Microfluidic control of cell pairing and fusion

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Original entry: Warren Lloyd Ung, APPHY 225, Fall 2009

"Microfluidic control of cell pairing and fusion"
Alison M. Skelley, Oktay Kirak, Heikyung Suh, Rudolf Jaenisch, and Joel Voldman.
Nature Methods (2009).

Soft Matter Keywords

Microfluidics, Cell Pairing, Cell Fusion

Figure 1: Microfluidic device for cell capture and pairing. (a) Schematic of device operation, (b, c, and d) SEM images of the device focusing on the cell trapping filter, (e) Two cell fusion arrays.

Summary

Skelley et al demonstrate a microfluidic device, which achieves relatively high rates of on-chip cell fusion. Their polydimethyl siloxane (PDMS) device (see Figure 1) implements a novel method for trapping pairs of desired cells in an array of weir filters. These pairs of cells are then fused using standard chemical and electrical methods. The overall efficiency of the fusion process is evaluated quantitatively and compared with current standards for cell fusion. Different methods for fusion, including electrical and chemical fusion techniques, are compared in the same device as well. Cell fusion is measured to be five times better in this microfluidic device than in a commercially available electrofusion chamber. In addition, cells remain viable as shown by subsequent culturing of cells fused within the device (refer to Figure 3).

Figure 2: Procedure for loading pairs of cells (a, b, and c) and overall image of the array filled with pairs of cells (d).
Figure 3: Fluorescence image of cells grown from those fused within the device. Doubly fluorescent red and green cells confirm the success of the fusion procedure.

Applications

Fusing cells together is interesting from a number of perspectives. One of the most prominent is the manufacture of hybridomas, a special kind of cell, which results from the fusion of a B-cell capable of producing antibodies against a particular antigen, with myeloma tumor cells, a cancerous kind of B-cell. Hybridomas retain the desirable quantities of both cells. They produce antibodies in large quantities and they are able to grow rapidly and indefinitely, meaning that they can be cultured. This allows antibody-producing cells to be immortalized, such as those from survivors of serious diseases, which have antibodies targeting those pathogens.

Another application of cell fusion is to study the way in which stem cells and germline cells can reprogram somatic cells - the normal cells found throughout the body - when these types of cells are fused together. This behaviour has previously been shown to occur, but the studies of the processes involved have been limited by the poor yields of cell fusion technology.

Soft Matter Discussion

The microfluidic device used for this cell fusion protocol uses clever design to create a cell filter which can capture cells (pictured in Figure 1) and pair them efficiently (shown in Figure 2). The filter is formed from an array of weir filters - essentially dams - which allow fluid to flow, but trap cells in place. When the weir is empty, fluid flow can pass over top of the weir, but when cells are in place, flow is diverted around the weir. This means that cells tend to flow into empty weirs, and once there, they are quite stable and other cells tend to flow past the occupied weir.

Each weir has two sides which can be used to capture cells under different flow conditions. The back of the weir can accommodate one cell at a time, while the front part of the weir can trap two cells at once. One type of cells is loaded into the weirs (as shown in Figure 2a) by flowing the first set of cells into the back-side capture areas of the weir (at this point the cell solution flows upward with respect to the orientation shown on Figure 2). Once the cells are captured, the flow is reversed, pushing the cells down into an adjacent weir (Figure 2b). Cells of a different type are introduced into this flow until two cells, one of each kind, occupy each weir. Once a pair of cells is captured, fusion can occur in a controlled manner. Since the cells, which are to be fused, are adjacent to one another, they will tend to fuse with one another when a suitable perturbation is applied. Current commercial methods have fairly abysmal yields, because they rely on random processes to bring the cells, which are to be fused, into close proximity.

Fusion is typically performed using either electrical or chemical methods. Both methods of fusion were investigated on this device, and compared with respect to their yields of fused cells by image analysis. The yield in terms of percentages of successful cell fusion were measured compared with similar commercial chemical and electrical cell fusion techniques. In each case, the microfluidic device showed a substantial increase in cell fusion efficiency.

From a purely soft matter perspective, the fusion of two cells can be thought of as merging two individual vesicles into one large vesicle. The chemical method demonstrated here uses polyethylene glycol (PEG) to induce a large osmotic pressure outside the cell. The cells shrink under the influence of the osmotic pressure, then grow back to full size when the PEG is removed from the system. In the case of electrofusion, electric fields are used to create transmembrane potentials, which may cause reversible holes in the plasma membrane by electroporation. Both of these methods essentially perturb the structure of the plasma membrane, which surrounds the cell. The hope is that, with two cells in close contact, these perturbations will lead the two plasma membranes to re-organize into a single membrane containing the contents of both cells.

Naturally, since cells are living and composed of intricate structures located within the cytoplasm, the process of cell fusion is a complex process. The more interesting aspects of cell fusion, including the internal re-arrangement of the hybrid cell, are mediated by the mechanisms of the cell. These mechanisms are the topic of ongoing study, as they can give rise to surprising hybrid behaviour, as in the case of hybridomas.

References

  1. Hybridoma Technology, Wikipedia, http://en.wikipedia.org/wiki/Hybridoma_Technology.