Microfluidic engineering of immune cells for improved therapeutic efficiency

Abstract

Disclosed herein is a microfluidic device for priming, collection and recovery of immune cells comprising: a) two separate fluidic channels: i) a first channel, wherein the first channel is infused with immune cells, and ii) a second channel, wherein the second channel acts as a collection reservoir for transmigrated cells, and; b) a microporous membrane interface, present between the first channel and the second channel, wherein the microporous membrane is functionalized on the first channel surface with adhesion molecules. Also disclosed are methods of creating enhanced cells, and using them in treatment of individuals in need thereof.

Description

[0001] MICROFLUIDIC ENGINEERING OF IMMUNE CELLS FOR IMPROVED THERAPEUTIC EFFICIENCY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 676,985, filed July 30, 2024, incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Current approaches for manipulation of therapeutic T cells rely on genetic modification, intricate manufacturing protocols, and extensive validation to ensure safety. These challenges greatly increase the required investment, market translation time, and final cost of the therapeutic cell product.

[0006] Microfluidic devices with multiple compartmentalized channels have been previously employed for studying and screening transmigration of cells, such as cancer cells and immune cells including T cells. While some of the reported microfluidic channels were constructed through embedding conventional Boyden chamber inserts directly onto of a microfluidic channel, other devices were composed of two channels interfacing with a microporous membrane layer in between. Another approach that has been reported for transmigration relies on formation of several channels in parallel divided by microfabricated posts, enabling a gel interface between channels and migration of cells within the porous gel media. Nevertheless, none of these microfluidic systems were designed and optimized to enhance the therapeutic efficiency of immune cells.

[0007] Thus, there is a need to address the aforementioned problems and other shortcomings associated with traditional microfluidic systems.

[0008] SUMMARY

[0009] The present invention relates to a microfluidic device and its use thereof. The present disclosure addresses at least a portion of the problems described above for the use of engineered immune cells, and their methods of making using the inventive microfluidic device disclosed herein.

[0010] In one aspect, the present invention provides a microfluidic device for priming, collection and recovery of immune cells comprising: a) two separate fluidic channels: i) a first channel, wherein the first channel is infused with immune cells, and ii) a second channel, wherein the second channel acts as a collection reservoir for transmigrated cells, and; b) a microporous membrane interface, present between the first channel and the second channel, wherein the microporous membrane is functionalized on the first channel surface with adhesion molecules. In some embodiments, the first channel has a width between 10-1000 pm, and a breadth of 50 pm. In some embodiments, the second channel has a width between 0. 1- 10 mm, and has a breadth of 1 .1 mm. Tn other embodiments, adhesion molecules can be selected from a group including but not limited to P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule- 1 (ICAM-1). In some embodiments, the second channel can comprise at least one transmigration enhancer. The transmigration enhancer can form a decreasing gradient towards the first channel, and can comprise a protein, peptide, or small molecule. The transmigration enhancer can comprise at least one chemokine, such as, but not limited to, CCL2, CCL3, CCL4, CCL5, CXCL9, and / or CXCL10. The transmigration enhancer can be loaded at intervals or continuously. In some embodiments, the immune cells can be infused into the first channel. Alternatively, the immune cells can be introduced without flow and allowed to settle and / or adhere passively. The immune cells can be flowed through the first channel, or can be flowed through at a wall shear stress between 0.1-10 dyne / cm2. The immune cells used in the device can include but are not limited to CD4 T-cells, CD8 T-cells, bulk CD3 cells, bulk or purified peripheral blood mononuclear cells, CAR T cells, monocytes, NK-cells, NK-T-cells, neutrophils or other therapeutic cells, such as mesenchymal stem cells.

[0011] In another aspect, the present invention provides a method of making a non-genetically engineered immune cell using the microfluidic device described herein. The non-genetically engineered immune cell can be selected from a group comprising of CD4 T-cells, CD8 T-cells, bulk CD3 cells, bulk or purified peripheral blood mononuclear cells, CAR T cells, monocytes, NK-cells, NK-T-cells, neutrophils or other therapeutic cells, such as mesenchymal stem cells. The non-genetically engineered immune cells can comprise increased motility in confined tumor environments compared to a control. In another embodiment, the non-genetically engineered immune cell can be a T-cell. Further, the non-genetically engineered T-cell can comprise decreased PD-1 expression compared to a control.

[0012] In a further aspect, disclosed herein is a method of treatment of a subject with cancer, comprising administering a non-genetically engineered immune cell to the subject, wherein the cell is produced using the microfluidic device described herein. In one embodiment, the non- genetically engineered immune cell can selected from a group comprising CD4 T-cells, CD8 T- cells, bulk CD3 cells, bulk or purified peripheral blood mononuclear cells, CAR T cells, monocytes, NK-cells, NK-T-cells, neutrophils or other therapeutic cells, such as mesenchymal stem cells. In another embodiment, the non-genetically engineered immune cell can comprise increased motility in confined tumor environments compared to a control. The non-genetically engineered immune cell can be a T-cell, for example. The non-genetically engineered T-cell can comprise decreased PD- 1 expression compared to a control.

[0013] Additional aspects and advantages of the disclosure will be set forth, in part, in the detailed description and any claims which follow, and in part will be derived from the detailed description or can be learned by practice of the various aspects of the disclosure. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0016] FIG. 1 (A-K) shows transmigration induces acute down-regulation of PD- 1 in CD8+ T cells. (A) Schematic of in vivo adoptive transfer experiment. CD8+ T cells were isolated from CD45.2+ OT-I mice, expanded ex vivo for 11 days, and intravenously injected into B16F10- OVA tumor-bearing CD45.1+ mice (IxlO5tumor cells implanted subcutaneously 7 days prior). Tumors were harvested at 2 h and 24 h postinjection for analysis. (B) Representative PD-1 expression in donor CD8+ T cells (CD45.1- CD45.2+) within tumors at 2 h and 24 h after adoptive transfer, compared to non-injected cells. (C) Summary of PD-1 expression on the donor T cells (n = 4 mice per group; mean ± SEM; **p < 0.01, unpaired t-test), indicating transient PD- 1 down-regulation at 2 h post- injection with recovery by 24 h. Gray shaded area indicates PD-1 expression in non-injected donor CD8+ T-cells. (D) (Left) Physiological steps of T cell transmigration in the tumor microenvironment: (1) rolling and tethering via adhesion molecules, (2) firm adhesion to the endothelium, and (3) chemokine-induced transmigration into the tumor. (Right) Design of microfluidic T cell chemokine-guided homing and infiltration platform (T- Chip), which recapitulates these processes in vitro. Expanded mouse CD8+ T cells are perfused over a porous membrane under controlled shear stress (0.75 dyn / cm2), and induced to transmigrate through 3 pm pores by surface-coated adhesion molecules (E / P selectin, ICAM-1, VCAM-1) and chemokines (CCL2-5, and CXCL9 / 10) supplied in the lower channel. (E) (Top) 3D schematic of T-Chip incorporating upper channel and lower channel, which are separated by a porous membrane, for cells perfusion and chemokine cues respectively. (Bottom) Bright-field images of porous membranes with low (6xl05pores / cm2) and high (2xl06pores / cm2) pore densities, each with 3 pm diameter pores. (F) Time-lapse bright-field images of individual mouse T cell active transmigrating through 3 pm pores at indicated time points (scale bar: 10 pm). (G) Quantification of transmigration efficiency (% of live cells collected from the lower channel) across low and high pore densities (n = 3 chips per group; mean ± SEM; *p < 0.05, unpaired t-test). (H) Transmigration efficiency according to presence or absence of surface- coated adhesion molecule (Adh. Mol.) and chemokine cues (C.C.) (n = 3 chips per group; mean ± SEM; **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey's multiple comparison test). (I) Representative Immunofluorescence images of PD- 1 expression and nucleus in parent and transmigrated CD8+ T cells recovered from the lower chamber (scale bar: 10 pm). Transmigrated cells exhibited substantially reduced surface PD-1 signal. (J) Representative surface PD-1 expression in parent cells (Control cells not infused in T-chip), transmigrated cells, and transmigrated cells without chemokine cues (Transmig. wo C.C.). (K) Quantification of (left) % PD-1+ cells and (right) PD-1 mean fluorescence intensity (MFI) from (J) (n = 3 chips per group; mean ± SEM; ****p < 0.0001, *p < 0.05, one-way ANOVA with Tukey's multiple comparison test) indicating that PD- 1 down- regulation is chemokine-independent.

[0017] FIG. 2 (A-F) shows transmigration through T-Chip reprograms CD8+ T cells by downregulating PD-1 and enhancing effector phenotype and function. (A) Representative expression of phenotypic and functional markers (PD-1, CXCR3, CD44, CD62L, Ki-67, and Granzyme B) in parent CD8+ T cells, parent cells exposed to chemokine cues without flow (Parent w C.C.), free-flowed cells (Free flow) recovered from the upper channel, and transmigrated cells (Transmig.) recovered from the lower channel of T-chip. (B) Quantification of marker-positive cells from (A) (n = 8-12 chips per group; mean ± SEM; one-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, ***!|;p < 0.0001). Transmigration significantly reduced PD-1+, CXCR3+, and CD62L+ populations while increasing CD44+ and Ki67+ populations. (C) Representative CD44 vs. CD62L plots showing memory phenotype subsets from (A). (D) Quantification of CD8+ T cell subsets defined by CD44 and CD62L expression: double-negative (- / -), naive (CD44-CD62L+), effector memory (TEM; CD44+CD62L-), and central memory (TCM; CD44+CD62L+) (n = 8-12 chips per group; mean ± SEM; *p < 0.05 to ****p < 0.0001, one-way ANOVA with Tukey's multiple comparison test). Transmigration increased TEM cells and reduced naive subsets. (E) Intracellular cytokine staining of IFN-y, IL-2, and TNF-a in OT-I parent and transmigrated CD8+ cells after 3 h co-culture with B16F10-OVA cells in the presence of 5 pg / ml Brefeldin A and with or without IFN-y (10 ng / ml) stimulation. (F) Quantification of cytokine-producing cells from (E) (n = 3-4 wells per group; mean + SEM; *p < 0.05, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey's multiple comparison test). Transmigration significantly enhanced IFN-y, IL-2, and TNF-a production, indicating a functionally rejuvenated effector phenotype.

[0018] FIG. 3 (A-I) shows (A) (Left) representative hright-field images, and (right) quantification of shape factor and cell size of parent and transmigrated CD8+ T cells. Transmigrated cells displayed increased morphological elongation and larger cell area, indicating an activated phenotype (n > 100 cells per group in one experiment; mean ± SEM; ****p < 0.0001, **p < 0.01, unpaired t-test). (B) (Left) Schematic diagram of T-cell migration assay and (Right) quantification of cell migration speed in 3D collagen gel (1.25 and 2 mg / mL). Transmigrated T cells exhibited significantly enhanced motility, particularly in 2mg / ml collagen gel (n = 20 cells per group in one experiment; mean ± SEM; **p < 0.01, unpaired t-test). (C) (Left) Schematic illustration and (Right) summary of in vitro tumor cell killing assay. Parent or transmigrated OT-I CD8+ T cells were co-cultured with B16F10-OVA tumor cells in the presence or absence of IFN-y (10 ng / ml) or anti PD-1 (aPDl, 10 pg / ml). Transmigrated T cells exhibited enhanced cytotoxicity, regardless of IFN-y and aPD-1 treatment (n = 3 wells per group; mean ± SEM; *p < 0.05, one-way ANOVA with Tukey's multiple comparison test). (D) Proliferation analysis of parent and transmigrated OT-I CD8+ T cells using Cell Trace Blue after 24 h co-culture with B16F10-OVA tumor cells with or without IFN-y (10 ng / ml) or aPD-1 (10 pg / ml) treatment. (E) Quantification of proliferating CD8+ T cells from (D) (n= 3 wells per group; mean ± SEM; **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparison test). Transmigrated T cells showed significantly higher proliferation rates, exceeding 70% in all conditions (± IFN-y, ± PD-1 blockade), indicating checkpoint-independent enhancement of proliferative potential (F) Schematic of in vivo peritumoral injection model. OT- I CD8+ T cells expanded ex vivo for 11 days were processed in T-chip and injected peritumorally into B16F10-OVA-bearing mice (IxlO5tumor cells implanted subcutaneously 7 days prior). Tumors were harvested for analysis 14 days post-injection. (G) Tumor volume over time (% change from initial volume) and quantification of tumor-infiltrating CD8+ donor T cells (CD45.1- CD45.2+) at endpoint (n = 3 mice per group, mean ± SEM; 2 mice in the control group died before day 14, leaving n = 1 for final tumor measurement; *p < 0.05, ****p < 0.0001, oneway ANOVA with Tukey's multiple comparison test). Transmigrated cells more effectively suppressed tumor growth, accumulated in tumors, and enhanced infiltration of host CD8+ T cells (CD45.1+ CD45.2-). (H) Immunohistochemistry staining for CD8 (magenta), CD45.2, CD31 and nucleus in xx-pm-thick sections of B16F10-OVA tumors harvested from (F) at endpoint (Scale bar = xx pm). (I) Quantification of donor CD8+ T cells in tumor-draining lymph node (TdLN), non-draining LN (NdLN), and spleen (n = 3-4 mice per group; mean ± SEM; **p < 0.01, ***p < 0.001, unpaired t-test). Transmigrated T cells exhibited increased trafficking to secondary lymphoid organs compared to parent cells.

[0019] FIG. 4 (A-J) shows transmigration through T-chip triggers acute and selective surface PD-1 loss in CD8+ T cells (A) Representative flow cytometry plots (left) and summary (right) of surface PD-1 expression of CD8+ T cells transmigrated through 3 pm and 5 pm pores compared to parent cells, indicating acute loss of PD-1 irrespective of pore size, (n = 3-5 chips per group; mean ± SEM; ****p < 0.0001, two-way ANOVA with Tukey's multiple comparison test). (B) Schematic illustration (left), representative flow cytometry plots (top right), and summary (bottom right) of surface PD-1 expression of CD8+ T cells physically pushed through 3 pm pores under high shear stress (144 dyne / cm2) showing no significant reduction compared to parent cells. (C) Representative flow cytometry plots (left) and summary (right) of surface PD-1 expression at day 0, 1 and 3 post-transmigration. PD-1 expression gradually recovered in transmigrated cells by day 3, while parent cells maintained high PD-1 levels, (n = 3-4 per group; mean ± SEM; ****p < 0.0001, one-way ANOVA with Tukey's multiple comparison test). (D) Representative flow cytometry plots (left) and summary (right) of PD- 1 expression on cell surface and whole cell (surface + intracellular) in parent and transmigrated mouse CD8+ T cells. Whole PD-1 staining was performed using the same surface antibody after cell permeabilization to detect both surface and intracellular PD-1. (E) Schematic illustration of labeling surface PD-1 on CD8+ T cells (pre-stained surface PD-1, pPD-1) via a fluorescently conjugated anti-PDl antibody prior to perfusion and transmigration in T-Chip for tracking PD-1 loss.

[0020] (F) Representative imaging flow cytometry images (left) showing pPD- 1 (red) and nucleus (magenta) in parent, non-migrated, and transmigrated CD8+ T cells (scale bar = 10 m). Quantification of pPDl-i- cells (right), showing acute pPD-1 loss after transmigration (n = 3-4 chips per group; mean + SEM; **p < 0.01 ***p < 0.001, one-way ANOVA with Tukey's multiple comparison test). (G) Analysis of pPD-1 localization using three compartmental masks: whole cell (WC), Nucleus (N), and surface-proximal (SP), calculated as WC minus N. The SP mask represents the membrane- adjacent region where PD-1 is typically localized in imaging flow cytometry images (top left). Representative imaging flow cytometry plots showing pPDl intensity in SP vs. WC mask for individual cells with linear regression (bottom left). Summary of linear regression slopes (right), indicating a lower surface-to- whole cell pPD-1 intensity slope, thus, surface-dominant PD-1 loss, (n = 3-4 chips per group; mean + SEM; ***p < 0.001, paired t-test). (H) Summary of pPD-1 expression in SPC mask from (G), supporting loss of surface- localized PD-1 after transmigration, (n = 3-4 chips per group; mean ± SEM; **p < 0.01, unpaired t-test). (I) Representative cross-sectional confocal images (left) of a transmigrating CD8+ T cell, pre-stained for surface PD-1 (red) and nucleus (blue), within a 3 pm pore (scale bar = 2 pm). Quantification (right) of pPD-1 intensity across pre- and post-transmigration cell bodies, (n = 20 cells per group in 2 experiments; mean ± SEM; ****p < 0.0001 , unpaired t-test). White arrow indicates migration direction. (J) Fluorometric quantification of pPD- 1 intensity in supernatant of parent cells in culture vs. transmigrated cells collected from channels of T-Chip, normalized to parent and expressed as fold change, indicating increased release of pPD-1 by transmigrated T cells, (n = 5 chips per group; mean + SEM; **p < 0.01, unpaired t-test).

[0021] FIG. 5 (A-H) shows transmigration drives proteasome-mediated degradation of PD- 1 in CD8+ T cells. (A) Volcano plot of differentially expressed genes (DEGs) from bulk RNA-seq comparing parent and transmigrated mouse CD8+ T cells (DESeq2). Dotted lines indicate significance thresholds: adjusted pvalue < 0.05 and Ilog 2 (FC)I > 1 (two-sided Wilcoxon ranksum test). Data points with extreme values (adjusted p-value < le-17 or Ilog2(fold change)! > 10) were clipped to the edge of the plot for visualization. Other genes were categorized: P value group (adjusted p-value < 0.05 and Ilog 2 (FC)I < 1), logaFC group (adjusted p-value < 0.05 and Ilog 2 (FC)I > 1), and NS (non-significant) group (adjusted p-value > 0.05 and Ilog 2 (FC)I < 1). (B) Dot plot displaying the top 20 enriched Gene Ontology (GO) biological processes. Each dot represents a GO term; dot size indicates the number of genes involved, and color reflects statistical significance (adjusted p-value). The x-axis represents the gene ratio (number of DEGs in the pathway / total number of genes in that pathway). Notably, “proteasome-mediated ubiquitin-dependent protein catabolic process” was the most significantly enriched term, indicating increased proteasomal degradation activity. (C) Expression of representative T cell exhaustion-related genes (Tox, Tim3 / Havcr2, Lag3, Pdcdl) from RNA-seq. Tox and Tim3 were unchanged, Lag3 was down-regulated and Pdcdl was up-regulated at the mRNA level. The discrepancy between increased Pdcdl mRNA and decreased surface PD-1 protein suggests a compensatory transcriptional response to acute PD-1 protein loss induced by transmigration. (D) GO subset enrichment analysis showing top 40 DEGs (ranked by absolute stat value) within “proteasome-mediated ubiquitin-dependent protein catabolic process” and “regulation of T cell activation” categories. Up-regulated genes included E3 ligases (Rnfl9a, Rnfl9bb, Smurfl, Smurf2, Arih2), the E3 adaptor Klhll5, and the deubiquitinase Usp9x, indicating activation of the ubiquitin-proteasome system. Co-occurring up-regulation of Tbx21, Ifng, and Cd44 further suggests that transmigration promotes PD-1 degradation and reprograms CD8+ T cells toward a functionally rejuvenated, effector-like state. (E) Schematic illustration of the proposed mechanism: transmigration induces PD-1 ubiquitination via an E3 ligase leading to proteasome- mediated degradation. (F) Representative surface PD-1 expression in transmigrated CD8+ T cells treated overnight with 100 pM Batimastat (a protease inhibitor) or lOpM MG-132 (a proteasome inhibitor). MG-132, but not Batimastat, prevented PD-1 loss in transmigrated cells, indicating that PD-1 loss is proteasome-dependent. (G) Representative expression of surface PD- 1 expression in (left) parent and (right) transmigrated CD8+ T cells, which were pre-treated overnight with increasing concentrations of MG-132 (0, 2.5, 5, 10 pM) prior to T-chip processing. (H) Summary of PD-1+ cells from (G) (n = 4-6 chips per group; mean } SEM; **p

[0022] < 0.01, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). Surface PD-1 loss in transmigrated cells was prevented in an MG-132 dose-dependent manner, while parent cells remained unaffected.

[0023] FIG. 6 (A-M) shows proteasome-dependent PD- 1 turnover is recapitulated in human CD8+ T cells following transmigration in T-chip. (A) Representative expression of surface PD- 1, CXCR3, CD45RA, and CCR7 in parent human CD8+ T cells (isolated from peripheral blood mononuclear cells), and transmigrated cells processed through 5 pm (Tr-5pm) or 3 pm (Tr-3pM) pores in the T-chip. (B) Quantification of PD-1+, CXCR3+, CD45RA+, and CCR7+ cells from (A). Transmigrated cells showed reduced PD-1 and CXCR3 expression (n = 4 chips per group; mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test). (C) Representative CD45RA vs CCR7 plots used for naive / memory subset classification. (D) (Left) Quantification of T cell subset frequencies from (C): naive (CD45RA+CCR7+), central memory (TCM; CD45RA-CCR7+), effector memory (TEM; CD45RA-CCR7-), and terminally differentiated effector memory (TEMRA; CD45RA+CCR7-) (n = 4 chips per group; mean ± SEM; *p < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Transmigration decreased TEMRA and increased TCM proportions, indicating a shift toward central memory phenotype. (Right) PD-1 expression across subsets (n = 4 chips per group; mean + SEM; **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test). PD-1 down-regulation occurred uniformly across all subsets, suggesting that PD-1 reduction is subset-independent. (E) Migration speed of parent and transmigrated T cells in 3D collagen matrices (1.25 or 2 mg / ml), indicating significantly higher motility of transmigrated T cells (n = 9-15 cells per group in one experiment; mean ± SEM; **p

[0024] < 0.01, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). (F) Representative imaging flow cytometry images showing surface pre-stained PD-1 (pPDl, yellow) and nucleus in parent, free-flowed, Tr-5pm, and Tr-3pm cells (scale bar = 7 pm). (G) Quantification of pPDl+ cells from (F), indicating acute loss of surface PD-1 following transmigration (n = 3-6 chips per group; mean ± SEM; ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). (H) (Left) Representative imaging flow cytometry plots showing pPDl intensity in cyto mask vs cell mask for individual cells, with linear regression. (Right) Quantification of linear regression slopes (n = 3 chips per group; mean ± SEM; *p < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The reduced slope in Tr-3pm cells indicates preferential loss of surface-localized PD-1. (I) Summary of cytoplasmic pPDl intensity from (F), showing significantly reduced signal in transmigrated cells compared to controls (n = 3 chips per group; mean ± SEM; **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test). (J) (Left) Representative cross-sectional confocal images of a human CD8+ T cell transmigrating through a 3 pm pore, which is stained for pPDl and nucleus (scale bar = 2 pm). (Right) Quantification pPDl intensity in pre- vs. post-transmigration cell bodies from cells transmigrating through 5 or 3 pm pores (n = 16 cells from 2 experiments; mean ± SEM; **p < 0.01, unpaired t-test). (K) Relative pPDl signal in culture supernatants from control and T-chip (5 or 3 pm pores) samples, indicating that transmigration promotes extracellular release of surface PD-1 (n = 3 chips per group; mean ± SEM; **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test). (L) Representative surface PD-1 expression in (left) parent and (right) Tr-3pm cells, which were pre-treated overnight with increasing concentrations of MG-132 (0, 2.5, 5, 10 pM) prior to T-chip processing. (M) Quantification of PD-1+ cell frequency from (L). MG-132 dose-dependently prevented PD-1 loss in Tr- 3 pm cells confirming proteasome-dependent PD-1 degradation in human CD8+ T cells (n = 4 chips per group; mean ± SEM; *p < 0.05, ***p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test within-group comparison and Sidak's multiple comparisons test for between- group comparison).

[0025] FIG. 7 (A-N) shows transmigration-induced PD-1 and Tim-3 down-regulation and phenotypic remodeling are recapitulated in tumor-infiltrating CD8+ T cells. (A) Schematic of mouse tumor infiltrating lymphocyte (TIL) experiment. Mouse TILs were isolated from B16F10- OVA tumors harvested 10 days after subcutaneous implantation (1x10scells), expanded ex vivo with high-dose IL-2 (6,000 lU / mL), processed through the T-chip (3 pm pores), and analyzed on day 28 post-expansion. (B) Representative expression of (Left) PD-1 and (Right) Tim-3 in expanded mouse CD8+ parent and transmigrated TILs. (C) Summary of PD-1 and Tim-3 expression in parent and transmigrated mouse CD8+ TILs (n = 5 chips per group from 5 independent tumor fragment pools; mean ± SEM; **p < 0.01, ***p < 0.001, unpaired ttest). Transmigration significantly reduced expression of both PD-1 and Tim-3. (D) Representative PD-1 vs. Tim-3 plots in parent and transmigrated mouse CD8+ TILs. (E) Quantification of PD- l / Tim-3 subsets from (D), indicating increased PD-l-Tim-3- and decreased PD-l+Tim-3-i- populations in transmigrated cells (n = 5 chips per group from 5 independent tumor fragment pools; mean ± SEM; **p < 0.01, ***p < 0.001, unpaired t-test). (F) Representative CD44 vs. CD62L plots in parent and transmigrated mouse CD8+ TILs. (G)Quantification of T cell subsets from (F): double-negative (- / -), naive (CD44-CD62L+), effector memory (TEM; CD44+CD62L- ), and central memory (TCM; CD44+CD62L+) populations (n = 5 chips per group from 5 independent tumor fragment pools; mean ± SEM; **p < 0.01, unpaired t-test). Transmigration increased TEM and reduced -I- populations. (H) Schematic of human TIL experiment. Human TILs expanded from surgically resected tumors with high- dose IL-2 (6,000 lU / mL) were processed through T-chip (3 pm pores) and analyzed. (I) Representative expression of (Left) PD- 1 and (Right) Tim-3 in parent and transmigrated human CD8+ TILs. (J) Summary of PD-1 and Tim-3 expressions in parent and transmigrated human CD8+ TILs. (n = 5 chips per group from 5 independent tumor fragments; mean ± SEM; *p < 0.05, unpaired t-test). Transmigration significantly reduced expression of both PD-1 and Tim-3 in human CD8+ TILs. (K) Representative PD-1 vs. Tim-3 plots in parent and transmigrated human CD8+ TILs. (L) Quantification of PD-l / Tim-3 subsets from (K), indicating increased PD-l-Tim-3- and reduced PD- l+Tim-3+ populations in transmigrated cells (n = 5 chips per group from 5 independent tumor fragments; mean ± SEM; *p < 0.05, **p < 0.01, unpaired t-test). (M) Representative CD45RA vs. CCR7 plots for memory subset classification in parent and transmigrated human CD8+ TILs. (N) Paired donor analysis of memory subsets from (M) (n = 5 chips per group from 5 independent tumor fragments; mean ± SEM; p-values from paired t-test). Transmigration reduced TCM (CD45RA-CCR7+) and modestly increased TEM (CD45RA-CCR7-) and TEMRA (CD45RA+CCR7-) populations. Although consistent trends were observed across donors, inter-donor heterogeneity limited statistical significance.

[0026] FIG. 8 (A-E) shows T-chip (also referred to herein as the microfluidic device, 100) assembly and fluid dynamics simulations of different configurations. FIG. 8A shows photograph of T-chip assembly without tubing connections. FIG. 8B shows schematic of different components employed in T-chip assembly. FIG. 8 (C-E) shows computation fluid dynamics simulation of top channels for different T-chip configurations, including 8 and 16 parallel top channels.

[0027] FIG. 9 (A-G) shows computational analyses of chemokine diffusion from bottom to top channels in T-chip. FIG. 9 (A-B) shows cross-sectional and isometric views of top and bottom channels shown over a typical computational diffusion result. The concentration difference across the membrane is plotted for different bottom channel thicknesses and two different pore density. Computationally calculated chemokine concentration from the top surface of the top channel to bottom surface of the bottom channel at different cross-sections across the channel length. Inset shows concentration change across the membrane. FIG. 9 (C-D) shows concentration difference across the membrane plotted against different positions across the top channel length at different time points after chemokine infusion for different pore densities. FIG. 9 (E-G) shows chemokine concentration profile in a multi-channel configuration and shows neighboring channels don’t affect each other at 30 min. time point.

[0028] FIG. 10 (A-C) shows transmigration enriches for live CD8+T cells and induces PD- 1 downregulation independently of memory phenotype. (A) Representative gating strategy for flow cytometry analysis of live CD8+T cells from parent and transmigrated groups. (B) Quantification of live cell percentages across parent, chemokine-exposed parent (Parent w. C.C.), non-migrated (perfused in T-chip and collected from the upper channel), and transmigrated conditions. Transmigrated cells exhibited significantly higher viability compared to all other groups (n = 8-12 chips per group; mean + SEM; one-way ANOVA with Tukey’s multiple comparisons test). (C) PD-1 expression across memory subsets defined by CD44 and CD62L (n = 8-12 chips per group; mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). While parent and control groups maintained uniformly high PD-1 expression, transmigrated cells showed markedly reduced PD-1+ frequency across all subsets. This indicates that PD-1 downregulation is not restricted to a specific differentiation state. Representative PD- 1 plots for each subset from the transmigrated group are shown on the right.

[0029] FIG. 11 (A-C) shows PDL1 expression on B16F10-OVA melanoma cells after exposure to increasing concentrations of IFN\ as shown in FIG. 11 A. FIG. 11B and 11C shows intracellular IFNy, IL-2, and TNFa secretion of live and dead parent and transmigrated OT-I CD8+T cells when interfaced with B16F10-OVA melanoma cells treated without or with IFNy. Error bars represent the SD of the mean. Statistical comparisons performed by two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001.

[0030] FIG. 12 (A-C) shows microfluidic convective filtration device and its components in FIG. 12A and FIG. 12B. FIG. 12C shows mean PD1 fluorescence values for parent and filtrated T cells at different flow rates. Error bars represent the SD of the mean.

[0031] FIG. 13 (A-C) shows percent tumor volume change and mouse weight of mice adoptively transferred with transmigrated and parent T cells at 5k and 15k doses, along with saline injected control group in FIG. 13A and FIG. 13B. FIG. 13C shows gating strategy for quantitative analyses of CD8+T cells.

[0032] FIG. 14 (A-B) shows transmigration induces transient downregulation of surface CXCR3 on CD8+T cells. (A) Representative flow cytometry plots and quantification of surface CXCR3 expression in parent versus transmigrated CD8+T cells processed in the absence of chemokine cues (Transmig. wo C.C.). Transmigration alone reduces surface CXCR3 levels, suggesting ligand-independent modulation (n = 3 chips per group; mean ± SEM; **p < 0.01 , unpaired t-test). (B) Time-course analysis of CXCR3 expression on parent and transmigrated cells. CXCR3 expression is gradually recovered to parental levels by 1 day post-transmigration (n = 4 chips per group; mean ± SEM; *p < 0.05, two-way ANOVA with Tukey’s multiple comparisons test).

[0033] FIG. 15 (A-D) shows Transmigration induces morphological remodeling in mouse but not human CD8+T cells. (A-B) Gating strategy for imaging flow cytometry analysis of mouse

[0034] (A) and human (B) CD8+ T cells. (C) Quantification of morphological parameters in mouse CD8+ T cells. Transmigrated cells exhibited increased cell area, reduced aspect ratio, and changes in nuclear size compared to parent cells (n > 1000 cells per group from 3 - 4 experiments; *p < 0.05, unpaired t-test). Gray dots indicate n = 50 randomly sampled individual cells to illustrate their distribution. (D) Morphological analysis of human CD8+T cells revealed no significant differences in cell body or nuclear geometry between parent and transmigrated groups (n > 1000 cells per group from 3 - 4 experiments). Gray dots indicate n = 50 randomly sampled individual cells to illustrate their distribution.

[0035] FIG. 16 (A-B) shows Tim-3 is transiently downregulated during tumor infiltration but not observed from in vitro transmigration of expanded CD8 T cells. (A) Tim-3 expression in donor OT-I CD8+ T cells (CD45.2+) recovered from tumors at 2 and 24 hours after adoptive transfer. CD8+ T cells were isolated from CD45.2+ OT-I mice, expanded ex vivo for 11 days, and intravenously injected into B16F10-OVA tumor-bearing CD45.1+ mice (1x10stumor cells implanted subcutaneously 7 days prior). Tumors were harvested at 2 h and 24 h post-injection for analysis. Tim-3 was markedly downregulated at 2 hours but partially restored by 24 hours, indicating a transient loss during early tumor infiltration (n = 4 mice per group; **p < 0.01, unpaired t-test). (B) Tim-3 expression in donor CD8+ T cells recovered from blood, tumordraining lymph node (TdLN), and spleen at 2 hours post-transfer.

[0036] FIG. 17 (A-D) shows transmigration selectively enriches proliferative and viable subsets of CD8+tumor-infiltrating lymphocytes (TILs) from both mouse and human tumors. (A-B) Gating strategy for flow cytometry analysis of CD8 TILs isolated from mouse (A) and human

[0037] (B) tumors. (C) In mouse CD8+TILs, transmigration led to strong enrichment of live cells and increased frequency of CD44+ populations. Notably, even in samples with low baseline Ki-67+frequency, transmigration enriched for highly proliferative Ki-67+cells, suggesting selective retention of functionally active subsets. CD62L and Granzyme B levels remained unchanged (5 chips per group from 5 independent tumor fragments; **p < 0.01, unpaired t-test). (D) Similarly, human CD8+TILs isolated through transmigration showed robust enrichment of live cells from samples with variable viability, accompanied by selective downregulation of CXCR3, with CCR7 and CD45RA expression largely maintained (5 chips per group from 5 independent tumor fragments; *p < 0.05, ***p < 0.001, paired t-test).

[0038] FIG. 18 (A-D) shows Transmigration selectively reduces PD-1 expression in human CAR-T cells across a range of CAR expression levels. (A) Representative flow cytometry plots of human CD8 CAR (anti-CD19) T cells showing CAR and PD-1 expression in parent and transmigrated populations. (B) Quantification of CAR and PD-1 expression on CD8+CAR-T cells. CAR expression was unaffected by transmigration, while PD-1+ frequency was significantly reduced (n = 4 - 6; *p < 0.05, unpaired t-test). (C) Representative flow plots showing stratification of CAR signal into five density groups (CAR 1-5) in parent and transmigrated cells. (D) PD-1 expression (top: % PD-1+; bottom: MFI) within each CAR density group. Transmigrated cells exhibited consistently lower PD-1 expression at all CAR levels, indicating that checkpoint regulation is uncoupled from CAR expression intensity (n = 4 per group; *p < 0.05, **p < 0.01, unpaired t-test).

[0039] DETAILED DESCRIPTION

[0040] Definitions

[0041] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0042] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0043] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0044] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0045] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0046] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0047] As used herein, the term “expand”, “expanding”, and any grammatical variations thereof as used herein, refers to the cellular processes of cell growth, proliferation, and / or differentiation, wherein the processes are allowed to occur naturally or are accelerated for the purpose of increasing cell numbers, cell size, cell function, cell subtype, and / or cell maturity.

[0048] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0049] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0050] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0051] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0052] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0053] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0054] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of’ shall mean excluding more than trace elements of other ingredients and substantia] method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0055] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0056] As used herein, “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue.

[0057] As used herein, “diagnose”, “diagnosed”, “diagnosing”, and any grammatical variations thereof as used herein, refers to the act of process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms.

[0058] As used herein, the term “buffer” refers to a solution consisting of a mixture of acid and its conjugate base, or vice versa. The solution is used as a means of keeping the pH at a nearly constant range to be used in a wide variety of chemical and biological applications.

[0059] A “chimeric antigen receptor” is an artificial T cell receptor used for immunotherapy. CAR are protein receptors that have been engineered to give T cells an enhanced ability to target a specific protein. CAR receptors are chimeric because the antigen binding and T cell activating functions have been combined into a single receptor. As used herein, the term “agent” refers to a living organism or biological substance, such as a bacterium, virus, protozoan, parasite, fungus, chemical, or toxin, that can be designed to purposefully fulfill a biological function or action.

[0060] The term “cancer” is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such as squamous cancers or adenomas)). It is used here to describe both solid tumors and hematologic malignancies, including epithelial (surface and glandular) cancers, soft tissue and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma.

[0061] The terms "cell," "cell line" and "cell culture" include progeny. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that has the same function or biological property, as screened for in the originally transformed cell, are included. The "host cells" used in the present invention generally are prokaryotic or eukaryotic hosts.

[0062] A “T cell” refers to a type of lymphocyte that is one of the most important white blood cells of the immune system. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. The immune-mediated cell death function of T cells is carried by two major subtypes: CD8+“killer” T cells and CD4+“helper T cells.

[0063] As used herein, the term “chemical compound” or “compound”, refers to a chemical substance consisting of two or more different types of atoms or chemical elements in a fixed stoichiometric proportion. These compounds have a unique and defined chemical structure held together in a defined spatial arrangement by chemical bonds. Chemical compounds can be held together by covalent bonds, ionic bonds, metallic ions, or coordinate covalent bonds.

[0064] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some aspects, the composition disclosed herein comprises non-engineered immune cells, whose therapeutic efficiency is enhanced by passing them through a microfluidic device holding immune-cell priming cytokines, as disclosed herein.

[0065] “Microfluidic device” refers to a miniaturized instrument or platform that manipulates small volumes of fluids (typically in the microliter to nanoliter range) through channels or chambers patterned on a substrate, often for the purpose of performing chemical, biological, or diagnostic assays. The microfluidic device may be fabricated from polymers, glass, silicon, or other materials using techniques such as soft lithography, injection molding, or 3D printing.

[0066] “Channel” refers to a defined conduit within the microfluidic device through which fluid can be introduced, directed, or removed. A channel may have any cross-sectional shape or geometry and may be open or enclosed. In the present invention, the device comprises at least a first channel and a second channel, which may be arranged laterally, vertically, or in a stacked configuration.

[0067] “Microporous membrane” (103) refers to a semipermeable structure positioned between two adjacent channels (e.g., the first and second channels), allowing the selective diffusion of soluble molecules and / or the migration of cells between compartments. The membrane may be made from polycarbonate, PDMS, PET, or other synthetic or natural materials. The membrane contains pores having a defined diameter, density, and spatial distribution, which may be fixed or tunable during manufacturing.

[0068] “Tunable pore diameter” refers to the ability to select or control the average diameter of pores within the microporous membrane, such as to accommodate different sizes of molecules or cells. Pore diameters may range from approximately 0.1 pm to 20 pm, depending on the application. This can be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pm, or any amount below, above, or in between these values.

[0069] “Pore density” refers to the number of pores per unit area of the microporous membrane, typically expressed as pores / cm2. This parameter may influence the overall permeability of the membrane and the rate of diffusion or cell transmigration.

[0070] “Diffusion” refers to the passive movement of molecules (e.g., chemokines, cytokines, drugs) across the microporous membrane, driven by concentration gradients. Diffusion enables chemical communication between compartments in the absence of active flow.

[0071] “Migration” refers to the active movement of cells across the microporous membrane in response to chemotactic, haptotactic, or mechanical cues. Migration may be tracked in real time or quantified post hoc using imaging or cell counting methods. As used herein, a cytokine refers to one or any number of substances or chemicals including, but not limited to interferons, interleukins, and growth factors, which are secreted by certain cells of the immune system and have an effect on other such cells. The cellular functions of cytokines include, but are not limited to pro-inflammation, anti-inflammation, proliferation, migration, wound healing, and cellular signaling.

[0072] General Description

[0073] Cellular therapies based on immune cells have been at the forefront of treatment of increasing number of maladies, including but not limited to cancer. Ability to withstand immunosuppressive and confined tissue microenvironment is a critical determinant of therapeutic efficiency for therapeutic immune cells. Disclosed herein is a bioinspired microfluidic device for non-genetical engineering of immune cells to enhance their mechanical and immunological properties.

[0074] Method of making a non-genetically engineered immune cell using the microfluidic device

[0075] Disclosed herein is a microfluidic device which is an instrument that uses very small amounts of fluid on a microchip to do certain laboratory tests. In the present invention, the microfluidic device incorporates two channels (a first channel 101, and a second channel 102), which can optionally be stacked. These channels can be separated by a microporous membrane (103), with tunable pore diameter and density, enabling diffusion of molecules and migration of cells across the membrane.

[0076] More specifically, Figure 8A-E depicts a microfluidic device (100) for priming, collection and recovery of immune cells comprising: a) two separate fluidic channels: i) a first channel (101), wherein the first channel is infused with immune cells, and ii) a second channel (102), wherein the second channel acts as a collection reservoir for transmigrated cells, and; b) a microporous membrane interface (103), present between the first channel and the second channel, wherein the microporous membrane is functionalized on the first channel surface with one or more adhesion molecules. Tubings (104) can provide input (104a) and output (104b) to the microfluidic device. The input tube (104a) to the first channel can provide a port for loading cells, such as high throughput adhesion of infused cells onto the microporous membrane interface (103). The input tube (104a) to the second channel can allow for loading of transmigration enhancers, such as chemokines. This can allow for gradient formation, as well as collection and recovery of primed immune cells through an output tube (104b). In a specific example, medical or pharmaceutical grade clear flexible silicone tubing (104) can be used. The input cells are transferred into the chip through this tubing from a syringe loaded in a syringe pump, although it can also be operated by a peristaltic pump, manually by hand, or even by passive techniques such as gravity or osmotic pressure. The cells can be perfused along the first channel (101), transmigrate into the second channel (102) at the membrane interface (103), and can then exit through outlet tubing (104b) connected to a collection reservoir (106). By way of example, for the second channel, a chemokine cocktail can be introduced through inlet tubing (104a) connected to that channel. The transmigrated cells and used chemokine cocktail are then perfused out through outlet tubing (104b) into a separate collection reservoir.

[0077] In some embodiments, the first channel has a “height” (on the “z” axis) of about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 21 pm, 22 pm, 23 pm, 24 pm, 25 pm, 26 pm, 27 pm, 28 pm, 29 pm, 30 pm, 31 pm, 32 pm, 33 pm, 34 pm, 35 pm, 36 pm, 37 pm, 38 pm, 39 pm, 40 pm, 41 pm, 42 pm, 43 pm, 44 pm, 45 pm, 46 pm, 47 pm, 48 pm, 49 pm, 50 pm, 51 pm, 52 pm, 53 pm, 54 pm, 55 pm, 56 pm, 57 pm, 58 pm, 59 pm, 60 pm, 61 pm, 62 pm, 63 pm, 64 pm, 65 pm, 66 pm, 67 pm, 68 pm, 69 pm, 70 pm, 71 pm, 72 pm, 73 pm, 74 pm, 75 pm, 76 pm, 77 pm, 78 pm, 79 pm, 80 pm, 81 pm, 82 pm, 83 pm, 84 pm, 85 pm, 86 pm, 87 pm, 88 pm, 89 pm, 90 pm, 91 pm, 92 pm, 93 pm, 94 pm, 95 pm, 96 pm, 97 pm, 98 pm, 99 pm, or 100 pm, or any amount below, above, or in between these values.

[0078] In some embodiments, the second channel has a “height” (on the “z” axis) of about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5 mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9 mm, 6 mm, 6.1 mm, 6.2 mm, 6.3 mm, 6.4 mm, 6.5 mm, 6.6 mm, 6.7 mm, 6.8 mm, 6.9 mm, 7 mm, 7.1 mm, 7.2 mm, 7.3 mm, 7.4 mm, 7.5 mm, 7.6 mm, 7.7 mm, 7.8 mm, 7.9 mm, 8 mm, 8.1 mm, 8.2 mm, 8.3 mm, 8.4 mm, 8.5 mm, 8.6 mm, 8.7 mm, 8.8 mm, 8.9 mm, 9 mm, 9.1 mm, 9.2 mm, 9.3 mm, 9.4 mm, 9.5 mm, 9.6 mm, 9.7 mm, 9.8 mm, 9.9 mm, or

[0079] 10 mm, or any amount above, below, or between these values.

[0080] In some embodiments, the microfluidic device has an overall width (on an “x” axis) of between 10 and 500 mm. 0.1 and 10 mm. Specifically, it can have width of 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 37 mm, 38 mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm 47 mm, 48 mm, 49 mm, 50 mm, 51 mm, 52 mm, 53 mm, 54 mm, 55 mm, 56 mm, 57 mm, 58 mm, 59 mm, 60 mm, 61 mm, 62 mm, 63 mm, 64 mm, 65 mm, 66 mm, 67 mm, 68 mm, 69 mm 70 mm, 71 mm, 72 mm, 73 mm, 74 mm, 75 mm, 76 mm, 77 mm, 78 mm, 79 mm, 80 mm, 81 mm, 82 mm, 83 mm, 84 mm, 85 mm, 86 mm, 87 mm, 88 mm, 89 mm, 90 mm, 91 mm, 92 mm 93 mm, 94 mm, 95 mm, 96 mm, 97 mm, 98 mm, 99 mm, 100 mm, 101 mm, 102 mm, 103 mm

[0081] 104 mm, 105 mm, 106 mm, 107 mm, 108 mm, 109 mm, 110 mm, 111 mm, 112 mm, 113 mm,

[0082] 114 mm, 115 mm, 116 mm, 117 mm, 118 mm, 119 mm, 120 mm, 121 mm, 122 mm, 123 mm,

[0083] 124 mm, 125 mm, 126 mm, 127 mm, 128 mm, 129 mm, 130 mm, 131 mm, 132 mm, 133 mm,

[0084] 134 mm, 135 mm, 136 mm, 137 mm, 138 mm, 139 mm, 140 mm, 141 mm, 142 mm, 143 mm,

[0085] 144 mm, 145 mm, 146 mm, 147 mm, 148 mm, 149 mm, 150 mm, 151 mm, 152 mm, 153 mm,

[0086] 154 mm, 155 mm, 156 mm, 157 mm, 158 mm, 159 mm, 160 mm, 161 mm, 162 mm, 163 mm,

[0087] 164 mm, 165 mm, 166 mm, 167 mm, 168 mm, 169 mm, 170 mm, 171 mm, 172 mm, 173 mm,

[0088] 174 mm, 175 mm, 176 mm, 177 mm, 178 mm, 179 mm, 180 mm, 181 mm, 182 mm, 183 mm,

[0089] 184 mm, 185 mm, 186 mm, 187 mm, 188 mm, 189 mm, 190 mm, 191 mm, 192 mm, 193 mm,

[0090] 194 mm, 195 mm, 196 mm, 197 mm, 198 mm, 199 mm, 200 mm, 201 mm, 202 mm, 203 mm,

[0091] 204 mm, 205 mm, 206 mm, 207 mm, 208 mm, 209 mm, 210 mm, 211 mm, 212 mm, 213 mm,

[0092] 214 mm, 215 mm, 216 mm, 217 mm, 218 mm, 219 mm, 220 mm, 221 mm, 222 mm, 223 mm,

[0093] 224 mm, 225 mm, 226 mm, 227 mm, 228 mm, 229 mm, 230 mm, 231 mm, 232 mm, 233 mm,

[0094] 234 mm, 235 mm, 236 mm, 237 mm, 238 mm, 239 mm, 240 mm, 241 mm, 242 mm, 243 mm,

[0095] 244 mm, 245 mm, 246 mm, 247 mm, 248 mm, 249 mm, 250 mm, 251 mm, 252 mm, 253 mm,

[0096] 254 mm, 255 mm, 256 mm, 257 mm, 258 mm, 259 mm, 260 mm, 261 mm, 262 mm, 263 mm,

[0097] 264 mm, 265 mm, 266 mm, 267 mm, 268 mm, 269 mm, 270 mm, 271 mm, 272 mm, 273 mm,

[0098] 274 mm, 275 mm, 276 mm, 277 mm, 278 mm, 279 mm, 280 mm, 281 mm, 282 mm, 283 mm,

[0099] 284 mm, 285 mm, 286 mm, 287 mm, 288 mm, 289 mm, 290 mm, 291 mm, 292 mm, 293 mm,

[0100] 294 mm, 295 mm, 296 mm, 297 mm, 298 mm, 299 mm, 300 mm, 301 mm, 302 mm, 303 mm,

[0101] 304 mm, 305 mm, 306 mm, 307 mm, 308 mm, 309 mm, 310 mm, 311 mm, 312 mm, 313 mm,

[0102] 314 mm, 315 mm, 316 mm, 317 mm, 318 mm, 319 mm, 320 mm, 321 mm, 322 mm, 323 mm,

[0103] 324 mm, 325 mm, 326 mm, 327 mm, 328 mm, 329 mm, 330 mm, 331 mm, 332 mm, 333 mm,

[0104] 334 mm, 335 mm, 336 mm, 337 mm, 338 mm, 339 mm, 340 mm, 341 mm, 342 mm, 343 mm,

[0105] 344 mm, 345 mm, 346 mm, 347 mm, 348 mm, 349 mm, 350 mm, 351 mm, 352 mm, 353 mm,

[0106] 354 mm, 355 mm, 356 mm, 357 mm, 358 mm, 359 mm, 360 mm, 361 mm, 362 mm, 363 mm,

[0107] 364 mm, 365 mm, 366 mm, 367 mm, 368 mm, 369 mm, 370 mm, 371 mm, 372 mm, 373 mm, 374 mm, 375 mm, 376 mm, 377 mm, 378 mm, 379 mm, 380 mm, 381 mm, 382 mm, 383 mm,

[0108] 384 mm, 385 mm, 386 mm, 387 mm, 388 mm, 389 mm, 390 mm, 391 mm, 392 mm, 393 mm,

[0109] 394 mm, 395 mm, 396 mm, 397 mm, 398 mm, 399 mm, 400 mm, 401 mm, 402 mm, 403 mm,

[0110] 404 mm, 405 mm, 406 mm, 407 mm, 408 mm, 409 mm, 410 mm, 411 mm, 412 mm, 413 mm,

[0111] 414 mm, 415 mm, 416 mm, 417 mm, 418 mm, 419 mm, 420 mm, 421 mm, 422 mm, 423 mm,

[0112] 424 mm, 425 mm, 426 mm, 427 mm, 428 mm, 429 mm, 430 mm, 431 mm, 432 mm, 433 mm,

[0113] 434 mm, 435 mm, 436 mm, 437 mm, 438 mm, 439 mm, 440 mm, 441 mm, 442 mm, 443 mm,

[0114] 444 mm, 445 mm, 446 mm, 447 mm, 448 mm, 449 mm, 450 mm, 451 mm, 452 mm, 453 mm,

[0115] 454 mm, 455 mm, 456 mm, 457 mm, 458 mm, 459 mm, 460 mm, 461 mm, 462 mm, 463 mm,

[0116] 464 mm, 465 mm, 466 mm, 467 mm, 468 mm, 469 mm, 470 mm, 471 mm, 472 mm, 473 mm,

[0117] 474 mm, 475 mm, 476 mm, 477 mm, 478 mm, 479 mm, 480 mm, 481 mm, 482 mm, 483 mm,

[0118] 484 mm, 485 mm, 486 mm, 487 mm, 488 mm, 489 mm, 490 mm, 491 mm, 492 mm, 493 mm,

[0119] 494 mm, 495 mm, 496 mm, 497 mm, 498 mm, 499 mm, or 500 mm, or any amount above, below, or between these values.

[0120] In some embodiments, the microfluidic device has an overall length (on a “y” axis) of between 10 and 250 mm. Specifically, it can be 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 37 mm, 38 mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, 50 mm, 51 mm, 52 mm, 53 mm, 54 mm, 55 mm, 56 mm, 57 mm, 58 mm, 59 mm, 60 mm, 61 mm, 62 mm, 63 mm, 64 mm, 65 mm, 66 mm, 67 mm, 68 mm, 69 mm, 70 mm, 71 mm, 72 mm, 73 mm, 74 mm, 75 mm, 76 mm, 77 mm, 78 mm, 79 mm, 80 mm, 81 mm, 82 mm, 83 mm, 84 mm, 85 mm, 86 mm, 87 mm, 88 mm, 89 mm, 90 mm, 91 mm, 92 mm, 93 mm, 94 mm, 95 mm, 96 mm, 97 mm, 98 mm, 99 mm, 100 mm, 101 mm, 102 mm, 103 mm, 104 mm, 105 mm, 106 mm, 107 mm, 108 mm, 109 mm, 110 mm, 111 mm, 112 mm, 113 mm, 114 mm, 115 mm, 116 mm,

[0121] 117 mm, 118 mm, 119 mm, 120 mm, 121 mm, 122 mm, 123 mm, 124 mm, 125 mm, 126 mm,

[0122] 127 mm, 128 mm, 129 mm, 130 mm, 131 mm, 132 mm, 133 mm, 134 mm, 135 mm, 136 mm,

[0123] 137 mm, 138 mm, 139 mm, 140 mm, 141 mm, 142 mm, 143 mm, 144 mm, 145 mm, 146 mm,

[0124] 147 mm, 148 mm, 149 mm, 150 mm, 151 mm, 152 mm, 153 mm, 154 mm, 155 mm, 156 mm,

[0125] 157 mm, 158 mm, 159 mm, 160 mm, 161 mm, 162 mm, 163 mm, 164 mm, 165 mm, 166 mm,

[0126] 167 mm, 168 mm, 169 mm, 170 mm, 171 mm, 172 mm, 173 mm, 174 mm, 175 mm, 176 mm,

[0127] 177 mm, 178 mm, 179 mm, 180 mm, 181 mm, 182 mm, 183 mm, 184 mm, 185 mm, 186 mm,

[0128] 187 mm, 188 mm, 189 mm, 190 mm, 191 mm, 192 mm, 193 mm, 194 mm, 195 mm, 196 mm,

[0129] 197 mm, 198 mm, 199 mm, 200 mm, 201 mm, 202 mm, 203 mm, 204 mm, 205 mm, 206 mm, 207 mm, 208 mm, 209 mm, 210 mm, 211 mm, 212 mm, 213 mm, 214 mm, 215 mm, 216 mm,

[0130] 217 mm, 218 mm, 219 mm, 220 mm, 221 mm, 222 mm, 223 mm, 224 mm, 225 mm, 226 mm,

[0131] 227 mm, 228 mm, 229 mm, 230 mm, 231 mm, 232 mm, 233 mm, 234 mm, 235 mm, 236 mm,

[0132] 237 mm, 238 mm, 239 mm, 240 mm, 241 mm, 242 mm, 243 mm, 244 mm, 245 mm, 246 mm,

[0133] 247 mm, 248 mm, 249 mm, or 250 mm in length, or any amount below, above, or in between these values.

[0134] In one specific example, the device has an overall size of 72 mm (length) x 112 mm (width) x 3.65 mm (height). The device can have a top cover (105a) and a bottom cover (105b). This is provided by way of example only, and it will be appreciated that this size can vary depending on individual needs.

[0135] In certain embodiments, the first channel comprises adhesion molecules to facilitate the selective capture, retention, or interaction with target cells). As used herein, "adhesion molecules" refer to a class of proteins or peptides that mediate binding between cells or between cells and the extracellular matrix, including but not limited to integrins, cadherins, selectins, immunoglobulin superfamily (IgSF) members, or engineered peptide sequences with specific binding affinity. These molecules may be immobilized on the surface of the device via covalent or non-covalent attachment methods, such as chemical cross-linking, adsorption, or incorporation into a polymeric matrix. The adhesion molecules may be presented in an oriented or non-oriented fashion to enhance their binding efficacy. When incorporated into the device, these molecules can be used to mimic native cell environments, promote tissue integration, or capture circulating cells (e.g., circulating tumor cells, immune cells, or stem cells). The spatial pattern, density, and composition of the adhesion molecules may be tuned to optimize performance for a given application.

[0136] The adhesion molecules may be selected from a group including, but not limited to, P- selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule- 1 (ICAM-1). These adhesion molecules are known to play critical roles in mediating cell-cell and cell-matrix interactions, particularly in immune surveillance, inflammation, and metastatic processes. P-selectin and E-selectin are typically expressed on activated endothelial cells and mediate the initial tethering and rolling of leukocytes along the vascular endothelium. VCAM-1 and ICAM-1 are members of the immunoglobulin superfamily and are involved in firm adhesion and transmigration of leukocytes through the endothelium via interaction with integrins such as VLA-4 and LFA-1, respectively. By incorporating these adhesion molecules into the first channel, it is possible to recapitulate aspects of the in vivo cellular microenvironment, enabling the selective recruitment, capture, or analysis of circulating cells of interest.

[0137] In some embodiments, the second channel can comprise at least one transmigration modulator. This can be either a transmigration enhancer, or a transmigration suppressor. As used herein, “transmigration” refers to the process by which cells move from the first channel to the second channel. Tn a microfluidic context, transmigration enhancers can be used to model physiological processes such as immune cell extravasation, tumor metastasis, or stem cell homing, or to isolate or analyze specific cell populations based on migratory behavior. The transmigration enhancer can form a decreasing gradient towards the first channel, and can comprise a protein, peptide, or small molecule. The transmigration enhancer can promote the directed movement of cells across the membrane.

[0138] One class of transmigration enhancers includes chemokines, which are small secreted proteins that bind to chemokine receptors on target cells and activate intracellular signaling cascades that direct cell migration. A chemokine gradient may be established within the device by delivering or immobilizing the chemokine in a defined spatial pattern, such as by microfluidic flow control or hydrogel encapsulation. The gradient may be linear, exponential, or stepwise, and may be static or dynamically maintained over time. Chemokines suitable for use in the device include members of the CC chemokine family, such as CCL1 (1-309), CCL2 (MCP-1), CCL3 (MIP-la), CCL4 (MIP-lp), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (eotaxin-1), CCL17 (TARC), CCL19 (MIP-30), CCL20 (MIP-3a), CCL21 (SLC), CCL22 (MDC), CCL25 (TECK), CCL27 (CTACK), and CCL28. Chemokines from the CXC family may also be used, including CXCL1 (GROa), CXCL2 (GROp), CXCL3 (GROy), CXCL5 (ENA-78), CXCL8 (IL-8), CXCL9 (MIG), CXCL10 (IP- 10), CXCL11 (I-TAC), CXCL12 (SDF- 1), CXCL13 (BCA-1), and CXCL16. Additional chemokines that may be used include the CX3C chemokine CX3CL1 (fractalkine) and the XC chemokines XCL1 (lymphotactin) and XCL2.

[0139] In addition to chemokines, other proteins may be used to facilitate or enhance transmigration, either by modifying the local microenvironment, activating signaling pathways, or modulating adhesion and permeability. Such proteins include cytokines and growth factors, such as tumor necrosis factor-alpha (TNF-a), interleukin- ip (IL- 1 P), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), stromal-derived factor- 1 (SDF-1), hepatocyte growth factor (HGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Endothelial adhesion proteins and extracellular matrix components may also be used to enhance transmigration, including intracellular adhesion molecule- 1 (ICAM-1), vascular cell adhesion molecule- 1 (VCAM-1), platelet endothelial cell adhesion molecule- 1 (PEC AM- 1 or CD31), junctional adhesion molecules (JAM-A and JAM-B), E-selectin, P-selectin, fibronectin, collagen (types I, III, and IV), laminin, and tenascin-C.

[0140] In certain embodiments, the transmigration enhancer may be a small molecule compound capable of modulating cell migration, chemotaxis, or endothelial permeability. Small molecules may act directly on target cells by engaging cell surface receptors, ion channels, or intracellular signaling pathways involved in cytoskeletal remodeling and directional motility. Alternatively, small molecules may act indirectly by altering the properties of the surrounding microenvironment, such as by loosening tight junctions, increasing endothelial permeability, or upregulating adhesion molecule expression. Non-limiting examples of small molecule transmigration enhancers include forskolin, which activates adenylate cyclase to increase intracellular cAMP; sphingosine- 1-phosphate (SIP), which modulates vascular integrity and immune cell trafficking; and chemotactic lipids such as leukotriene B4 (LTB4) or plateletactivating factor (PAE). Additional compounds that may be used include synthetic receptor agonists or antagonists targeting CXCR4, CCR5, or other chemokine receptors; ROCK inhibitors that modulate actomyosin contractility; or matrix metalloproteinase (MMP) activators that degrade extracellular barriers. These small molecules may be introduced into one or more compartments of the microfluidic device in soluble form or immobilized on surfaces or within hydrogels to establish stable concentration gradients. In certain implementations, combinations of small molecules and protein-based transmigration enhancers may be used synergistically to model or manipulate specific migratory behaviors.

[0141] I think instead of or in addition to using transmigration enhancers, the second channel may comprise at least one transmigration suppressor.

[0142] A "transmigration suppressor" refers to any protein, peptide, small molecule, or environmental factor that inhibits or restricts cell movement across the membrane. The incorporation of a transmigration suppressor may be used to create an inhibitory microenvironment that mimics immunosuppressive or tumor-associated settings. This configuration enables the selective evaluation, enrichment, or analysis of cells capable of overcoming suppressive cues, thereby facilitating the identification and isolation of highly migratory or resilient cell subsets.

[0143] Examples of transmigration suppressors include, but are not limited to, transforming growth factor-beta (TGF-P), interleukin- 10 (IL-10), prostaglandin E2 (PGE2), indoleamine 2,3- dioxygenase (IDO), and other immunoregulatory cytokines or small molecules known to negatively modulate cell motility or activation. Additional suppressors may include extracellular matrix components that increase physical barrier properties, tight junction proteins that reduce permeability, or pharmacological inhibitors targeting actomyosin contractility and cytoskeletal remodeling (e.g., ROCK activators or myosin light chain kinase activators).

[0144] Similar to enhancers, suppressors can be introduced into the device in soluble form, immobilized on surfaces, or encapsulated within hydrogels to establish defined spatial or temporal gradients. These suppressors may be loaded continuously or at defined intervals, analogous to the loading strategies described for enhancers. By adjusting the type, concentration, and presentation mode of the suppressor, the microfluidic device can be used to model restrictive microenvironments and assess cellular capabilities for transmigration under inhibitory conditions.

[0145] The combination of transmigration enhancers and suppressors in the same or separate channels can be used to create complex, physiologically relevant chemotactic or mechanotactic landscapes. This approach allows for precise modulation of cell behavior, providing a versatile platform for studying immune cell function, tumor cell invasion, or therapeutic cell resilience in various microenvironmental contexts.

[0146] These molecules may be immobilized or presented in soluble form to modulate transmigration through the microfluidic device. The extent and kinetics of transmigration may be quantified using optical, electrical, or biochemical detection methods, and the migrated cell populations may be isolated for downstream analysis. The transmigration modulator can be loaded at intervals or continuously. When loaded at intervals, it can occur every 1, 5, 10, 30, or 60 seconds, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, or every 20, 30, 40, 50, or 60 minutes, or every 2, 3, 4, 5, 6, 7, or 8 hours, or every 12, 18, 24, 30, 36, or 48 hours, or any amount below, above, or in between these values.

[0147] Various cells can be infused into the first channel, then transmigrated through the device. An example of the cell type that can be used is an immune cell. Immune cells contemplated herein include, but are not limited to, neutrophils, monocytes, macrophages, dendritic cells, natural killer (NK) cells, T lymphocytes, B lymphocytes, and their respective subtypes. Neutrophils, as first responders in the innate immune system, may be used to model acute inflammatory responses or chemotaxis toward bacterial components. Monocytes and macrophages, which can be polarized into pro-inflammatory (Ml) or anti-inflammatory (M2) phenotypes, are useful for studying tissue infiltration, phagocytosis, and cytokine secretion. Dendritic cells may be incorporated to study antigen presentation or migration toward lymphoid tissue analogs. Natural killer (NK) cells can be used to model cytotoxic activity against tumor cells or virally infected cells. T lymphocytes may include bulk CD3 cells, CD4+ helper T cells, CD8+ cytotoxic T cells, regulatory T cells (Tregs), and yd T cells, which may be used to investigate activation, trafficking, or immune surveillance. B lymphocytes, including naive, memory, and plasma cells, may be introduced to study antibody production or interactions with antigen-presenting cells. In some embodiments, immune cells may be primary human cells, immortalized cell lines, or genetically modified cells expressing fluorescent markers, reporters, or synthetic receptors such as chimeric antigen receptors (CARs). These immune cells may be introduced into the device as single populations or as part of a mixed cellular environment to mimic complex tissue or tumor microenvironments. Also disclosed are any other type of therapeutic cells, such as mesenchymal stem cells.

[0148] In some embodiments, the immune cells may be infused into the first channel of the microfluidic device using controlled fluid flow. Alternatively, the immune cells may be introduced into the channel under static conditions and allowed to settle by gravity and / or adhere passively to the channel surface or to any cellular or biomimetic coating disposed therein. When introduced under flow conditions, the immune cells may be delivered at a flow rate sufficient to generate a defined wall shear stress, which may be selected to mimic physiological or pathophysiological conditions. For example, the immune cells may be flowed through the first channel at a wall shear stress between 0.1 dyne / cm2and 10 dyne / cm2, such as between 0.1 and 1.0 dyne / cm2, between 1.0 and 2.0 dyne / cm2, between 2.0 and 5.0 dyne / cm2, or between 5.0 and 10.0 dyne / cm2. The precise shear stress may be selected based on the desired biological response, such as to promote rolling adhesion (e.g., at lower shear), firm adhesion and spreading (e.g., at intermediate shear), or selective detachment or migration (e.g., at higher shear). In some implementations, a pulsed or oscillatory flow pattern may be applied to simulate dynamic in vivo environments. Flow may be driven by pressure, syringe pumps, or electroosmotic forces, and may be unidirectional or recirculating. Under static conditions, immune cell attachment may be enhanced by surface functionalization of the channel with adhesion molecules, extracellular matrix proteins, or other biological cues. In either case, the immune cells may be monitored in real time using microscopy, optical sensors, or other detection modalities to assess cell behavior, transmigration, activation status, or interactions with other cell types or stimuli present in the device.

[0149] The immune-mechanical priming process in the developed microfluidic platform induces reinvigorated phenotype with decreased PD-1 expression (a key surface receptor in immunosuppression) and increased motility in confined extracellular environments. The described priming process was tested and validated using both mouse CD8+ T cells and human CD8+ and CD4+ chimeric antigen receptor (CAR)- T cells. Furthermore, therapeutic efficiency of microfluidic primed T cells was shown in a mouse melanoma tumor model, where primed CD8 T cells suppressed tumor growth significantly compared to saline control and unprimed parent CD8 T cells.

[0150] Method of Making Immune Cells

[0151] In another aspect, the present invention provides a method of making a non-genetically engineered immune cell using the microfluidic device described herein. The method comprises introducing immune cells, such as T lymphocytes, into one or more channels of the device under conditions designed to modulate cellular phenotype or behavior without the use of exogenous genetic modification. In some embodiments, the immune cells are exposed to defined chemical, physical, or mechanical stimuli within the microfluidic environment that mimic key features of the tumor microenvironment or inflammatory tissue. Such conditions may include specific shear stress profiles, chemokine gradients, extracellular matrix compositions, or interactions with cocultured stromal or endothelial cells. These stimuli may be used individually or in combination to induce a phenotypic shift in the immune cells, enhancing desirable attributes such as migratory capacity, persistence, or resistance to immunosuppressive signals. Also disclosed herein are cells made by this process.

[0152] The non-genetically engineered immune cells produced by this method can exhibit increased motility in confined environments, such as those characteristic of solid tumors, compared to untreated or conventionally cultured control cells. This enhanced motility may be observed using in vitro assays that model interstitial confinement or extracellular matrix density, or may be quantified by measuring cellular velocity, directional persistence, or deformation capacity during transmigration through constricted spaces. The improved migratory phenotype may enable the immune cells to more effectively infiltrate tumor tissue in vivo, thereby enhancing their therapeutic potential in adoptive cell transfer therapies, immune surveillance, or tissue regeneration.

[0153] In some embodiments, the immune cell is a non-genetically engineered T lymphocyte. The T cell may be derived from a peripheral blood mononuclear cell (PBMC) population, a purified CD3+ fraction, or a subset thereof, such as CD4+ helper T cells or CD8+ cytotoxic T cells. The microfluidic conditioning may be applied at any stage of the T cell activation or expansion process, and may optionally be performed in the presence of antigen-presenting cells or soluble activating agents. Following microfluidic treatment, the T cells may exhibit changes in surface marker expression, functional capacity, or resistance to tumor-mediated inhibition. In a further embodiment, the non-genetically engineered T cell produced by the disclosed method can comprise decreased expression of programmed cell death protein 1 (PD-1) compared to a control T cell cultured under conventional conditions. PD-1 is an inhibitory immune checkpoint receptor that is upregulated in response to chronic stimulation and is often exploited by tumor cells to suppress T cell activity. Reduction of PD-1 expression without the use of gene editing or transduction may offer a safer and more regulatory-compliant approach to improving T cell efficacy. In certain embodiments, the reduction in PD-1 expression is sustained for at least 24, 48, or 72 hours post-treatment, and may be measured using flow cytometry, quantitative PCR, or protein-based detection methods. The resulting T cells may demonstrate enhanced cytotoxicity, cytokine secretion, or tumor cell killing when applied in therapeutic contexts.

[0154] Method of Treating Sub jects in Need Thereof

[0155] In a further aspect, the present invention provides a method for treating a subject diagnosed in need thereof. This can be, for example cancer. By way of example, the method can comprise administering to the subject an immune cell population, wherein the immune cells are produced using the microfluidic device and methods described herein. The subject may first undergo diagnosis and staging of cancer using clinical, imaging, or histopathological techniques, after which a treatment regimen incorporating the immune cell therapy may be initiated. The immune cells are conditioned within the microfluidic device to enhance therapeutic properties, such as tumor infiltration, persistence, and resistance to immunosuppression, without the use of viral transduction, CRISPR / Cas systems, or other forms of genetic modification. Alternatively, the cells may have undergone these modifications and are then further refined using the microfluidic device disclosed herein.

[0156] In one embodiment, the non-genetically engineered immune cells administered to the subject may be selected from a group including CD4+ helper T cells, CD8+ cytotoxic T cells, bulk CD3+ T cells, or total peripheral blood mononuclear cells (PBMCs), either unprocessed or enriched for specific subpopulations. The immune cells may also include monocytes, natural killer (NK) cells, NKT cells (a hybrid lymphoid population expressing both T cell and NK cell markers), neutrophils, or other functionally active leukocyte subsets. In certain implementations, the therapeutic cells may include mesenchymal stem cells (MSCs), which, although not classical immune cells, possess immunomodulatory, anti-inflammatory, and regenerative properties that can support antitumor activity and tissue repair. It is contemplated that combinations of these cell types may also be employed to achieve synergistic immune responses, particularly in tumors with heterogeneous microenvironments or variable immune infiltration profiles. In another embodiment, the immune cells administered to the subject exhibit enhanced biological performance as a result of microfluidic conditioning. For example, the immune cells may demonstrate significantly increased motility in confined or mechanically resistant tumor environments compared to untreated or conventionally cultured control cells. Tumor tissues often present dense extracellular matrix components and narrow interstitial spaces that impede immune cell migration. Cells conditioned in the described microfluidic device are exposed to mechanical cues, chemotactic gradients, or surface interactions that promote cytoskeletal adaptation, polarization, and increased deformability, thereby improving their ability to infiltrate and migrate within solid tumors.

[0157] The immune cells may also demonstrate altered expression of key immunoregulatory receptors. In particular, non-genetically engineered T cells produced using the device may exhibit decreased expression of programmed cell death protein 1 (PD-1), an inhibitory immune checkpoint receptor that dampens T cell activity in response to chronic antigen stimulation. Reduced PD- 1 expression may result in enhanced T cell activation, prolonged persistence in the tumor microenvironment, and improved cytotoxicity against cancer cells, without the need for genetic knockout or antibody-mediated checkpoint blockade. This intrinsic checkpoint modulation may lower the risk of systemic immune-related adverse events and reduce the complexity and cost of treatment.

[0158] As disclosed herein, the method of treatment using these non-genetically engineered immune cells offers several clinical advantages. By avoiding genetic modification, the cells retain their endogenous gene regulation, may present fewer off-target effects, and are potentially more acceptable from a regulatory and ethical standpoint. The enhanced motility and reduced expression of inhibitory receptors lead to increased therapeutic efficacy, characterized by improved tumor infiltration, greater on-target cytolytic activity, and resistance to exhaustion. Furthermore, these properties may allow for dose reductions, decreased manufacturing time, and improved patient safety by minimizing the risks of cytokine release syndrome or autoimmune toxicity.

[0159] The non-genetically engineered immune cells may be administered to the subject via intravenous infusion, intratumoral injection, or regional perfusion, depending on the tumor type and location. In some embodiments, the treatment may be administered in combination with additional therapeutic agents, such as immune checkpoint inhibitors, cytokines, monoclonal antibodies, chemotherapy, or radiation, in order to enhance antitumor responses or facilitate immune cell recruitment. The timing, dosage, and frequency of administration may be optimized based on patient response and the pharmacokinetics of cell persistence and activity in vivo. EXAMPLES

[0160] To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0161] EXAMPLE 1

[0162] In a first example, These systems have generally dealt with: 1) systematic investigation of T cell migration in response to fine-tuned extracellular cues, or 2) screening migration potential of T cell populations under various drug combinations for cancer therapy. On the other hand, the microfluidic platform described in our invention disclosure is designed and optimized for non-genetical engineering of T cells through transmigration in a bio-inspired blood vessel tumor interface. Other work aiming to engineer phenotype and functional capabilities of therapeutic T cells so far have utilized nanopatterned polyacrylamide gel substrates or 3D microporous gels. Employment of substrates with nanogroove patterns have been shown to enhance migration capabilities of T cells both in vitro and 3D tumor microenvironment. On the other hand, use of collagen gels with tunable viscoelastic properties has been shown to generate distinct phenotypes of T cells when activated within these 3D gel scaffolds. Fundamentally different from these reports, our invention aims to tune not only the migration capability but also immunological phenotype of therapeutic T cell products to overcome mechanical barriers and immunosuppressive environment of solid tumors typical of many cancers.

[0163] EXAMPLE 2

[0164] In a second example, the disclosed microfluidic technology incorporates two stacked channels: (1) A top channel with a 50 pm height (can be tuned between 20-200 pm) to enable high throughput adhesion of infused cells on the microporous membrane interface and (2) a bottom channel with a thickness of 1.1 mm (can be tuned between 0.25-5 mm) enabling chemokine loading and gradient formation, as well as collection and recovery of primed immune cells. The microporous membrane is functionalized with various adhesion molecules including but not limited to P-selectin, E-selectin, vascular cell adhesion molecule- 1 (VCAM-1), and intracellular adhesion molecule- 1 (ICAM-1). Chemokine cocktail includes but not limited to CCL2, CC13, CCL4, CCL5, CXCL9, and CXCL10. Immune cells are infused into the top channel at a wall shear stress between 0.25-2 dyne / cm2 and fresh chemokine solution is loaded into the bottom channel every 15-60 min. Microfluidic perfusion is run depending on the required number of cells are primed and collected from the bottom channels.

[0165] The presented technology achieves enhanced therapeutic T cell phenotype through microfluidic priming without the need for any genetic modification, thus ensuring safe and cost- effective solution.

[0166] For human scale clinical therapies, the described invention should be scaled to enable high throughput operation. Furthermore, for improved reliability and in line integration with the current cell manufacturing protocols, automation of the microfluidic device should be achieved.

[0167] Eastly, it should be understood that while the present disclosure has been provided in detail with respect to certain illustrative and specific aspects thereof, it should not be considered limited to such, as numerous modifications are possible without departing from the broad spirit and scope of the present disclosure as defined in the appended claims.

[0168] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0169] REFERENCES Adu-Berchie, K., Liu, Y., Zhang, D. K. Y., Freedman, B. R., Brockman, J. M., Vining, K. H., Nerger, B. A., Garmilla, A. &Mooney, D. J. Generation of functionally distinct T- cell populations by altering the viscoelasticity of their extracellular matrix. Nat Biomed Eng 7, 1374-1391, (2023). de Haan, L., Suijker, J., van Roey, R., Berges, N., Petrova, E., Queiroz, K., Strijker, W., Olivier, T., Poeschke, O., Garg, S. &van den Broek, L. J. A Microfluidic 3D Endothelium-on-a-Chip Model to Study Transendothelial Migration of T Cells in Health and Disease. Int J Mol Sci 22, (2021). Kwasny, D., Kiilerich-Pedersen, K., Moresco, J., Dimaki, M., Rozlosnik, N. &Svendsen, W. E. Microfluidic device to study cell transmigration under physiological shear stress conditions. Biomed Microdevices 13, 899-907, (2011). Lee, J. Y., Buzney, C. D., Poznansky, M. C. &Sackstein, R. Dynamic alterations in chemokine gradients induce transendothelial shuttling of human T cells under physiologic shear conditions. J Leukoc Biol 86, 1285-1294, (2009). Lee, J., Kim, S. E., Moon, D. &Doh, J. A multilayered blood vessel / tumor tissue chip to investigate T cell infiltration into solid tumor tissues. Lab Chip 21, 2142-2152, (2021). Pavesi, A., Tan, A. T., Koh, S., Chia, A., Colombo, M., Antonecchia, E., Miccolis, C., Ceccarello, E., Adriani, G., Raimondi, M. T., Kamm, R. D. &Bertoletti, A. A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors. JCI Insight 2, (2017). Ren, X., Getschman, A. E., Hwang, S., Volkman, B. F., Klonisch, T., Levin, D., Zhao, M., Santos, S., Liu, S., Cheng, J. &Lin, F. Investigations on T cell transmigration in a human skin-on-chip (SoC) model. Lab Chip 21, 1527-1539, (2021). Schreiber, T. H., Shinder, V., Cain, D. W., Alon, R. &Sackstein, R. Shear flowdependent integration of apical and subendothelial chemokines in T-cell transmigration: implications for locomotion and the multistep paradigm. Blood 109, 1381-1386, (2007). Tabdanov, E. D., Rodriguez-Merced, N. J., Cartagena-Rivera, A. X., Puram, V. V., Callaway, M. K., Ensminger, E. A., Pomeroy, E. J., Yamamoto, K., Lahr, W. S., Webber, B. R., Moriarity, B. S., Zhovmer, A. S. &Provenzano, P. P. Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments. Nat Communl2, 2815, (2021).

Claims

CLAIMSWhat is claimed is:

1. A microfluidic device for priming, collection and recovery of immune cells comprising: a. two separate fluidic channels: i. a first channel, wherein the first channel is infused with immune cells, ii. a second channel, wherein the second channel acts as a collection reservoir for transmigrated cells, and; b. a microporous membrane interface, present between the first channel and the second channel, wherein the microporous membrane is functionalized on the first channel surface with adhesion molecules.

2. The microfluidic device of claim 1, wherein the first channel has a width between 10-1000 pm.

3. The microfluidic device of any one of claims 1-2, wherein the first channel has a breadth of 50 pm.

4. The microfluidic device of claim 1, wherein the second channel has a width between 0.1-10 mm.

5. The microfluidic device of any one of claim 1 or claim 4, wherein the second channel has a breadth of 1.1 mm.

6. The microfluidic device of any one of claims 1-5, wherein the adhesion molecules are selected from a group including but not limited to P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule-1 (ICAM- 1).

7. The microfluidic device of claim 1, wherein the second channel comprises at least one transmigration enhancer.

8. The microfluidic device of claim 1, wherein the transmigration enhancer forms a decreasing gradient towards the first channel.

9. The microfluidic device of any one of claims 1-7, wherein the transmigration enhancer comprises a protein, peptide, or small molecule.

10. The microfluidic device of claim 9, wherein the transmigration enhancer comprises at least one chemokine.

11. The microfluidic device of claim 10, wherein the at least one chemokine comprises CCL2, CCL3, CCL4, CCL5, CXCL9, and / or CXCL10.

12. The microfluidic device of any one of claims 1-11, wherein the transmigration enhancer is loaded at intervals or continuously.

13. The microfluidic device of claim 1, wherein the immune cells are infused into the first channel.

14. The microfluidic device of claim 13, wherein the immune cells are introduced without flow and allowed to settle and / or adhere passively.

15. The microfluidic device of claim 13, wherein the immune cells are flowed through the first channel.

16. The microfluidic device of claim 15, wherein the cells are flowed through at a wall shear stress between 0.1-10 dyne / cm2.

17. The microfluidic device of any one of claims 1-16, wherein immune cells comprise CD4 T-cells, CD8 T-cells, bulk CD3 cells, bulk or purified peripheral blood mononuclear cells, CAR T cells, monocytes, NK-cells, NK-T-cells, neutrophils or mesenchymal stem cells.

18. A method of making a non-genetically engineered immune cell using the microfluidic device of claims 1-17.

19. The method of claim 18, wherein the non-genetically engineered immune cell is selected from a group comprising CD4 T-cells, CD8 T-cells, bulk CD3 cells, bulk or purified peripheral blood mononuclear cells, CAR T cells, monocytes, NK-cells, NK-T-cells, neutrophils or mesenchymal stem cells.

20. The method of any one of claims 18-19, wherein the non-genetically engineered immune cell comprises increased motility in confined tumor environments compared to a control.

21. The method of any one of claims 18-20, wherein the non-genetically engineered immune cell is a T-cell.

22. The method of claim 21, wherein the non-genetically engineered T-cell comprises decreased PD-1 expression compared to a control.

23. A method of treatment of a subject with cancer, comprising administering a non- genetically engineered immune cell to the subject, wherein the cell is produced using the microfluidic device of any of claims 1-17.

24. The method of claim 23, wherein the non-genetically engineered immune cell is selected from a group comprising CD4 T-cells, CD8 T-cells, bulk CD3 cells, bulk or purified peripheral blood mononuclear cells, CAR T cells, monocytes, NK-cells, NK-T-cells, neutrophils or mesenchymal stem cells.

25. The method of any one of claims 23-24, wherein the non-genetically engineered immune cell comprises increased motility in confined tumor environments compared to a control.

26. The method of any one of claims 23-25, wherein the non-genetically engineered immune cell is a T-cell.

27. The method of claim 26, wherein the non-genetically engineered T-cell comprises decreased PD-1 expression compared to a control.

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