Microfluidic device for rapid enrichment of Anti-tumor t-cells and methods of use thereof

Abstract

The present disclosure provides devices and methods for the identification of high affinity, tumor specific T-cells. The present disclosure further provides microfluidic devices for the identification of tumor specific T-cells. Aspects of the disclosure further relate to populations of tumor specific T-cells and methods for treating cancer.

Description

TITLE OF THE INVENTIONMICROFLUIDIC DEVICE FOR RAPID ENRICHMENT OF ANTI TUMOR T- CELLS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 618,637, filed January 8, 2024, and U.S. Provisional Appl. Ser. No. 63 / 706,139, filed October 11, 2024, the entire disclosure of each of which is incorporated herein by reference.STATEMENT OF GOVERNMENT RIGHTS

[0001] This invention was made with government support under CA283852 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] This present disclosure relates to the field of cancer therapeutics, and more specifically to devices and methods for the rapid identification of tumor specific T-cells.BACKGROUND OF THE INVENTION

[0003] Immunotherapies for cancer rely on the use of T-cells that can specifically identify and target tumor cells. This technique has been highly effective in the treatment of some cancers, including melanoma. High-affinity T-cells have been found to better infiltrate tumors and reduce the number of interactions required to activate a T-cell response, and thus may improve the efficacy of tumor cell targeting. Existing methods for identifying potent anti-tumor T-cells, however, are time consuming (requiring weeks to months) and require complicated screening procedures and sophisticated instrumentation.

[0004] Functional cellular studies can be used to study the T-cell response after interacting with a tumor cell, including T-cell cytokine secretion and activation, as well as tumor cell death. Though these methods identify the T-cells that are functionally active against a tumor, they cannot be completed on a large-scale, as each potential epitope is screened individually. Assays can also be performed with peptide MHC complex (pMHC) tetramers to detect specific T-cell receptors; however, this method does not determine the functional response of T-cellsand requires prior knowledge of specific molecular targets on tumor cells to screen for the corresponding T-cell receptors.

[0005] There is a need in the art for the development of improved devices and methods permitting rapid identification of high-affinity anti-tumor T-cells that are compatible with both adherent and nonadherent tumor cells. The present disclosure provides such devices and methods.SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a population of tumor cells into a microfluidic device, wherein at least one cell of the population comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; c) introducing a sample comprising a population of T-cells into the microfluidic device; and d) performing a wash step to remove T- cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex. In one embodiment, the method may further comprise performing an additional wash step to remove tumor cells of the population that remain mobilized within the microchannel prior to step (c) above. In another aspect, the present disclosure provides a method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a sample comprising a population of tumor cells and a population of T-cells into a microfluidic device, wherein at least one cell of the population of tumor cells comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one micro well; and c) performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.

[0007] In another embodiment, each microwell of the plurality of microwells has a diameter of about 10 pm to about 60 pm. In yet another embodiment, there is about 60 pm to about 100 pm of space between each microwell of the plurality of microwells. The microchannel, in one embodiment, comprises at least about 10,000 microwells or at least about 60,000 microwells.In another embodiment, the methods of the present disclosure may further comprise collecting the tumor cell / T-cell complex. A plurality of tumor cells of the population, in yet another embodiment, comprise the magnetic label. In still yet another embodiment, applying the magnetic force to the microfluidic device immobilizes a plurality of tumor cells of the population to a plurality of microwells. In one embodiment, a plurality of tumor cells of the population of tumor cells and a plurality of T-cells of the population of T-cells form a plurality of tumor cell / T-cell complexes. The time period required to complete the method, in one embodiment, is less than about 1 hour. In another embodiment, the methods of the present disclosure are capable of being completed in less than about 1 hour. In yet another embodiment, the wash step or the additional wash step comprises washing the microchannel at a flow rate of about 0.01 mL / min to about 0.5 mL / min. The introducing the sample comprising the population of T-cells, in still yet another embodiment, comprises introducing the sample at a flow rate of about 0.5 pL / min to about 5.0 pL / min.

[0008] In one embodiment, the population of tumor cells is a population of tumor cells derived from a lung cancer, a non-small cell lung cancer, a colorectal cancer, a pancreatic cancer, an appendiceal cancer, a small bowel adenocarcinoma, a hepatobiliary cancer, a gynecological malignancy, a hematopoietic cancer, a breast cancer, a bladder cancer, a prostate cancer, or a skin cancer. The population of tumor cells, in another embodiment, is a population of human tumor cells. The population of T-cells, in yet another embodiment, is a population of human T-cells. In still yet another embodiment, the population of tumor cells and the sample comprising the population of T-cells are derived from the same subject. The subject, in one embodiment, is a human subject. In another embodiment, the population of tumor cells and the sample comprising the population of T-cell are derived from different subjects.

[0009] In yet another aspect, the present disclosure provides, a microfluidic device comprising: a) a sample reservoir; b) an outlet; c) a microchannel in fluid communication with the sample reservoir and the outlet, the microchannel comprising a plurality of microwells, wherein each micro well of the plurality of microwells has a diameter of about 10 pm to about 60 pm, and wherein there is about 60 pM to about 100 pM of space between each micro well of the plurality of microwells; and d) a magnetic field source configured to apply a magnetic field to at least a portion of the plurality of microwells. In one embodiment, the microchannel comprises a cellulose acetate microwell substrate. In another embodiment, the microchannel comprises at least about 10,000 or at least about 60,000 microwells. The microchannel, in yet another embodiment, is about 10 mm to about 20 mm in length, about 1.0 mm to about 4.0 mm inwidth, or about 100 pm to about 500 pm in height. The sample reservoir, in still yet another embodiment, is about 5 mm to about 20 mm in diameter. In one embodiment, the outlet is about 0.5 mm to about 10 mm in diameter.

[0010] In still yet another aspect, the present disclosure provides a method of fabricating a microchannel comprising a plurality of microwells, the method comprising fabricating the plurality of microwells using a CO2 laser cutter and a rastering speed of about 50% maximum laser speed to about 100% maximum laser speed at about 15% to about 35% power, wherein each microwell of the plurality of microwells has a diameter of about 10 pm to about 60 pm, and wherein there is about 60 pM to about 100 pM of space between each micro well of the plurality of microwells. In one embodiment, the microchannel comprises a cellulose acetate microwell substrate. The microchannel, in another embodiment, comprises at least about 10,000 or at least about 60,000 microwells.

[0011] In one aspect, the present disclosure provides a method of isolating a cell-cell complex, the method comprising: a) introducing a first population of cells into a microfluidic device, wherein at least one cell of the first population comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one cell of the first population to at least one microwell; c) introducing a sample comprising a second population of cells into the microfluidic device; and d) performing a wash step to remove cells of the second population that remain mobilized within the microchannel, wherein at least one cell of the first population and at least one of the second population form a cell-cell complex. In another aspect, the present disclosure provides a method of isolating a cell-cell complex, the method comprising: a) introducing a sample comprising a first population of cells and a second population of cells into a microfluidic device, wherein at least one cell of the first population comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of micro wells; b) applying a magnetic force to the microfluidic device to immobilize at least one cell of the first population to at least one micro well; and c) performing a wash step to remove cells of the second population that remain mobilized within the microchannel, wherein at least one cell of the first population and at least one cell of the second population form a cell-cell complex.

[0012] In another aspect, the present disclosure provides a method of isolating a tumor antigen specific T-cell, the method comprising: a) immobilizing at least one tumor cell of a population of tumor cells in a fluid chamber of a microfluidic device; b) introducing a sample comprisinga population of T-cells into the fluid chamber; c) generating an AC electrothermal flow inside the fluid chamber; and d) performing a wash step to remove T-cells of the population that remain mobilized within the fluid chamber, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex. In one embodiment, the method may further comprise performing an additional wash step to remove tumor cells of the population that remain mobilized within the fluid chamber prior to step (b). In another embodiment, a plurality of tumor cells of the population of tumor cells and a plurality of T-cells of the population of T-cells form a plurality of tumor cell / T-cell complexes. The microfluidic device, in yet another embodiment, comprises planar electrodes and generating the AC electrothermal flow comprises stimulating the electrodes at about 5 Vpp to about 100 Vpp or about 10 kHZ to about 1 MHz. In still yet another embodiment, the wash step or the additional wash step comprises washing the fluid chamber using a shear flow of about 0.05 dyn / cm2to about 1.0 dyn / cm2.

[0013] In yet another aspect, the present disclosure provides a microfluidic device comprising a) a sample reservoir; b) an outlet; c) a fluid chamber coupling the sample reservoir to the outlet, wherein the fluid chamber is configured to allow for immobilization of a plurality of cells of a first population of cells; and d) a pair of electrodes positioned within at least a portion of the fluid chamber, wherein the pair of electrodes are configured to produce an alternating current (AC) electrothermal flow of fluid in the fluid chamber when an alternating current signal is applied across the pair of electrodes, thereby facilitating formation of cell-cell complexes, each cell complex comprising an immobilized cell of the first population of cells and a cell of a second population of cells introduced into the fluid chamber after the plurality of cells of the first population of cells are immobilized in the fluid chamber. In one embodiment, the pair of electrodes are disposed on a surface of the fluid chamber. The fluid chamber, in another embodiment, is about 10 mm to about 50 mm in length, about 1.0 mm to about 20 mm in width, or about 0.5 mm to about 5.0 mm in height. In yet another embodiment, the sample reservoir is about 5 mm to about 20 mm in diameter. The outlet, in still yet another embodiment, is about 0.5 mm to about 10 mm in diameter.

[0014] In still yet another aspect, the present disclosure provides a method of isolating a cellcell complex, the method comprising: a) immobilizing at least one cell of a first population of cells in a fluid chamber of a microfluidic device; b) introducing a sample comprising a second population of cells into the fluid chamber; c) generating an AC electrothermal flow inside the fluid chamber; and d) performing a wash step to remove cells of the second population thatremain mobilized within the fluid chamber, wherein at least one cell of the first population and at least one cell of the second population form a cell-cell complex.

[0015] In one aspect, the present disclosure provides a method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a population of tumor cells into a microfluidic device, wherein at least one cell of the population comprises a magnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; c) introducing a sample comprising a population of T-cells into the microfluidic device; d) generating an AC electrothermal flow inside the microchannel; and e) performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0017] FIG. 1 shows a representative microfluidic device of the present disclosure. FIG. 1A shows a photograph of the microfluidic device. The inset shows an optical micrograph of the microwell array. FIG. IB shows an expanded view of the microfluidic device.

[0018] FIG. 2 shows characteristic properties of a representative microwell array of the present disclosure. FIG. 2A shows microwell diameter vs. laser rastering speed. Each data point represents the mean ± standard deviation (SD) of measurements from 30 distinct microwells. The inset images show optical micrographs of microwells fabricated at different rastering speeds. Scale bars, 50 pm. FIG. 2B shows the 3D profile of microwells fabricated at 25% power and 58% rastering speed.

[0019] FIG. 3 demonstrates OVA cell immobilization in microwells. FIG. 3A shows representative images of fluorescently stained LLC-OVA cells labeled with magnetic beads immobilized in microwells before exposure to flow at a rate of 0.5 mL / min. Scale bars, 200 pm. FIG. 3B shows representative images of fluorescently stained LLC-OVA cells labeledwith magnetic beads immobilized in microwells after exposure to flow at a rate of 0.5 mL / min. Scale bars, 200 pm. FIG. 3C shows the percentage of bead-LLC-OVA cell conjugates remaining in micro wells after exposure to fluid flow at different flow rates.

[0020] FIG. 4 demonstrates OT-I T-Cell-LLC-OVA interactions within a representative microfluidic device of the present disclosure. FIG. 4A shows representative images of fluorescently stained immobilized LLC-OVA tumor cells and OT-I T-cells before exposure to 0.5 mL / min of flow. Scale bars, 200 pm. FIG. 4B shows representative images of fluorescently stained immobilized LLC-OVA tumor cells and OT-I T-cells after exposure to 0.5 mL / min of flow. Scale bars, 200 pm.

[0021] FIG. 5 shows a photograph of a representative microfluidic device of the present disclosure. Scale bar, 6 mm.

[0022] FIG. 6 shows a representative fabrication process flow for a microfluidic device of the present disclosure.

[0023] FIG. 7 shows a schematic illustration of a representative microfluidic system of the present disclosure.

[0024] FIG. 8 shows schematic illustrations depicting immobilization of LLC tumor cells (FIG. 8A), incubation of OT-1 T cells (FIG. 8B), alternating current electric field (ACEF) enhancement (FIG. 8C), and post-incubation shear flow (FIG. 8D).

[0025] FIG. 9 shows Ansys Fluent simulation results of the temperature contour (FIG. 9A) and streamlines within the chamber (FIG. 9B) with ACEF (25 Vpp, 200 kHz).

[0026] FIG. 10 shows the fraction of LLC tumor cells immobilized in the fluid chamber following increasing shear flow. Each data point represents the mean + standard deviation (SD) of 3 independent measurements. Inset shows immobilized LLC tumor cells after shear flow. Scale bar, 100 pm.

[0027] FIG. 11 shows the fraction of OT-1 T cells attached to LLC tumor cells following shear flow. Each bar represents the mean ± SD of 3 independent measurements, p < 0.05 - two experimental groups show a statistically significant difference.DETAILED DESCRIPTION OF THE INVENTION

[0028] The present disclosure provides devices and methods for the rapid identification of high-affinity tumor specific T-cells that are compatible with both adherent and non-adherenttumors. The devices and methods of the present disclosure provide a significant advance in the art by minimizing the time required (less than about 1 hour) for the identification of high- affinity, tumor specific T-cells and maximizing the number of tumor cell / T-cell interactions that can be isolated within a microchannel. The process of immobilizing tumor cells, provided by the present disclosure, comprising the use of magnetic beads can be completed within 1 minute, with is significantly faster than allowing tumor cells to naturally adhere to the surface (Ashby et al., Adv Healthc Mater. , vol 11, 2022). As there are approximately 60,000 wells within the microchannel provided in one embodiment of the present disclosure, and a majority of those wells contain both tumor cells and T-cells, the devices and methods of the present disclosure allow for the high throughput identification of high-affinity T-cells. Additionally, the use of microwells allows weakly bound cells to be easily removed using fluid flow and the remaining T-cells to be positioned within close proximity of the tumor cells for a defined period of time. The T-cell / tumor cell complexes can also be easily removed from the channel by removing the magnetic force from microfluidic device, thus allowing them to be collected for further analysis.

[0029] The microwells of the microfluidic device of the present disclosure may, in some embodiments, be fabricated using a standard CO2 laser cutter, eliminating the need for cleanroom facilities and complex fabrication procedures. Furthermore, this fabrication method results in the production of microwells with uniform dimensions that can be tuned by altering the laser settings, making them adaptable for other cellular studies. The present disclosure demonstrates that magnetic bead-labeled tumor cells can be rapidly (~1 minute) immobilized into microwells using a magnet and remain immobilized even when exposed to fluid flow at flow rates up to 0.5 mL / min. Experiments performed using this device with LLC-OVA tumor cells and OT-I T-cell revealed its ability to remove weakly bound T-cells, thus allowing for the localized incubation of the two cell types to study their interaction. In addition to its speed and simplicity, the devices and methods of the present disclosure are compatible with both adherent and nonadherent tumor cells, making it a versatile platform for multiple applications, including T-cell / tumor cell interaction studies and T-cell screening.

[0030] The embodiments of the disclosure described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Instead, the embodiments selected for description have been chosen to enable one skilled in the art to practice the invention. It should be understood that the concepts presented herein may be used in various applications andshould not be limited to use in the specific embodiments depicted in the drawings or described herein.A. Microfluidic Devices

[0031] FIG. 1A shows a photograph of the microfluidic device 101 comprising a reservoir 102 in fluid communication with a microchannel 103 and an outlet 104. The microchannel 103 fluidically couples the reservoir 102 to the outlet. The microchannel 103 comprises a plurality of microwells 105. In the example of FIG. 1A, the microwells 105 are patterned in a bottom surface of the microchannel 103. FIG. IB shows an expanded view of a microfluidic device 101 assembled by attaching a microwell-patterned substrate 106 to a PMMA substrate 107 using double-sided adhesive tape 108. In certain embodiments, a microfluidic device of the present disclosure may further comprise a means for generating a magnetic force, such as a magnetic field source (e.g., a magnet, an electric conductor carrying a current, a coil, a solenoid, or a toroid). The means for generating a magnetic force may be in direct contact with the microfluidic device or may be external to the micro fluidic device but in sufficient proximity to the device to exert a magnetic force on the device. As described further below, the magnetic force may be applied to the microfluidic device, and more particularly to the microwells of the microfluidic device, to immobilize magnetic bead-labeled cells in the microwells.

[0032] In certain aspects, the present disclosure provides a microfluidic device comprising: a) a sample reservoir; b) an outlet; c) a microchannel in fluid communication with the sample reservoir and the outlet, the microchannel comprising a plurality of microwells, wherein each microwell of the plurality of microwells has a diameter of about 10 pm to about 60 pm, and wherein there is about 60 pM to about 100 pM of space between each microwell of the plurality of microwells; and d) a magnetic field source configured to apply a magnetic field to at least a portion of the plurality of microwells, wherein each micro well of the plurality of micro wells has a diameter of about 10 pm to about 60 pm, about 20 pm to about 60 pm, or about 30 pm to about 60 pm, including all ranges and values derivable therebetween, and wherein there is about 60 pm to about 100 pm of space between each microwell (center to center) of the plurality of microwells, including all ranges and values derivable therebetween. In certain embodiments, the microchannel may comprise a microwell substrate. The microwell substrate may be, in some embodiments, fabricated using any substrate compatible with CO2 laser etching. Non-limiting examples of such substrates include cellulose acetate, poly(methyl methacrylate) (PMMA), poly(ethylene terephthalate) (PET), poly (imides) (PI), poly(lactic acid), cellulose acetate propionate, cellulose films, polyamide (PA), poly vinylidene fluoride(PVDF), polyvinyl alcohol (PVA), polypropylene (PP), polysulfone (PSF), polyethersulfone (PES), polyethylene (PE), and glass. In some embodiments, the microwell substrate may overlay a second substrate. The second substrate may be any substrate compatible with microfluidic applications including, but not limited to, poly(methyl methacrylate) (PMMA), (poly)dimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyimide polyethylene (PE), SU-8 resin, cyclic olefin copolymer (COC), and polyethylene glycol diacrylate (PEGDA).

[0033] In certain embodiments, a microchannel of the present disclosure may comprise at least about 10,000, at least about 15,000, at least about 20,000, at least about 25,000, at least about 30,000, at least about 35,000, at least about 40,000, at least about 45,000, at least about 50,000, at least about 55,000, or at least about 60,000 microwells, including all ranges and values derivable therebetween.

[0034] In particular embodiments, the microchannel may be about 10 mm to about 20 mm, about 10 mm, about 11 mm, about 12 mm, about 13mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, or about 20 mm in length, including all ranges and values derivable therebetween. The microchannel of the present disclosure, in certain embodiments, may be about 1.0 mm to about 4.0 mm, about 1.0 mm, about 2.0 mm, about 3.0 mm, or about 4.0 mm in width, including all ranges and values derivable therebetween. In some embodiments, a microchannel of the present disclosure may be about 100 pm to about 500 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, or about 500 pm in height, including all ranges and values derivable therebetween. The microfluidic device of the present disclosure, in many embodiments, may comprise a sample reservoir that is about 5 mm to about 20 mm, about 5 mm, about 10 mm, about 15 mm, or about 20 mm in diameter, including all ranges and values derivable therebetween. In particular embodiments, the microfluidic device of the present disclosure may comprise an outlet that is about 0.5 mm to about 10 mm, about 0.5 mm, about 1.0 mm, about 2.0 mm, about 3.0 mm, about 4.0 mm, about 5.0 mm, about 6.0 mm, about 7.0 mm, about 8.0 mm, about 9.0 mm, or about 10.0 mm in diameter, including all ranges and values derivable therebetween.

[0035] In certain embodiments, a fluid chamber of the present disclosure may be about 10 mm to about 50 mm, about 15 mm to about 45 mm, about 20 mm to about 40 mm, about 25 mm to about 40 mm, about 30 mm to about 40 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm about inlength, including all ranges and values derivable therebetween. A fluid chamber of the present disclosure, in some embodiments, may be about 1.0 mm to about 20 mm, about 5.0 mm to about 15 mm, about 1.0 mm, about 2.0 mm, about 3.0 mm, about 4.0 mm, about 5.0 mm, about 6.0 mm, about 7.0 mm, about 8.0 mm, about 9.0, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, or about 20 mm in width, including all ranges and values derivable therebetween. In particular embodiments, a fluid chamber of the present disclosure may be about 0.5 mm to about 5.0 mm, about 0.5 mm to about 4.0 mm, about 1.0 mm to about 2.0 mm, about 1.2 mm to about 2.0 mm, about 1.4 mm to about 2.0 mm, about 1.6 mm to about 2.0 mm, or about 1.8 mm to about 2.0 mm in height, including all ranges and values derivable therebetween.

[0036] In order to allow for the immobilization of magnetically labeled cells in the microwells of a microfluidic device of the present disclosure, a means for generating a sufficiently large magnetic force and suitable magnetic labels must be available. Any means for generating a magnetic force known in the art may be used according to the devices and methods of the present disclosure. For example, a source of an electric fields may be used as a means for generating a magnetic force. Non-limiting examples of sources of electric fields include, a moving charge, an electric conductor carrying a current, a coil, a solenoid, a toroid, a magnet bar, a round magnet, or a horseshoe magnet. Extraction and purification of biomolecules is often achieved using functionalized magnetic beads. During extraction, the targeted biomolecules bind specifically to the surface of the beads through chemical moieties. After immobilizing the magnetic beads in the microwells with a magnetic force, undesirable biomolecules and fluids are removed, usually with a pipette or by-passing fluid flow through the microchannel. Optimal extractions are defined as ones with a maximum retention of desired biomolecules, and a maximum removal of biomolecules that remain mobilized.

[0037] In some embodiments of the present disclosure, tumor cells may be labeled using tumor antigen specific antibodies labeled with magnetic beads. Methods for labeling antibodies with magnetic beads are known in the art and any such method may be used according to the embodiments of the present disclosure. Tumor antigens that antibodies may directed may include any antigen expressed on the surface of a tumor cell. Such antigens are known in the art and any such antigen may be used according to the embodiments of the present disclosure. Non-limiting examples of such antigens include oncofetal antigens, oncoviral antigens, and overexpressed or accumulated antigens. Examples of such antigens include, but are not limited to, GD2 ganglioside, CA-125, CD51, EpCAM, vascular endothelial growth factor receptor 2(VEGFR2), carcinoembryonic antigen (CEA), mesothelin, epidermal growth factor receptor (EGFR), ERRB2 (HER2), tumor-associated glycoprotein 72 (TAG-72), gelatinase B, HLA- DR, activin receptor-like kinase 1, PDL-1, CD319, phosphotidylserine, CD22, B-cell maturation antigen (BCMA), fibroblast growth factor receptor 2 (FGFR2), ICAM-1, CD44, CD19, CD20, CD30, CD25, PD-1, endoglin, Lewis-Y antigen, R0R11, glypican 3, PTK7, TRAIL-R2, CD70, CD40, CD221, CD38, DLL4, PCDP1, ERRB3 (HER3), SLAMF7, CSF1R, HGFR, AXL, TWEAK Receptor, MUC5AC, CD3, integrin av 6, folate receptor 1, TYRP1, IL-3 receptor, CD80, IGF-1 receptor, MUC1, CA-IX, GPNMB, VEGR1, MCAM, SDC1, GUCY2C, CD152, CCR5, CD37, CD33, KIR2D, CD56, CD23, TRAIL-R1, CD74, GD3 ganglioside, CCR4, C242 antigen, PDGFRA, CD276, CD79B, vimentin, fibronectin extra domain B, LAG3, Notch receptor, and CD134.

[0038] FIG. 5 shows a photograph of another example microfluidic device 501 of this disclosure. The microfluidic device 501 comprises a sample well 502 in fluid communication with a chamber 503, which is fabricated by bonding a poly(methyl methacrylate) (PMMA) rectangular chamber (10-mm-wide x 35-mm-long x 1.9-mm-high) to a plasma-treated polystyrene substrate and covering the rectangular chamber with a sheet or layer of PMMA. The chamber 503 is also in fluid communication with an outlet 504, such that the chamber 503 fluidically couples the sample well 501 to the outlet 504. Planar electrodes are disposed on a bottom surface of the microfluidic device 501, such that the planar electrodes 505 are in electrical communication with fluid placed in or flowing through the microchannel 504. The sample well 502 (10-mm-diameter) is attached to the inlet of the chamber 503 and is used to contain cell solutions, which may be withdrawn through the chamber 503 via the outlet 504 using a syringe pump or another appropriate mechanism. The planar electrodes 505 (1 mm- wide) were screen-printed on the bottom surface of the microfluidic device 501 using Ag / AgCl ink. As explained further elsewhere in this disclosure, cells (e.g., tumor cells) can be immobilized on a surface of the chamber 503 (e.g., using magnetic force-assisted mobilization as described above with respect to FIGS. 1-4 or using another approach). After the cells are immobilized, cells from a different population (e.g., T-cells) may be introduced to the chamber 503. An AC electrical signal provided to the planar electrodes 505 induce AC electrothermal flow which speeds up the formation of cell-cell complexes between the immobilized cells and the newly introduced cells.

[0039] In some embodiments, planar electrodes may be screen-printed using any material known in the art to be useful for manufacturing electrodes. Non-limiting examples of suchmaterials include carbon, silver, gold, platinum, zinc, silver chloride, and dielectric. In certain embodiments, planar electrodes may be deposited onto the substrate via a metal evaporator or sputtering using metals that include but are not limited to chromium, titanium, gold, silver, and platinum.

[0040] FIG. 6 shows a representative fabrication process for a microfluidic device of the present disclosure. In certain embodiments, the rectangular chamber may be cut from 1 ,5-mm- thick poly(methyl methacrylate) (PMMA) using a CO2 laser cutter. The substrate, in some embodiments, may be laser-cut from 1-mm-thick polystyrene. In particular embodiments, Ag / AgCl electrodes may be screen-printed onto the substrate. The PMMA chamber, in certain embodiments, may be attached to the substrate using laser-cut double-sided adhesive. In some embodiments, the sample well (inlet) and O-ring (outlet) may be attached to the rectangular chamber by epoxy glue.

[0041] FIG. 7 shows an illustration of a representative microfluidic system of the present disclosure comprising a microfluidic device 701 comprising electrodes 702. The microfluidic device 701 is in fluid communication with a syringe pump 703. The electrodes 702 are in electrical connection with a function generator 704. In some embodiments, a device of the present disclosure may comprise a fluid chamber connected to a syringe pump using a 0.15- mm internal diameter tubing. Cell solutions may then be drawn through the fluid chamber. The electrodes, in certain embodiments, may be connected to the function generator using 0.2 mm copper electrical wires or using other appropriate electrical connections and contacts.

[0042] Any means for generating AC electrothermal flow known in the art may be used according to the devices and methods of the present disclosure. In particular, any means of applying an external electric field to a fluid thereby causing Joule heating and temperature gradients may be used according to the devices and methods of the present disclosure. For example, the means for applying the external electric field may include a function generator, such as the example function generator 704 of FIG. 7, and / or other electronic component(s), such as power supplies, signal generators, etc., in any suitable configuration for applying an AC electrical signal to the electrodes 702. In certain embodiments, an AC electric field may be applied to a conductive fluid within a frequency range of about 10 kHz to about 1 MHz, about 50 kHz to about 900 kHz, about 50 kHz to about 800 kHz, about 50 kHz to about 700 kHz, about 50 kHz to about 600 kHz, about 50 kHz to about 500 kHz, about 50 kHz to about 400 kHz, about 100 kHz to about 300 kHz, about 10 kHz, about 50 kHz, about 100 kHz, about 150 kHz, about 200 kHz, about 250 kHz, about 300 kHz, about 350 kHz, about 400 kHz, about450 kHz, about 500 kHz, about 550 kHz, about 600 kHz, about 650 kHz, about 700 kHz, about 750 kHz, about 800 kHz, about 850 kHz, about 900 kHz, about 950 kHz, or about 1 MHz, including all ranges and variable derivable therebetween. In certain embodiments the AC electric field may be applied at a voltage peak-to-peak (Vpp) of about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 20 to about 30, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100, including all ranges and variable derivable therebetween.B. Methods for Rapid Identification of High Affinity, Tumor- Specific T-cells

[0043] In certain aspects, the present disclosure provides a method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a population of tumor cells into a microfluidic device, wherein at least one cell of the population comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; c) introducing a sample comprising a population of T-cells into the microfluidic device; and d) performing a wash step to remove T- cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex. In certain embodiments, the method may further comprise performing an additional wash step to remove tumor cells of the population that remain mobilized within the microchannel prior to step (c) above. In particular aspects, the present disclosure provides a method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a sample comprising a population of tumor cells and a population of T-cells into a microfluidic device, wherein at least one cell of the population of tumor cells comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one micro well; and c) performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.

[0044] In some embodiments, the method may further comprise collecting the tumor cell / T- cell complex. Following collection of the tumor cell / T-cell complex, high affinity, tumor-specific T-cells may be isolated, analyzed, and / or expanded for use in cancer immunotherapy. In certain embodiments, the T-cell receptor sequences may be cloned and then introduced into a cell to produce a high affinity, tumor specific engineered T-cell or engineered T-cell population for cancer immunotherapy.

[0045] In particular, embodiments, a plurality of tumor cells of the population comprise the magnetic label. The applying of the magnetic force, in certain embodiments, to the microfluidic device immobilizes a plurality of tumor cells of the population to a plurality of microwells. In some embodiments, wherein a plurality of tumor cells of the population of tumor cells and a plurality of T-cells of the population of T-cells form a plurality of tumor cell / T-cell complexes. In particular embodiments, the time period required to complete the method is less than about 1 hour. In some embodiments, a first wash step or a second wash step comprises washing the microchannel at a flow rate of about 0.01 mL / min to about 0.5 mL / min, about 0.01 mL / min, about 0.05 mL / min, about 0.1 mL / min, about 0.2 mL / min, about 0.3 mL / min, about 0.4 mL / min, or about 0.5 mL / min or higher, including all ranges and values derivable therebetween. In certain embodiments, introducing the sample comprising the population of T-cells comprises introducing the sample at a flow rate of about 0.5 pL / min to about 5.0 pL / min, about 0.5 pL / min to about 4.0 pL / min, about 0.5 pL / min to about 3.0 pL / min, or about 0.5 pL / min to about 2.0 pL / min, including all ranges and values derivable therebetween. A first was step or a second wash step, in certain embodiments, may comprise washing using a shear flow of about 0.05 dyn / cm2to about 1.0 dyn / cm2, about 0.10 dyn / cm2to about 0.90 dyn / cm2, about .10 dyn / cm2to about 0.80 dyn / cm2, about 0.10 dyn / cm2to about 0.70 dyn / cm2, about 0.10 dyn / cm2to about 0.60 dyn / cm2, about 0.10 dyn / cm2to about 0.50 dyn / cm2, about 0.20 dyn / cm2to about 0.40 dyn / cm2, about 0.20 dyn / cm2to about 0.30 dyn / cm2, about 0.30 dyn / cm2to about 0.60 dyn / cm2, about 0.40 dyn / cm2to about 0.60 dyn / cm2, about 0.10 dyn / cm2, about 0.15 dyn / cm2, about 0.20 dyn / cm2, about 0.25 dyn / cm2, about 0.30 dyn / cm2, about 0.35 dyn / cm2, about 0.40 dyn / cm2, about 0.45 dyn / cm2, about 0.50 dyn / cm2, about 0.55 dyn / cm2, about 0.60 dyn / cm2, about 0.65 dyn / cm2, about 0.70 dyn / cm2, about 0.75 dyn / cm2, about 0.80 dyn / cm2, about 0.85 dyn / cm2, about 0.90 dyn / cm2, about 0.95 dyn / cm2, or about 1.0 dyn / cm2, including all ranges and values derivable therebetween.

[0046] The population of tumor cells used according to the methods of the present disclosure may be a population of tumor cells derived from any cancer type, including tumor cells that are adherent and tumor cells non-adherent. Non-limiting examples of such cancers include lung cancer, non-small cell lung cancer, colorectal cancer, pancreatic cancer, appendicealcancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological malignancy, hematopoietic cancer, breast cancer, bladder cancer, prostate cancer, and skin cancer. The population of tumor cells, in one embodiment, is a population of human tumor cells. The population of T-cells, in another embodiment, is a population of human T-cells. In some embodiments, the population of tumor cells and the sample comprising the population of T- cells are derived from the same subject. In one embodiment, the subject is a human subject. In yet another embodiment, the population of tumor cells and the sample comprising the population of T-cell are derived from different subjects.

[0047] A person of ordinary skill in the art will understand that the methods and microfluidic devices of the present disclosure may be adapted to isolate any cell-cell complex or to analyze any cell-cell interaction. In some aspects, the present disclosure provides a method of isolating a cell-cell complex, the method comprising: a) introducing a first population of cells into a microfluidic device, wherein at least one cell of the population comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one cell of the first population to at least one microwell; c) introducing a sample comprising a second population of cells into the microfluidic device; and d) performing a wash step to remove cells of the second population that remain mobilized within the microchannel, wherein at least one cell of the first population and at least one of the second population form a cellcell complex. In particular aspects, the present disclosure provides a method of isolating a cellcell complex, the method comprising: a) introducing a sample comprising first population of cells and a second population of cells into a microfluidic device, wherein at least one cell of the first population comprises a magnetic label, and wherein the microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one cell of the first population to at least one microwell; and c) performing a wash step to remove cells of the second population that remain mobilized within the microchannel, wherein at least one cell of the first population and at least one cell of the second population form a cell-cell complex.

[0048] In some embodiments, isolation of a cell-cell complex is further improved by combining magnetic force-assisted cell immobilization with AC electrothermal flow-assisted complex formation. In some aspects, the present disclosure provides a method of isolating a tumor antigen specific T-cell. The method comprises a) introducing a population of tumor cells into a microfluidic device, wherein at least one cell of the population comprises amagnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; c) introducing a sample comprising a population of T-cells into the microfluidic device; and d) generating an AC electrothermal flow inside the microchannel; and performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.C. Therapeutic Compositions and Methods

[0049] Following collection of the tumor cell / T-cell complex utilizing the methods of the present disclosure, high affinity, tumor- specific T-cells may be isolated, analyzed, and / or expanded for use in cancer immunotherapy. In certain embodiments, the T-cell receptor sequences may be cloned and then introduced into a cell to produce a high affinity, tumor specific engineered T-cell or engineered T-cell population for cancer immunotherapy.

[0050] Neoantigen targeting has shown great promise for inducing antitumor immune responses in cancer patients. Cytotoxic T-lymphocytes that recognize antigenic peptides derived from mutated proteins presented at the tumor cell surface by HLA class I molecules have been shown to cause regression of large tumors. Furthermore, T-cell receptors derived from such antigen-specific T-cells can be isolated, cloned, and utilized to create engineered T- cells. However, as a natural consequence of high HLA diversity and the specific nature of most tumor-associated mutations, very few neoantigen targets are shared among patients.

[0051] Adoptive T-cell therapy (ACT) refers to the practice of administering tumor-specific T-cells to a patient with cancer. The goal of adoptive T-cell therapy is that the transferred cells will recognize and kill the cancer cells without causing significant damage to healthy tissues. Adoptive T-cell therapy may utilize, for example, T-cells isolated from the patient or a donor and expanded in the laboratory prior to transfer. Adoptive T-cell therapy may also utilize genetically modified T-cells that express, for example, chimeric antigen receptors (CARs) or T-cell receptors (TCRs). Adoptive T-cell therapy using CARs and TCRs has demonstrated impressive results in several clinical trials. The efficacy of adoptive T-cell therapy is determined, at least in part, by the TCRs’ interaction with peptide-major histocompatibility complexes (pMHCs), which comprise a peptide bound to an MHC molecule.

[0052] TCRs for use in adoptive T-cell therapy must be matched to a patient’s human leukocyte antigen (HLA) allele. The human class I MHC protein and the human class II MHC protein are each encoded by 3 gene regions. Human class I MHC is encoded by HLA- A, HLA- B, and HLA-C, and human class II MHC is encoded by HLA-DR, HLA-DP, and HLA-DQ. There is a continuing need in the art for the identification of novel cancer specific TCRs for use in cancer treatment.

[0053] T-cell receptors comprise two different polypeptide chains, termed the T-cell receptor a (TCRa) and p (TCR ) chains, linked by a disulfide bond. Each TCRa and TCRP chain comprises a variable antigen binding region, and the structure of these chains is similar to that of a Fab fragment of an immunoglobulin molecule. The TCRa and TCRP chains are responsible for the antigen recognition demonstrated by most T-cells, although a minority of T-cells instead comprise an alternative but structurally similar TCR, which comprises a pair of polypeptide chains designated TCRy and TCRS. TCR and TCRyS receptors differ from membrane-bound immunoglobulins that serve as B-cell receptors in the following ways: 1) TCRs have only one antigen binding site, whereas immunoglobulins have two; and 2) TCRs are never secreted, whereas immunoglobulins may be secreted as an antibody.

[0054] Both chains of a TCR comprise an amino-terminal variable (V) region, a constant (C) region, and a short hinge region containing a cysteine residue that forms the interchain disulfide bond. The TCR V region shares homology with the immunoglobulin V domain, and the TCR C region shares homology with the immunoglobulin C domain. Each TCR chain comprises a hydrophobic transmembrane domain that spans the lipid bilayer and ends in a short cytoplasmic tail. In some embodiments, a TCR of the present disclosure may be a single chain TCR. Methods for producing single chain TCRs are known in the art and any such method may be used according to the embodiments of the present disclosure. Non-limiting examples of single chain TCRs are described in Knies, et al. , Oncotarget 7(16): 21199-21221 and U.S. 10,538,573.

[0055] The three-dimensional structure of the TCR has been determined. The TCR chains fold in a manner similar to a Fab fragment, although the final 3D structure appears a little shorter and wider. There are, however, some distinct differences between TCRs and Fab fragments. The most striking difference is in the Ca domain, which folds differently than any other immunoglobulin-like domain. The half of the domain that is juxtaposed with the CP domain forms a P sheet similar to that found in other immunoglobulin-like domains, but the other half of the domain is formed of loosely packed strands and a short segment of a helix. Theintramolecular disulfide bond of the Cot domain joins a P strand to this segment of a helix, however, in most immunoglobulin-like domains the intramolecular disulfide bond joins two strands.

[0056] There are also differences in domain interaction between TCRs and immunoglobulins. The interface between the V and C domains of both TCR chains is more extensive than that demonstrated by antibodies, which may decrease the flexibility of the hinge region between the TCR domains. Furthermore, the interaction between the Ca and CP domains is distinct in that it is assisted by carbohydrate, with a sugar group from the Ca domain making a number of hydrogen bonds to the CP domain. Finally, a comparison of the variable binding sites shows that, although the complementarity-determining region (CDR) loops align fairly closely with those of antibody molecules, there is some displacement. This displacement is particularly prevalent in the V a CDR2 loop, which is oriented at roughly a right angle compared to the equivalent loop of an antibody V domain. This displacement is a result of a shift in the P strand that anchors one end of the loop from one face of the domain to the other. A strand displacement also causes a change in the orientation of the VP CDR2 loop in two of the seven VP domains for which structures are known.

[0057] In some embodiments, a tumor cell or T-cell population of the present disclosure may be combined with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” or “adjuvant” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the tumor cell or T-cell populations of the present disclosure, or other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or pharmaceutical compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an agent may be added to a therapeutic composition or pharmaceutical composition to modify for example the cellular target, cellular localization, or cellular uptake of a tumor specific T-cell as described herein. Such an agent may include any excipient, diluent, carrier, or adjuvant that is acceptable for pharmaceutical use. Such an agent may be non-naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the therapeutic or pharmaceutical composition.

[0058] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a composition comprising a T-cell or T-cell population of the present disclosure. In some embodiments, a therapeutic composition has the activity of specifically recognizing and binding to a tumor cell in a subject as described herein. In one embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor growth or tumor progression in a subject. In another embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor size in a subject.

[0059] A compound or composition of the present disclosure is meant to encompass a composition suitable for administration to a subject, such as a mammal, particularly a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the composition are pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, intraarticular, intraarterial, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalation, vaginal, intraosseous, trans nasal, injection, microneedle, topical, and transdermal. The appropriate dosage of a composition, as described herein, may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, clinical history, response to the treatment, and the discretion of the attending physician. In some embodiments, therapeutic compositions provided by the present disclosure may include various "unit doses." A unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose. In one embodiment, an effective amount of T-cell or T-cell population as described herein may be represented as a daily effective amount, a twice daily effective amount, a bi-daily effective amount, a weekly effective amount, a monthly effective amount, or a bi-monthly daily effective amount.

[0060] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment(alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0061] As used herein, “subject” or “patient” refers to animals, including humans, from which populations of tumor cells or populations of T-cells are derived, or who are treated with the therapeutic compounds or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs and the like. In particular embodiments, a subject in need of therapy may be any subject who is afflicted with or at risk of developing cancer.

[0062] A composition, as described herein, may include, in particular embodiments, a combination of therapeutic agents. In some embodiments, a composition as described here may be administered as a single composition or as more than one composition. Different compositions as provided herein, in certain embodiments, may be administered by the same route of administration or by different routes of administration.

[0063] A peptide or polynucleotide molecule for use according to the compositions of the present disclosure may, in some embodiments, be a recombinant peptide or nucleic acid. As used herein, the term “recombinant” refers to a polynucleotide molecule, protein, or cell that is not naturally present, or is not naturally present in the same form or structure and was created by human intervention. In one embodiment, a recombinant polynucleotide may be a DNA molecule or may be an RNA molecule. A recombinant polynucleotide molecule or a recombinant polypeptide molecule or protein may comprise, in certain embodiments, a combination of two or more polynucleotide or polypeptide sequences that do not naturally occur together in the same manner, such as a polynucleotide molecule or protein that comprises at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other. As used herein the term “heterologous” refers to a polynucleotide molecule or protein that is not naturally present or is not naturally present in the same form or structure and was created by human intervention. For example, a heterologous polynucleotide molecule or protein may not naturally occur in the cell being transformed or may be expressed in a manner or genomic context that differs from the natural expression pattern or genomic context found in the cell being transformed. The heterologous polynucleotide molecule or protein, in some embodiments, may be overexpressed in the cell being transformed. In certain embodiments, a recombinant polynucleotide molecule, protein, construct, or vector may comprise any combination of two or more polynucleotide or protein sequences in the samemolecule that are heterologous to one another, such that the combination is man-made and not normally found in nature. As used herein, the phrase “not normally found in nature” means not found in nature without human intervention. A recombinant polynucleotide or protein molecule, may comprise, for example, polynucleotide or protein sequences that are separated from other polynucleotide or protein sequences that exist in proximity to each other in nature. A recombinant polynucleotide or protein molecule may also comprise, for example, polynucleotide or protein sequences that are adjacent to or contiguous with other polynucleotide or protein sequences that are not naturally in proximity with each other. Such a recombinant polynucleotide molecule, protein, or expression construct may also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered or constructed outside of a cell. For example, a recombinant polynucleotide molecule may comprise any engineered or man-made plasmid, vector, or expression construct, and may include a linear or circular DNA molecule. Such plasmids, vectors, and expression constructs may comprise, for example, various maintenance elements including, but not limited to, a heterologous promoter sequence, a prokaryotic origin of replication, or a selectable marker.

[0064] In certain embodiments, the compositions and methods for treating an individual described herein may be combined with any other composition or method of treatment known in the art. The compositions and methods may be administered in any suitable manner known in the art. For example, a first and a second therapeutic agent or inhibitor may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, a first and a therapeutic agent or inhibitor may be administered in separate compositions. In certain embodiments, a first and a second cancer treatment or inhibitor may be administered in the same composition.

[0065] Non-limiting examples of additional treatment modalities that may be included in combination with the compositions and methods provided herein include a therapeutic agent or surgery. In specific embodiments, the methods and compositions of the present disclosure may be combined with other therapies directed towards the treatment of cancer as described herein.D. Methods of Producing a Microwell Array

[0066] In certain aspects, the present disclosure provides methods of fabricating a microchannel comprising a plurality of microwells, the method comprising fabricating the plurality of micro wells using a CO2 laser cutter and a rastering speed of about 1% maximumlaser speed to about 100% maximum laser speed, including all ranges and values derivable therebetween, at about 1% to about 100% power, including all ranges and values derivable therebetween, wherein each microwell of the plurality of microwells has a diameter of about 10 pm to about 60 pm, including all ranges and values derivable therebetween, and wherein there is about 60 pm to about 100 pm of space between each microwell of the plurality of micro wells (center to center), including all ranges and values derivable therebetween. A person of ordinary skill in the art will appreciate that the rastering speed and power may be chosen based on the material used to create the plurality of microwells having a diameter of about 10 pm to about 60 pm. In some embodiments, the material chosen to create the plurality of microwells is PMMA or a cellulose acetate and the rastering speed is about 50% maximum laser speed to about 100% maximum laser speed, including all ranges and values derivable therebetween and the power is about 15% to about 35% power, including all ranges and values derivable therebetween. In certain embodiments, the microwell substrate may be fabricated using any substrate compatible with CO2 laser etching. Non-limiting examples of such substrates include cellulose acetate, poly(methyl methacrylate) (PMMA), polyethylene terephthalate) (PET), poly(imides) (PI), poly(lactic acid), cellulose acetate propionate, cellulose films, polyamide (PA), polyvinylidene fluoride (PVDF), polyvinyl alcohol (PVA), polypropylene (PP), polysulfone (PSF), polyethersulfone (PES), polyethylene (PE), and glass. In some embodiments, the microwell substrate may overlay a second substrate. The second substrate may be any substrate compatible with microfluidic applications including, but not limited to, poly(methyl methacrylate) (PMMA), (poly)dimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyimide polyethylene (PE), SU-8 resin, cyclic olefin copolymer (COC), and polyethylene glycol diacrylate (PEGDA).

[0067] The term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word "comprising" or other open language in the claims, the words "a" and "an" denote "one or more," unless specifically noted otherwise. The terms "comprise," "have," and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.Similarly, any system or method that "comprises," "has," or "includes" one or more components is not limited to possessing only those components and covers other unlisted components.

[0068] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0069] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0070] The following examples are included to illustrate embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents or components that are chemically, physiologically, and / or structurally related may be substituted for the agents or components described herein and the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.Example 1: Materials and Methods.

[0071] Design and Fabrication of the Microfluidic Device. A microfluidic device provided by the present disclosure, in certain embodiments, comprises of a PMMA microchannel (length = 15 mm, width = 2.5 mm, height = 150 pm) containing a dense array of circular microwells connected to a 10-mm diameter sample reservoir and outlet (FIG. 1A), which are fabricated using a CO2 laser cutter (Universal Laser Systems). Microwells are formed via laser etching through a stainless-steel mesh (opening size = 43 pm, McMaster Carr) placed over a 100 pm- thick cellulose acetate substrate. The device is assembled by attaching themicrowell-patterned substrate to the PMMA substrate using double-sided adhesive tape (FIG. IB). Imaging and device characterization were performed using a Keyence VHX-7000 digital microscope. A microfluidic device of the present disclosure, in certain embodiments, may comprise a fluid chamber fabricated by bonding a poly(methyl methacrylate) (PMMA) rectangular chamber (10-mm-wide x 35-mm-long x 1.9-mm-high) to a plasma-treated polystyrene substrate, a 10-mm-diameter well attached to the inlet of the chamber, used to contain cell solutions, which are withdrawn through the fluid chamber through the outlet using a syringe pump (FIG. 5). In particular embodiments, planar electrodes (1 mm-wide) may be screen-printed on the bottom of the fluid chamber using Ag / AgCl ink (FIG. 5).

[0072] Cell Staining. To facilitate visualization of the cells, LLC-OVA tumor cells and OT- I CD8+T-cells were stained with CFSE (Invitrogen) and CMTPX (Invitrogen) fluorescent stains, respectively. The cells were first washed twice with phosphate buffered saline (PBS). For every million cells in the suspension, 100 pL of 5 pM dye was added and the cells were incubated in the dark for 15 minutes. Following incubation, the dye was quenched with cold, complete media and the stained cells were washed twice before resuspension in media.

[0073] Magnetic Bead Labeling. LLC-OVA cells, from a murine lung cancer cell line, were used for T-cell interaction studies. The cells were tagged with 2.8 pm magnetic beads (Dynabeads, ThermoFisher Scientific). Prior to cell tagging, magnetic beads were prepared by incubating 250 pg of carboxylated beads with 50 pL of 10 mg / mL l-Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) / N-Hydroxy succinimide (NHS) in 2-(N- morpholino) ethanesulfonic acid (MES) buffer (pH=5.5) for 1 hour, washed, and then incubated with 7.5 pg of anti-H2kB antibody (Biolegend) for 15 hours. The beads were then washed and incubated in 3% bovine serum albumin (BSA) for 1 hour to block the remaining surface and stored in 100 pL of 0.1M PBS at 4°C until use. LLC-OVA cells were labeled with beads by adding 50 pL of the anti-H2kB IgG-conjugated beads to 1 million cells and incubating on a tilt shaker for 30 minutes. The beads were then washed to remove unbound cells and the bead-tagged LLC-OVA cells were resuspended in 100 pL of complete RPMI- 1640 media.

[0074] Immobilization of Tumor Cells in Microwells. 15 pL of the bead-LLC-OVA cell conjugates were dispensed into the microchannel using a micropipette through the channel outlet. The bead-cell conjugates were allowed to settle into the wells for 1 minute, after which the microchannel was placed on a neodymium magnet (McMaster Carr), causing the LLC- OVA cells to be magnetically immobilized in the microwells. A syringe pump (KD Scientific)was connected to the outlet of the channel using 1 mm-diameter tubing and was used to withdraw RPMI-1640 media from the sample reservoir, thus causing it to flow through the microchannel. A flow rate of 0.01 mL / min was used to wash away weakly bound bead-cell conjugates or unlabeled LLC-OVA cells. The flow rate was subsequently increased to expose the immobilized bead-cell conjugates to increasing amounts of shear stress.

[0075] T-Cell Interaction Studies. 15 pL of murine OT-I T-cells in suspension (10 million cell / mL) were dispensed into the sample reservoir and withdrawn through the channel at a flow rate of 1 pL / min using a syringe pump. When the channel was filled, the flow was stopped to allow the T-cells to settle to the bottom of the channel and interact with the immobilized tumor cells. After 30 minutes, the sample reservoir was filled with RPMI-1640 media, which was withdrawn through the channel to apply shear stress to the T-cells. Cells were imaged using a Zeiss Axiolmager M2 microscope.Example 2: Characterization of Micro wells

[0076] Using a laser etching technique, a dense microwell array was rapidly fabricated (< 30 sec / cm2) in thin (100 pm) cellulose acetate sheets with high reproducibility (< 10% variability in well dimensions) across an area of ~ 10 cm2. Microwell dimensions were linearly correlated with the laser rastering speed, enabling the fabrication of wells with diameters ranging from 30 pm (100% laser speed) to 60 pm (60% laser speed) at 25% power (FIG. 2A). Higher laser power or slower laser rastering speed produced larger and deeper microwells compared to lower laser power or faster rastering speeds. The microwells were uniformly spaced ~80 pm apart with a consistent well depth across the rastered array (FIG. 2B).

[0077] This method allows for the rapid fabrication of microwell arrays using a standard CO2 laser cutter, broadening its accessibility to researchers and scientists without access to cleanroom facilities or experience with microfabrication. Furthermore, the fabrication parameters, including the laser rastering speed / power and dimensions of the stainless-steel mesh, can be varied to produce microwells with different diameters and depths for other applications. The cost of fabricating microwells using this approach is lower compared with microfabrication-based methods, as the mesh is not damaged during the microwell fabrication process and can be reused indefinitely.Example 3: LLC-OVA Cell Immobilization in Microwells

[0078] Microwells for T-cell interaction studies were fabricated with 25% laser power and 58% laser rastering speed, resulting in microwells with a diameter of ~60 pm and depth of ~50 pm. A larger well diameter was used, as this results in less space between wells, and increased the likelihood that bead-cell conjugates will settle into the micro wells rather than in the spaces between wells. Additionally, it was observed that using deeper wells allowed the bead-cell conjugates to remain in the wells in the presence of shear flow while significantly minimizing the occurrence of clumping of bead-cell conjugates in the presence of large paramagnetic forces. Bead-cell conjugates settled into > 90% of the wells, as indicated by the cells presenting in a grid-like pattern (FIG. 3A).

[0079] The effectiveness of this magnetic bead-based cell immobilization method was evaluated by generating fluid flow in the channel with flow rates from 0.01 mL / min to 0.5 mL / min. At the lowest flow rate (0.01 mL / min), bead-cell conjugates that were not immobilized within microwells and unlabeled LLC-OVA cells were readily washed away. As the flow rate within the channel was increased, it was observed that bead-cell conjugates remained stationary within the wells and a negligible number of bead-cell conjugates became detached and washed away. The number of bead-cell conjugates remaining in the channel after flow at different flow rates was quantified. Using a one-way ANOVA with a Tukey’s post hoc comparison, it was found that there was no significant difference in the number of bead-cell conjugates that remained in the microchannel before fluid flow and after exposure to flow at flow rates of 0.25 mL / min and 0.5 mL / min (FIG. 3B, FIG. 3C). These results indicate that tumor cells can be rapidly (1 minute) and effectively immobilized within microwells using magnetic beads and an external magnet, even when exposed to flow rates up to 0.5 mL / min.Example 4: OT-I T-Cell-LLC-OVA Interactions - Magnetic Force

[0080] OT-I T-cells were selected as a proof-of-concept cell line to study interactions with LLC-OVA cells. Using the microfluidic device shown in FIG. 1A with immobilized bead- LLC-0 V A cells in micro wells, OT-I T-cells were pumped into the channel at a flow rate of 1 pL / min. After 10 pL of the cell suspension was introduced into the channel, the flow was stopped to allow the T-cells to settle to the bottom of the channel. Similar to the tumor cells, the T-cells quickly settled into the microwells within 1 minute. Using a 1: 1 ratio of LLC-21OVA cells-to-OT-I T-cells, -85% of the wells were filled with T-cells, which presented in a grid-like pattern similar to the LLC-OVA cells (FIG. 4A).

[0081] After allowing the cells to incubate for 30 minutes, flow was initiated to remove weakly bound T-cells. At a low flow rate (0.01 mL / min), T-cells residing outside of the wells were washed away. As the flow rate was increased to 0.5 mL / min, the majority of the T-cells remained in the micro wells (FIG. 4B), demonstrating that they were bound to the immobilized LLC-OVA tumor cells.Example 5: OT-I T-Cell-LLC-OVA Interactions - AC Electrothermal Flow

[0082] The device shown in FIG. 5 was used to isolate SIINFEKL-specific CD8+(OT-1) T- cells, which target ovalbumin (OVA) expressing cell lines, including Lewis Lung Carcinoma (LLC) tumor cells. LLC cells (~700k cells) were immobilized in the fluid chamber by seeding the cells into the chamber and allowing them to attach overnight, a shear flow of 0.25 dyn / cm2was applied to remove unbounded cells (FIG. 8A). Next, OT-1 T-cells (500k cells / mL) were introduced into the chamber and co-incubated with the immobilized LLC cells for 30 min (FIG. 8B). ACEF was generated inside the chamber by stimulating the electrodes with an AC signal (25 Vpp, 200 kHz) for 1.5 min, followed by 8.5 min of static incubation (FIG. 8C), which was repeated 3 times. After ACEF-enhanced incubation, a shear flow of 0.50 dyn / cm2was applied to remove weakly-bound cells (FIG. 8D).

[0083] Ansys Fluent numerical simulations were performed, which showed that a -6.2 K / mm temperature gradient was generated by AC electric field-induced Joule heating, resulting in the generation of swirling microflows in the chamber (FIG. 9). Experiments to characterize the adhesion strength of immobilized LLC tumor cells revealed that -100% and 80% of LLC cells remained attached following the application of shear flows at 1 dyn / cm2and 2.5 dyn / cm2, respectively (FIG. 10). Lastly, the effectiveness of ACEF in isolating OT-1 T-cells was compared to cell isolation without ACEF. A 2.2-fold improvement in the yield of OT-1 T- cells was observed by employing ACEF (FIG. 11). The present disclosure demonstrates the effectiveness of ACEF in enhancing the isolation of anti-tumor T-cells for the first time, which can facilitate the screening of potent anti-tumor T-cells leading to more effective immunotherapies .

Claims

CLAIMS1. A method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a population of tumor cells into a microfluidic device, wherein at least one cell of the population comprises a magnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of micro wells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; c) introducing a sample comprising a population of T-cells into the microfluidic device; and d) performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.

2. The method of claim 1, the method further comprising performing an additional wash step to remove tumor cells of the population that remain mobilized within the microchannel prior to said step (c).

3. The method of claim 1, wherein each microwell of the plurality of micro wells has a diameter of about 10 pm to about 60 pm, or wherein there is about 60 pm to about 100 pm of space between each microwell of the plurality of micro wells.

4. The method of claim 1, wherein the microchannel comprises at least about 10,000 microwells or at least about 60,000 microwells.

5. The method of claim 1, further comprising collecting the tumor cell / T-cell complex.

6. The method of claim 1 , wherein a plurality of tumor cells of the population comprise the magnetic label.

7. The method of claim 1, wherein applying the magnetic force to the microfluidic device immobilizes a plurality of tumor cells of the population to a plurality of microwells.

8. The method of claim 1, wherein a plurality of tumor cells of the population of tumor cells and a plurality of T-cells of the population of T-cells form a plurality of tumor cell / T-cell complexes.

9. The method of claim 1, wherein a time period required to complete said method is less than about 1 hour.

10. The method of claim 2, wherein the wash step or the additional wash step comprises washing the microchannel at a flow rate of about 0.01 mL / min to about 0.5 mL / min.

11. The method of claim 1 , wherein said introducing the sample comprising the population of T-cells comprises introducing the sample at a flow rate of about 0.5 pL / min to about 5.0 pL / min.

12. The method of claim 1, wherein the population of tumor cells is a population of tumor cells derived from a lung cancer, a non-small cell lung cancer, a colorectal cancer, a pancreatic cancer, an appendiceal cancer, a small bowel adenocarcinoma, a hepatobiliary cancer, a gynecological malignancy, a hematopoietic cancer, abreast cancer, a bladder cancer, a prostate cancer, or a skin cancer.

13. The method of claim 1, wherein the population of tumor cells is a population of human tumor cells.

14. The method of claim 1, wherein the population of T-cells is a population of human T- cells.

15. The method of claim 1, wherein the population of tumor cells and the sample comprising the population of T-cells are derived from the same subject.

16. The method of claim 15, wherein the subject is a human subject.

17. The method of claim 1, wherein the population of tumor cells and the sample comprising the population of T-cell are derived from different subjects.

18. A microfluidic device comprising: a) a sample reservoir; b) an outlet; c) a microchannel in fluid communication with the sample reservoir and the outlet, the microchannel comprising a plurality of microwells, wherein each microwell of the plurality of microwells has a diameter of about 10 pm to about 60 pm, and whereinthere is about 60 p M to about 100 p M of space between each microwell of the plurality of micro wells; and d) a magnetic field source configured to apply a magnetic field to at least a portion of the plurality of micro wells.

19. The microfluidic device of claim 18, wherein said microchannel comprises a cellulose acetate microwell substrate.

20. The microfluidic device of claim 18, wherein the microchannel comprises at least about 10,000 or at least about 60,000 microwells.

21. The microfluidic device of claim 18, wherein the microchannel is about 10 mm to about 20 mm in length, about 1.0 mm to about 4.0 mm in width, or about 100 pm to about 500 pm in height.

22. The microfluidic device of claim 18, wherein the sample reservoir is about 5 mm to about 20 mm in diameter.

23. The microfluidic device of claim 18, wherein the outlet is about 0.5 mm to about 10 mm in diameter.

24. A method of fabricating a microchannel comprising a plurality of microwells, the method comprising fabricating the plurality of microwells using a CO2 laser cutter and a rastering speed of about 50% maximum laser speed to about 100% maximum laser speed at about 15% to about 35% power, wherein each microwell of the plurality of microwells has a diameter of about 10 pm to about 60 pm, and wherein there is about 60 pM to about 100 pM of space between each microwell of the plurality of microwells.

25. The method of claim 24, wherein said microchannel comprises a cellulose acetate microwell substrate.

26. The method of claim 24, wherein the microchannel comprises at least about 10,000 or at least about 60,000 microwells.

27. A method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a sample comprising a population of tumor cells and a population of T-cells into a microfluidic device, wherein at least one cell of the population of tumor cells comprises a magnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of micro wells;b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; and c) performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.

28. A method of isolating a cell-cell complex, the method comprising: a) introducing a first population of cells into a microfluidic device, wherein at least one cell of the first population comprises a magnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of micro wells; b) applying a magnetic force to the microfluidic device to immobilize at least one cell of the first population to at least one microwell; c) introducing a sample comprising a second population of cells into the microfluidic device; and d) performing a wash step to remove cells of the second population that remain mobilized within the microchannel, wherein at least one cell of the first population and at least one of the second population form a cell-cell complex.

29. A method of isolating a cell-cell complex, the method comprising: a) introducing a sample comprising a first population of cells and a second population of cells into a microfluidic device, wherein at least one cell of the first population comprises a magnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of microwells; b) applying a magnetic force to the microfluidic device to immobilize at least one cell of the first population to at least one microwell; and c) performing a wash step to remove cells of the second population that remain mobilized within the microchannel, wherein at least one cell of the first population and at least one cell of the second population form a cell-cell complex.

30. A method of isolating a tumor antigen specific T-cell, the method comprising: a) immobilizing at least one tumor cell of a population of tumor cells in a fluid chamber of a microfluidic device; b) introducing a sample comprising a population of T-cells into the fluid chamber; c) generating an AC electrothermal flow inside the fluid chamber; and d) performing a wash step to remove T-cells of the population that remain mobilized within the fluid chamber, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.

31. The method of claim 30, the method further comprising performing an additional wash step to remove tumor cells of the population that remain mobilized within the fluid chamber prior to said step (b).

32. The method of claim 30, wherein a plurality of tumor cells of the population of tumor cells and a plurality of T-cells of the population of T-cells form a plurality of tumor cell / T-cell complexes.

33. The method of claim 30, wherein the microfluidic device comprises planar electrodes and generating the AC electrothermal flow comprises stimulating the electrodes at about 5 Vppto about 100 Vppor about 10 kHZ to about 1 MHz.

34. The method of claim 31, wherein the wash step or the additional wash step comprises washing the fluid chamber using a shear flow of about 0.05 dyn / cm2to about 1.0 dyn / cm2.

35. The method of claim 30, wherein the population of tumor cells is a population of tumor cells derived from a lung cancer, a non-small cell lung cancer, a colorectal cancer, a pancreatic cancer, an appendiceal cancer, a small bowel adenocarcinoma, a hepatobiliary cancer, a gynecological malignancy, a hematopoietic cancer, a breast cancer, a bladder cancer, a prostate cancer, or a skin cancer.

36. The method of claim 30, wherein the population of tumor cells is a population of human tumor cells.

37. The method of claim 30, wherein the population of T-cells is a population of human T- cells.

38. The method of claim 30, wherein the population of tumor cells and the sample comprising the population of T-cells are derived from the same subject.

39. The method of claim 38, wherein the subject is a human subject.

40. The method of claim 30, wherein the population of tumor cells and the sample comprising the population of T-cell are derived from different subjects.

41. A microfluidic device comprising: a) a sample reservoir; b) an outlet; c) a fluid chamber coupling the sample reservoir to the outlet, wherein the fluid chamber is configured to allow for immobilization of a plurality of cells of a first population of cells; and d) a pair of electrodes positioned within at least a portion of the fluid chamber, wherein the pair of electrodes are configured to produce an alternating current (AC) electrothermal flow of fluid in the fluid chamber when an alternating current signal is applied across the pair of electrodes, thereby facilitating formation of cell-cell complexes, each cell complex comprising an immobilized cell of the first population of cells and a cell of a second population of cells introduced into the fluid chamber after the plurality of cells of the first population of cells are immobilized in the fluid chamber.

42. The microfluidic device of claim 41, wherein the pair of electrodes are disposed on a surface of the fluid chamber.

43. The microfluidic device of claim 41 , wherein the fluid chamber is about 10 mm to about 50 mm in length, about 1.0 mm to about 20 mm in width, or about 0.5 mm to about 5.0 mm in height.

44. The microfluidic device of claim 41, wherein the sample reservoir is about 5 mm to about 20 mm in diameter.

45. The microfluidic device of claim 41, wherein the outlet is about 0.5 mm to about 10 mm in diameter.

46. A method of isolating a cell-cell complex, the method comprising: a) immobilizing at least one cell of a first population of cells in a fluid chamber of a microfluidic device; b) introducing a sample comprising a second population of cells into the fluid chamber; c) generating an AC electrothermal flow inside the fluid chamber; and d) performing a wash step to remove cells of the second population that remain mobilized within the fluid chamber, wherein at least one cell of the first population and at least one cell of the second population form a cell-cell complex.

47. A method of isolating a tumor antigen specific T-cell, the method comprising: a) introducing a population of tumor cells into a microfluidic device, wherein at least one cell of the population comprises a magnetic label, and wherein said microfluidic device comprises a microchannel comprising a plurality of micro wells; b) applying a magnetic force to the microfluidic device to immobilize at least one tumor cell of the population to at least one microwell; c) introducing a sample comprising a population of T-cells into the microfluidic device; d) generating an AC electrothermal flow inside the microchannel; and e) performing a wash step to remove T-cells of the population that remain mobilized within the microchannel, wherein at least one tumor cell of the population of tumor cells and at least one T-cell of the population of T-cells form a tumor cell / T-cell complex.