Methods for Cell Separation and Cytometry Based on Directed Cell Rolling on Patterned Substrates

Applications

Cell type separation and cytometry via cell rolling can be used for a variety of purposes including disease diagnosis, biological research, and therapeutics. Situations that require the separation of heterogeneous cell populations run the gamut from medical practices such as blood cell counting to therapeutic procedures such as the isolation of pure stem cells and isolation and quality control of T-cells for immunotherapies.

Problem Addressed

Current cell separation strategies, including FACS, affinity columns, magnetic bead based separation, and rosette separation, involve multiple processing steps that are expensive and time consuming. They also often involve cell modifications or labels that are undesirable, especially in cell therapeutics. Ideally, a new separation tool would facilitate rapid, accurate separation of cells into multiple populations while requiring minimal cell handling and would be compatible with manufacture of cell therapeutics. Similarly, cytometric methods that are real-time, inline, and quantitative with minimal variability across operators and locations are lacking, with the need being especially acute in the manufacture of cell therapeutics. 

Technology

This invention takes advantage of receptor-ligand interaction between surface-immobilized affinity molecules (such as selectins or other molecules) and the surface of cells that results in cell rolling. Rolling is exhibited by cells such as leukocytes, hematopoietic stem cells, metastatic cancer cells, and platelets. These cells transiently adhere to surfaces coated with selectin and travel along them with more ease than along uncoated surfaces. Cells directed along a particular flow trajectory on the selectin-coated surface will deviate slightly from their original direction when they reach the edge of the selectin-coated surface, so as to remain on the higher-affinity selectin coating. The angle of deviation depends on the interaction between the cells and selectin, which varies broadly among different cell types. By pattering a variety of angles of selectin bands along a rolling surface, cells can be sorted based on their rolling properties into different wells. The sorted cells can be used immediately without further processing. This manner of separation affords not only the separation of rolling from non-rolling cells, but also the separation of different subpopulations of rolling cells. Furthermore, the continuous-flow separation design is easy to implement and scale for parallel operation. For instance, the separation flow chambers may be linked in series to a common cell inlet and buffer inlet, as well as channels of outlets for sorted cells, to increase throughput. In addition, the outlet channels may have volumetric designs to quantify the amount of successfully sorted cells. The same method can be used for cell cytometry, by simply observing the trajectories of cells flowing across the patterned surfaces in the device.

Advantages

  • Quick separation or analysis of cell subpopulations 
  • Continuous-flow  operation
  • Easy implementation and scaling
  • Label-free and quantitative analysis of cells
  • Minimum cell handling 
  • No pre- or post-sort cell modifications or processing necessary