Separation of Proteins and Cells

Since the transport of proteins across the interface in a PEG-dextran system follows comparatively simple dynamics, the study of transport processes of larger and more complex objects was self-evident. In this chapter the continuous separation of proteins from cells from lymphoblastoid cell lines (LCL-cells) is described. While proteins overcome the phase boundary and, supported by an electric field, leave the phase they have been initially dissolved in almost completely, lymphoblastoid cells are retained. They are unable to cross the phase boundary if the field strength stays below a threshold value.

12.1 Cells in Aqueous Two-Phase Systems

Contrary to proteins cells are relatively large particles. One of the difficulties in partitioning mammalian cells in macroscopic systems lies in the gathering of particles onto the interface of two phases by sedimentation or flotation, since cell densities are comparable to ATPSs made of PEG and dextran [27,226,227].

Furthermore, the partition behavior of cells in ATPSs is slightly different to the behavior of proteins. Often almost 100% of the cells prefer either the PEG or the dextran-phase [27], cf.

Fig. 12-1. For example, the affinity of cells to the phases can be influenced by adding sodium chloride (NaCl). At low concentrations of NaCl cells often prefer the PEG-phase but with an increasing amount of NaCl the affinity turns into a high preference to the dextran-phase [27,28].

Fig. 12-1: Partitioning of LCL cells (fluorescent dots) in an ATPS consisting of PEG and dextran. Here, all cells are found inside PEG bubbles demonstrating that cells often strongly prefer one of the two phases. In the left picture one bubble is marked by a dotted line.

But also the molecular weights of the used polymers can have a strong influence on the partitioning behavior [28]. For the separation experiments of proteins and cells described here, the standard PEG-dextran system (A) was applied, where almost 100% of the LCL cells prefer the PEG-phase.

12.2 Active Separation

For an active separation immiscible aqueous phases (type A) are injected separately into the main channel that is part of the microfluidic system described above. The lower PEG-phase, cf. Fig. 12-2, partly contains a mixture of BSA molecules and lymphoblastoid cells. In order to avoid possible gravity effects the cell migration is perpendicular to the direction of gravity.

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Fig. 12-2: (a) Schematic showing the transport of proteins and cells in a tri-laminated two-phase system. Below a critical electric field strength the cells are trapped at the two-phase boundary. (b) Entrance of the main channel. Cells (fluorescent dots) and proteins (background fluorescence) are located in the lower PEG-phase.

The total flow rates used in the experiments were 0.05 and 0.03 ml h^-1 for the upper and lower PEG-phases and 0.008 ml h^-1 for the dextran-phase, respectively. A detailed description of the microfluidic chip was already given in chapter 7. The labeling procedure of the proteins and cells can be found in chapter 2.

12.3 Results

At the entrance of the main channel both proteins and cells are dissolved in the same PEG-phase, cf. Fig. 12-2 and Fig. 12-3. Without an electric field and once the different phases get in contact, diffusion of proteins into the dextran-phase is observed. Halfway along the channel the cells are randomly distributed within the preferred PEG-phase while the proteins diffuse not only into the neighboring dextran-phase but also into the gel matrix below.

After applying an electric field the proteins start to move into the central dextran-phase and finally, near the outlet of the channel, into the upper PEG-phase and gel matrix. On the contrary, the cells are retained by the phase boundary and stay in the preferred PEG-phase. A further increase of electric field strength forces them to cross the phase boundary continuing their electrophoretic migration towards the positive electrode. Below this critical field strength, 100% of the cells are found in their preferred PEG-phase allowing a continuous separation of LCL-cells from proteins. A typical result of such an experiment at 4 Vdc is shown in Fig. 12-3. Already near the entrance, cf. Fig. 12-3 (a), parts of the proteins have been already transported into the dextran-phase. Near the outlet, Fig. 12-3 (c), a complete depletion of the phase from proteins has been reached while the cells have been trapped in their preferred phase and collected at the phase boundary.

In order to visualize the transport behavior of the proteins the fluorescence intensities across the channel at different positions are combined in one graph, see Fig. 12-4. If no electric field is applied the proteins mainly stay in their initial phase and slightly diffuse into the ambient liquid. In case of an applied electric field and halfway along the channel the major part of the proteins has passed the first phase boundary. Near the outlet, cells and proteins are separated almost completely.

Separation of Proteins and Cells

Fig. 12-3: Separation of lymphoblastoid cells from proteins in a PEG-dextran system. The cells are visible as bright spots, while the background fluorescence is due to proteins. At 4 Vdc

and halfway along the channel (b) the proteins have been partially transported into the dextran-phase, by contrast cells are retained at the phase boundary. (c) At the end of the channel cells have been trapped in their preferred phase and collected at the phase boundary while the proteins have already crossed the intermediate dextran-phase and reached the upper PEG-phase of the trilaminated flow.

But the separation depends not only on the applied electric field but also on the flow velocity and the width of the lamella the sample molecules are initially dissolved in. Both, a lower flow velocity and a smaller lamella width would lead to an accelerated separation within the main channel. Furthermore, cell positions in regard to the phase boundary have been analyzed, see Fig. 12-5. Near the outlet the maximum measured distance between the center of a cell and the phase boundary is reduced to approximately 30 µm. But most of the cells are located directly at the boundary and are attached to it, see Fig. 12-3. In Fig. 12-5 (right) the distribution of cells in five snapshots, within a lamella of about 100 µm and near the outlet, is analyzed. If no electric field is applied cells are randomly distributed across their initial lamella. In contrast, as soon as a field of 4.5 Vdc is applied, approximately 70% of the cells can be found within a range of 10 µm from the phase boundary.

Fig. 12-4: Left: Fluorescence intensity profiles of BSA molecules across the channel near the inlet of the channel, halfway along the channel and near the outlet when no electric field is applied. The first phase boundary is located at 0 µm. Right: Fluorescence intensity profiles at 4 Vdc, where at the outlet the proteins have left their initial phase almost completely.

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Fig. 12-5: Left: Maximum distance of a cell from the phase boundary (y=0) at values of the applied voltage. At 4.5 Vdc the cells start to penetrate the boundary and resume their electrophoretic motion. Below 4.5 Vdc nearly all cells are lined up at the phase boundary like pearls on a string. Right: Number of cells at different distances from the boundary, with and without an electric field.

12.4 Conclusion

The investigated PEG-dextran aqueous two-phase system allows a very gentle separation of cells and proteins. Additionally, this microfluidic setup combines the separation process with an enrichment of the desired cells directly at the phase boundary. Since also cell lysis due to high electric field strengths is avoided, it is found to be superior to typical electric field flow fractionation systems. This makes such a microfluidic device a promising candidate for further types of separation, purification and enrichment processes of biomolecules.