Microfluidic device for cell separation and uses thereof

• US 20070231851A1
• Filed: 03/21/2007
• Published: 10/04/2007
• Est. Priority Date: 09/27/2002
• Status: Abandoned Application

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Toner, Mehmet, Kapur, Ravi, Truskey, George
• Application Number:
  US11/726,276
• Publication Number:
  US 20070231851A1
• Time in Patent Office: Days
• US Class Current: 435/29
• 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 microflow apparatus comprising:

a body having a randomized flow path which comprises an inlet means, an outlet means, and a microchannel arrangement extending between said inlet and outlet means, wherein said microchannel arrangement includes a plurality of transverse separator posts being integral with a base surface of said microchannel and projecting therefrom, wherein said posts are arranged in a pattern capable of providing said randomized flow path.

2. The apparatus of claim 1, wherein said posts are aligned substantially perpendicular to said base surface.

3. The apparatus of claim 1, wherein said posts have at least two different cross sectional sizes.

4. The apparatus of claim 1, wherein the surface of said microchannel is coated with a hydrophilic layer.

5. The apparatus of claim 1, wherein the surface of said microchannel is coated with a sequestering agent.

6. The apparatus of claim 1, wherein the surface of said microchannel is coated with a sequestering agent selected from the group consisting of antibody, antigen, receptor, ligand, oligonucleotide, and peptide.

7. The apparatus of claim 1, wherein a sequestering agent is coupled to the surface of said microchannel by a linker.

8. The apparatus of claim 1, wherein a sequestering agent is coupled to the surface of said microchannel by a hydrophilic linker or a layer of hydrogel.

9. The apparatus of claim 1 wherein said inlet means includes a well capable of holding a liquid sample.

10. The apparatus of claim 1 wherein said microchannel is sealed with a plate affixed to the free ends of said posts.

11. The apparatus of claim 1, wherein said microchannel comprises an optically transparent base surface and can be viewed by optical detection means.

12. The apparatus of claim 1, wherein said microchannel is sealed with a plate affixed to the free ends of said posts and wherein said base surface of said microchannel and said plate are optically transparent.

13. A kit comprising the apparatus of claim 1 and an instruction for coating the surface of said microchannel with a sequestering agent.

14. A kit comprising the apparatus of claim 1, wherein the surface of said microchannel is coated with a sequestering agent.

15. A method of capturing a target molecule in a sample comprising causing a body of liquid containing said sample to flow through said microchannel of the apparatus of claim 1, wherein the surface of said microchannel is coated with a sequestering agent capable of binding to the target molecule.

16. A method of detecting a target molecule in a sample comprising:

causing a body of liquid containing said sample to flow through said microchannel of the apparatus of claim 1, wherein the surface of said microchannel is coated with a sequestering agent capable of binding to the target molecule and detecting the target molecule.

17. The method of claim 16, wherein the target molecule is a cell associated with a condition.

18. The method of claim 16, wherein the target molecule is a cancer or tumor cell.

19. The method of claim 16, wherein the target molecule is a fetal cell.

20. The method of claim 16, wherein the target molecule is a target cell and the ratio of number of target cells and number of total cells in the sample is at least 1:

10⁹.

21. A microflow apparatus for separating biomolecules, such as cells, from a bodily fluid or other liquid, which apparatus comprises:

a body having a flow path defined therein through which a bodily fluid containing target biomolecules can be caused to flow, the flow path having inlet means, outlet means, and a microchannel arrangement extending between said inlet and outlet means, which microchannel arrangement includes a collection region with a plurality of transverse separator posts located in said region, said posts being integral with a base surface of said microchannel and projecting therefrom, said posts being located in an irregular pattern extending laterally across said flow path in said collection region so as to interrupt straight-line flow and streamlined flow of liquid through said region, and said surfaces of said collection region including said posts carrying sequestering agents that will bind with target biomolecules, whereby disruption of streamlined flow throughout said collection region as a result of said pattern of said posts creates effective capture of target biomolecules in said collection region.

22. The apparatus of claim 21, wherein said sequestering agents on said surfaces in said collection region are coupled thereto by hydrophilic linkers or a hydrogel coating so that they will bind effectively with ligands on the exterior surfaces of target cells in the bodily liquid.

23. The apparatus according to claim 21, wherein said substrate is flat and has two major flat surfaces, wherein said flow path is defined in one of said surfaces, wherein a flat plate is affixed to said one surface to close said flow path in said collection region, and wherein free ends of said posts are affixed to said flat plate.

24. The apparatus according to claim 21, wherein said substrate and/or a flat plate which closes said collection region is optically transparent so that optical detection means positioned adjacent said collection region can monitor parameters of biomolecules captured within said region and provide an output signal corresponding to a monitored parameter.

25. The apparatus of claim 21, wherein said collection region has a hydrophilic coating on all surfaces thereof, to which coating said sequestering agents are directly or indirectly bound.

26. The apparatus of claim 21, wherein said posts are arranged in a random pattern throughout said region and are spaced at least about 60 μ

m from the closest adjacent post.

27. A method for separating biomolecules, such as cells, from a bodily fluid or other liquid, which method comprises:

causing a bodily fluid containing target biomolecules to flow downstream along a flow path from an inlet to an outlet, which flow path comprises a channel arrangement that includes a first microchannel having a collection region and separating target biomolecules from the flowing liquid by (a) interrupting straight-line flow of the liquid through said collection region by blocking such flow with a plurality of separator posts located in said region, which posts are integral with a base surface of said microchannel and project therefrom, said posts extending transverse to said flow path and being located in an irregular pattern that extends laterally across said region and disrupts streamlined flow therethrough, and (b) capturing target biomolecules in the flowing liquid on surfaces in said collection region by binding the target biomolecules thereto via sequestering agents carried on surfaces of said collection region, which capture is effective because of the disruption of streamlined flow throughout said collection region.

28. The method of claim 27, wherein a sample of said bodily liquid is placed into a well at said inlet and drawn through said channel by vacuum.

29. The method according to claim 27, wherein a second group of target biomolecules are separated in a second collection region located downstream from the first collection region, which second region also contains a set of separator posts, which second region surfaces carry different sequestering agents than those in said first collection region.

30. The method according to claim 27, wherein said flow path is defined in a body which is optically transparent and wherein optical detection means positioned adjacent the flow path monitors parameters of biomolecules captured by separator posts within the flow path and provides an output signal corresponding to a monitored parameter.

31. The method of claim 27, wherein said target biomolecules are cells and said sequestering agents in said collection region are coupled thereto by hydrophilic linkers of a hydrogel coating so that they effectively bind with ligands on exterior surfaces of the target cells.

32. The method according to claim 27, wherein, following said capture by said sequestering agents, said flow path is washed to remove nonspecifically bound biomolecules, and following said washing, said captured cells are released from said collection region and collected downstream for analysis.