Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells

Abstract

Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm × 7.1 mm Ø) with immobilized concanavalin A was used. Chromatographic columns (113 mm × 7.1 mm Ø) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl α-d-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.

Introduction

Despite being one of the most powerful and widely used techniques in the separation of biomolecules, affinity chromatography is a relatively novel approach among existing methods for the specific separation of different population of cells [1], [2], [3], [4], [5]. Although several studies on cell fractionation using affinity columns have been reported in the late seventies and early eighties [6], [7], [8], [9], [10], [11] a real breakthrough of the technology was never observed. The four main obstacles on the way of designing a versatile chromatographic procedure for an efficient cell affinity separation are the large size of cells and hence their negligible diffusivity; complex structure and chemistry of cell surface that leads to high non-specific adsorption [12], [13]; a multivalent nature of specific binding that highly complicates elution of bound cells [2], [14], [15] and the mixed mode of interactions that may take place between the affinity support and the cell surface. Low diffusivity of the cells could be circumvented when using expanded bed adsorption chromatography (EBAC). Although a few examples on cell fractionation by EBAC have recently been reported [2], [16], it is doubtful that this technique will find wide application for cell separation as it does not overcome the problem of recovering cells bound to affinity surface. In the published protocols, the detachment of bound cells was achieved by removing the adsorbent from the column and agitating the slurry in order to elute bound cells using mechanical shear, the procedure resulting in reduced viability of the cells [2].

Different strategies have been tested in an attempt to overcome the problem of strong multipoint attachment of cells to affinity surfaces. For example, in a membrane-based chromatographic system bound B-cells were released by transmembrane diffusion of hydrochloric acid (pH 1) into a flow of neutralizing normal saline [1]. In approaches that do not require such drastic elution conditions cell detachment was achieved by the use of ligands immobilized through cleavable bonds [10], the passage of air–liquid interfaces [17] or by using flow-induced shear forces [18]. The latter leads to a pronounced dilution of the preparation of eluted cells and also involves the risk of cell damage.

Affinity cryogel monoliths have been recently introduced as chromatographic adsorbent for the separation of biological nanoparticles (bacterial and mammalian cells, viruses, plasmids, cell organelles, inclusion bodies [3], [5], [19], [20], [21], [22]). Polyacrylamide-based cryogels are produced when polymerization proceeds at subzero temperatures when most of the solvent, water, is frozen while the dissolved monomers are concentrated in small nonfrozen regions. Ice crystals perform as porogen, forming after melting a system of large, 10–100 μm, interconnected pores [3]. Macropores filled with liquid, e.g. running buffer, constitute about 90% of the swollen cryogel monolith [23] and provide unhindered passage of cells through native cryogel monoliths without affinity ligands [24] or of cells not having receptors for the immobilized affinity ligands [5]. Various coupling chemistries can be used for the immobilization of ligands on the cryogel adsorbents [25]. Cryogels are highly elastic and about 70% of the total liquid inside can be removed by mechanical compression. The compressed cryogels swell again when contacted with liquid and restore the original shape within less than a minute. Recently, we have developed a completely new method of eluting cryogel-bound bioparticles by elastic deformation of cryogels [14], [26]. This phenomenon was demonstrated for a variety of bioparticles of different sizes and for different ligand–receptor pairs (IgG–protein A, carbohydrate–ConA, metal ion–chelating ligand) and is believed to be due to breaking of polyvalent bonds between bound particles and affinity cryogel matrix and the change in the distance between affinity ligands when the elastic matrix is deformed [14]. The release of affinity bound cells by mechanical compression of a cryogel monolithic adsorbent is a unique and efficient way of cell detachment. This detachment strategy and the continuous macroporous structure make cryogels very attractive for application in cell chromatography.

Due to the capillary forces which keep liquid inside the interconnected macropores, cryogel monoliths have drainage-protecting properties and can be used in a screening 96-minicolumn plate format in which the monoliths are inserted into open-ended wells of a microtiter plate [26], [27].

The objective of this work was to demonstrate the possibility of an efficient chromatographic cell separation on an example of two types of model cells, Escherichia coli and Saccharomyces cerevisiae, using ConA-cryogel column (composed of six cryogel monoliths 18.8 mm × 7.1 mm stacked on top of each other). The optimization of the separation conditions was performed using native and ConA-cryogel monoliths in a 96-minicolumn plate format. Moreover, ConA-cryogel monoliths in this format were used for the analysis of cell content in the chromatographic fractions using a method of integrated isolation and assay of the metabolic activity of cells [26], [28].