Field of the Invention

[0001 ] The present invention generally relates to a microfluidic chip, and more particularly, relates to a microfluidic chip that can isolate and/or capture circulating tumor cells, erythrocytes, leukocyte and other rare cells from a biological fluid.

Background of the Invention

[0002] Rare cell refers to a cell that is sparse but which has an important biological function or which has clinical detection significance. Examples of rare cells include human peripheral blood circulating tumor cells, circulating endothelial cells, and tumor stem cells. Circulating tumor cells (CTCs) are one kind of representative rare cell and refer to tumor cells that have spontaneously escaped or disseminated from a tumour as a result of a diagnostic procedure or from tumor lesions into peripheral blood. CTCs can develop into metastatic tumors under certain conditions. Of significance is the fact that in 90% of cancer patients, the cause of death is metastases.

[0003] Clinical evidence shows that detection of circulating tumor cells in peripheral blood can be used to monitor the prognosis of a solid tumor and predict chemotherapy effectiveness in cancer patients. For an early stage of cancer, the circulating tumor cells can serve as an important early diagnostic predictor and can also provide a clinical reference for a target population.

[0004] The content of circulating tumor cells in peripheral blood is extremely rare (only a few to dozens of circulating tumor cells in 10 mL whole blood), as compared to about 1.0 x lO ⁹ leukocytes and 5.0x 10 ^{1 1} erythrocytes in peripheral blood. Therefore, it is important to quickly and efficiently isolate circulating tumor cells to permit accurate counting, and molecular and functional analysis. Methods of enriching circulating tumor cells exist and include the following: 1) the CellSearch™ system which has been approved by the U.S. FDA for the detection of circulating tumor cells in the peripheral blood of patients with metastatic breast, prostate and colorectal cancer. This system captures circulating tumor cells with magnetic particles that are labelled with an EpCAM antibody; however, the efficiency of this method is low due to nonspecific adsorption causing CTCs to lose their activity. The CTCs cannot be cultured again, and signal transduction functional analysis cannot be conducted; 2) a membrane-like technique for enriching circulating tumor cells separates CTCs based on a physical difference between circulating tumor cells and other cells. The disadvantages of this method include: cell clogging occurs easily, efficiency and accuracy are low, and contamination by a large number of similar-sized leukocytes that are also captured; and 3) microfluidic chips have been used to enrich circulating tumour cells but exhibit disadvantages and require further development.

[0005] In view of the foregoing, it would be desirable to develop an improved method of analyzing circulating tumor cells within a sample.

Summary of the Invention

[0006] An integrated microfluidic chip comprising a centrifugal pre-isolating structure and a multi-stage sieve structure has now been developed that permits isolation and capture of target cells, such as circulating tumor cells or other rare cells, from a biologic sample in an efficient manner.

[0007] Thus, in one aspect, a microfluidic chip useful to separate different sized cells in a biological sample is provided. The microfluidic chip comprises a matrix mounted on a support substrate, the matrix comprising:

i) a centrifugal pre-isolation structure comprising a sample inlet to receive a sample and direct the sample to a first end of a spiral channel through a screen portion comprising 2 or more screen structures, wherein each screen structure comprises multiple adjacent columns separated by inter-column spaces through which the sample flows, and the spiral channel comprises first and second outlet channels at a second end of the spiral channel, the first outlet channel configured to collect large-sized particles from the spiral channel and the second outlet channel configured to collect small-sized particles from the spiral channel; and

ii) a multi-stage sieve structure adapted to receive sample at different levels from the first and second outlet channels of the centrifugal pre-isolation structure, and to receive buffer through buffer inlets formed in the sieve structure to facilitate flow of the sample within the sieve structure from the first and second outlets, said sieve structure comprising multiple stacked cell isolation sieves from top to bottom of the sieve structure, said cell isolation sieves being separated by cell collection rows, each sieve being composed of multiple adjacent columns separated by inter column spaces which decrease in size from a top sieve to a bottom sieve, said sieve structure further comprising outlets to collect separated cells. [0008] In another aspect, a method of isolating target cells which are at least about 12-

35pm in size from a biological sample is provided comprising the steps of:

i) inject the sample at a pressure of 50-l 80Kpa into a centrifugal pre-isolation structure via a sample inlet which directs the sample through a screen portion to a spiral channel, wherein the screen portion comprises 2 or more screen structures, each screen structure comprising multiple adjacent columns separated by inter-column spaces through which the sample flows;

ii) directing large-sized cells and particles to a first outlet channel of the pre-isolation structure, and small-sized particles to a second outlet channel;

iii) feeding cells from the first and second outlets of the spiral channel into a multi-stage sieve structure at different levels, and flowing the cells through a series of cell isolation sieves separated by cell collection rows, said cell isolation sieves being stacked from top to bottom of the sieve structure, by introducing buffer at buffer inlets in the multi-stage sieve structure at a pressure suitable to further separate the cells based on size, wherein each sieve comprises multiple columns and inter-column spaces that decrease in size from the top to the bottom of the multi-stage sieve structure; and

iv) collecting cells separated in the sieve structure at outlets adapted to collect small, intermediate and large cells, respectively.

[0009] These and other aspects of the invention are described by reference to the following figures.

Brief Description of the Figures

[0010] Figure 1 illustrates a microfluidic chip in accordance with an embodiment of the invention;

[001 1] Figure 2 illustrates an expanded view of a volute screen structure of a microfluidic chip in accordance with an embodiment; and

[0012] Figure 3 is a partial cutaway of a multistage sieve structure of a microfluidic chip in accordance with an embodiment.

Detailed Description of the Invention

[0013] An integrated microfluidic chip 20 as shown in Figure 1 is provided for use to isolate target cells from a sample such as whole blood, plasma, cerebrospinal fluid, pleural effusion, ascites, urine, amniotic fluid, and like. Target cells generally include cells which are at least about 12-35 mhi in size and which exhibit poor deformability, such as circulating tumor cells and other rare cells including magnocellular cells. The microfluidic chip, thus, is useful to separate large target cells from other cells within a given sample, such as leukocytes, erythrocytes and the like.

[0014] The microfluidic chip 20 is generally composed of a matrix structure (1) and a supporting substrate (1.1) that supports the matrix structure (1) and is bonded to the matrix structure. Alternatively, the matrix and supporting substrate are integrally formed, manufactured by any monolithic or unibody technique as would be known to one of skill in the art. The matrix (1) and supporting substrate (1.1) are each generally made of a material that is biocompatible, biosafe and transparent, such as a polymeric material. Suitable materials for the matrix structure include, but are not limited to, polymer dimethyl siloxane (PDMS), silica, photoresist such as SU8, polymethylmethacrylate (PMMA), polycarbonate (PC), copolymers of cycloolefm (COC)or polystyrene (PS). The substrate may be made of similar materials, e.g. polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefm (COC), polystyrene (PS), or glass.

[0015] The matrix structure (1) comprises a centrifugal pre-isolation structure (30) and a multi-stage sieve structure (40).

[0016] The centrifugal pre-isolation structure (30) comprises a central sample inlet (2) that directs sample through a volute screen portion (3) to a spiral channel (4), which extends in either a clockwise or counter-clockwise direction. Together the volute screen portion (3) and the spiral channel (4) function to separate the sample into cells of different sizes. As shown in Figure 2, the volute screen portion (3) comprises two or more screen structures (3.1 and 3.2). The distance between each screen structure is less than or equal to 150 pm. Each screen structure comprises multiple adjacent columns having a length ranging from 3-300 pm, and each column being separated by an inter-column space which is less than or equal to about 50 pm. The columns are oriented to form a ring. The sample solution enters the chip through inlet (2) and is passed through the inter-column spaces of each screen structure. The columns are shaped to prevent the sample (such as whole blood) from coagulating. For example, the cross section of each column may be triangular, quadrilateral, square, rectangular, trapezoid, pentagonal, hexagonal, circular or irregular-shaped. [0017] The sample solution enters the chip through inlet (2) and is passed through the inter column spaces of each screen structure (3.1 and 3.2) of the volute screen portion (3), and then flows through the spiral channel (4). The spiral channel (4) comprises about 2 to 15 rounds or spirals. The diameter of the channel ranges from 10 pm to 300 pm. The flow within the spiral channel is controlled to facilitate separation of cells in the sample. For example, the flow is such that large-sized cells/particles are propelled towards the outer edge of the spiral channel, while smaller sized cells/particles in the stream accumulate along the inner edge of the spiral channel. The pressure required to result is cell separation will vary with certain properties (such as viscosity) of the sample; however, a pressure in the range of about 50-180 Kpa at inlet (2) will result in cell separation within a sample. The large-sized cells are collected in a first collection channel (13), while small-sized cells are collected in a second collection channel (14).

[0018] The volute screen portion (3) and spiral channel (4) are also generally made of a biocompatible polymer such as, but not limited to, PDMS, SU8, PMMA, PC, COC and PS.

[0019] The partially separated cells are then fed into the multi-stage sieve structure (40) from the centrifugal pre-isolation structure (30) and undergo repeated filtration through multiple sieve layers (12) of the sieve structure (40) such that cells of different sizes are separated and collected at different cell collection zones.

[0020] The sieve structure (40) comprises multiple stacked cell isolation sieves from top to bottom of the sieve structure (40). Each sieve is separated from adjacent sieves by a cell collection row (1 1). Each sieve is composed of multiple columns (15), which are about 10-100 pm high, and inter-column spaces (16), as in the cross-sectional view of the multi-stage sieve structure (40) shown in Figure 3. The number of columns (15) in each sieve (12) ranges from 100 to 40000, and the distance between columns (i.e. inter-column spaces (16)) of each sieve is 2 pm to 300 pm. Generally, the inter-column spaces (16) in each sieve (12) decreases from the top of the sieve structure (40) to the bottom of the sieve structure, e.g. the inter-column spaces (16) in the upper portion of the sieve structure (40) may be greater than 100 pm (large sieves), while the intercolumn spaces (16) in the lower portion of the sieve structure may be less than 100 pm (intermediate to small sieves). The number of sieves in the sieve structure (40) ranges from 10 to 10000 in total, and the distance between sieves (or the size of the cell collection rows 1 1) ranges from 5 mhi to 300 pm. The cross section of the columns may be triangular, circular, quadrilateral, square, trapezoidal, pentagonal, hexagonal, or other regular or irregular shape.

[0021] The distribution and size of the columns in the multi-stage sieve structure (40) are provided to guide different sized cells to an appropriate cell collection outlet. Thus, small-sized cells such as erythrocytes are guided towards the erythrocyte outlet (8), and while mid-sized cells are guided towards the intermediate-sized leukocyte outlet (9). Likewise, large sized tumour cells are prevented from flowing into either of outlets (8) or (9), but are guided by both the design and buffer flow (controlled by the pressures at each of the buffer inlets) to either be maintained in cell collection rows (1 1), or collected at the CTC outlet (10).

[0022] In particular, the initial size-separated sample is fed into the multi-stage sieve structure (40) of the microfluidic chip from the first and second collection channels (13 and 14) of the centrifugal pre-isolation structure (30). As shown in Figure 1 , cell collection channels (13) and (14) feed separated cells into the sieve structure (40) at different levels within the sieve structure. The first collection channel (13), which collects large-sized cells, feeds these cells into the top or upper portion of the sieve structure (40) so that the cells can proceed through large sieves (12) having larger inter-column spaces (16). These cells are captured in a cell collection row (1 1) when they come to a sieve having inter-column spaces (16) which are smaller than the diameter of the cells themselves. These cells may be retained in the collection row or washed into the CTC outlet (10). Any small cells inadvertently captured with the large cells in first collection channel will readily continue to pass through the sieves (12) for collection in the appropriate cell collection outlet, (8) or (9), and thus, will not contaminate the large target cells. The second collection channel (14) of the centrifugal pre-isolation structure (30), which collects smaller cells, feeds these cells into the sieve structure (40) at an intermediate level that bypasses the largest sieves and directs the cells through intermediate and small sieves (12), e.g. sieves in the lower portion of the sieve structure (40). Intermediate-sized cells will be captured in a cell collection row (1 1) when they can come to a sieve with inter-column spaces (16) that are smaller than the cells themselves, and can then be retained in the cell collection row or directed to outlet (9). Small cells will flow through the sieves for collection in outlet (8).

[0023] During this process, erythrocytes and other small-sized cells which are readily deformable (e.g. cells less than 10 pm in size) proceed through the sieve layers (12) and are removed from the structure (40) through a collection row or zone comprising the erythrocyte outlet (8). Intermediate-sized cells such as leukocytes (e.g. less than 20 pm) proceed through many but not all of the sieve layers (12) and when they can no longer flow through the sieve layers (12), they collect within a collection zone or row (1 1) and are removed by buffer flow from the structure (40) through the leukocyte outlet (9). Circulating tumor cells and other rare cells (large-sized, magnocellular nucleus that possess poor deformability) are captured much sooner in the process and collect in a middle to upper collection zone or row (1 1), and may optionally be removed from the structure through the CTC outlet (10).

[0024] Cell buffer solution is introduced into the multi-stage sieve structure (40) through inlets (5), (6) and (7) to facilitate cell flow and cell separation within the structure (40). In one embodiment, the sieve structure (40) comprises a first buffer inlet (5) proximal to the spiral channel outlets (13/14), a second buffer inlet (6) located underneath the sieves or at the bottom of the multi stage sieve structure (40) and a third buffer (7) inlet located at the top of the sieve structure (40).

[0025] Cell buffer solutions that may be used include solutions which are biocompatible and suitable for use with cells, i.e. any buffer adjusted to physiological pH, such as Dulbecco's phosphate-buffered saline. The different buffer inlets provide flow in various directions (e.g. up, down, left and right), and provide the ability to adjust pressure and flow rates applied to different regions of the multi-stage sieve structure (40). Generally, large target cells are successfully isolated from sample with buffer flows resulting from controlled pressures as follows: a pressure in the range of about 20-120 Kpa at buffer inlet (5), 30-150 Kpa at buffer inlet (6), and 40-250 Kpa at buffer inlet (7).

[0026] Target cells such as circulating tumor cells and other rare cells (large-sized, magnocellular cells of poor deformability) collected in a collection area (1 1) within the sieve structure (40) may be quantified directly within the microfluidic chip using a microscope.

[0027] Alternatively, the cells may be collected within the CTC outlet (10) and detectably labelled to permit their identification and/or quantification using techniques well established in the art. Various labels may be used for this purpose, including immunological labels incorporating a detectable tag such as a chromogenic, chemiluminescent, fluorescent, or radioisotope tag, or an enzyme label (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and b-galactosidase) that emits a signal on addition of a substrate to generate a chromogenic, chemiluminescent or fluorescent signal.

[0028] In one embodiment, target cells are identified using an immunological label, i.e. an antibody that binds with the target cell. Antibodies to a target cell, including polyclonal or monoclonal antibodies, may be prepared using well-established hybridoma technology developed, for example, by Kohler and Milstein (Nature 256, 495-497(1975)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a target cell. The term“antibody” as used herein is intended to include fragments thereof which also specifically react with a target cell, as well as chimeric antibody derivatives, i.e., antibody molecules resulting from the combination of a variable non-human animal peptide region and a constant human peptide region. Antibody labels to certain target cells may also be commercially available (e.g. at Abeam Inc., Thermo Fisher Scientific, Sigma Aldrich, Santa Cruz Biotechnology, etc.). The selected antibody is made detectable by labelling with an appropriate tag as above. Examples of suitable fluorescent tags include, but are not limited to, ethidium bromide, fluorescein, allophycocyanin, phycoerythrin, 4',6-diamidino-2-phenylindole (DAPI) and green fluorescent protein.

[0029] The immunological label is introduced into the microfluidic chip through the appropriate inlets in the microfluidic chip, e.g. inlets (5), (6) and/or (7) and incubated for a period of time to permit labelling following fixing of the cells with a fixing agent (e.g. 4% neutral paraformaldehyde solution, 40% formalin, 80% ethanol, glutaraldehyde) and treatment with a surfactant. Unbound antibody is washed from the chip with buffer. After staining is completed, the microfluidic chip is observed to quantify the detectable tag, e.g. fluorescence, chemiluminescence, radioactivity, or other tag, using appropriate technology.

[0030] In one embodiment, the microfluidic chip incorporates a digital imaging device, such as a charge-coupled device (CCD), electron multiplying charge-coupled device (EMCCD) or a complementary metal oxide semiconductor (CMOS), that converts the signal emitted by the detectable tag into an electrical output to provide a visual image.

[0031] The present microfluidic chip provides improved separation of target cells, such as large circulating tumor cells and other large cells, from a biological sample, that is efficient and accurate, resulting in separated target cells with low levels of contaminating cells. [0032] Embodiments of the invention are described in the following specific example which is not to be construed as limiting.

Example

[0033] A human whole blood sample (1 -15 ml) without any preparation, i.e. no lysis of erythrocytes, dilution of blood, or filtration, was introduced into a microfluidic chip as shown in Fig. 1 at the inlet (2). The sample was introduced into the centrifugal pre-isolation structure (30) at inlet (2) at a pressure in the range of 50-180 Kpa. The sample was flowed through the volute screen portion (3) and spiral channel (4) of structure (30). Large-sized cells were collected in channel (13) and directed into the multi-stage sieve structure (40) of the chip, while smaller cells were collected and entered the multi-stage sieve structure (40) through channel (14). Cell buffer solutions were introduced into the multistage sieve structure through inlets (5), (6) and (7) at pressures of 20-120 Kpa at inlet (5), 30-150 Kpa at inlet (6) and 40-250 Kpa at inlet (7).

[0034] The sample was flowed through the multistage sieve structure (40) and was filtered through the multiple sieves (12). Cells were collected when passage through sieves was no longer possible. Erythrocytes and other cells (small size, of strong deformability) were removed through outlet (8); leukocytes (intermediate size) were removed via outlet (9); circulating tumor cells and other rare cells (large-sized, magnocellular nucleus and of poor deformability) were captured, and collected within a cell collection area (1 1) of the sieve structure.

[0035] The collected large cells were optionally stained and counted in the microfluidic chip using the following method:

1 ) DPBS buffer was injected into the microfluidic chip through inlets (5) and (7), and rinsed the chip for 2 minutes;

2) a fixative solution, neutral paraformaldehyde solution (4%), was then introduced into the chip via inlets (5) and (7) and the cells were fixed for 10-20 minutes;

3) the fixative solution was removed from the chip by rinsing with DPBS buffer for 2 minutes through inlets (5) and (7);

4) Triton X 100 working solution was injected into the microfluidic chip through inlets (5) and (7) to penetrate the cell membrane for 10 minutes; 5) antibodies of EpCAM-APC, CK18-FITC, CD45-PE, DAPI, etc. were diluted with buffer to a working concentration (e.g. EpCAM-APC: 4pg/ml; CK18-FITC: 6.25pg/ml, CD45-PE: 0.25 pg/ml, DAPI: 0.5 pg/ml), and then the antibody-buffer mix was introduced into the chip through inlets (5) and (7). The chip was incubated at 4-37° C for 1-12 hours;

6) DPBS buffer was introduced into the microfluidic chip through inlets (5) and (7) to wash any non-binding antibody;

7) following the above staining protocol, the microfluidic chip was observed under the fluorescence microscope with different wavelengths.

Results:

[0036] Using the present microfluidic chip, tumour cells were successfully isolated from other cells in the blood sample, and identified.