Current affinity capture products are based on magnetic particles much smaller than the target cell. These range in size from 50 nm up to 4.7 µM. They are designed to sediment slowly to increase the likelihood for the antibody-coated particle to encounter the target cell. This places a limitation on the volume of the magnetic core that can be used. The low density makes it difficult for the particle to migrate through viscous primary tissues such as blood and the lower the magnetic material content the less likely the particle is to respond to a distant magnetic field. Small aperture flow through capture devices have been developed to achieve capture of these cell/small particle complexes, but they have slow sample processing times and limited volume capability.
Previous investigations reported in the scientific literature on cell capture using antibody coated beads considerably larger than the cells themselves showed very poor performance. However, CellCap has now proven that this approach is indeed possible and confers a number of key advantages for processing large volumes of complex and viscous materials. CellCap's 'big beads' have an ultra-dense core and are designed to sediment rapidly through homogenized tissue and whole blood; in doing so they encounter and bind target cells. They work very effectively in a roller bottle or rotating 'end over end' formats. The CellCap 'big beads' are combined with the molecular zipper surface chemistry which greatly increases the yield of captured cells.
In the molecular zipper the large contact area with the big bead results in greater numbers of antibodies contacting and binding to cell surface antigens. Once bound, the cell is held with very high avidity as it is much less likely that shear forces can break all the antibody bonds.
Stem cell on surface of capture bead
Mesenchymal stem cell from rat adipose captured on surface of bead using CD90 antibodies. Stem cells were subsequently released and shown to be viable in culture. This experiment was performed using a roller bottle. Alternative capture approaches under investigation rely on recirculating sample through the capture beads to increase capture of low abundance cells.
Click on right arrow to see an illustration of recirculating column format
Certain applications work with the cells remaining attached to the big beads. Analysis using in situ lysis followed by PCR is a good example. Others, such as flow cytometry or implantation experiments require release of the cells from the big beads, which can be achieved with a simple change of buffer with no impact on cell viability.
We are currently investigating the capacity limitation of the system for large numbers of cells in clinically significant sample volumes and also its ability to capture very small numbers of cells for diagnostic applications. To date we have depleted over 15 million T-cells from 15 mls of whole blood in a 15 minute incubation on a roller bottle. For large sample volume applications we have depleted 100,000 stem cells from 100 ml of FCS. At the lower end of the scale we have captured and released 5,000 rat stem cells from 1 ml of rat adipose SVF. Work is ongoing to investigate how few cells can be captured with a target of 5 cells from 10 ml of blood, which would be significant for the field of Circulating Tumour Cell detection.
For very low cell numbers we anticipate requiring a recirculation of the sample to provide multiple passes to capture all the cells from larger sample volumes. The animation above illustrates the capture and release in a vertical, expanded bed recirculation device.