Improved sedimentation field-flow fractionation separation channel for concentrated cellular elution

Highlights

• Evolution of SdFFFF channel: insertion of an outlet stream splitter and downscaling.

• Respect of channel flow path and “Hyperlayer” elution mode for cell.

• Splitter cell recovery (>90–95%) increased with external field.

• Improved cancer stem cells sorting: 10 fold enrichment.

Abstract

SdFFF is now commonly used for cell sorting. Nevertheless, as with many other separation methods, SdFFF Hyperlayer elution leads (1) to sample dilution resulting in cell loss which could restrict further use; and (2) to a high output flow rate impacting detector sensitivity and selectivity. In order to limit these problems, we proposed modifications of the SdFFF separation channel consisting both in downscaling and the insertion of an outlet stream splitter. This last system corresponded to a strip which divides the flow rate output into two parts, one containing concentrated cells in a reduced volume and flow rate, the other containing the excess mobile phase useless for further cell manipulation, detection and characterization. For the first time we have shown that splitter implementation and downscaling respected channel flowing and resulted in Hyperlayer elution of around 95% of cells in less than 50% of input flow rate. Improved cell sorting was demonstrated by enrichment (∼10 times) of cancer stem cells from WiDr cells with two times less quantity of injected cells.

Introduction

Developed in the late 60s by Giddings [1], field-flow fractionation (FFF) methodology is described as one of the most versatile separation techniques [2]. This chromatographic-like separation family, in particular sedimentation-FFF (SdFFF), appears to be well suited for isolation and characterization of micron-sized species such as cells. Indeed, SdFFF is a gentle, non-invasive and tagless method [3], [4]. These advantages are based on the drastic limitation of cell-solid phase interactions by the use of (1) an empty ribbon-like separation channel without a stationary phase and (2) the “Hyperlayer” elution mode, a size/density driven separation mechanism [5], [6], [7], [8], [9], [10], [11]. The principle of cell separation is based on physical criteria such as size and density [3], [4], [5], [6], [7], [12], and depends on the differential elution of species submitted to the combined action of (1) a parabolic profile generated by flowing a mobile phase through the channel and (2) an external field applied perpendicularly to the flow direction [7]. In SdFFF, a multigravitational external field is generated by rotation of the separation channel in a rotor basket, constituting one of the most complex instrument used in FFF separation [7], [12]. In the last decade, our laboratory has developed prototypes [13], [14] and applications for cell sorting in new fields such as neurology and cancer stem cells (CSC) [12], [15], [16], [17], [18], [19], [20].

Nevertheless, as with many separation methods, to be efficient SdFFF leads to diluted samples, amplified by the use of an empty channel. Unfortunately, this can result in injurious dispersion of rare cells (i.e. CSC present in a tumor biopsy) as well as increased cell loss during further preparation, culture or characterization steps. Furthermore, SdFFF is only a separation method, and cannot detect or characterize particles. Then, dilution and high flow rate also impact detector sensitivity and selectivity. In this work, the separation channel was modified to decrease output flow rate and cell dilution.

As previously demonstrated in different FFF devices, one solution could be the insertion of an outlet stream splitter. It could be described as a strip, a blade which divided the flow stream at the outlet of the FFF channel. In such a way the concentrated cells would be splitted away from the excess of the mobile phase [21], [22], [23], [24]. Indeed, size and density based cell separation takes place in the first part of separation channel, according to the “Hyperlayer” elution mode. When separation is complete, the splitter divided the channel flow into two parts, one from the accumulation wall side containing the maximum of sorted cells in a reduced volume enhancing detection and further uses, the second from the depletion wall containing the excess mobile phase, useful for efficient cell sorting, but useless for subsequent cell culture, characterization or detection. Then, the device always operates such as an SdFFF device and should not be assimilate as a centrifugal SPLITT device which differs by channel design, operation mode and number of separation stages [2], [25], [26], [27], [28], [29]. The implementation of this splitter led to an increased void volume which was counterbalanced by the use of a downscaled channel [30]. After examining the impact of the splitter on flow path and void volume, we studied the efficiency of this new channel to sort cancer stem cells from colon WiDr cancer cell line. Our results showed that it was possible to collect nearly 95% of living cells in a 50% reduced volume compared to a classical channel, with effective CSC sorting.