Vladimir I. Lozinsky

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.

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.

Study of cryostructurization of polymer systems VII. Structure formation under freezing of poly(vinyl alcohol) aqueous solutions

Rheological properties of the cryogels produced by freezing of concentrated aqueous solutions of poly(vinyl alcohol) have been studied. These properties were shown to depend on the polymer concentration in the initial solution, on PVA molecular weight, cryostructurization duration and temperature. Electron microscopy demonstrates the heterogeneous porous structure of these cryogels and the dependence of the observed pattern on the conditions of formation of the studied objects.

Poly(vinyl alcohol) cryogels employed as matrices for cell immobilization. 2. Entrapped cells resemble porous fillers in their effects on the properties of PVA-cryogel carrier

Abstract Poly(vinyl alcohol) cryogels which are prepared by freeze-thawing of concentrated aqueous solutions of the polymer are used as carriers for cell immobilization. Biomass entrapment causes a variation in the mechanical properties of the composites obtained. With rheological testing and scanning electron microscopy, it was shown that cells which manifested the reinforcing action in respect to the carrier resembled in their “physical behavior” simple model inorganic fillers of definite porosity. This porosity allows macromolecules of gel-forming polymer to penetrate into the inner regions of the particles and promote the formation of tight contacts between the matrix and dispersed phase, such as entrapped cells in particular. The immobilized cultures and model fillers were as follows: Citrobacter intermedius, Zymomonasas mobilis , and Pseudomonas sp. bacteria, Saccharomyces cerevisiae native and modified yeast cells, titanium dioxide, silica gels, and controlled pore glass.

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

Abstract 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.

Study of cryostructuration of polymer systems. XII. Poly(vinyl alcohol) cryogels: Influence of low-molecular electrolytes

The influence of the presence of low-molecular electrolytes in initial solutions of poly(vinyl alcohol) (PVA) on the results of cryotropic gelation of the polymer (gelation caused by the freezing thawing) and on the swelling characteristics of PVA cryogels prepared in a salt-free medium were studied. The reinforcing ability of the electrolytes with respect to the gel strength for both alkaline element cations and simple low-molecular anions has been shown to be in agreement with the positions of these ions in corresponding lyotropic (Hofmeister) series. Namely, the ions (chaotropic ones), which are capable to interfere the H-bonding, disturbed somewhat the cryotropic gel-formation of PVA and facilitated the marked additional swelling of cryogels preliminary prepared in pure water medium, whereas the ions (antichaotropic ones), which are capable to promote the H-bonding, caused the formation of reinforced cryogels and resulted in the shrinking of cryogels prepared without salt additives. More pronounced effects were observed for anions as compared with cations. Some anomalous swelling behavior of PVA cryogel in Cs+-containing solutions was supposed to be associated with the formation of weak chelates. In addition to antichaotropic inorganic salts like NaF, rather high salting-out effects in respect to PVA were exhibited by wellsoluble amino acid salts: glycine zwitter-ions, lysine monochlorohydrate, and monosodium aspartate.

A Brief History of Polymeric Cryogels

Polymeric cryogels, the gels formed in moderately frozen gelling systems, have been empirically known for many decades, but systematic scientific research on various cryogels and the peculiarities of cryotropic gel formation only commenced at the beginning of the 1980s. This historical review briefly describes the principal stages of the studies on these very interesting gel materials. It also discusses some mechanisms of their formation, as well as summarizes published data on the main representatives of chemically crosslinked (covalent), ionically linked, and noncovalent (physical) cryogels.

On the possibility of mechanodestruction of poly(vinyl alcohol) molecules under moderate freezing of its concentrated water solutions

SummaryThe freezing of the concentrated PVA water solutions results in obtaining (after thawing) hydrogels of non-covalent nature. In the course of a single freezing-thawing cycle there is no any noticeable destruction or covalent cross-linking of PVA macromolecules.

Basic Principles of Cryotropic Gelation

Polymeric cryogels are the gel systems formed in moderately frozen solutions or colloidal dispersions of precursors potentially capable of gelling. Polymeric cryogels are of growing practical interest in various applied areas. The fabrication of any cryogel includes the following necessary stages: preparation of the feed system, its freezing, incubation of the gelation system in a frozen state, and thawing of the frozen sample. The nature of gel precursors, their concentration in the initial feed, and the conditions of each of the stages affect the physicochemical properties and porous morphology of the resulting cryogels. Certain specific effects are inherent in the processes of cryotropic gel formation, namely, apparent decrease in the critical concentration of gelation, acceleration of gel formation over a certain range of negative temperatures, a bell-shaped temperature dependence of the cryotropic gelation efficiency, and generation of the specific porosity peculiar to cryogels. This chapter presents the basic principles of cryotropic gelation processes and also discusses the factors influencing the properties of various types of cryogels.