Akshay Srivastava

Cell separation using cryogel-based affinity chromatography.

In cell affinity chromatography, type-specific cell separation is based on the interaction between cell-surface receptors and an immobilized ligand on a stationary matrix. This protocol describes the preparation of monolithic polyacrylamide and polydimethylacrylamide cryogel affinity matrices that can be used as a generic type-specific cell separation approach. The supermacroporous monolithic cryogel has highly interconnected large pores (up to 100 μm) for convective migration of large particles such as mammalian cells. In this protocol, they are functionalized to immobilize a protein A ligand by a two-step derivatization of epoxy-containing cryogel monolith (reaction with ethylenediamine and glutaraldehyde). Target cells were labeled with specific antibodies and then they were captured in the cryogel through affinity with protein A. These specifically captured cells were recovered in high yields while retaining their viability by mechanical squeezing of the spongy and elastic cryogel matrices. The suggested cell separation protocol takes <30 min for complete separation on a preprepared protein A–immobilized cryogel column.

Synthesis and Characterization of a Temperature-Responsive Biocompatible Poly(N-vinylcaprolactam) Cryogel: a Step Towards Designing a Novel Cell Scaffold

A poly(N-vinylcaprolactam) (PVCl) cryogel and poly(N-vinylcaprolactam)-co-gelatin interpenetrating cryogel network were synthesized and characterized with respect to physical and biological properties. The PVCl cryogel was synthesized in 5% dimethyl sulfoxide (DMSO) containing aqueous medium and PVCl-co-gelatin interpenetrating cryogel network was synthesized in water as solvent. Both these cryogel networks have good physical morphology as confirmed by scanning electron microscopy. The porosity of these cryogels were characterized by various methods like, adsorption of water and cyclohexane and confirmed by analysis on mercury porosimeter and nitrogen adsorption studies. The porosity of PVCl and PVCl-co-gelatin cryogels was 96% and 98%, respectively, and the permeability of the two types of cryogels was 1.01 × 10−12 m4/Ns and 1.66 × 10−12 m4/Ns, respectively. The effective diffusion coefficients (D eff) of bovine serum albumin (BSA) in PVCl cryogel and PVCl-co-gelatin cryogel were 3.5 × 10−7 cm2/s and 3.4 × 10−7 cm2/s, respectively. These materials were further characterized to demonstrate its interaction with biological system. The blood compatibility studies showed minimal hemolysis (4–6%) caused by these materials and a very low adsorption of BSA (0.001–0.002 mg/g dry scaffold). However, the fetal bovine serum (FBS) adsorption studies demonstrate the protein binding at 37°C. Furthermore, cytotoxicity test and the fibroblast cell adhesion studies showed the potential of these PVCl-based cryogels for suitable biomaterial applications.

Monosaccharide-Responsive Phenylboronate-Polyol Cell Scaffolds for Cell Sheet and Tissue Engineering Applications

Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.