adsorption medium.

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). The newly developed platform based on selected bacteriophages immobilized within macropores of the monolithic cryogels presents a convenient alternative to antibodies for fast and selective development of the specific adsorbent.

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. Due to the large pore size in macroporous monoliths the particulate material present in non-clarified feeds did not block the columns. The captured plasmid DNA was eluted with 1M NaCl as particulate-free preparation with significantly reduced content of protein and RNA as compared to the applied lysate.

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. Bound lactoferrin was eluted using 1M NaCl with a purity of >95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.

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. Due to the large interconnected pores it was possible to use cryogel columns at flow rates as high as 10 ml/min. The columns packed with Sepharose CL-4B with immobilized PEI, PMB and lysozyme were impossible to use at these high flow rates due to the collapse of the bed. The dynamic capacities of the cryogel columns were nearly independent of the flow rate. In the presence of EDTA, BEs were quantitatively captured from mixtures with a model protein, bovine serum albumin (BSA) at pH 7.2 with practically no protein losses. At pH 3.6 BEs were captured directly from non-clarified E. coli cell lysate resulting in more than 10(4) times BEs clearance.

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. The porous structure of the pAAm-cryogels was visualized using environmental scanning electron microscopy (ESEM) that allowed monitoring of the dehydration process in pAAm-cryogels. The accessibility of ligands covalently coupled to the polymer backbone for low molecular weight target, Cu() ions, and high molecular weight target, the protein lysozyme, was assessed for pAAm-cryogels produced from feeds with different monomer concentration. The amount of bound Cu() ions increased linearly with increasing monomer concentration in the reaction feed, suggesting that all ligands are equally accessible for small targets. On the contrary, lysozyme binding demonstrated a clear maximum at monomer concentration about 18% suggesting that only ligands present at the surface of pore walls are accessible for high molecular weight targets.

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. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries.

High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format.

Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples.

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.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. 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. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the Cu(2+)-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, 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.

Polymeric cryogels as promising materials of biotechnological interest.

Cryogels are gel matrices that are formed in moderately frozen solutions of monomeric or polymeric precursors. Cryogels typically have interconnected macropores (or supermacropores), allowing unhindered diffusion of solutes of practically any size, as well as mass transport of nano- and even microparticles. The unique structure of cryogels, in combination with their osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of biological nanoparticles (plasmids, viruses, cell organelles) and even whole cells. Polymeric cryogels are efficient carriers for the immobilization of biomolecules and cells.

Affinity fractionation of lymphocytes using a monolithic cryogel.

A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60-70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.

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.

Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels.

The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 microm) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)6-tagged single chain (sc) Fv antibody fragments, (His)6-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)6-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques.

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.

Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles.

Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 microm. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.

Addition of Polybrene improves stability of organophosphate hydrolase immobilized in poly(vinyl alcohol) cryogel carrier.

Organophosphate hydrolase, covalently attached to the beads of poly(vinyl alcohol) cryogel in the presence of Polybrene, was fivefold more stable in 15% (v/v) ethanol solution than the free enzyme. Immobilized biocatalyst, prepared with an addition of Polybrene, retained a half of its initial activity in 50% (v/v) aqueous ethanol solution, 90% of activity during 10 working cycles of Paraoxon hydrolysis and 85% of activity after storage in the 50 mM CHES buffer (pH 9.0) at room temperature for 2 months.

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 host's neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.

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. The cryogels were prepared in plastic carriers, resulting in desired mechanical stability and named as macroporous gel particles (MGPs). Sorption and desorption experiments for different metals (Cu²+, Zn²+, Cd²+, and Ni²+ with IDA adsorbent and Cu²+ and Zn²+ with TBA adsorbent) were carried out in batch and monolithic modes, respectively. Obtained capacities with Cu²+ were 74 µmol/mL (TBA) and 19 µmol/mL gel (IDA). The metal removal was higher for pH values between pH 3 and 5. Both adsorbents showed improved sorption at lower temperatures (10°C) than at higher (40°C) and the adsorption significantly dropped for the TBA adsorbent and Zn²+ at 40°C. Desorption of Cu²+ by using 1 M HCl and 0.1 M EDTA was successful for the IDA adsorbent whereas the desorption with the TBA adsorbent needs further attention. The result of this work has demonstrated that MGPs are potential treatment alternatives within the field of environmental separations and the removal of heavy metals from water effluents.