Prior generation optical cell sorting methods

Optical flow sorters measure and select user-defined cell types by illuminating individual cells with a laser and detecting the emitted light. The emitted light is spectrally separated (or separated by "color") to identify the cells of interest.  In droplet-based flow sorters, a cell of interest is selected by applying an electrical charge to a fluid stream (containing the sample).    An electrically-charged droplet is then produced containing one or more cells.  The resulting charged droplet travels through an electric field between two high voltage deflection plates of opposite polarities. These droplets (containing the cells of interest) are eventually deflected into a collection tube for further use.

Droplet sorters rely on flow sorting technology initially developed in the 1960's. While quite powerful, the prior generation technology imposes a number of limitations on its users.   First and foremost, there is only a single fluid stream used.    This means that only one cell at a time can be analyzed.   This limits the number of cells that can be identified and sorted for further use.   Second, sorting speed is increased by increasing the velocity of the fluid stream (along with the frequency of droplet generation).   The velocity is increased by applying significant pressure to the fluid stream.  As a result, high shearing forces are generated that may compromise cell function and viability.  Cell sorting recovery and purity also decrease as velocity is increased, due to an inability to discriminate adjacent cells.  These issues with droplet-based sorters limit their practical sorting speed to about 20,000 cells/sec at a 10% error rate (i.e. - where two or more particles are too close together to be accurately distinguished).   These limitations exclude the use of droplet sorters for a majority of cell therapy applications, due to the large number of cells required to obtain a therapeutic dose.

Moreover, droplet sorters create aerosols which may impose a significant hazard to the environment, the operator and/or other cell samples. When the cell sample contains human or potentially infectious material, these aerosols can be hazardous to nearby personnel. Existing droplet sorters are not designed with a closed or disposable fluidic pathway and do not have an acceptable level of safety for human therapeutic applications. 

Other Cell Sorting Methods

Other, non-optical cell sorting methods use magnetic fields or differences in particle buoyancy and density.  Magnetic cell sorting uses antibody-coated (magnetic) particles that bind to a specific cell type.   When the particles pass by a magnetic field, the desired cells are separated.  Density gradient cell sorting uses centrifugation to separate desired cells.  Other cell sorting methods use sedimentation, affinity adsorption or affinity extraction to select desired cells.

Magnetic cell sorting is frequently used in human therapeutic applications.    Magnetic cell sorting methods can occur under aseptic conditions and can supply sufficient cells for therapeutic use.   However, magnetic cell sorters are unable to sort cells on the basis of cell size, morphology or intracellular markers.   Furthermore, multiple sequential sorting steps are required to purify cell populations that are identified by multiple surface antigens.   This reduces the recovery of cells and increases the number of cell manipulation steps.   Finally, it has been extremely difficult to reliably sort cells based upon a threshold-sensitive level of antigen expression (low, mid, high) using magnetic cell separation technologies.  

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