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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Swelling behavior of poly(vinyl alcohol) cryogels employed as matrices for cell immobilization

Poly(vinyl alcohol) cryogels are prepared from aqueous solutions of the polymer by freezing and thawing and are employed as matrices for cell immobilization. The swelling behavior of these macroporous gel carriers in pure water and in solutions of certain compounds (salts, amino acids, and glucose) was studied to elucidate the osmotic properties of the cryogels during long-term exposure to aqueous media. It was shown that after the initial sol fraction was washed out, the residual gel matrix possessed high stability even at extreme pH conditions (acid or alkali concentration up to 1.0 mol l⁻¹) or in the presence of strong chaotropic salts such as sodium rhodanide. Although the macroporous supermolecular structure of the carriers under consideration underwent certain changes as a result of aging processes during prolonged washing of the gel, the high porous morphology of the material was retained.     

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.     

Pt-based electro-catalytic materials derived from biosorption processes and their exploitation in fuel cell technology

Yeast-based biomass, immobilised in polyvinyl alcohol (PVA) cryogels, was used as a biosorbant material for the recovery of platinum (PtCl₆²⁻) from aqueous solutions. The resulting biomass-Pt matrices were then employed directly as an electro-catalytic anode in a fuel cell configuration to generate electrical energy from renewable sources such as glucose and ethanol. We suggest an integrated strategy incorporating the derivation of a high-value product from a bioremediative process with a view towards producing energy from renewable fuels such as glucose and ethanol.     

Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.     

Comparison of citric acid production byAspergillus niger immobilized in gels and cryogels of polyacrylamide

Aspergillus niger was immobilized in cryogels and in conventional gels of polyacrylamide. The growth of cells entrapped in two kinds of gels and the production of citric acid by the immobilized cells were investigated and compared. Cells immobilized in cryogels were more suitable for citric acid production.     

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. The equilibrium swelling ratio of the DMTLT cross-linked pNIPA cryogels at temperatures below lower critical solution temperature (LCST) of pNIPA, was over ten times higher than that of the gels synthesized at room temperature from the same feed composition. The MBAAm cross-linked pNIPA cryogels synthesized in water exhibited the highest equilibrium swelling and the fastest response. The critical transition temperature, T c, was lower ( T c approximately 31 degrees C) for pNIPA-cryogels synthesized in dioxane/water media or cross-linked with DMTLT as compared to MBAAm cross-linked pNIPA cryogels synthesized in water (T c approximately 33 degrees C). Scanning electron microscopy (SEM) revealed different porous structure and pore surface morphology depending on the cross-linker (MBAAm or DMTLT) and the solvent (water or dioxane/water) used. Gels and cryogels were also characterized by SAXS, showing that the nanostructure of the samples is related to swelling.     

Monolithic cryogels made of agarose-chitosan composite and loaded with agarose beads for purification of immunoglobulin G

In order to obtain a novel absorbent with high adsorption capacity for the purification of immunoglobulin G (IgG), continuous supermacroporous agarose beads embedded agarose-chitosan composite monolithic cryogels (agarose-chitosan cryogels) were prepared by cryo-copolymerization of agarose-chitosan blend solutions with glutaraldehyde as the crosslinker in the presence of agarose beads. After coupling 2-mercaptopyridine onto divinylsulfone-activated matrix, the obtained cryogels were used for the purification of IgG. The microstructure morphologies of the cryogels were analyzed by scanning electron microscopy. The results showed that the obtained cryogels possess interconnected pores of 10-100 μm size. The specific surface area was 350 m(2)/g with maximum adsorption capacity of IgG 71.4 mg/g. The cryogels showed workable stability, and can be reused at least 15 times without significant loss in adsorption capacity. IgG purity after one-step purification from human plasma was monitored by electrophoresis and the average recovery was estimated to be 90%.     

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.     

Transforming growth factor beta stimulates fibroblast-collagen matrix contraction by different mechanisms in mechanically loaded and unloaded matrices

Studies were carried out to test the idea that transforming growth factor beta (TGFbeta) stimulated fibroblast contraction of collagen matrices by different mechanisms depending on mechanical loading on the cells and matrix. Under mechanically unloaded conditions (floating matrices), TGFbeta stimulated contraction directly as an agonist and indirectly by preactivating cells to express the myofibroblast phenotype. Increased contraction of floating matrices by preactivated cells appeared to result in part from an autocrine mechanism. Under mechanically loaded conditions (stressed matrices), TGFbeta had no direct agonist effect on contraction. Fibroblasts preactivated to become myofibroblasts showed increased ability to transfer tension to stressed matrices, and tension persisted even after the cells' actin cytoskeleton was disrupted. Our findings are consistent with the idea that fibroblasts activated to become myofibroblasts in vitro have increased contractile activity and indicate that multiple mechanisms that differ depending on mechanical loading on the cells and matrix are involved.     

The specific biopanning of single-domain antibody against haptens based on a functionalized cryogel

Phage display technology is commonly applied for high-throughput screening of single-domain antibodies (sdAbs), and the problem of non-specific adsorption caused by carrier proteins seriously affects the biopanning of single-domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)-functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR-specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre-incubation were applied to the biopanning process through single-factor experiments. Results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single-domain antibodies to haptens.     

Immobilization of hydrocarbon-oxidizing bacteria in poly(vinyl alcohol) cryogels hydrophobized using a biosurfactant

A simple biosurfactant-based hydrophobization procedure for poly(vinyl alcohol) (PVA) cryogels was developed allowing effective immobilization of hydrocarbon-oxidizing bacteria. The resulting partially hydrophobized PVA cryogel granules (granule volume 5 microl) contained sufficient number (6.5 x 10(3)) of viable bacterial cells per granule, possessed high mechanical strength and spontaneously located at the interface in water-hydrocarbon system. Such interfacial location of PVA granules allowed high contact of immobilized biocatalyst with hydrophobic substrate and water phase, thus providing bacterial cells with mineral and organic nutrients. As a result, n-hexadecane oxidation efficiency of 51% after 10-day incubation was achieved using immobilized biocatalyst. PVA cryogels with increased hydrophobicity can be used for immobilization of bacterial cultures performing oxidative transformations of water-immiscible organic compounds. Immobilization of in situ biosurfactant producing Rhodococcus bacteria into PVA cryogel is discussed. PVA cryogel granules with entrapped alkanotrophic rhodococcal cells were stable after 10-month storage at room temperature.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.     

Adsorption and elution behaviors of bovine serum albumin in metal-chelated affinity cryogel beds

Metal-chelated supermacroporous cryogels are effective supports for affinity chromatographic separation of biomolecules in downstream processes. In this work, polyacrylamide cryogel beds were prepared in glass columns (16 mm inner diameter) and coupled with iminodiacetic acid (IDA). These cryogels were loaded with Zn²⁺ and Ni²⁺ and the so-called Zn²⁺-IDA-cryogels and Ni²⁺-IDA-cryogels were obtained. Permeabilities and height equivalent to theoretical plates (HETPs) of these cryogel beds were measured and the cryogel structure was analyzed using scanning electron microscopy (SEM). Bovine serum albumin (BSA) was employed as a model protein to elucidate the adsorption and elution behaviors of these cryogels under various conditions, such as different flow rate, solution pH, and composition of the eluents. The results showed that the Zn²⁺-IDA-cryogels and Ni²⁺-IDA-cryogels in this study had interconnected supermacropores and high water permeabilities (∼10⁻¹¹ m²). The loading flow velocity had a weak influence on the breakthrough curves and binding capacities for BSA, while the solution pH had an evident effect on the binding capacities for BSA in these cryogels. Maximum binding capacity for BSA was observed near the isoelectric point of BSA. The bound BSA can be eluted effectively using an imidazole solution. A low-eluting flow rate was found to be beneficial to the elution process. Possible mechanisms were proposed and discussed.     

Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production

In this study, a new cell immobilization technique is presented. Cells of Clostridium acetobutylicum DSM 792 form a macroporous aggregate through cryogelation with concomitant crosslinking together with activated polyethyleneimine (PEI) and poly(vinyl alcohol) (PVA). The cell based cryogel presents a highly porous, elastic structure with walls consisting of densely packed crosslinked cells. The immobilization process maintained the viability of cells as new bacterial cells were observed when gel-plugs were incubated in liquid medium, glucose was consumed and solvent production was observed. Solvent production was improved 2.7-fold in immobilized cells in comparison to free cells. It was possible to reuse the gel-plugs 3–5 times in partial or completely fresh medium, reaching a maximum butanol concentration in the broth of 18.2 g/l and yield of 0.41 (g/g) in one of the cycles. The use of cells based cryogels can be a good alternative for improvement of acetone-butanol-ethanol (ABE) process as cells are immobilized in a macroporous structure with low limitations for mass transfer with potential for high yield production.     

Degradation of thiodiglycol, the hydrolysis product of sulfur mustard, with bacteria immobilized within poly(vinyl) alcohol cryogels

Alcaligenes xylosoxidans subsp. xylosoxidans (SH91) capable of biodegradation of thiodiglycol (TDG) were immobilized in poly(vinyl) alcohol (PVA) cryogels. Cryoimmobilized biocatalyst was formed as spherical granules with a diameter of 0.5 mm; the biomass concentration inside the gel matrix was as high as 10% (w/w). The immobilized cells were capable of rapid degradation of TDG in tap water or potassium phosphate buffer (100 mM, pH 8.0) containing only (NH4)2 SO4. The immobilized biocatalyst did not show any substrate inhibition up to 200 mM TDG, and retained 100% activity during three months of continuous use in a repeated-batch bioreactor.     

Fibroblast quiescence in floating collagen matrices: decrease in serum activation of MEK and Raf but not Ras

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors.     

Immobilization of champagne yeasts by inclusion into cryogels of polyvinyl alcohol for preventing cell escape from carrier matrix

Wine champagnizing, a process involving the use of champagne yeasts immobilized by inclusion into cryogels of polyvinyl alcohol, has been studied. Treatment of yeast cells with the autoregulatory factor d1 was proposed as a means of preventing the cell escape from the carrier matrix. Such a treatment inhibited growth and proliferation processes in yeasts cells, without affecting the activity of fermentation; the resulting champagne had the same organoleptic and chemical characteristics as its counterparts obtained using traditional techniques.     

Oxidized Dextran as Crosslinker for Chitosan Cryogel Scaffolds and Formation of Polyelectrolyte Complexes between Chitosan and Gelatin

Macroporous scaffolds composed of chitosan and using oxidized dextran as a crosslinker are produced through cryogelation. Introducing gelatin as a third component into the structure results in the formation of mesopores in the pore walls, which are not seen if gelatin is excluded. The mesoporous structure is explained by the formation of polyelectrolyte complexes between chitosan and gelatin before crosslinking takes place. The scaffolds exhibit highly elastic properties withstanding compressions up to 60%. The in vitro biocompatibility of the cryogels is evaluated using fibroblasts from a mouse cell line (L929) and it is seen that the cells adhere and proliferate on the scaffolds. The mesoporous structure seems to have a positive effect on proliferation.