Apparatus for Magnetic Separation of Cells

• US 20120045828A1
• Filed: 10/28/2011
• Published: 02/23/2012
• 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: Davis, Ronald W., Jeffrey, Stefanie S., Mindrinos, Michael N., Pease, R. Fabian, Powell, Ashley Ann, Hajhossein Talasaz, AmirAli
• Granted Patent: US 9,267,943 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• 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 device for capturing and isolating cells comprising:

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

(b) a magnetic member having a non-adherent sleeve preventing direct contact of the magnetic member with the cells;

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

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

(e) a mechanism for producing a magnetic field from the magnetic member 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.

2. The device of claim 1 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.

3. The device of claim 1 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.

4. 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 actuator 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) movement of the magnetic member into and out of the container to retrieve target cells from the mixed cell population.

5. The device of claim 4 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.

6. The device of claim 4 where the magnetic member has a tapered tip.

7. The device of claim 4 wherein the magnetic member possesses a field strength at the tip of at least 0.5 Tesla.

8. The device of claim 7 where the magnetic member is a rare earth magnet.

9. The device of claim 7 where the magnetic member has a pull strength of at least 70 pounds.

10. The device of claim 7 where the magnetic member has a surface field strength of 0.2 to 1 Tesla.

11. The device of claim 4 where the magnetic member comprises neodymium, iron and boron.

12. The device of claim 4 where the sleeve consists essentially of a material selected from the group consisting of vinyl polymer, paramagnetic metal, ceramic material, and polyHEMA.

13. The device of claim 4 where the magnetic member consists essentially of neodymium, iron and boron alloy.

14. The device of claim 4 where the actuator causes orbital movement to contact the magnetic member with a majority of cells.

15. The device of claim 4 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.

16. The device of claim 15 where there are separate containers for holding sample, wash solution and target cells.

17. The device of claim 15 where the containers comprise a material which is non-adherent to target cells.

18. 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) movement of the magnetic member into and out of the container to retrieve target cells from the mixed cell population.

19. The device of claim 18 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.

20. The device of claim 18 wherein the container defines an open pool for holding the sample.

21. The device of claim 18 wherein the actuator comprises a component which causes the magnetic member to sweep through the pool in concentric circles.

22. The device of claim 18 comprising only one magnetic member in one container.

23. The device of claim 18 comprising multiple magnetic members controlled by a single actuator.

24. The device of claim 18 further comprising a probe for extracting a single target cell from a collection of target cells isolated after removal of contaminant cells.

25. The device of claim 24 comprising a computer programmed with image recognition software and controlling the probe.

26. The device of claim 18 further comprising a magnet for removing target cells from the sleeve.

27. A composition comprising an isolated population of viable circulating tumor cells (“

CTCs”

) having at least ten cells and having at least 90% purity of CTCs

28. The composition of claim 27 wherein the CTCs are breast cancer cells.

29. The composition of claim 27 further comprising a mesenchymal-like cancer stem cell.