Rare cell isolation device and method of use thereof
This invention concerns patentable devices configured to capture cancer stem cells and cell clusters. After capturing such cells and/or clusters, the cancerous cells are subjected to whole genome sequencing. The resulting genomic sequence information is then compared to that for “normal” or non-diseased tissue (obtained, for example, from either the same patient, or a population sample, etc.) in order to identify the specific genetic mutation(s) present in the CSCs. Further analysis then correlates the genetic mutations with cell growth signaling pathways typically found with tumor metastases. Armed with this information, an oncologist can then develop a specifically targeted therapy that utilizes approved drugs or drug candidates undergoing clinical testing to address the identified driver mutations and thus effect a “targeted” therapy tailored to the particular patient's disease.
1. A microfluidic device for capturing a rare cell or rare cell cluster or a bulk tumor cell from a sample having a nonporous substrate that comprises downstream of a sample addition port and arrayed in series:
a) a plurality of capture zones connected by one or more valved microfluidic channels, wherein each capture zone comprises a releasable cell capture reagent that specifically binds a rare cell surface marker, the cell capture reagent being immobilized on the nonporous substrate, wherein the one or more microfluidic channels connecting a first capture zone and a second capture zone of the plurality of capture zones contains a rare cell or rare cell cluster or bulk tumor cell retention element, wherein the plurality of capture zones contain one or more herringbone geometries, wherein the plurality of capture zones are contained in an aligned high-low herringbone configuration (HLHC), and wherein the HLHC comprises channels that are offset from one another and which fluidly connect with one another in an alternating pattern to produce an increased channel surface area for binding; and
b) a detector for detecting the rare cell or rare cell cluster or bulk tumor cell bound by the cell capture reagents,
wherein the sample is added to the microfluidic device at a flow rate greater than or equal to 27.5 μL/min and the microfluidic device is configured to detect one or more of a rare cell, a rare cell cluster or a bulk tumor cell, simultaneously, and to maintain the rare cell or rare cell cluster or bulk tumor cell alive and viable, and
wherein the sample is selected from the group consisting of whole blood, blood fractions such as serum and plasma, urine, sweat, lymph, feces, ascites, seminal fluid, sputum, nipple aspirate, post-operative seroma, wound drainage fluid, saliva, synovial fluid, ascites fluid, bone marrow aspirate, cerebrospinal fluid, nasal secretions, amniotic fluid, bronchoalveolar lavage fluid, pleural effusion, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, or tonsil cells.
2. The microfluidic device of claim 1 , wherein each capture zone comprises a different releasable cell capture reagent that specifically binds a different rare cell surface marker.
3. The microfluidic device of claim 1 , wherein the device comprises four or more capture zones, and wherein at least one capture zone comprises a releasable cell capture reagent that specifically binds a non-rare cell surface marker.
4. The microfluidic device of claim 1 , wherein the releasable cell capture reagents each comprises an antibody, an antigen-binding antibody fragment or an antigen specific aptamer.
5. The microfluidic device of claim 1 , wherein one or more of the releasable cell capture reagents comprise a detectable label.
6. The microfluidic device of claim 1 , wherein at least one capture zone includes a dissolvable matrix and at least one capture zone includes a non-dissolvable matrix.
7. The microfluidic device of claim 6 , wherein the dissolvable matrix is dissolvable by a chelating agent, enzyme or combination thereof.
8. The microfluidic device of claim 6 , wherein the dissolvable matrix is non-covalently bound alginate and the non-dissolvable matrix is covalently bound alginate.
9. The microfluidic device of claim 1 , wherein the rare cell surface marker is selected from the group consisting of CD44, CD47, MET, EpCAM, CD34, CD38, CD90, CD19, Stro-1, CD105, CD133, ESA, CD24, ALDH, ALDH1, CD 166, SP, CD20, CD117, A2β1, EGFR, HER2, ERCC1, CXCR4, E-Cadherin, Mucin-1, Cytokeratin, PSA, PSMA, RRM1, Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1, EML4, Leukocyte Associated Receptor (LAR), and any combination thereof.
10. The microfluidic device of claim 1 , wherein the microfluidic device is configured to further capture a bulk tumor cell simultaneously with the rare cell or rare cell cluster.
11. The microfluidic device of claim 10 , wherein the rare cell, the rare cell cluster, or bulk tumor cell are harvested by dissolving the non-covalently bound alginate, or are harvested directly from a capture zone using a needle punch.
12. The microfluidic device of claim 1 , wherein the microfluidic device is configured to control the fluid flow of the sample to effectuate isolation and harvest of live and viable rare cell, rare cell cluster, or bulk tumor cell.
13. The microfluidic device of claim 1 , wherein the microfluidic device is configured to detect one or more of a rare cell, a rare cell cluster or a bulk tumor cell by microscopy or flow cytometry.
14. The microfluidic device of claim 1 , wherein the microfluidic device is configured to isolate the rare cell, rare cell cluster, bulk tumor cell, or bulk tumor cell for analysis by one or more of image analysis, cell number analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassays, nuclear exclusion analysis, whole genome sequencing, or a combination thereof, wherein the isolated rare cell, rare cell cluster, or bulk tumor cell is alive and viable and/or wherein the isolated cell is suitable the analysis which requires living cells.