Microstructure for particle and cell separation, identification, sorting, and manipulation
The invention relates to microscale cell separating apparatus which are able to separate cells on the basis of size of the cells, interaction of the cells with surfaces of the apparatus, or both. The apparatus comprises a stepped or sloped separation element ( 16 ) interposed between an inlet region ( 20 ) and an outlet region ( 22 ) of a void that can be filled with fluid. The void can be enclosed within a cover ( 12 ) and fluid flow through the void engages cells with the separation element. Only cells which have (or can deform to have) a characteristic dimension smaller than or equal to the distance between a step and the cover or body can pass onto or past a step. Modifications of surfaces within the apparatus can also inhibit passage of cells onto or past a step.
1. A microscale apparatus for separating particles, the apparatus comprising a body, a cover, and a separation element,
the body and cover defining a void having an inlet region, an outlet region, and a surface,
the separation element i) being disposed in the void, ii) having a plurality of steps including a first step and a second step, and iii) defining a narrow passageway that fluidly connects the inlet and outlet regions in a fluid path,
the narrow passageway including a first passageway and a second passageway,
the first passageway fluidly connecting the inlet region and the second passageway, being bounded by the first step and the surface of the void, and having a height defined by the distance between the first step and the surface of the void, and
the second passageway fluidly connecting the first passageway and the outlet region, being bounded by the second step and the surface of the void, and having a height defined by the distance between the second step and the surface of the void, wherein the width of the narrow passageway at the portion of the second step nearest the inlet region in the fluid path is greater than the height of the second passageway,
the height of the second passageway being smaller than the height of the first passageway.
2. The apparatus of claim 1 , wherein the separation element is attached to at least one of the body and the cover.
3. The apparatus of claim 2 , wherein the separation element is integral with at least one of the body and the cover.
4. The apparatus of claim 1 , wherein one of the cover and the body defines a fluid inlet port in fluid communication with the inlet region and one of the cover and the body defines a fluid outlet port in fluid communication with the outlet region.
5. The apparatus of claim 4 , further comprising a fluid displacement device for providing fluid to the fluid inlet port.
6. The apparatus of claim 1 , wherein the void is filled with fluid.
7. The apparatus of claim 6 , wherein the particles are cells and the cells are disposed in the inlet region.
8. The apparatus of claim 1 , wherein the height of each of the first and second passageways is in the range from 0.1 micrometer to 1000 micrometers.
9. The apparatus of claim 8 , wherein the height of each of the first and second passageways is in the range from 0.5 micrometer to 25 micrometers.
10. The apparatus of claim 8 , wherein the height of each of the first and second passageways is in the range from 1 micrometer to 16 micrometers.
11. The apparatus of claim 8 , wherein the height of each of the first and second passageways is in the range from 1 micrometer to 10 micrometers.
12. The apparatus of claim 8 , wherein the height of each of the first and second passageways is in the range from 1 micrometer to 5 micrometers.
13. The apparatus of claim 1 , wherein one of the body, the cover, and the separation element defines a fluid channel for withdrawing fluid from the void at a step of the separation element.
14. The apparatus of claim 1 , further comprising a device for detecting a particle in the void at a step of the separation element.
15. The apparatus of claim 1 , further comprising a device for manipulating a particle in the void at a step of the separation element.
16. The apparatus of claim 15 , wherein the device is a device for killing a cell.
17. The apparatus of claim 16 , wherein the device is a heating element.
18. The apparatus of claim 1 , wherein a surface of the separation element has an antibody attached thereto.
19. An apparatus comprising two apparatus of claim 1 , wherein the outlet region of the first apparatus is in fluid communication with the inlet region of the second apparatus.
20. The apparatus of claim 1 , wherein each of the first step and the second step is defined by a pair of planar surfaces of the separation element that meet at a right angle.
21. The apparatus of claim 1 , wherein the separation element has at least one additional step in addition to the first and second step, each additional step defining an additional passageway that i) fluidly connects the second step and the outlet region ii) is bounded by the additional step and the surface of the void, and iii) has a height defined by the distance between the additional step and the surface of the void, the height of each additional passageway being smaller than the height of all other passageways between it and the inlet region.
22. The apparatus of claim 1 , wherein the height of the first passageway is about 12-16 micrometers and the height of the second passageway is about 5.5 to 8.5 micrometers.
23. A kit comprising a body, a cover, and a separation element,
the body and cover being adapted to fit one another and, when assembled, to define a void having an inlet region, an outlet region, and a surface,
the separation element i) being disposable in the void of the assembled body and cover, ii) having a plurality of steps including a first step and a second step, and iii) defining a narrow passageway that fluidly connects the inlet and outlet regions in a fluid path when the body and cover are assembled with the separation element disposed in the void such that,
the narrow passageway includes a first passageway and a second passageway,
the first passageway fluidly connecting the inlet region and the second passageway, being bounded by the first step and the surface of the void, and having a height defined by the distance between the first step and the surface of the void, and
the second passageway fluidly connecting the first passageway and the outlet region, being bounded by the second step and the surface of the void, and having a height defined by the distance between the second step and the surface of the void, wherein the width of the narrow passageway at the portion of the second step nearest the inlet region in the fluid path is greater than the height of the second passageway,
the height of the second passageway being smaller than the height of the first passageway.
24. A method of separating particles, the method comprising providing the particles to the inlet region of a microscale apparatus, the apparatus comprising a body, a cover, and a separation element,
the body and cover defining a void having an inlet region, an outlet region, and a surface,
the separation element being disposed in the void, having a plurality of steps including a first step and a second step, and iii) defining a narrow passageway that fluidly connects the inlet and outlet regions in a fluid path,
the narrow passageway including a first passageway and a second passageway,
the first passageway fluidly connecting the inlet region and the second passageway, being bounded by the first step and the surface of the void, and having a height defined by the distance between the first step and the surface of the void, and
the second passageway fluidly connecting the first passageway and the outlet region, being bounded by the second step and the surface of the void, and having a height defined by the distance between the second step and the surface of the void, wherein the width of the narrow passageway at the portion of the second step nearest the inlet region in the fluid path is greater than the height of the second passageway,
the height of the second passageway being smaller than the height of the first passageway,
passing a fluid from the inlet region into the outlet region by way of the narrow passageway, whereby particles are separated based on a characteristic dimension of the individual particles, and
thereafter collecting separated particles.
25. The method of claim 24 , wherein the fluid is passed from the inlet region to the outlet region using a fluid displacement device to add fluid to the inlet region by way of a fluid inlet port that extends through at least one of the body and the cover and that fluidly communicates with the inlet region.
26. The method of claim 24 , wherein the separated particles are collected from a step of the separation element.
27. The method of claim 24 , wherein the particles provided to the inlet region are cells of a blood sample and the collected particles are stem cells.
28. The method of claim 27 , wherein the blood sample is a cord blood sample.
29. The method of claim 27 , wherein the height of the second passageway is sufficient to permit passage of blood platelets therethrough.
30. The method of claim 24 , wherein the particles are cells.
31. The method of claim 24 , wherein the particles provided to the inlet region are cells of a blood sample obtained from a pregnant woman and the collected particles are fetal cells.
32. The method of claim 24 , wherein the particles provided to the inlet region are cells of a blood sample obtained from a pregnant woman and the collected particles are fetal stem cells.
33. The method of claim 24 , wherein the height of the first passageway is about 12-16 micrometers and the height of the second passageway is about 5.5 to 8.5 micrometers.