Preparation of Cryogels
Gelation at subzero temperatures (cryotropic gelation) is a versatile technology platform that allows for preparation of macroporous monoliths (cryogels), from a variety of synthetic and natural precursors and a wide range of porosity. Cryogels are produced in semi-frozen aqueous medium when growing ice crystals perform as pore-forming agent and template large interconnected pores after melting. In a semi-frozen medium, though looking like a solid block, it actually consists of two main parts: the system of growing ice crystals and the small regions surrounded by ice crystals (so-called non-frozen liquid microphase). While all reagents are concentrated in the non-frozen liquid microphase, some part of the solvent (water) remains non-frozen and provides the solutes accumulated into non-frozen liquid microphase with sufficient molecular or segmental mobility for reactions to perform. A unique property of acceleration of chemical reactions performed in non-frozen liquid microphase compared to the chemical reaction in bulk solution is often observed within a defined range of negative temperatures. After melting solvent crystals, a system of large interconnected pores is formed. Thus the shape and size of the crystals formed determine the shape and size of the pores formed after defrosting the sample. Due to the pronounced concentration of reagents in the non-frozen liquid microphase (so-called, effect of cryo-concentrating), the pore walls in cryogels are formed from the highly concentrated polymer phase thus rendering the cryogels with high mechanical stability. Cryogels are prepared through different routes of gel formation as hydrogen bonding, chemical cross-linking of polymers and free radical cross-linking polymerization, mainly in an aqueous medium. The gel surface chemistry is designed depending on particular application. By keeping the proper control over all parameters, the cryogels are produced reproducibly with tailored properties. Cryogels are principally differing from the conventional gels prepared in non-frozen medium specifically due to the system of interconnected macropores (frequently endowing the cryogels with sponge morphology) and due to the structure of pore walls which are formed from highly concentrated polymer phase. The presence of large (10-200 µm) interconnected pores in the cryogels specified their main applications as suitable macroporous continuous phase for processing the particulate fluids as suspensions of viruses, microbial and mammalian cells, and as 3D scaffolds in tissue-engineering.
Macroporous Cryogel stained with FITC
Macroporous cryogel with doublemacroporous polymer networks stained with Rhodamine (redcolored) and FITC (greencolored)
Cryogel cell scaffolds and bioreactors
The cryogels meet most of the requirements to be suitable as supports for cell cultivation. The open-porous structure of 3D cryogel scaffolds with bioactive surfaces and tissue-like elasticity ensure proper microenvironment for the living cells. Size of pores, surface chemistry and elasticity of pore walls can be varied to a large extent when preparing the cryogel scaffolds for cell culture application. The cryogel scaffolds are prepared from different low- and high-molecular weight precursors both of synthetic and natural origin. By introducing special functionalities as extracellular matrix (ECM) recognition elements or bioactive substances which induce tissue in-growth, it is possible to design the 3D cryogel biomimetics with bioactive surfaces. Different types of cells were shown to grow and proliferate on 3D cryogel scaffolds specially designed for a standard 96-well plate. Such a platform represents a unique in vitro model for cell-based assays with potential in drug screening. The cryogelation approach allows for the broad design of cryogel scaffolds with open permeable structure, required gel surface chemistry and optimal mechanical strength.
The cryogel monolithic bioreactors were shown have low back pressure and enhanced mass transport of substrates and metabolites to and from the entrapped cells. Thus, hybridoma cells and human colon cancer HCT116 cells were effectively attached and cultured in gelatin-coated cryogel monolith bioreactors for long-term continuous production of different therapeutics.
CD34 human myeloid leukemia KG-1 cells labeled with anti-CD34 are bound to protein A cryogel column
E.coli cells, bound to ion-exchange cryogel column
Cryogels for cell separation
Cryogel monolith affinity columns at present are the only available macroporous adsorbents suitable for chromatography of cells. Due to the presence of large (10-200 µm) interconnected pores, the mass-transport in cryogels is due to convection allowing for free passage of different-sized solutes and bioparticles with size up to 10 µm. When there are no specific ligands on the surface of cryogels, the cells freely pass through such monolithic columns (so-called, naked columns) without blocking the pores. The large size of interconnected pores in the cryogel monoliths allowed for red blood cells to pass freely through the naked cryogel columns with no lysis. However, when specific ligands are presented on the surface of the pores, the microbial and mammalian cells can bind and be eluted from the cryogel columns with high yield and retained viability using the conventional elution methods. High elasticity of cryogels allows for the innovative and principally different way (compared to the conventional elution methods) for the elution of affinity bound cells, i.e. chemical (or specific) elution accompanied by an extensive mechanical compression of the cryogels monoliths resulting in efficient detachment of bound cells with high recovery and retained viability. This novel efficient elution strategy together with continuous macroporous structure of cryogel monoliths makes these adsorbents very attractive for application in affinity cell separation. The main strategy for the separation of mammalian cells implies first labeling the cells of interest with specific antibodies followed by application to Protein A-cryogel column and, eventually, release of affinity bound labeled cells via mechanical compression of protein A-cryogel monolith.
Cryogels in biocatalysis
The unique porous structure of cryogels together with elasticity and high mechanical stability have motivated their wide use as matrices for cell immobilization. Depending on pore size of cryogels and potential applications of immobilized cell biocatalysts (ICBs), two main approaches for cell immobilization are used, namely immobilization via mechanical entrapment into cryogels and immobilization to the surfaces of pores within cryogels via specific interactions or through adsorption. Cryogels can be prepared as beads, sheets, monoliths or can be designed inside an open-ended protective plastic housing. Due to the high stability of cryogel matrix, the most of the ICB withstand long storage in a dried state and maintained high cell activity after re-activation in a nutrient medium.
The ICBs can be regenerated by simple incubation in the growth medium to restore bioconversion activity. Long term use of the same ICB is important for the development of an efficient industrial process. Another important concern is the preservation of the ICBs to keep them without contamination for prolonged time. From these perspectives, microorganisms selected from extreme environments (extremophiles), are of the best choice for the preparation of ICB. Thus, PVA-entrapped alkaliphilic Bacillus agaradhaerens, showed high stability at storage without any additives and activity of the stored ICB was completely revived by activation in the culture medium. An excellent stability of cryogels allowed for using ICBs in a water-poor medium. For example, the PVA-cryogel-entrapped cells of Bacillus pseudofirmus AR-199 with β-galactosidase activity were used for continuous synthesis of alkyl galactosides by transglycosylation.
Dried Cryogel monoliths. Diameter in swollen state 10 mm, volume 5 ml.
Cryogel beads. Diameter 3 mm