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

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Shows (A) P-Selectin immobilized on a polystyrene substrate using microfluidic technology to create edges followed by adsorption of BSA-FITC to reveal the design. Stripes were 100 μm wide. (B) Tracks of rolling HL-60 cells which were flowed at concentration of 1×106 cells/mL over the substrate at a shear rate of 2 dyn/cm2. Tracks were obtained by processing 194 images acquired at 0.5 Hz using a Matlab code. Cells can be seen to interact and roll only on the selectin stripe. (C) A magnified image of the inset showing representative tracks reveals that cells roll in the direction of fluid flow within the P-selectin stripe, but change direction and roll along the edge upon encountering the edge (marker O). Cells within the P-Selectin stripe that do not encounter the edge (marker □) roll in the direction of the fluid flow, and not in the direction of the stripe. Other cells can be seen rolling on the edge (marker ×). The direction of cell rolling is determined by the edge, and not by the shape of the coated area on which the cells roll.Depicts a schematic of an example of an edge design that would result in net displacements of two cell types in opposite directions. The surfaces comprises two different kinds of edges that make different angles with respect to the direction of flow. The first edge encountered by the cells makes an angle such that both cell types can follow it. The second edge is inclined at a larger angle or has receptors such that only one cell type (dashed line) can roll along that edge. A spatial variation in the above repeating design obtained by changing the second edge gradually over a large area can be used for focusing of a particular cell type.Illustrates that cell separation may be performed using flow chambers with selectin edges at varying angles (A) or a constant angle (B). Chamber length (L), width (w), cell inlet width (winlet), and chamber height (h) are design parameters that may be particularly relevant. As shown, in one embodiment, ten devices may be used in parallel for cell separation to increase throughput (C).Depicts a schematic and an image illustrating immobilization of P-selectin to create edges. A silicone rubber mask was placed on a glass substrate (A), and P-selectin was coated on the exposed area of the substrate by physisorption (B). The silicone mask was then removed from the substrate (C), and BSA was used to block the areas that were not coated with P-selectin (D). Use of fluorescein-labeled BSA enabled visualization of the P-selectin arrangement using an epifluorescence microscope (E). HL-60 cells adhered selectively to the P-selectin region, confirming coating of some areas of the substrate with P-selectin. Scale bar: 100 μmShows photographs illustrating that a P-selectin edge directs motion of rolling cells. Rolling HL-60 cells that encountered the edge of a P-selectin-coated area making an angle to the fluid flow direction were forced to roll along the edge. The motion of a cell forced to roll along the edge is compared with another cell rolling in the direction of fluid flow, highlighted by circles. The edge succeeded in changing the direction of motion of the rolling cell by 8.6°, resulting in effectively displacing the cell by 0.15 mm from its original position for every 1 mm of length along the direction of flow. Wall shear stress was 1.9 dyn/cm2.
Categories
Inventors
Professor Robert Langer
Department of Chemical Engineering, MIT
External Link (langer-lab.mit.edu)
Professor Rohit Karnik
Department of Mechanical Engineering, MIT
External Link (web.mit.edu)
Seungpyo Hong
Department of Chemical Engineering, MIT
Ying Mei
Department of Chemical Engineering, MIT
Professor Daniel Anderson
Department of Chemical Engineering, MIT
External Link (anderson-lab.mit.edu)
Jeffrey Karp
Department of Health Sciences and Technology, MIT
External Link (www.karplab.net)
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Cell Rolling Separation

US Patent Pending

Cell Rolling Separation

US Patent 8,986,988

Cell Rolling Separation

US Patent 9,555,413
Publications
Cell Sorting By Deterministic Cell Rolling
Lab on a Chip, April 21, 2012, p. 1427-1430
A Cell Rolling Cytometer Reveals the Correlation Between Mesenchymal Stem Cell Dynamic Adhesion and Differentiation State
Lab on a Chip, Jan 7, 2014, p. 161-166
Affinity Flow Fractionation of Cells via Transient Interactions with Asymmetric Molecular Patterns
Scientific Reports, July 31, 2013

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 heterogenous 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 expensive equipment or multiple processing steps that are expensive and time consuming. They also often involve cell modifications that could potentially change the characteristics of the original cell. Despite the laborious nature of these methods, they are still quite often not specific enough. Ideally, a new separation tool would facilitate rapid, accurate separation of cells into multiple populations while requiring minimal cell handling. 

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. 

Advantages

  • Quick separation of cell subpopulations 
  • Continuous-flow separation 
  • Easy implementation and scaling
  • Minimum cell handling 
  • No pre- or post-sort cell modifications or processing necessary