Microfluidic device for cell separation and uses thereof

• US 8,895,298 B2
• Filed: 09/29/2003
• Issued: 11/25/2014
• 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:
  US10/529,453
• Publication Number:
  US 20060134599A1
• Time in Patent Office: 4,075 Days
• Field of Search: None
• US Class Current: 435/325
• 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 producing a cell population enriched in a first type of cell larger than an adult, enucleated red blood cell, said method comprising:

continuously flowing a blood sample of a specific volume through a channel in a microfluidic device comprising;

(i) continuously flowing the blood sample past a series of obstacles in the channel, the obstacles fixed in position separated by gaps arranged so that flow of the blood sample past the obstacles directs adult, enucleated red blood cells and cells smaller than adult, enucleated red blood cells in a first direction and directs cells larger than adult, enucleated red blood cells in a second direction to produce a first sample enriched in cells larger than adult, enucleated red blood cells; and

without stopping the flow of the first sample,

(ii) continuously flowing the first sample past obstacles that each comprise one or more binding moieties that preferentially bind to the first type of cell in the first sample, thereby producing a population enriched in the first cell type.

2. The method of claim 1, further comprising prior to, during, or after steps (i) and (ii) contacting said sample with

a solution that preferentially lyses cells of a second type, to cause greater lysis of cells of the second type compared to cells of the first type.

3. The method of claim 1, further comprising after steps (i) and (ii) collecting one or more cells of said first type.

4. The method of claim 2, wherein said solution comprises NaHCO₃ and acetazolamide.

5. The method of claim 2, further comprising the step, after said lysis, of diluting the product of said lysis with a diluent.

6. The method of claim 1, wherein said binding moiety comprises an anti-CD71, an anti-CD36, an anti-GPA, or an anti-CD45 antibody, or a combination thereof.

7. The method of claim 1, wherein said first type of cell is a fetal red blood cell.

8. The method of claim 1, wherein at least 60% of cells of said first type in said sample are bound to said obstacles of step (ii).

9. The method of claim 1, wherein said obstacles of step (ii) are ordered in a two-dimensional array.

10. The method of claim 1 further comprising after step (ii) releasing cells bound to said obstacles.

11. The method of claim 10, wherein said releasing comprises applying a shear force or lysing said bound cells.

12. The method of claim 10, further comprising analyzing the cellular contents of said cells after said releasing.

13. The method of claim 12, wherein said analyzing comprises Fluorescence In Situ Hybridization (FISH).

14. The method of claim 12, wherein said analyzing comprises nucleic acid analysis.

15. The method of claim 1, wherein said first type of cell comprises fetal cells, epithelial cells, tumor cells, stem cells, bacteria, protozoa, or fungi.

16. The method of claim 10, further comprising identifying one or more cells of said first type after said releasing.

17. The method of claim 10, wherein said binding moiety comprises an antibody.

18. The method of claim 17, wherein said antibody is a fetal-cell specific, epithelial-cell specific, tumor-cell specific, stem-cell specific, bacteria specific, protozoan specific, or fungal specific antibody.

19. The method of claim 1 further comprising staining cells bound to said obstacles of step (ii) to identify cells bound thereto.

20. The method of claim 19, further comprising analyzing the cellular contents of said cells during or after said staining.

21. The method of claim 20, wherein said analyzing comprises nucleic acid analysis.

22. The method of claim 19, wherein said staining comprises Fluorescence In Situ Hybridization (FISH).

23. The method of claim 19, wherein said first type of cell comprises fetal cells, epithelial cells, or tumor cells.

24. The method of claim 19, further comprising identifying one or more cells of said first type during or after said staining.

25. The method of claim 19, wherein said binding moiety comprises an antibody.

26. The method of claim 25, wherein said antibody is a fetal-cell specific, epithelial-cell specific, tumor-cell specific, stem-cell specific, bacteria specific, protozoan specific, or fungal specific antibody.