Methods and Apparatus for Magnetic Separation of Cells

• US 20090220979A1
• Filed: 12/11/2008
• Published: 09/03/2009
• Est. Priority Date: 12/12/2007
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Jeffrey, Stefanie S., Mindrinos, Michael N., Powell, Ashley Ann, Talasaz, AmirAli Hajhossein, Davis, Ronald W., Pease, R. Fabian
• Granted Patent: US 8,071,395 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/286 disposed at the inner circumference of a recipient, e.g. magnetic drain bolt
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Abstract

Described here is an automated robotic device that isolates circulating tumor cells (CTCs) or other biological structures with extremely high purity. The device uses powerful magnetic rods covered in removable plastic sleeves. These rods sweep through blood samples, capturing, e.g., cancer cells labeled with antibodies linked to magnetically responsive particles such as superparamagnetic beads. Upon completion of the capturing protocol, the magnetic rods undergo several rounds of washing, thereby removing all contaminating blood cells. The captured target cells are released into a final capture solution by removing the magnetic rods from the sleeves. Additionally, cells captured by this device show no reduced viability when cultured after capture. Cells are captured in a state suitable for genetic analysis. Also disclosed are methods for single cell analysis. Being robotic allows the device to be operated with high throughput.

Claims

1. A method for capturing and isolating cells comprising:

(a) mixing a sample comprising rare target cells and contaminant cells with magnetically responsive particles having an affinity to the rare target cells to produce a sample solution having magnetically responsive particles bound to rare target cells;

(b) contacting a magnetic member having a non-adherent sleeve with the sample solution;

(c) producing a continuous relative motion between the magnetic member and the sample solution while the magnetic member produces a magnetic field across the non-adherent sleeve, such that the magnetically responsive particles bound to rare target cells are selectively captured onto the non-adherent sleeve;

(d) contacting the magnetic member having the captured rare target cells with a recovery solution;

(e) substantially removing the magnetic field produced by the member across the non-adherent sleeve whereby the magnetically responsive particles bound to rare target cells are released into the recovery fluid.

2. The method of claim 1 wherein the non-adherent sleeve comprises a sleeve over the magnetic member, and step (e) of substantially removing magnetic field across the non-adherent sleeve comprises removing the magnetic member from at least a portion of the sleeve.

3. The method of claim 1 wherein the magnetic member comprises an electromagnet, and step (e) of substantially removing the magnetic field across the non-adherent sleeve comprises de-magnetizing the electromagnet.

4. The method of claim 1 wherein the magnetic member is held stationary in a fluid flow channel and the continuous relative motion between the magnetic member and the sample solution is produced by flowing the sample solution past the magnetic member.

5. The method of claim 1 where the continuous relative motion between the magnetic member and the sample solution produces a velocity in the non-turbulent flow regime.

6. The method of claim 1 wherein the continuous relative motion between the magnetic member and the sample solution has a Reynolds number of 0.1 to 100.

7. The method of claim 1 wherein the continuous relative motion between the magnetic member and the sample solution has velocity of 0.1 mm/sec to 1 mm/sec.

8. The method of claim 1 wherein the magnetic member has a surface field strength of 0.2 Tesla to 1 Tesla.

9. The method of claim 1 wherein the magnetic member has a surface field strength of 0.2 Tesla to 1 Tesla and the continuous relative motion between the magnetic member and the sample solution has velocity of 0.1 mm/sec to 1 mm/sec.

10. The method of claim 1 wherein the continuous relative motion is applied such that a majority of the sample solution has access to the magnetic member.

11. The method of claim 1 wherein the non-adherent sleeve comprises a polymer selected from the group consisting of vinyl, chlorofluorocarbon polymers and silicone.

12. The method of claim 1 wherein the non-adherent sleeve comprises polyvinyl chloride (PVC).

13. The method of claim 1 further comprising contacting the magnetic member, having captured rare target cells, with a wash fluid after step (c).

14. A method for capturing and isolation of cells comprising:

(a) contacting a magnetic member having a non-adherent sleeve with a sample solution comprising rare target cells and contaminant cells wherein the rare cells constitute less that 0.1% of the cells in the sample and wherein some or all of the rare target cells are bound to magnetically responsive particles;

(b) moving the magnetic member relative to the sample solution to capture rare target cells bound to magnetically responsive particles; and

(c) releasing the rare target cells from the non-adherent sleeve into a recovery solution, whereby the capture rate is greater 35%, and the purity is greater than 99%.

15. The method of claim 14 wherein the sample solution is blood and the rare cells are present in the sample solution at less than 1 in 50 million, and the capture rate is greater than 75% and the purity is greater than 90%.

16. The method of claim 14 wherein the sample solution is blood and the rare cells are present in the sample solution at less than 1 in 500 million, and the capture rate is greater than 35% and the purity is greater than 25%.

17. The method of claim 14 wherein greater than 80% of the recovered rare target cells are intact.

18. The method of claim 14 wherein greater than 80% of the recovered rare target cells are viable.

19. A device for capturing and isolating cells comprising:

(a) a sample vessel for holding a sample solution comprising rare target cells and contaminant cells wherein some or all of the rare target cells are bound to magnetically responsive particles;

(b) a magnetic member having a non-adherent sleeve;

(c) a recovery vessel for holding a recovery solution;

(d) an actuator for causing continuous relative movement of the magnetic member (i) between containers, (ii) into and out of the vessels, and (iii) at least two directions within the sample vessel to provide continuous relative motion between the magnetic member and the sample solution to provide selective capture of magnetically responsive particles having bound rare cells; and

(e) a mechanism for producing a magnetic field across the non-adherent sleeve while the magnetic member is in the sample solution, and for substantially removing the magnetic field across the non-adherent sleeve while the non-adherent sleeve is in the recovery solution.

20. The device of claim 19 wherein the non-adherent sleeve comprises a sleeve over the magnetic member, and the mechanism for substantially removing the magnetic field across the non-adherent sleeve comprises moving the magnetic member out of at least a portion of the sleeve.

21. The device of claim 19 wherein the magnetic member comprises an electromagnet, and the mechanism for substantially removing the magnetic field across the non-adherent sleeve comprises demagnetizing the magnetic member.

22. A device for capture and isolation of target cells from a mixed cell population wherein the target cells are labeled with magnetically responsive material to form labeled target cells, comprising:

(a) a magnetic member having a tip end and another end connected to an actuator;

(b) a sleeve between the magnetic member and the mixed cell population to prevent direct contact of cells with the magnetic member, said sleeve being nonmagnetic and nonadherent to cells in the mixed cell population;

(c) said sleeve being separable from the magnetic member by the actuator, which causes movement between the magnetic member and the sleeve, whereby the magnetic member is moved more or less into the sleeve; and

(d) said actuator being constructed and arranged for causing in the device (i) relative stirring movement between the magnetic member and the mixed cell population to contact the magnetic member with a majority of cells in the mixed cell population, and (ii) causing movement of the magnetic member into and out of the container to retrieve target cells from the mixed cell population.

23. The device of claim 22 wherein the magnetic member is a rod having a diameter of at least 4 mm to produce a high magnetic field strength and having a tapered tip for producing a high magnetic gradient.

24. The device of claim 22 where the magnetic member has a tapered tip.

25. The device of claim 22 wherein the magnetic member possesses a field strength at the tip of at least 0.5 Tesla.

26. The device of claim 25 where the magnetic member is a rare earth magnet.

27. The device of claim 25 where the magnetic member has a pull strength of at least 70 pounds.

28. The device of claim 25 where the magnetic member has a surface field strength of 0.2 to 1 Tesla.

29. The device of claim 22 where the magnetic member comprises neodymium, iron and boron.

30. The device of claim 22 where the sleeve consists essentially of a material selected from the group consisting of vinyl polymer, paramagnetic metal, ceramic material, and polyHEMA.

31. The device of claim 22 where the magnetic member consists essentially of neodymium, iron and boron alloy.

32. The device of claim 22 where the actuator causes orbital movement to contact the magnetic member with a majority of cells.

33. The device of claim 22 further comprising one or more containers for holding the sample, for holding a wash solution and for holding target cells after isolation from the mixed cell population.

34. The device of claim 33 where there are separate containers for holding sample, wash solution and target cells.

35. The device of claim 33 where the containers comprise a material which is non-adherent to target cells.

36. A device for capture and isolation of target cells in a sample having a mixed cell population of target cells and contaminant cells, wherein the target cells are labeled with magnetically responsive material to form labeled target cells, comprising:

(a) a container for holding the sample;

(b) a magnetic member having a tip;

(c) a sleeve between the magnetic member and the mixed cell population to prevent direct contact of cells with the magnetic member, said sleeve being nonmagnetic and nonadherent to cells in the mixed cell population;

(d) said sleeve being separable from the magnetic member by an that actuator causes movement between the magnetic member and the sleeve whereby the magnetic member is moved more or less into the sleeve; and

(e) said actuator being constructed and arranged for causing (i) relative stirring movement in the container between the magnetic member and the mixed cell population to contact the magnetic member with a majority of cells in the mixed cell population, and (ii) causing movement of the magnetic member into and out of the container to retrieve target cells from the mixed cell population.

37. The device of claim 36 further comprising a container which comprises a portion having a magnet opposite the magnetic member, for attracting isolated labeled target cells from the magnetic member in forming an isolated population.

38. The device of claim 36 wherein the container defines an open pool for holding the sample.

39. The device of claim 36 wherein the actuator comprises a component which causes the magnetic member to sweep through the pool in concentric circles.

40. The device of claim 36 comprising only one magnetic member in one container.

41. The device of claim 36 comprising multiple magnetic members controlled by a single actuator.

42. The device of claim 36 further comprising a probe for extracting a single target cell from a collection of target cells isolated after removal of contaminant cells.

43. The device of claim 42 comprising a computer programmed with image recognition software and controlling the probe.

44. The device of claim 36 further comprising a magnet for removing target cells from the sleeve.

45. A method for capture and isolation of intact, rare target cells in a sample having a mixed cell population of target cells and contaminant cells, wherein the target cells are labeled with magnetically responsive material to form labeled target cells, comprising:

(a) labeling the sample with a label specific for target cells, said label being magnetically responsive, to form a labeled sample;

(b) contacting the labeled sample with a member extending into the sample and comprising a strong magnet covered by a nonmagnetic, nonadherent sleeve;

(c) sweeping the member through the sample to cause cells to attach to the sleeve;

(d) washing the sleeve with cells attached to remove unlabeled cells;

(e) separating the magnet from the sleeve, whereby labeled cells are removed from the sleeve; and

(f) collecting labeled cells to form a composition comprising beads and labeled cells.

46. The method of claim 45 further comprising the step of isolating a single labeled cell from the beads.

47. The method of claim 45 where the label is an antibody against an cell surface marker selected from the group consisting of HLA, EpCAM, CD44, and CD96

48. The method of claim 45 where the sample is human peripheral blood.

49. The method of claim 45 where the target cells are CTCs.

50. The method of claim 45 where the strong magnet is a rare earth magnet having an axial dipole whereby magnetic particles are concentrated at one elongated end of the magnet.

51. The method of claim 45 wherein the nonadherent sleeve is a polymer.

52. The method of claim 45 wherein the polymer is a polymer selected from the group consisting of polyvinyl chloride, chlorofluorocarbon polymers or silicone.

53. The method of claim 45 further comprising the step of applying a magnetic field opposite the sleeve after the magnet has been removed to assist in removing labeled cells from the sleeve.

54. The method of claim 45 further comprising multiple washing steps as recited in step (d).

55. The method of claim 45 further comprising the step of extracting intact genetic material from isolated target cells.

56. The method of claim 45 further comprising the step of testing for expression of certain genes in extracted genetic material.

57. The method of claim 56 wherein the genes are selected from the group consisting of GAPDH Beta Actin, Big ribosome protein (RPLPO), CRYAB, EGFR, FOXA1, CD44, ESR1 (ER) PGR (PR), and mutated forms of Myc, Ras, BRCA1, BRCA2, APC, and p53.

58. The method of claim 56 comprising the testing for the expression of vimentin, where increased expression of vimentin indicates a mesenchymal CTC.

59. A composition comprising an isolated population of viable CTCs having at least 10 cells of at least 90% purity of CTCs

60. The composition of claim 59 wherein the CTCs are breast cancer cells.

61. The composition of claim 59 further comprising an MSC.