Cell rolling separation

• US 8,986,988 B2
• Filed: 09/29/2008
• Issued: 03/24/2015
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Hong, Seungpyo, Mei, Ying, Anderson, Daniel Griffith, Langer, Robert S., Karnik, Rohit Nandkumar, Karp, Jeffrey Michael, Bose, Suman
• Primary Examiner(s): Ketter, Jim
• Assistant Examiner(s): GALISTEO GONZALEZ, ANTONIO
• Application Number:
  US12/680,249
• Publication Number:
  US 20100304485A1
• Time in Patent Office: 2,367 Days
• Field of Search: 435/325, 435/372
• US Class Current: 435/325
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2501/585 Integrins
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

The present invention provides systems for cell separation based on cell rolling on surfaces along edges of regions coated with cell adhesion molecules. A variety of designs of coated regions and edges are disclosed.

Claims

1. A method comprising steps of:

providing a surface that is at least partially coated with an ordered layer of cell adhesion molecules

wherein the surface comprises at least one edge between an area coated with the ordered layer and another area that is not coated with the ordered layer; and

wherein the area that is coated with the ordered layer of cell adhesion molecules is entirely covered with the ordered layer of cell adhesion molecules; and

flowing a cell stream that contains a population of cells across the surface in a direction which forms a non-zero angle α

_s with the at least one edge, wherein at least one cell in the population of cells comprises a surface moiety that is recognized by the cell adhesion molecules and wherein the at least one cell rolls for a period of time in a direction that is α

_s to the direction of flow as a result of rolling along at least a portion of the at least one edge.

2. The method of claim 1 further comprising separating the at least one cell from the remainder of the population of cells.

3. The method of claim 1, wherein the edge is characterized by a sharpness corresponding to a change from about 10% to about 90% density of cell adhesion molecules over a distance of less than about 5 μ

m, wherein the percentage density of cell adhesion molecules is measured as compared to the maximum density of cell adhesion molecules in the coated area adjacent to the edge.

4. The method of claim 1, wherein the surface comprises a plurality of edges.

5. The method of claim 1, wherein the surface comprises a plurality of coated areas.

6. The method of claim 5, wherein each coated area defines a strip with two edges.

7. The method of claim 6, wherein the two edges of each strip are substantially parallel.

8. The method of claim 7, wherein the strips are substantially parallel to each other.

9. The method of claim 8, wherein the strips are separated from each other by a gap having a substantially fixed distance w_g between adjacent strips and the strips each have substantially the same width w_s.

10. The method of claim 9, wherein w_s, is in the range of from about 0.01 μ

m to about 10 mm.

11. The method of claim 9, wherein the at least one cell has an average diameter d and wherein w_s<

3d.

12. The method of claim 9, wherein w_g is in the range of from about 0.2 μ

m to about 10 mm.

13. The method of claim 1, wherein the population of cells includes at least one subpopulation of cells with a common characteristic, and in the step of flowing, at least one cell in the subpopulation rolls for a period of time in a direction that is α

_s to the direction of flow as a result of rolling along at least a portion of the at least one edge.

14. The method of claim 13, wherein substantially all cells from the subpopulation roll for a period of time in a direction that is α

_s to the direction of flow as a result of rolling along at least a portion of the edge.

15. The method of claim 14, wherein the cells in the subpopulation all comprise a surface moiety that is recognized by the cell adhesion molecules.

16. The method of claim 15, wherein the surface moiety is selected from the group consisting of ligands of P-selectin, ligands of E-selectin, ligands of L-selectin, and combinations thereof.

17. The method of claim 15, wherein the surface moiety is selected from the group consisting of P-selectin ligand-1 (PSGL-1), glycosylation-dependent cell adhesion molecule-I (GlyCAM-1), CD15, CD34, CD44, E-selectin ligand-I (ESL-1), and combinations thereof.

18. The method of claim 15, wherein the surface moiety is selected from the group consisting of VLA-4, gp200, and combinations thereof.

19. The method of claim 15, wherein the cells in the subpopulation are all stem cells.

20. The method of claim 19, wherein the cells in the subpopulation are stem cells selected from the group consisting of mesenchymal stem cells, hematopoietic stem cells, and embryonic stem cells.

21. The method of claim 15, wherein the cells in the subpopulation are all cancer cells.

22. The method of claim 15, wherein the cells in the subpopulation are all progenitor cells.

23. The method of claim 15, wherein the cells in the subpopulation are all of a cell type selected from the group consisting of:

red blood cells, neutrophils, lymphocytes, monocytes, white blood cells, and combinations thereof.

24. The method of claim 1, wherein the cell adhesion molecules are selected from the group consisting of selectins, integrins, cadherins, immunoglobulin cell adhesion molecules, and combinations thereof.

25. The method of claim 1, wherein the cell adhesion molecules comprise one or more extracellular matrix cell adhesion molecules.

26. The method of claim 1, wherein the ordered layer of cell adhesion molecules further comprises antibodies.

27. The method of claim 1, wherein the ordered layer of cell adhesion molecules further comprises one or more cell modifying ligands.

28. The method of claim 1, wherein the at least one cell rolls in a direction that is α

_s to the direction of flow at an average speed of at least about 0.1 μ

m/s.

29. The method of claim 1, wherein the shear stress on cells flowed over the surface is in a range between about 0.05 dyn/cm² to about 50 dyn/cm².

30. The method of claim 1, wherein the at least one cell contacts the surface area with a cell contact radius (r_rcontact) of at least about 0.25 μ

m.

31. The method of claim 1, further comprising a step of treating the population of cells before the step of flowing with an agent that modifies cell deformability.

32. The method of claim 13, further comprising collecting the subpopulation of cells.

33. The method of claim 1, wherein the surface further comprises an element to capture the at least one cell on the surface.

34. The method of claim 6, wherein the surface comprises a plurality of strips.

35. The method of claim 34, wherein the surface comprises at least 5 strips.

36. The method of claim 34, wherein the surface comprises at least 7 strips.

37. The method of claim 34, wherein the surface comprises at least 13 strips.

38. The method of claim 5, wherein the plurality of coated areas provide different kinds of edges that make different angles with respect to the direction of flow.

39. The method of claim 5, wherein the plurality of coated areas are defined by shapes selected from squares, rectangles, triangles, polygons, ellipses, circles, arcs, waves, and any combination thereof.

40. The method of claim 1, wherein the at least one edge is substantially linear.

41. The method of claim 1, wherein the at least one edge comprises a curved portion.

42. The method of claim 1, wherein the at least one edge comprises both linear and curved portions.

43. The method of claim 1, wherein α

_s is more than about 5 degrees.

44. The method of claim 1, wherein α

_s is less than about 30 degrees.

45. The method of claim 10, wherein w_s is less than about 50 μ

m.

46. The method of claim 29, wherein the shear stress on cells flowed over the surface is in a range between about 0.2 dyn/cm² to about 5 dyn/cm².

47. A method comprising steps of:

providing a three dimensional surface that is at least partially coated with an ordered layer of cell adhesion molecules, and

flowing a cell stream that contains a population of cells, simultaneously with a buffer stream that does not contain cells, across the surface in such conditions to create a stagnation line of no flow, wherein the direction of flow forms a non-zero angle α

_s with the stagnation line, wherein at least one cell in the population of cells comprises a surface moiety that is recognized by the cell adhesion molecules and wherein at least one cell in the population of cells rolls at least part of the time along the stagnation line in a direction that is α

_s to the direction of flow.

48. A method comprising steps of:

providing a surface that is at least partially coated with an ordered layer of cell adhesion molecules, wherein the surface comprises at least one edge between an area coated with the ordered layer and another area that is not coated with the ordered layer; and

flowing a first cell stream that contains a population of cells, simultaneously with a buffer stream that does not contain cells, across the surface in a direction which forms a non-zero angle α

_s with the at least one edge, wherein at least one cell in the population of cells comprises a surface moiety that is recognized by the cell adhesion molecules and wherein the at least one cell rolls for a period of time in a direction that is α

_s to the direction of flow as a result of rolling along at least a portion of the at least one edge.

49. The method of claim 48, further comprising separating the at least one cell from the remainder of the population of cells.

50. The method of claim 48, wherein the edge is characterized by a sharpness corresponding to a change from about 10% to about 90% density of cell adhesion molecules over a distance of less than about 5 μ

m, wherein the percentage density of cell adhesion molecules is measured as compared to the maximum density of cell adhesion molecules in the coated area adjacent to the edge.

51. The method of claim 48, wherein the surface comprises a plurality of edges.

52. The method of claim 48, wherein the surface comprises a plurality of coated areas.

53. The method of claim 48, wherein each coated area defines a strip with two edges.

54. The method of claim 53, wherein the two edges of each strip are substantially parallel.

55. The method of claim 54, wherein the strips are substantially parallel to each other.

56. The method of claim 48, wherein the population of cells includes at least one subpopulation of cells with a common characteristic, and in the step of flowing, at least one cell in the subpopulation rolls for a period of time in a direction that is α

_s to the direction of flow as a result of rolling along at least a portion of the at least one edge.

57. The method of claim 56, wherein substantially all cells from the subpopulation roll for a period of time in a direction that is α

_s to the direction of flow as a result of rolling along at least a portion of the edge.

58. The method of claim 57, wherein the cells in the subpopulation all comprise a surface moiety that is recognized by the cell adhesion molecules.

59. The method of claim 58, wherein the surface moiety is selected from the group consisting of ligands of P-selectin, ligands of E-selectin, ligands of L-selectin, and combinations thereof.

60. The method of claim 58, wherein the cells in the subpopulation are all stem cells.

61. The method of claim 58, wherein the cells in the subpopulation are all cancer cells.

62. The method of claim 58, wherein the cells in the subpopulation are all progenitor cells.

63. The method of claim 58, wherein the cells in the subpopulation are all of a cell type selected from the group consisting of:

red blood cells, neutrophils, lymphocytes, monocytes, white blood cells, and combinations thereof.

64. The method of claim 48, wherein the cell adhesion molecules are selected from the group consisting of selectins, integrins, cadherins, immunoglobulin cell adhesion molecules, and combinations thereof.

65. The method of claim 48, wherein the cell adhesion molecules comprise one or more extracellular matrix cell adhesion molecules.

66. The method of claim 48, wherein the ordered layer of cell adhesion molecules further comprises antibodies.

67. The method of claim 48, wherein the ordered layer of cell adhesion molecules further comprises one or more cell modifying ligands.

68. The method of claim 56, further comprising collecting the subpopulation of cells.