Recovery of rare cells using a microchannel apparatus with patterned posts
A microflow apparatus for separating or isolating cells from a bodily fluid or other liquid sample uses a flow path where straight-line flow is interrupted by a pattern of transverse posts. The posts are spaced across the width of a collection region in the flow path, extending between the upper and lower surfaces thereof; they have rectilinear surfaces, have arcuate cross-sections, and are randomly arranged so as to disrupt streamlined flow. Sequestering agents, such as Abs, are attached to all surfaces in the collection region via a hydrophilic coating, preferably a hydrogel containing isocyanate moieties or a PEG or polyglycine of substantial length, and are highly effective in capturing cells or other targeted biomolecules as a result of such streamlined flow disruption.
1. A microflow apparatus comprising:
a body having a randomized flow path which comprises an inlet, an outlet, and a microchannel arrangement extending between said inlet and outlet to form a collection region, the flow path designed to support a fluid flow rate of 0.01 to 100 mm/second,
wherein:
said microchannel arrangement includes a plurality of transverse separator posts arranged in a random and irregular pattern, wherein said random and irregular pattern has the characteristics of 1) the posts having at least three different cross-sectional sizes in the range of about 70 to about 130 microns in diameter being integral with a base surface of said microchannel and projecting therefrom, 2) random arrangement of relative spacing of the posts, and 3) the combination of spacing among the posts and different cross-sectional sizes of posts resulting in the posts occupying about 15% to about 25% of the total volume of the collection region,
the randomized flow path is configured for cell capture, and
when in operation said microchannel is connected to a pump to support the continuous flow of liquid sample at a flow rate of 0.01 to 100 mm/second.
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 are arranged in a random and irregular pattern generated by a mathematical algorithm.
4. The apparatus of claim 1 , wherein said posts are separated by a minimum spacing of 50 microns.
5. The apparatus of claim 1 , wherein said posts are arranged in a random and irregular pattern across the entire width of said collection region.
6. The apparatus of claim 1 , wherein the randomized flow path provided by said posts promotes swirling and eddies.
7. The apparatus of claim 1 , wherein the surface of said microchannel is coated with a hydrophilic layer.
8. The apparatus of claim 1 , wherein the surface of said microchannel is coated with a hydrophilic layer of at least about 1 micron thick comprising isocyanate-functional polymer of PEG, PPG, or a copolymer thereof.
9. The apparatus of claim 1 , wherein the surface of said microchannel is coated with a sequestering agent.
10. 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.
11. The apparatus of claim 1 , wherein a sequestering agent is coupled to the surface of said microchannel by a linker.
12. 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.
13. The apparatus of claim 1 , wherein said microchannel is sealed with a plate.
14. The apparatus of claim 13 , wherein said posts are affixed to said plate at their distal ends.
15. The apparatus of claim 13 , wherein said plate is optically transparent.
16. The apparatus of claim 1 , wherein said microchannel comprises an optically transparent base surface.
17. The apparatus of claim 1 , wherein said inlet includes a well capable of holding a liquid sample.
18. A kit comprising the apparatus of claim 1 , wherein the surface of said microchannel is coated with a sequestering agent.
19. A method of capturing a target cell 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 cell.
20. A method of capturing a target cell 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 cell and detecting the target cell.
21. The method of claim 20 , wherein the target is a cell associated with a condition.
22. The method of claim 20 , wherein the target is a cancer or tumor cell.
23. The method of claim 20 , wherein the target is a fetal cell.
24. The method of claim 20 , wherein the body of liquid containing said sample flows through said microchannel at a speed of about 0.5 mm to about 5 mm per second.
25. The method of claim 20 , wherein the body of liquid containing said sample flows through said microchannel at a speed of about 0.5 μL per minute to about 10 μper minute.
26. The method of claim 20 , wherein the body of liquid containing said sample flows through said microchannel at a speed of about 10 μL per minute.
27. The apparatus of claim 1 , wherein the surface of said microchannel is coated with a hydrophilic layer of at least about 1 micron thick comprising linear PEG or PPG having a N-hydroxy-succinimidyl ester moiety at a first end and a maleimidyl moiety at a second end opposite to said first end.
28. The apparatus of claim 1 , wherein the collection region is about 100 microns high and about 2 to 4 mm wide.
29. A microflow apparatus comprising:
a body having a randomized flow path which comprises an inlet, an outlet, and a microchannel arrangement extending between said inlet and outlet to form a collection region, the flow path designed to support a fluid flow rate of 0.01 to 100 mm/second,
wherein:
said microchannel arrangement includes a plurality of transverse separator posts being integral with a base surface of said microchannel and projecting therefrom and arranged in a random and irregular pattern, wherein said random and irregular pattern is designed according to the following parameters 1) the posts having at least three different cross-sectional sizes in the range of about 70 to about 130 microns in diameter, 2) random arrangement of relative spacing of the posts, and 3) the combination of spacing among the posts and different cross-sectional sizes of posts resulting in the posts occupying about 15% to about 25% of the total volume of the collection region,
the randomized flow path is configured for cell capture, and
when in operation said microchannel is connected to a pump to support the continuous flow of liquid sample at a flow rate of 0.01 to 100 mm/second.