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1. (WO2019028445) DEVICES AND METHODS FOR SEPARATING CIRCULATING TUMOR CELLS FROM BIOLOGICAL SAMPLES
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We claim:

1. A multi-stage microfluidic device for enriching circulating tumor cells in a biological sample, the device comprising:

(i) a first stage comprising a first end, a second end, a first microfluidic channel fluidly connecting the first end and the second end, a first fluid inlet fluidly connected to the first microfluidic channel at the first end, and one or more filters along a length of the first microfluidic channel; wherein the first fluid inlet is configured to receive the biological sample; and wherein the one or more filters are configured to remove a first plurality of waste particles from the biological sample;

(ii) a second stage comprising a third end, a fourth end, a second microfluidic channel fluidly connecting the third end and the fourth end, a second fluid inlet fluidly connected to the second microfluidic channel at the third end, and a first fluid outlet fluidly connected to the second microfluidic channel at the fourth end; wherein the second fluid inlet is configured to receive a sheathing fluid; wherein the first fluid outlet is configured to receive a second plurality of waste particles from the biological sample; and

(iii) a third stage comprising a fifth end, a sixth end, a third microfluidic channel fluidly connecting the fifth end and the sixth end; a second fluid outlet fluidly connected to the third microfluidic channel at the sixth end, and one or more circulating tumor cell outlets fluidly connected to the third microfluidic channel at the sixth end; wherein the second fluid outlet is configured to receive a third plurality of waste particles from the biological sample; and wherein the one or more circulating tumor cell outlets are configured to receive a majority of the circulating tumor cells from the biological sample.

2. The microfluidic device according to claim 1 , further comprising one or more magnetic sources, wherein the one or more magnetic sources cause one or both of:

(a) a non-uniform magnetic field along a length of the second microfluidic channel having a component sufficiently perpendicular to the second microfluidic channel to cause magnetic particles in the second microfluidic channel to be deflected into the first fluid outlet; and

(b) a focusing magnetic field having a field maximum along a length of the third microfluidic channel sufficient to cause magnetic particles in the third microfluidic channel to be focused toward a center of the third microfluidic channel.

3. The microfluidic device according to claim 1 or claim 2, wherein the device comprises a first magnet array and a second magnet array;

wherein the third stage is sandwiched between the first magnet array and the second magnet array;

wherein the first magnet array and the second magnet array are oriented to repel each other; and

wherein the third stage is oriented such that the length of the third microfluidic channel is centrally aligned between the first magnet array and the second magnet array.

4. The microfluidic device according to claim 1 , wherein one or more of the first microfluidic channel, the second microfluidic channel, and the third microfluidic channel have a thickness of about 10 μm to about 10000 μm, about 10 μm to about 1000 μm, about 10 μm to about 500 μm, about 150 μm to about 350 μm, about 220 μm to about 280 μm, or about 250 μm.

5. The microfluidic device according to claim 1 , wherein the second stage has a width of about 50 μm to about 10000 μm, about 500 μm to about 5000 μm, about about 1200 μm to about 2000 μm, about 1400 μm to about 1800 μm, or about 1600 μm.

6. The microfluidic device according to claim 1 , wherein the third stage has a width of about 50 μm to about 10000 μm, about 500 μm to about 5000 μm, about 800 μm to about 1600 μm, about 1000 μm to about 1400 μm, or about 1200 μm.

7. The microfluidic device according to claim 1 , wherein the majority of the circulating tumor cells comprises about 90%, about 92%, about 95%, about 97%, or more of the circulating tumor

cells as compared to a total number of circulating tumor cells present in the biological sample inserted into the first fluid inlet when in operation.

8. The microfluidic device according to claim 7, wherein the biological sample comprises whole blood, wherein the whole blood comprises a plurality of components.

9. The microfluidic device according to claim 8, wherein the plurality of components comprises magnetically labelled white blood cells, and wherein at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are not collected in the one or more circulating tumor cell outlets as compared to a total number of white blood cells present in the whole blood inserted into the first fluid inlet when in operation.

10. The microfluidic device according to claim 8, wherein the plurality of components comprises magnetically labelled white blood cells, and wherein at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are collected in one or more of the filters, the first fluid outlet, and the second fluid outlet as compared to a total number of white blood cells present in the whole blood inserted into the first fluid inlet when in operation.

11. The microfluidic device according to claim 7, wherein the plurality of components comprise unlabeled rare cells and at least 90%, 92%, 95%, or more of the unlabeled rare cells are collected in the one or more circulating tumor cell outlets as compared to a total number of unlabeled rare cells present in the whole blood inserted into the first fluid inlet when in operation.

12. A method of enriching circulating tumor cells in a biological sample comprising a plurality of components, the method comprising introducing the biological sample into the first fluid inlet of a microfluidic device according to any one of claims 1-11 at a flow rate sufficient to cause the biological sample to flow along the first microfluidic channel, the second microfluidic channel, and the third microfluidic channel such that a majority of the circulating tumor cells from the biological sample are collected in the one or more circulating tumor cell outlets.

13. The method according to claim 12, wherein the biological sample is whole blood.

14. The method according to claim 12, wherein the biological sample comprises about 50 to about 250 circulating tumor cells per milliliter of the biological sample.

15. The method according to claim 12, wherein the flow rate is about 6 milliliters to about 25 milliliters of the biological sample per hour.

16. The method according to claim 12, wherein the circulating tumor cells are selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.

17. A single-stage microfluidic device for enriching circulating tumor cells in a biological sample, the device comprising a first stage comprising:

a first end,

a second end,

a microfluidic channel fluidly connecting the first end and the second end,

a fluid inlet fluidly connected to the microfluidic channel at the first end,

three fluid outlets each fluidly connected to the microfluidic channel at the second end, and

a magnet along a length of the microfluidic channel to create a non-uniform magnetic field along the microfluidic channel;

wherein the microfluidic channel has a length of about 1 cm to about 100 cm;

wherein the microfluidic channel has a width of about 50 μπι to about 10000 μπι;

wherein the microfluidic channel has a thickness of about 10 μm to about 10000 μm; and

wherein a gradient of magnetic field flux density of the magnet is about 0.001 T/m to 1000 T/m.

18. A method of enriching circulating tumor cells in a biological sample comprising a plurality of components, the method comprising introducing the biological sample and a biocompatible ferrofluid into the fluid inlet of a microfluidic device according to claim 17 at a flow rate sufficient to cause the biological sample to flow along the microfluidic channel, wherein a majority of the circulating tumor cells from the biological sample are collected in one of the outlets.

19. The method according to claim 18, wherein the flow rate is about 10 μί to about 600 piper minute.

20. The method according to claim 18, wherein the biological sample is whole blood.

21. The method according to claim 18, wherein the biological sample comprises about 50 to about 250 circulating tumor cells per milliliter of the biological sample.

22. The method according to claim 18, wherein the flow rate is about 6 milliliters to about 25 milliliters of the biological sample per hour.

23. The method according to claim 18, wherein the circulating tumor cells are selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.

24. A method of enriching circulating tumor cells in a sample of whole blood, wherein the whole blood comprises unlabeled rare cells and white blood cells, the method comprising:

(i) adding a plurality of magnetic beads to the sample to produce a magnetically labeled sample, wherein at least some of the white blood cells are associated with the magnetic beads;

(ii) filtering the magnetically labeled sample in a microfluidic device to produce a filtered sample by removing large cell debris from the magnetically labeled sample;

(iii) separating at least a portion of the white blood cells that are associated with the magnetic beads by flowing the filtered sample through a sheath flow in a nonuniform magnetic field to produce a first enriched sample; and

(iv) isolating a majority of the unlabeled rare cells by magnetic flow focusing the first enriched sample in a microfluidic channel.