Microfluidic device for cell separation and uses thereof

• US 8,372,579 B2
• Filed: 05/08/2007
• Issued: 02/12/2013
• Est. Priority Date: 09/27/2002
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Toner, Mehmet, Truskey, George, Kapur, Ravi
• Primary Examiner(s): Ware, Debbie K
• Application Number:
  US11/800,940
• Publication Number:
  US 20070264675A1
• Time in Patent Office: 2,107 Days
• Field of Search: None
• US Class Current: 435/2
• CPC Class Codes: B01L 1/52 Transportable laboratories; Field kits
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• 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: B01L 9/54 Supports specially adapted for pipettes and burettes automated pipetting stations G01N35/10
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 5/0087 : Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: G01N 1/40 : Concentrating samples
• CPC Class Codes: G01N 1/405 : by adsorption or absorption
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 2015/1028 : Sorting particles
• CPC Class Codes: G01N 2333/70582 : CD71
• CPC Class Codes: G01N 2333/70589 : CD45
• CPC Class Codes: G01N 2333/70596 : Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54386 : Analytical elements
• CPC Class Codes: G01N 33/56966 : Animal cells
• CPC Class Codes: G01N 33/574 : for cancer
• CPC Class Codes: Y10T 29/49982 : Coating
• CPC Class Codes: Y10T 428/24744 : Longitudinal or transverse tubular cavity or cell

Abstract

The invention features methods for separating cells from a sample (e.g., separating fetal red blood cells from maternal blood). The method begins with the introduction of a sample including cells into one or more microfluidic channels. In one embodiment, the device includes at least two processing steps. For example, a mixture of cells is introduced into a microfluidic channel that selectively allows the passage of a desired type of cell, and the population of cells enriched in the desired type is then introduced into a second microfluidic channel that allows the passage of the desired cell to produce a population of cells further enriched in the desired type. The selection of cells is based on a property of the cells in the mixture, for example, size, shape, deformability, surface characteristics (e.g., cell surface receptors or antigens and membrane permeability), or intracellular properties (e.g., expression of a particular enzyme).

Claims

1. A method of diagnosing cancer in a patient, the method comprising:

flowing a blood sample from a patient through a device comprising a plurality of obstacles on a substrate, wherein the obstacles are each coupled to one or more binding moieties that selectively bind to an epithelial cell or a tumor cell indicative of cancer in the patient, wherein said flowing selectively enriches said epithelial cells or tumor cells by a factor of at least 1,000 in said blood sample.

2. The method of claim 1, wherein said blood sample is flowed at a rate of at most 15 μ

l/sec.

3. The method of claim 1, wherein said device has sensitivity or specificity greater than 75%.

4. The method of claim 1, wherein said device retains at least 60% of said epithelial cells or tumor cells in said blood sample.

5. The method of claim 1, wherein said device enriches said epithelial cells or tumor cells in said blood sample by a factor of at least 100,000.

6. The method of claim 1, further comprising performing nucleic acid analysis on said enriched epithelial cells or tumor cells.

7. The method of claim 5, further comprising performing nucleic acid analysis on said enriched epithelial cells or tumor cells.

8. The method of claim 1, further comprising using fluorescence in situ hybridization (FISH) analysis to identify epithelial cells or tumor cells present in the enriched cell population.

9. The method of claim 5, further comprising using fluorescence in situ hybridization (FISH) analysis to identify epithelial cells or tumor cells present in the enriched cell population.

10. The method of claim 1, wherein the binding moiety is an antibody.

11. The method of claim 10, wherein the antibody selectively binds to a cell surface receptor.

12. The method of claim 1, wherein the distance between obstacles varies in a flow direction.

13. The method of claim 1, wherein the obstacles are arranged in an equilateral triangular array.

14. The method of claim 1, wherein the flowing comprises flowing at a flow rate of 1.0 to 20 μ

l/sec.

15. The method of claim 1, wherein the flowing comprises flowing at a flow rate that provides a wall shear stress on cells less than 1 dyne/cm².

16. The method of claim 1, wherein red blood cells are not enriched in said blood sample.

17. The method of claim 1, wherein the plurality of obstacles comprises an array that comprises a first subarray and a second subarray, and wherein the obstacles in the first subarray have a different feature than the obstacles in the second subarray.

18. The method of claim 13, wherein the obstacles in the equilateral triangular array of obstacles are arranged in a staggered array.

19. The method of claim 1, wherein the epithelial cells or tumor cells are living cells.

20. The method of claim 1, wherein the obstacles are spaced 10 to 100 microns apart.

21. The method of claim 1, wherein the obstacles are arranged in rows aligned normal to a direction of flow of the blood sample through the device and obstacles in one row are shifted in a direction normal to the flow with respect to obstacles in an adjacent row.

22. The method of claim 1, wherein the device is configured in the form of a flow chamber, and wherein the substrate forms a bottom surface of the flow chamber.

23. The method of claim 22, wherein the device further comprises a top surface.

24. The method of claim 22, wherein the device comprises a transparent window.

25. The method of claim 23, wherein the obstacles contact both the top and bottom surfaces of the flow chamber.

26. The method of claim 23, wherein the obstacles are distributed on the bottom surface of the flow chamber and have a height that leaves a gap between top ends of the obstacles and the top surface.