T cell activation and expansion method based on adaptive liquid-liquid interface and application thereof

Abstract

The application belongs to the technical field of cell culture, and discloses a T cell activation and expansion method based on self-adaptive liquid-liquid interface and application thereof. The application provides application of a liquid-liquid interface culture system in T cell activation and expansion, wherein the liquid-liquid interface comprises a two-dimensional liquid-liquid interface and a liquid-liquid interface microsphere, both of which can form a protein nanofilm at an oil-water interface, and the protein nanofilm is used to load molecules providing T cell receptor activation signals and / or molecules providing T cell costimulatory signals, so as to construct a self-adaptive T cell activation interface with dynamic molecular display capability. Compared with a traditional rigid solid culture dish and a magnetic bead system, the liquid-liquid interface has fluidity, molecular migration and interface responsiveness, and is more conducive to T cell receptor aggregation, formation of an immunological synapse-like structure and synergistic integration of T cell receptor activation signals and costimulatory signals.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for T cell activation and expansion based on an adaptive liquid-liquid interface and its application. Background Technology

[0002] In vitro activation and expansion of T cells are core steps in tumor immunotherapy, adoptive cell therapy, CAR-T cell preparation, and basic immunological research. Currently, in vitro activation and expansion of T cells commonly utilize commercially available activation systems, with mainstream products broadly categorized into three types: magnetic microsphere-based activation materials, soluble nanomatrix / polymer-based activation materials, and non-magnetic bead-based soluble antibody complex activation reagents. These commercial systems primarily achieve T cell activation and expansion by providing CD3 and CD28 signals, but they differ in material morphology, interfacial properties, ligand fluidity, mechanical properties, and biomimetic immune activation capabilities.

[0003] Among them, Thermo Fisher Scientific's Dynabeads™ Human / Mouse T-Activator CD3 / CD28 is a typical magnetic microsphere activation material. Essentially, it consists of inert supercis-polystyrene microspheres with surface-coupled anti-CD3 and anti-CD28 antibodies; taking the mouse product as an example, the particle diameter is approximately 4.5 μm. This type of material has advantages such as high activation efficiency, mature operation, and good reproducibility. However, because it is a rigid magnetic plastic microsphere, it usually requires post-processing using a magnetic separation device. Furthermore, its structure, elasticity, and size are not matched to cells, resulting in relatively fixed antibody distribution, making flexible control and personalization impossible. The interface lacks membrane-like fluidity and adaptability, thus still differing from natural antigen-presenting cells.

[0004] Miltenyi Biotec's T Cell TransAct™ is a soluble polymer nanomatrix activation material that mediates T cell activation through CD3 / CD28 agonists. The product description emphasizes its polymer nanomatrix structure, which allows for gentle and efficient activation of resting T cells. Furthermore, Miltenyi utilizes Anti-Biotin MACSiBead particles loaded with biotinylated antibodies to mimic antigen-presenting cells. While these systems offer good biocompatibility and process adaptability, they still rely on artificially constructed immobilized or semi-immobilized stimulatory units and cannot simultaneously achieve the membrane-like fluidity, rearrangement capability, and geometrical biomimicry of natural APCs.

[0005] STEMCELL Technologies' ImmunoCult™ Human CD3 / CD28 or CD3 / CD28 / CD2 TCell Activator belongs to the class of magnetic bead-free soluble antibody complex activators. It can activate and expand T cells under conditions without magnetic beads or feeder cells, and is typically used in conjunction with serum-free / heterogeneous component-free expansion media. This type of system avoids the magnetic bead separation step and has good biocompatibility; however, it essentially still provides stimulation through soluble antibody complexes and lacks a biomimetic activation interface that can mimic the granular contact and dynamic interfacial rearrangement of natural APCs.

[0006] It can be seen that existing magnetic beads and solid planar systems are all high-rigidity materials, whose structure, elasticity, and size are mismatched with cells, making them unable to be flexibly controlled and customized, and unable to effectively simulate the dynamic formation process of innate immune synapses. Existing methods often focus more on amplification efficiency and ease of operation, while paying insufficient attention to the regulation of T cell fate by interfacial mechanical properties, ligand mobility, and dynamic immune synapse-like structures. Under conditions of high stiffness, immobilized ligand distribution, or continuous strong stimulation, T cells may be more likely to differentiate into terminal effector states or exhaustion-like states, which is not conducive to maintaining a high proportion of memory stem cell-like T cells or central memory T cells. Existing biomimetic systems are often complex, which is not conducive to standardization and promotion. There is a lack of a culture platform that is both operationally feasible and can regulate T cell activation, amplification, and functional fate through interfacial properties.

[0007] The physiological activation of T cells in vivo depends not only on chemical signals such as CD3 / CD28, but also on dynamic processes such as receptor-ligand aggregation at the contact interface, TCR microcluster formation, cytoskeleton traction, and interfacial feedback. Natural antigen-presenting cell membranes possess fluidity, flexibility, and molecular rearrangement capabilities, an environment that traditional solid planes or magnetic bead surfaces struggle to provide. Furthermore, while existing technologies can effectively expand T cells, they often focus more on increasing quantity, neglecting cell quality, particularly the proportion of central memory T cells, the proportion of memory stem cell-like T cells, in vivo persistence, and anti-tumor durability. Existing research and application experience indicate that T cell products with higher proportions of memory stem cell-like T cells and central memory T cells generally exhibit superior proliferative potential, post-infusion persistence, and therapeutic efficacy. Therefore, improving the memory phenotype while ensuring activation and expansion is a crucial technical challenge in this field.

[0008] In recent years, liquid-liquid interface culture systems have attracted attention as a dynamic biomimetic material system. This system consists of an upper aqueous phase and a lower oil phase, forming an interface with interfacial tension and a certain degree of fluidity. Existing liquid-liquid interface technologies have been used in stem cell culture, droplet microcarrier construction, and other fields, demonstrating the significant cell regulation potential of the liquid-liquid interface itself. In particular, droplet microcarrier technology shows that by dispersing oil-phase droplets in an aqueous phase, uniformly sized spherical liquid-liquid interface structures can be formed, and their size can be controlled through methods such as printing, microfluidics, droplet addition, emulsification, or oscillation. However, existing publicly available technologies lack a platform-based approach that unifies two-dimensional liquid-liquid interfaces and liquid-liquid interface microspheres under the same technical principle, specifically for T cell activation and expansion. Especially lacking is a technical solution for constructing protein nanofilms on the surface of two-dimensional liquid-liquid interfaces or liquid-liquid interface microspheres, and loading T cell receptor activation signaling molecules and / or T cell co-stimulatory signaling molecules onto these protein nanofilms, thereby mimicking the contact between natural APCs and T cells, receptor aggregation, co-stimulatory synergy, formation of immune synapse-like structures, and promoting the formation of memory phenotypes.

[0009] This invention is hereby proposed. Summary of the Invention

[0010] Given that existing T-cell activation systems primarily rely on rigid solid interfaces or magnetic bead interfaces, struggle to mimic the dynamic membrane interface characteristics of natural APCs in contact with T cells, have a low proportion of memory stem cell-like T cells and central memory T cells, and require further improvement in in vivo persistence and anti-tumor function, this invention provides a T-cell activation and expansion platform and method based on an adaptive liquid-liquid interface. The platform comprises both a two-dimensional liquid-liquid interface and liquid-liquid interface microspheres. The two-dimensional liquid-liquid interface provides a planar, APC-like stimulation interface, while the liquid-liquid interface microspheres provide a particulate, APC-like stimulation interface; both are essentially dynamic stimulation systems centered on a liquid-liquid interface. This platform enables T cells to achieve effective activation and expansion while simultaneously increasing the proportion of memory stem cell-like T cells and central memory T cells, and enhancing in vitro killing function and in vivo anti-tumor activity.

[0011] The first objective of this invention is to provide an application of a liquid-liquid interface culture system in the activation and expansion of T cells.

[0012] A second aspect of the present invention is to provide a method for T cell activation and expansion.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an application of a liquid-liquid interface culture system in T cell activation and expansion; wherein the liquid-liquid interface comprises a two-dimensional liquid-liquid interface or liquid-liquid interface microspheres. The two-dimensional liquid-liquid interface is constructed from an upper aqueous phase and a lower oil phase. A protein nanofilm is formed at the interface of the two-dimensional liquid-liquid interface. The protein nanofilm is loaded with molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulation signals. The liquid-liquid interface microspheres are formed by dispersing an oil phase in an aqueous phase, and a protein nanofilm is formed at the oil-water interface of the liquid-liquid interface microspheres. The protein nanofilm is loaded with molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulation signals.

[0014] Compared to existing rigid magnetic beads, the liquid-liquid interface microspheres of this invention exhibit stronger dynamic biomimicry; compared to simple two-dimensional interfaces, the microsphere form is more suitable for simulating the contact between natural antigen-presenting cells and T cells. By simulating the membrane-like dynamic contact of antigen-presenting cells through an oil / water interface with fluidity, molecular mobility, and interfacial responsiveness, it can be used to regulate T cell activation, expansion, memory differentiation, and anti-tumor functions.

[0015] In some embodiments of the present invention, the molecule providing the T cell receptor activation signal includes at least one of anti-CD3 antibody, anti-TCR antibody, peptide-MHC complex, CD3 binding molecule, TCR binding molecule or functional fragment thereof.

[0016] In some embodiments of the present invention, the final concentration of the molecule providing the T-cell receptor activation signal in the aqueous phase is 0.1-50 μg / mL; further, 1-30 μg / mL; even further, 5-30 μg / mL, such as any value of 5, 10, 15, 20, 25 or 30 μg / mL or a range formed by any two of them.

[0017] In some embodiments of the present invention, the molecule providing T cell co-stimulation signal includes at least one of anti-CD28 antibody, CD80, CD86, or 4-1BBL.

[0018] In some embodiments of the present invention, the final concentration of the molecule providing the T cell co-stimulatory signal in the aqueous phase is 0.1-50 μg / mL; further, 1-30 μg / mL; even further, 5-30 μg / mL, such as any value of 5, 10, 15, 20, 25 or 30 μg / mL or a range formed by any two of them.

[0019] Under the combined stimulation of T cell receptor activation signaling molecules and T cell co-stimulatory signaling molecules, the liquid-liquid interface culture system can provide a dynamic stimulation interface with fluidity and rearrangement, which is conducive to T cell receptor aggregation, formation of immune synapse-like structures and integration of co-stimulatory signals, thereby promoting T cell activation and expansion, and helping to increase the proportion of memory stem cell-like T cells and / or central memory T cells, thus enhancing anti-tumor function.

[0020] In some embodiments of the present invention, the concentration ratio of the T cell receptor activation signaling molecule to the T cell co-stimulatory signaling molecule is 1:(1-2).

[0021] In some embodiments of the present invention, the liquid-liquid interface culture system further includes auxiliary interface functional molecules.

[0022] The auxiliary interface functional molecules include molecules that can enhance effective contact, receptor aggregation, interfacial dwell time, or the formation of immune synapse-like structures between T cells and the liquid-liquid interface, such as anti-CD2 antibodies, anti-LFA-1 antibodies, ICAM-1, CD58, and proteins with specific functional peptides. In this invention, the role of these molecules is not simply to promote adhesion, but to assist in mimicking the interfacial contact and signaling processes between natural APCs and T cells.

[0023] In some embodiments of the present invention, the auxiliary interface functional molecule includes at least one of anti-CD2 antibody, anti-LFA-1 antibody, ICAM-1, cell adhesion molecule, extracellular matrix protein or serum albumin.

[0024] In some embodiments of the present invention, the cell adhesion molecule or extracellular matrix protein includes a protein, polypeptide or derivative thereof containing an RGD sequence, and more preferably includes at least one of fibronectin, hydrin, osteopontin, fibrinogen, laminin, collagen or gelatin.

[0025] In some embodiments of the present invention, the serum albumin includes at least one of bovine serum albumin, human serum albumin, or recombinant serum albumin.

[0026] In some embodiments of the present invention, the oil phase comprises a fluorinated liquid that is immiscible or poorly miscible with the aqueous phase and is suitable for cell culture or biological interface construction.

[0027] In some embodiments of the present invention, the fluorinated liquid is at least one of FC-40, FC70, HFE-7500 or HFE-7100.

[0028] In some embodiments of the present invention, the volume ratio of the aqueous phase to the oil phase is (20-1):1, such as any ratio of 1:1, 2:1, 5:1, 10:1, 15:1 or 20:1 or any range of both.

[0029] The physical and biochemical properties of the interface can be further modulated by adjusting the composition of the oil phase, the components of auxiliary interface functional molecules, the density and ratio of molecules that activate T cell receptors and provide T cell co-stimulatory signals.

[0030] In some embodiments of the present invention, the aqueous phase is an aqueous system.

[0031] In some embodiments of the present invention, the aqueous system includes at least one of water, PBS buffer, physiological saline, cell culture medium, protein-containing aqueous solution, antibody-containing aqueous solution, or cytokine-containing aqueous solution.

[0032] In some embodiments of the present invention, the method for forming the microspheres includes at least one of printing, microfluidics, drop addition, emulsification, oscillation, stirring, membrane emulsification, or ultrasonic emulsification.

[0033] In some embodiments of the present invention, the diameter of the microspheres is 5-200 μm; further, 7-50 μm; even further, 8-25 μm; and most preferably 8-15 μm.

[0034] The liquid-liquid interface microspheres in this invention are preferably sized similarly to natural antigen-presenting cells, which can simulate the contact mode between antigen-presenting cells and T cells, thereby simulating the activation of T cells. This is closer to the dynamic interface contact in the natural T cell activation process than traditional rigid magnetic beads.

[0035] In some embodiments of the present invention, when the liquid-liquid interface is a two-dimensional liquid-liquid interface, the liquid-liquid interface culture system is prepared by the following method: an oil phase and an aqueous phase containing molecules that provide T cell receptor activation signals and / or provide T cell co-stimulatory signals are sequentially added to a container, and incubated to form an initial interface layer loaded with signal molecules at the oil-water interface; then a solution containing serum albumin and / or other auxiliary interface functional molecules is added, and incubated again to form a protein nanofilm; the aqueous phase is partially or completely replaced with a culture medium containing cytokines to obtain the two-dimensional liquid-liquid interface culture system.

[0036] In some preferred embodiments of the present invention, the incubation time is 2-12 hours; more specifically, 4-8 hours.

[0037] In some preferred embodiments of the present invention, the re-incubation time is 1-5 hours; more specifically, 1-2 hours.

[0038] In some preferred embodiments of the present invention, the final concentration of the auxiliary interface functional molecule in the solution is 0.1-10 mg / mL; further, it is 0.1-5 mg / mL, such as any value or a range formed by any two of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mg / mL.

[0039] In some preferred embodiments of the present invention, the container includes containers commonly used for cell culture, which can be selected according to the actual situation.

[0040] In some embodiments of the present invention, when the liquid-liquid interface is a liquid-liquid interface microsphere, the liquid-liquid interface culture system is prepared by the following method: dispersing an oil phase into an aqueous phase by means of a printing device, a microfluidic device, dropwise addition, emulsification, oscillation, stirring, membrane emulsification, or ultrasonic emulsification to form liquid-liquid interface microspheres in the aqueous phase; wherein, the aqueous phase contains molecules that provide T cell receptor activation signals and / or provide T cell co-stimulatory signals, causing the molecules to self-assemble at the oil-water interface of the liquid-liquid interface microspheres to form a protein nanofilm; subsequently, the formed liquid-liquid interface microspheres are transferred to a solution containing serum albumin and / or other auxiliary interface functional molecules for incubation, so that serum albumin and / or other auxiliary interface functional molecules are adsorbed onto the protein nanofilm; the obtained liquid-liquid interface microspheres are transferred to a culture medium containing cytokines to obtain the liquid-liquid interface microsphere culture system.

[0041] In some preferred embodiments of the present invention, the incubation time is 1-5 hours; more specifically, 1-2 hours.

[0042] Compared with existing methods, the present invention obtains an interface loaded with T cell activation signaling molecules by means of liquid-liquid interface self-assembly, avoiding complex and cumbersome chemical modification processes.

[0043] The liquid-liquid interface culture system of this invention can increase the ratio of memory stem cell-like T cells and central memory T cells, and enhance the in vitro killing function and / or in vivo anti-tumor function of T cells.

[0044] This invention constructs a two-dimensional liquid-liquid interface system consisting of an upper aqueous phase and a lower oil phase in a cell culture container, or constructs a liquid-liquid interface microsphere system formed by oil phase droplets dispersed in an aqueous phase. T cell activation signaling molecules and / or co-stimulatory signaling molecules are provided in the interface region, enabling T cells to acquire activation signals during contact with the liquid-liquid interface. The liquid-liquid interface system includes two basic morphologies: a two-dimensional liquid-liquid interface and liquid-liquid interface microspheres. The two-dimensional liquid-liquid interface is a planar dynamic stimulation interface; the liquid-liquid interface microspheres are particulate dynamic stimulation interfaces, preferably with a size close to the cellular scale of natural antigen-presenting cells, thereby simulating the local contact, receptor aggregation, co-stimulation, and immune synapse-like structure formation processes between antigen-presenting cells and T cells.

[0045] Unlike traditional rigid magnetic beads or solid planes, the liquid-liquid interface in this invention possesses a certain degree of fluidity, molecular migration, and cellular responsiveness, enabling local molecular rearrangement and interfacial responses during T cell contact, spreading, membrane protrusion activation, receptor binding, and cytoskeleton traction. When T cells contact the liquid-liquid interface, the TCR / CD3 complex, CD28 co-stimulatory receptors, and other membrane receptors on the T cell membrane surface interact with anti-CD3 antibodies, anti-CD28 antibodies, and interfacial accessory proteins at the interface. During cell-interface contact, T cell receptor binding, changes in membrane tension, cytoskeleton traction, and the fluidity of the interfacial protein layer collectively drive local migration of interfacial molecules, resulting in relative enrichment of anti-CD3 antibodies and / or anti-CD28 antibodies at the T cell edge, cell-interface contact ring, receptor aggregation region, or immune synapse-like contact region.

[0046] The molecular movement and rearrangement enhance local receptor-ligand interactions on T cells, increase the aggregation of TCR / CD3 signaling molecules, and promote the synergistic input of CD3-related signals and CD28 co-stimulatory signals in the same contact area. Therefore, the liquid-liquid interface of this invention provides T cells with a dynamic stimulatory microenvironment close to the natural antigen-presenting cell membrane, which is beneficial for the formation of immune synapse-like structures, early T cell activation, subsequent expansion, and memory phenotype formation. Thus, the T cell states include resting state, early activation state, spreading state, receptor aggregation state, cytoskeleton rearrangement or traction state, and different differentiation states. The dynamic response includes, but is not limited to, the local enrichment, speckled aggregation, or rearrangement of anti-CD3 antibodies, anti-CD28 antibodies, serum albumin, or other interfacial helper molecules in the cell contact area.

[0047] A second aspect of the present invention provides a method for T cell activation and expansion comprising contacting T cells with a liquid-liquid interface containing molecules described in the first aspect of the present invention that provide T cell receptor activation signals and / or provide T cell co-stimulatory signals, and culturing them in a culture medium containing cytokines.

[0048] In some embodiments of the present invention, when the liquid-liquid interface is a two-dimensional liquid-liquid interface, the method includes the following steps: partially or completely replacing the aqueous phase with a culture medium containing cytokines, seeding T cells into the aqueous phase of the culture medium containing cytokines, so that the T cells come into contact with the two-dimensional liquid-liquid interface and are cultured; or When the liquid-liquid interface is a liquid-liquid interface microsphere, the method includes the following steps: adding the liquid-liquid interface microsphere to a culture medium containing cytokines, seeding T cells into the culture medium containing the liquid-liquid interface microsphere, and bringing the T cells into contact with the liquid-liquid interface microsphere and culturing them.

[0049] In some embodiments of the present invention, the cytokines include interleukin-type cytokines.

[0050] In some embodiments of the present invention, the interleukin cytokines include at least one of IL-2, IL-7, IL-15 or IL-21.

[0051] In some preferred embodiments of the present invention, the cytokines include IL-2, or a combination of IL-7 and IL-15, or a combination of IL-7, IL-15 and IL-21.

[0052] In some embodiments of the present invention, the final concentration of the cytokine in the culture medium is 10-300 IU / mL; further, it is 10-100 IU / mL, such as any value or range formed by any combination of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 IU / mL.

[0053] In some embodiments of the present invention, the final concentration of the cytokine in the culture medium is 0.1-100 ng / mL by mass concentration; further, it is 1-50 ng / mL, such as any value of 1, 5, 10, 20, 30, 40 or 50 ng / mL or a range formed by any two of them.

[0054] In some embodiments of the present invention, the seeding density of the T cells is (0.1-100) × 10⁻¹⁰. 6 cells / mL; further, (0.5-10) × 10 6 cells / mL.

[0055] In some embodiments of the present invention, the culture temperature is 37°C.

[0056] In some embodiments of the present invention, the culture time is 2-14 days; more specifically, 2-10 days, such as any value or a range formed by any two of 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0057] In some embodiments of the present invention, the culture process involves changing the medium using a culture medium containing cytokines.

[0058] In some embodiments of the present invention, the fluid replacement is a half-volume fluid replacement.

[0059] In some embodiments of the present invention, the T cells include peripheral blood T cells, umbilical cord blood T cells, tumor-infiltrating T cells, αβ T cells, γδ T cells, and CD4+ T cells. + T cells, CD8 + T cells, natural T cells, and / or genetically modified T cells.

[0060] In some embodiments of the present invention, the gene-modified T cell contains an introduced exogenous gene.

[0061] In some embodiments of the present invention, the genetically modified T cell comprises any one or more of the following: a gene segment encoding a chimeric antigen receptor (CAR), a gene segment encoding a T cell receptor that specifically recognizes tumor antigens, and a gene segment encoding a fusion protein of the T cell receptor.

[0062] This invention transforms T cell activation from a "rigid solid surface / magnetic bead surface" to an "adaptive liquid-liquid interface," which is conducive to the formation of a high proportion of memory stem cell-like T cells and central memory T cells, and enhances in vitro killing function and in vivo anti-tumor activity.

[0063] In some embodiments of the present invention, the culture medium includes common culture media for culturing T cells, such as a complete culture formed by adding supplementary components required for T cell culture to a basal medium. These supplementary components include serum or serum substitutes, glutamine, antibiotics, interleukins, and other components that promote T cell survival, activation, and proliferation. A conventional selection can be made according to the specific circumstances.

[0064] The beneficial effects of this invention are: This invention provides an application of a liquid-liquid interface culture system in T cell activation and expansion. The liquid-liquid interface comprises a two-dimensional liquid-liquid interface and liquid-liquid interface microspheres, both of which can form protein nanofilms at the oil-water interface. These protein nanofilms are then loaded with molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulatory signals, thereby constructing an adaptive T cell activation interface with dynamic molecular display capabilities. Compared to traditional rigid solid culture dishes and magnetic bead systems, the liquid-liquid interface of this invention exhibits fluidity, molecular mobility, and interfacial responsiveness, which is more conducive to T cell receptor aggregation, the formation of immune synapse-like structures, and the synergistic integration of T cell receptor activation signals and co-stimulatory signals. Without altering the basic stimulus framework of molecules providing T cell receptor activation signals / providing T cell co-stimulatory signals (such as CD3 / CD28), the proportion of memory stem cell-like T cells and central memory T cells can be increased, while reducing the tendency for T cell exhaustion.

[0065] This invention can achieve synergistic regulation of the physical, biochemical, geometric properties and molecular display capabilities of the interface by adjusting the composition of the oil phase, the composition of the aqueous phase, the interfacial protein components, the types, densities and ratios of auxiliary interfacial functional molecules, T cell receptor activation signaling molecules and co-stimulatory signaling molecules, as well as the size of the liquid-liquid interfacial microspheres. Compared with traditional solid planar or magnetic bead systems, this invention has better overall controllability, biomimicry and potential for personalized customization.

[0066] This invention provides a method for T cell activation and expansion, which enables T cells to be effectively activated and expanded while increasing the proportion of memory stem cell-like T cells and central memory T cells, and enhancing in vitro killing function and in vivo anti-tumor activity. Experimental results show that T cells obtained using the method provided by this invention exhibit excellent function in in vitro killing experiments and in vivo anti-tumor experiments. Attached Figure Description

[0067] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the platform for activating T cells at the liquid-liquid interface according to the present invention.

[0068] Figure 2 The image shows a phase-contrast micrograph of mouse T cells in their activated state at a two-dimensional liquid-liquid interface (i.e., Example 1), with a scale bar of 500 μm.

[0069] Figure 3 The image shows a phase-contrast micrograph of mouse T cells in the vicinity of the liquid-liquid interface microsphere (i.e., Example 2), with a scale bar of 25 μm.

[0070] Figure 4The image shows the dynamic enrichment of antibodies at the two-dimensional liquid-liquid interface and the formation of Jurkat T cell immune synapse-like structures in Example 1. The scale bar is 5 μm. The cell nucleus is marked in blue, CD3ζ in green, the cytoskeleton in red, and antibody molecules at the liquid-liquid interface in purple.

[0071] Figure 5 This figure compares the proportion of TCM in mouse T cells on day 3 under different activation conditions. In the figure, express P <0.001, Control represents the unstimulated blank group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0072] Figure 6 This figure shows a comparison of CD69 expression in mouse T cells 24 h after different activation conditions. In the figure, ns represents no significant difference, Control represents the unstimulated blank group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0073] Figure 7 This figure shows a comparison of CD25 expression in mouse T cells 48 hours after different activation conditions. In the figure, represent P <0.05, represent P <0.01, Control represents the unstimulated blank group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0074] Figure 8 This figure compares the CFSE proliferation efficiency of mouse T cells on day 3 under different activation conditions. In the figure, represent P <0.05, represent P <0.001, Control represents the unstimulated blank group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0075] Figure 9This figure shows a comparison of the fold expansion of mouse T cells on day 5 under different activation conditions. In the figure, ns represents no significant difference, Control represents the unstimulated control group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0076] Figure 10 This figure compares the proportion of Treg cells in mouse T cells on day 7 under different activation conditions. In the figure, represent P <0.05, represent P <0.01, Control represents the unstimulated blank group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0077] Figure 11 This figure compares the proportion of exhausted cells in mouse T cells on day 10 under different activation conditions. In the figure, represent P <0.01, represent P <0.0001, Control represents the unstimulated blank group, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, TCP represents the culture plate coated with anti-CD3 / CD28 antibody group, and ALI represents the two-dimensional liquid-liquid interface group.

[0078] Figure 12 This image shows the in vitro killing effect of OT-1 T cells expanded using the method in Example 1 on B16-OVA cells. In the image, represent P <0.001, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, and ALI represents the two-dimensional liquid-liquid interface group.

[0079] Figure 13 This image shows the in vivo antitumor effect of OT-I T cells expanded using the method in Example 1 in a B16-OVA subcutaneous tumor-bearing model. In the image, represent P <0.001, Dynabeads represents the anti-CD3 / CD28 magnetic bead group, and ALI represents the two-dimensional liquid-liquid interface group. Detailed Implementation

[0080] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0081] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0082] Terminology Explanation: An adaptive liquid interface (ALI) is a liquid-liquid interface consisting of an aqueous phase and an oil phase that is immiscible or poorly miscible with the aqueous phase. The adaptive liquid interface includes two forms: two-dimensional liquid-liquid interfaces and liquid-liquid interface microspheres. This interface possesses certain fluidity, molecular mobility, interfacial responsiveness, and cell contact responsiveness. Unlike the relatively fixed stimuli molecules on the surface of traditional solid culture plates or magnetic beads, the liquid-liquid interface of this invention can undergo a certain degree of interfacial molecular movement, local rearrangement, and spatial redistribution based on the contact state of T cells, receptor binding state, cell spread, membrane protrusion function, and cytoskeleton traction state, thereby providing T cells with a dynamic stimuli microenvironment different from that of rigid solid surfaces.

[0083] The "adaptability" described in this invention is not limited to changes in the overall morphology of the interface, but also includes changes in the local enrichment, speckled aggregation, rearrangement, or spatial distribution of protein nanofilms, T cell receptor activation signaling molecules, T cell co-stimulation signaling molecules, serum albumin, or other auxiliary interface functional molecules at the interface according to the T cell state.

[0084] Two-dimensional liquid-liquid interface: refers to a relatively continuous planar liquid-liquid interface formed by an upper aqueous phase and a lower oil phase in a culture vessel. This two-dimensional liquid-liquid interface can be obtained by contacting and incubating the upper aqueous phase and the lower oil phase, forming a protein nanofilm at the oil-water interface. The protein nanofilm may be loaded with molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulatory signals, and may further include serum albumin or other auxiliary interface functional molecules. T cells are mainly located in the upper aqueous phase and interact with the protein nanofilm at the interface between the two phases. This configuration is consistent with the liquid-liquid interface culture system structure in the applicant's published patents, both being a two-phase interface system of "upper aqueous phase + lower oil phase".

[0085] Liquid droplet microspheres: These are spherical or near-spherical liquid-liquid interface structures formed by dispersing oil droplets in an aqueous phase. A protein nanofilm forms at the oil-water interface, and these microspheres can be loaded with molecules that provide T-cell receptor activation signals, molecules that provide T-cell co-stimulatory signals, and / or auxiliary interfacial functional molecules. The size of these liquid-liquid interface microspheres is preferably close to that of natural antigen-presenting cells, used to simulate the granular contact, receptor aggregation, co-stimulatory synergy, and interfacial signal transduction processes between antigen-presenting cells and T cells.

[0086] APC mimicry function: refers to the function of mimicking the contact, receptor aggregation, co-stimulatory synergy, and immune synapse-like structure formation processes between natural antigen-presenting cells (APCs) and T cells through two-dimensional liquid-liquid interfaces or liquid-liquid interface microspheres with cell-scale size, surface capable of loading molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulatory signals, and with fluidity and adaptability.

[0087] Anti-CD3 antibody: refers to an antibody that can bind to CD3 molecules or CD3 complexes on the surface of T cells, and is used to provide T cell receptor activation signals to T cells.

[0088] Anti-CD28 antibody: refers to an antibody that can bind to the CD28 molecule on the surface of T cells and is used to provide co-stimulatory signals to T cells.

[0089] BSA, Bovine Serum Albumin: In this invention, it can be used as an interface aid, a component of protein nanofilms, or an interface stabilizing component to assist in the formation, stabilization, or regulation of the distribution of interfacial molecules at the oil-water interface.

[0090] FC40: A transparent and colorless fluorocarbon oil that can be used as the lower oil phase in liquid-liquid interface systems. TCM cells: T central memory cells, typically exhibiting strong proliferative potential, good long-term survival, and persistence in vivo. In mouse systems, they are often identified through CD44-positive and CD62L-positive populations. In human T cell systems, they can be identified through a combination of markers such as CCR7, CD45RA, CD45RO, and CD62L.

[0091] TSCM cells, or T stem cell memory cells, are generally considered to have stronger self-renewal potential and better long-term persistence. They are identified by a combination of biomarkers, including CD45RA, CCR7, CD62L, CD27, CD28, CD95, Sca-1, CD122, and CD127.

[0092] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0093] Example 1 A method for T cell activation and expansion based on FC40 liquid-liquid interface (illustration shown) Figure 1 (As shown), including the following steps: (1) Select an appropriate cell culture container as the culture system carrier. Here, a 96-well cell culture plate is selected as the culture system carrier. (2) Add 100 μL of FC40 to the container as the lower oil phase; (3) Prepare the upper aqueous phase, which includes an aqueous solution of anti-CD3 antibody (Biolegend, 100340) and anti-CD28 antibody (Biolegend, 102116) with a total concentration of 5 μg / mL, and the antibody concentration ratio is 1:1; (4) Slowly add 150 μL of the upper aqueous phase to the lower oil phase and incubate overnight at 4°C. Add 2 mg / mL BSA aqueous solution by half-volume replacement (i.e., remove about 1 / 2 volume of the upper aqueous phase and add an equal volume of BSA aqueous solution), and continue incubation at 37°C and 5% CO2 for 2 h to form a stable protein nanofilm (i.e., liquid-liquid interface protein film) at the liquid-liquid interface. Then, replace the upper aqueous phase with T cell culture medium containing a final concentration of 30 IU / mL IL-2 by half-volume replacement. The T cell culture medium is RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 2 mM L-glutamine or GlutaMAX, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES and 50 μM β-mercaptoethanol; (5) The isolated mouse T cells were added to the upper aqueous phase, with a cell density of 1×10⁻⁶ cells / cells. 6 cells / mL, cultured at 37℃ and 5% CO2; (6) During the culture process, the culture system is replaced with half the volume of medium every 1-2 days, that is, about 1 / 2 volume of supernatant culture medium is removed from the culture system and an equal volume of fresh T cell culture medium is added to maintain the nutrients and cytokine concentration required for cell growth. (7) After 48 hours of culture, place the pipette tip close to the protein membrane at the liquid-liquid interface, avoiding puncture or significant disturbance to the lower oil phase. Slowly aspirate and release the upper culture medium, gently pipetting 10 times to detach the T cells attached to the protein membrane at the liquid-liquid interface. Then collect the upper cell suspension, centrifuge at 300 ×g for 5 min, discard the supernatant, and resuspend in PBS or T cell culture medium to obtain T cells activated at the liquid-liquid interface, which can be used for subsequent flow cytometry phenotype, in vitro killing, and in vivo antitumor function evaluation.

[0094] In this embodiment, FC40 is preferably used as the lower oil phase. The inventors have found that under these conditions, T cell activation can be effectively promoted, and a higher proportion of central memory T cells can be obtained.

[0095] After culture, the two-dimensional liquid-liquid interface region was observed and photographed using an inverted phase-contrast microscope. The results showed that mouse T cells underwent significant morphological changes and aggregation at the two-dimensional liquid-liquid interface, manifested as increased cell volume, increased intercellular contact, and the formation of local cell clusters, suggesting that this two-dimensional liquid-liquid interface can effectively induce mouse T cell activation. Phase-contrast micrographs of the activated state of mouse T cells at the aforementioned two-dimensional liquid-liquid interface are shown below. Figure 2 As shown.

[0096] Example 2 A method for T cell activation and expansion based on liquid-liquid interface microspheres includes the following steps: (1) Prepare an aqueous solution containing anti-CD3 antibody and anti-CD28 antibody. The anti-CD3 antibody (Biolegend, 100340) and anti-CD28 antibody (Biolegend, 102116) aqueous solution have an antibody concentration ratio of 1:1. Use this aqueous solution as the aqueous phase for later use. (2) FC40 was selected as the oil phase, and the volume ratio of the oil phase to the aqueous phase was 1:20. The oil phase was prepared into dispersed droplets by printing device, microfluidic device, drop addition or emulsification method (for specific methods, refer to "Seemann R, Brinkmann M, Pfohl T, Herminghaus S. Droplet based microfluidics. Rep Prog Phys. 2012;75(1):016601. doi:10.1088 / 0034-4885 / 75 / 1 / 016601IF: 20.7 Q1"). The droplets were incubated for 1 hour to form liquid-liquid interface microspheres in the aqueous phase. The surface of the liquid-liquid interface microspheres was formed by the liquid-liquid interface formed by the oil phase and the aqueous phase. Anti-CD3 antibody and anti-CD28 antibody could be adsorbed, assembled or enriched in the interface region to form an interface protein layer loaded with T cell activation signals. The diameter of the liquid-liquid interface microspheres is 8-15 μm to simulate the granular contact scale between natural antigen-presenting cells and T cells. (3) The formed liquid-liquid interface microspheres were transferred to an aqueous system containing 2 mg / mL BSA aqueous solution and incubated at 37 °C for 2 h to further form a stable interface layer on the surface of the microspheres and to locate the activating molecules in the interface region. (4) Collect the prepared liquid-liquid interface microspheres and add them to T cell culture medium containing 30 IU / mL IL-2. Co-culture them with mouse T cells in 96-well cell culture plates. The T cell density is 1×10⁻⁶. 6 The microspheres at the liquid-liquid interface were cultured at 37°C and 5% CO2 to achieve T cell activation and expansion. (5) During the culture process, the culture system is replaced with half the volume of medium every 1-2 days, that is, about 1 / 2 volume of supernatant culture medium is removed from the culture system and an equal volume of fresh T cell culture medium is added to maintain the nutrients and cytokine concentration required for cell growth. (6) After 48 hours of culture, T cells were collected by centrifugation or other gentle separation methods for later use (which can be used for subsequent flow cytometry phenotype, in vitro killing and in vivo anti-tumor function evaluation).

[0097] In this embodiment, the size of the liquid-liquid interface microspheres is preferably close to that of natural antigen-presenting cells to simulate the granular contact, local receptor aggregation, and dynamic activation process between antigen-presenting cells and T cells. Compared with two-dimensional liquid-liquid interfaces, liquid-liquid interface microspheres are more advantageous in simulating the activation effect of natural antigen-presenting cells on T cells in terms of geometric scale, curved surface contact mode, and granular stimulation mode. Mouse T cells can undergo local aggregation, cell volume increase, and cell cluster formation near the liquid-liquid interface microspheres. Phase contrast microscopy images of their activation-related morphological states are shown below. Figure 3 As shown.

[0098] Comparative Example 1 This comparative example provides a method for T cell activation and expansion using commercially available anti-CD3 / CD28 magnetic microspheres, including the following steps: (1) Take the isolated mouse T cells and resuspend them in complete T cell culture medium. The complete T cell culture medium is RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 2 mM L-glutamine or GlutaMAX, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES and 50 μM β-mercaptoethanol. Add 30 IU / mL IL-2 to the culture medium as needed for the experiment; (2) Mouse T cells were seeded into 96-well cell culture plates at a cell density of 1×10⁻⁶ cells / well. 6 -1.5×10 6 cells / mL, preferably 1×10⁻⁶ 6 cells / mL; (3) Take Thermo Fisher Scientific's Dynabeads™ Mouse T-Activator CD3 / CD28 magnetic microspheres and mix them thoroughly before use to form a homogeneous suspension. Following the manufacturer's instructions, the magnetic microspheres can be pre-washed as needed for the experiment and resuspended in T-cell complete culture medium. (4) Add Dynabeads™ Mouse T-Activator CD3 / CD28 magnetic microspheres to the T cell suspension so that the ratio of magnetic microspheres to T cells is 1:1; (5) The culture system was placed at 37℃ and 5% CO2 to induce T cell activation and expansion. During the culture process, cell morphology, cell size, cell aggregation and culture medium color changes were observed daily, and cells were counted periodically; (6) When the cell density exceeds 2.5 × 10 6When the cell count reaches 0.5 × 10⁶ cells / mL, or the culture medium turns noticeably yellow, readjust the cell count to 0.5 × 10⁶ cells / mL. 6 -1×10 6 The cells / mL were increased, and fresh T-cell complete culture medium containing 30 IU / mL IL-2 was added for further culture.

[0099] (7) After 48 hours of culture, the cell suspension was transferred to centrifuge tubes and placed on a magnetic separator for 1-2 minutes to allow the magnetic microspheres to accumulate on one side of the tube wall under the influence of the magnetic field. The supernatant containing T cells was then carefully aspirated and transferred to a new centrifuge tube to remove the magnetic microspheres. The obtained T cells can be used for subsequent flow cytometry phenotypic analysis, cell proliferation detection, in vitro killing experiments, and in vivo antitumor function evaluation.

[0100] In this comparative example, the Dynabeads™ Mouse T-Activator CD3 / CD28 consists of commercially available magnetic microspheres with surface-conjugated anti-CD3 and anti-CD28 antibodies, effectively providing TCR stimulation and co-stimulatory signals to induce T cell activation and expansion. This system is mature, has high activation efficiency, and good reproducibility; however, it is essentially a rigid magnetic plastic microsphere, resulting in a relatively fixed antibody distribution. The stimulation interface lacks membrane-like fluidity, rearrangement, and cellular responsiveness, and post-culture processing typically requires a magnetic separation device. Therefore, this system differs from the T-cell activation platform of this invention, which features a dynamic liquid-liquid interface and an adaptive interface layer.

[0101] Comparative Example 2 This comparative example provides a method for T cell activation and expansion based on a commercial polymer nanomatrix, including the following steps: (1) Take isolated human peripheral blood mononuclear cells or purified human T cells and resuspend the cells in a culture medium suitable for T cell expansion. The complete T cell culture medium is RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 2 mM L-glutamine or GlutaMAX, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES and 50 μM β-mercaptoethanol. Add 30 IU / mL IL-2 to the culture medium as needed for the experiment; (2) Human T cells are seeded into cell culture plates, cell culture flasks, or cell culture bags, with a cell density preferably of 0.5 × 10⁻⁶. 6 -2×10 6 cells / mL, more preferably 1×10⁻⁶ 6 cells / mL; (3) Take Miltenyi Biotec's T Cell TransAct™ activating reagent, mix gently before use, and avoid vigorous shaking to prevent reagent aggregation or bubble formation. The T Cell TransAct™ is a colloidal polymer nanomatrix structure with CD3 and CD28 agonists coupled to it, which can provide TCR stimulation signals and co-stimulation signals to T cells; (4) Add T Cell TransAct™ to the T cell suspension according to the manufacturer’s instructions or recommended dosage, and mix gently to ensure that the activating agent is evenly dispersed in the culture system; (5) The culture system was placed at 37℃ and 5% CO2 to induce T cell activation. During the culture process, cell morphology, cell aggregation and culture medium color changes were observed, and the medium was replenished, changed or expanded according to the cell proliferation. (6) After culturing for 2-3 days, count the cells and add fresh T cell culture medium and cytokines according to the cell density to maintain the cells within a suitable expansion density range. During subsequent culture, count the cells and add culture medium every 2-3 days until the required number of cells for the experiment is reached; (7) After 48 hours of culture, excess activating reagents can be removed by centrifugation, medium washing, or other gentle treatments, and T cells can be collected. The obtained T cells can be used for subsequent flow cytometry phenotypic analysis, cell proliferation detection, in vitro killing experiments, gene transduction experiments, and functional evaluation.

[0102] In this comparative example, T Cell TransAct™ delivers CD3 / CD28 activation signals via a polymer nanomatrix, inducing T cell activation and expansion under conditions without feeder cells or antigens. This system offers advantages such as ease of use, good biocompatibility, and adaptability to cell engineering processes. However, its stimulation units primarily rely on artificial polymer nanomatrixes, lacking the dynamic protein layer formed at the oil-water / liquid-liquid interface, the mobility of interfacial molecules, and the cellular traction response. It cannot simultaneously mimic the membrane-like fluidity, local rearrangement ability, and micron-sized granular contact structure of natural antigen-presenting cells. Therefore, this system differs significantly from the adaptive liquid-liquid interface microsphere activation platform described in this invention.

[0103] Comparative Example 3 This comparative example provides a method for T cell activation and expansion using a commercially available soluble antibody complex, comprising the following steps: (1) Take isolated human peripheral blood mononuclear cells or purified human T cells and resuspend the cells in a culture medium suitable for human T cell culture and expansion. The culture medium can be ImmunoCult™-XF T Cell Expansion Medium. Add 30 IU / mL IL-2 to the culture medium as needed for the experiment; (2) Human T cells were seeded into cell culture plates, cell culture flasks or cell culture bags at a cell density of 1×10⁻⁶ cells / mL. 6 cells / mL; (3) Take STEMCELL Technologies' ImmunoCult™ Human CD3 / CD28 T Cell Activator or ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator and mix gently before use. The activator is a soluble antibody complex that can bind to and cross-link CD3 and CD28 on the surface of T cells, thereby providing the initial stimulation signal and co-stimulation signal required for T cell activation; (4) Add ImmunoCult™ T Cell Activator to the T cell suspension according to the manufacturer's recommended dosage. For example, when using ImmunoCult™ Human CD3 / CD28 T Cell Activator, add it to the cell suspension at a ratio of 25 μL / mL and mix gently to ensure that the soluble antibody complex is evenly distributed in the culture system; (5) The culture system is placed at 37℃ and 5% CO2 to activate T cells. The activation culture time is preferably 1-3 days, more preferably 2 days. During the culture process, cell morphology, cell aggregation and changes in the color of the culture medium are observed; (6) After 2 days of culture, count the viable cells and replenish the culture medium containing cytokines with fresh T cells according to the cell density. During subsequent expansion, count the cells and replenish the culture medium every 2-3 days to maintain a suitable cell density and cytokine concentration. Culture can continue until the required number of cells for the experiment is obtained, preferably for 7-12 days; (7) After culture, T cells are collected by centrifugation and washed with fresh culture medium or buffer to remove residual soluble antibody complexes and culture medium components. The obtained T cells can be used for subsequent flow cytometry phenotypic analysis, cell proliferation detection, in vitro killing experiments, gene transduction experiments, and functional evaluation.

[0104] In this comparative example, ImmunoCult™ T Cell Activator is a soluble antibody complex-based T cell activation reagent. This system avoids the magnetic bead separation step, is easy to operate, and has good adaptability to cell culture processes. However, this system mainly relies on the cross-linking of soluble antibody complexes with T cell surface receptors, lacking micron-sized particulate contact structures, and also lacking the membrane-like fluidity, molecular mobility, local accumulation capacity, and cellular responsive rearrangement characteristics of a liquid-liquid interface. Therefore, this system is difficult to simulate the dynamic interfacial contact process between natural antigen-presenting cells and T cells, and is fundamentally different from the liquid-liquid interface microsphere activation platform described in this invention.

[0105] Comparative Example 4 This comparative example provides a method for T cell activation and expansion using anti-CD3 and anti-CD28 antibodies immobilized on the surface of a culture plate, including the following steps: (1) Preparation of antibody coating solution. Take anti-mouse CD3 antibody and anti-mouse CD28 antibody, dilute with sterile PBS or carbonate buffer to obtain a coating solution containing anti-CD3 antibody and anti-CD28 antibody. The total concentration of the anti-CD3 antibody and anti-CD28 antibody can be 10 μg / mL, and the concentration ratio of the two is 1:1; (2) Add the above antibody coating solution to a sterile cell culture plate, ensuring it fully covers the bottom of the plate. For example, in a 96-well plate, add 100 μL of antibody coating solution to each well. Then, incubate the plate overnight at 4°C or at 37°C for 1-3 h to allow the anti-CD3 antibody and anti-CD28 antibody to adsorb and immobilize on the surface of the plate. (3) After coating, remove the antibody coating solution from the wells and gently wash the culture plate 1-3 times with sterile PBS to remove unbound free antibodies. (4) Take the isolated mouse spleen T cells and resuspend them in complete T cell culture medium. The complete T cell culture medium is RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 2 mM L-glutamine or GlutaMAX, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES and 50 μM β-mercaptoethanol. Recombinant mouse IL-2 is added to the culture medium as needed to make the final IL-2 concentration 30 IU / mL; (5) Mouse T cells were seeded into the culture plates coated with anti-CD3 and anti-CD28 antibodies, at a cell density of 0.5 × 10⁻⁶ cells / mL. 6 -2×10 6 cells / mL, preferably 1×10⁻⁶6 cells / mL. After inoculation, gently shake the culture plate to ensure the cells are evenly distributed on the coated surface; (6) The culture system was placed at 37℃ and 5% CO2 to induce T cell activation and expansion. During the culture process, cell morphology, cell aggregation, changes in culture medium color, and changes in cell density were observed daily. Depending on the cell proliferation, half of the medium was changed every 1-2 days, i.e., about 1 / 2 volume of supernatant culture medium was aspirated and an equal volume of fresh T cell complete culture medium containing 30 IU / mL IL-2 was added. (7) After culture, collect suspended and weakly attached T cells by gentle pipetting or pipetting, and transfer the cell suspension to centrifuge tubes. After low-speed centrifugation, discard the supernatant and wash 1–2 times with PBS or complete T cell culture medium. The obtained T cells can be used for subsequent flow cytometry phenotypic analysis, cell proliferation detection, in vitro killing experiments and other functional evaluations.

[0106] In this comparative example, anti-CD3 and anti-CD28 antibodies were immobilized on the surface of a TCP culture plate via physical adsorption, thereby providing TCR stimulation and co-stimulatory signals to T cells. This system is simple to operate and low in cost, and can serve as a control for traditional two-dimensional rigid surface activation methods. However, the culture plate surface is a rigid solid interface, with relatively fixed antibody distribution, lacking membrane-like fluidity, local migration ability, and cellular responsive rearrangement characteristics. Furthermore, its planar structure makes it difficult to simulate the granular curved surface contact between natural antigen-presenting cells and T cells. Therefore, this system differs significantly from the adaptive liquid-liquid interface described in this invention in terms of interfacial mechanics, molecular mobility, and biomimetic activation mechanism.

[0107] Effect Example 1. Confocal imaging analysis of local antibody enrichment and immune synapse-like rearrangement in the T cell contact region induced by adaptive liquid-liquid interface. The interface antibody was labeled with Alexa Fluor 647, T cell CD3ζ was labeled with Alexa Fluor 488, T cell F-actin was labeled with Alexa Fluor 594, and cell nuclei were stained with DAPI. The specific procedure is as follows: A two-dimensional liquid-liquid interface was constructed in a 96-well plate according to the method in Example 1. Alexa Fluor 647 fluorescent secondary antibody (abcam, ab150119) was added using a half-displacement method and incubated at room temperature for 2 hours. After washing three times with T cell culture medium, the isolated mouse spleen T cells were resuspended in complete T cell culture medium and seeded onto the aforementioned two-dimensional adaptive liquid-liquid interface, allowing the T cells to settle naturally and come into contact with the liquid-liquid interface. The culture system was incubated at 37°C and 5% CO2 for 3 hours. After incubation, some of the supernatant culture medium was gently aspirated to avoid disturbing the oil-water interface and the T cells on the interface. 4% paraformaldehyde fixative was slowly added to the culture system, and fixation was performed at room temperature for 10-20 minutes. After fixation, the cells were gently washed three times with PBS. Cells were then permeabilized with PBS containing 0.1%–0.3% Triton X-100 and incubated at room temperature for 5–15 min. After permeabilization, the cells were washed with PBS and blocked with PBS containing 1% BSA, incubated at room temperature for 30 min. After blocking, anti-CD3ζ primary antibody (Beyotime, AF0096) was added and incubated at room temperature for 2 h. After washing, Alexa Fluor 488-labeled fluorescent secondary antibody (abcam, 150077) was added and incubated at room temperature in the dark for 30–60 min to label T cell CD3ζ. Then, Alexa Fluor 594-labeled phalloidin (abcam, ab176757) was added and incubated at room temperature in the dark for 40 min to label T cell F-actin. DAPI staining solution (Beyotime, C1005) was then added and incubated at room temperature in the dark for 5 min to label cell nuclei. After staining, the cells were gently washed with PBS, keeping the samples moist with PBS or anti-fluorescence quenching mounting solution.

[0108] T cells were cultured using the method described in Example 1. After the T cells came into contact with the liquid-liquid interface, fluorescence images of each channel were acquired using a confocal microscope.

[0109] Test results as follows Figure 4As shown, the liquid-liquid interface antibody was labeled with Alexa Fluor 647, T cell CD3ζ with Alexa Fluor 488, and F-actin with Alexa Fluor 594. Cell nuclei were stained with DAPI. Confocal imaging results showed that at 0 h, the interface antibody signal was relatively uniform overall, with T cell CD3ζ and F-actin mainly distributed peripherally. After 3 h of activation, T cells significantly spread at the liquid-liquid interface, CD3ζ was enhanced in the cell-interface contact area, F-actin showed peripheral rearrangement, and the interface antibody showed significant local enrichment near the T cell contact area. These results suggest that antibodies in the adaptive liquid-liquid interface can undergo local redistribution during T cell contact and activation, and are spatially correlated with T cell receptor aggregation and cytoskeleton rearrangement.

[0110] 2. Flow cytometry-based detection of TCM-like memory T cell phenotype The isolated mouse spleen T cells were seeded into the following groups: unstimulated control group (i.e., the unstimulated control group did not contain anti-CD3 antibody, anti-CD28 antibody, anti-CD3 / anti-CD28 magnetic beads, culture plate coated with antibody, or adaptive liquid-liquid interface stimulation material, and the T cells were cultured only in complete medium containing IL-2), anti-CD3 / anti-CD28 magnetic bead group (i.e., the method of Comparative Example 1), culture plate coated with anti-CD3 / CD28 antibody group (i.e., the method of Comparative Example 4), and adaptive liquid-liquid interface group (i.e., the method of Example 1). The cells were cultured at 37°C and 5% CO2. After 72 hours of culture, the cells were collected, washed 1-2 times with PBS, and then flow cytometry antibodies CD44-APC (Biolegend, 103027) and CD62L-PE (Biolegend, 161203) were added. The cells were incubated at 4°C in the dark for 30 minutes, washed, and then analyzed using a flow cytometer (Beckman CytoFLEX). For mouse T cells, CD3... + CD44 and CD62L were detected in the population, with CD44 being the most prominent. + CD62L + As TCM-like cells, the proportion of the above-mentioned memory-like T cells in different activation systems was compared to evaluate the regulatory effect of the adaptive liquid-liquid interface platform of the present invention (i.e., the method of Example 1) on T cell memory differentiation and cell quality.

[0111] The comparison results of the proportion of TCM in mouse T cells after 72 h under different activation conditions are as follows: Figure 5 As shown, the above results indicate that the adaptive liquid-liquid interface group significantly improves CD44 + CD62L +The proportion of TCM-like T cells was significantly better than that of the anti-CD3 / anti-CD28 magnetic bead group and the culture plate coated with anti-CD3 / CD28 antibody group.

[0112] 3. Flow cytometry analysis of the expression of CD69, an early activation marker, and CD25, a mid-stage activation marker, in mouse T cells under different activation conditions. Mouse T cells were cultured according to the grouping method described above. After 24 h of culture, T cells were collected from the unstimulated blank group, the anti-CD3 / anti-CD28 magnetic bead group, the culture plate coated with anti-CD3 / CD28 antibody group, and the adaptive liquid-liquid interface group, respectively. The collection method was the same as above, avoiding vigorous pipetting or excessive centrifugation to prevent affecting cell state. After collection, an appropriate amount of cells was added to a flow cytometry tube, and flow cytometry antibody CD69-FITC (Biolegend, 104505) was added for surface staining. After mixing, the cells were incubated at 4°C in the dark for 30 min. After staining, the cells were washed 1-2 times with PBS or flow cytometry staining buffer to remove unbound antibodies, and the cells were resuspended before analysis. The results of CD69 expression in mouse T cells 24 h after different activation conditions are shown below. Figure 6 As shown.

[0113] Mouse T cells were cultured according to the grouping method described above. After 48 h of culture, T cells were collected from the unstimulated blank group, the anti-CD3 / anti-CD28 magnetic bead group, the culture plate coated with anti-CD3 / CD28 antibody group, and the adaptive liquid-liquid interface group, respectively. The collection method was the same as above, avoiding vigorous pipetting or excessive centrifugation to prevent affecting cell state. After collection, an appropriate amount of cells was added to a flow cytometry tube, and flow cytometry antibody CD25-FITC (Biolegend, 102005) was added for surface staining. After mixing, the cells were incubated at 4°C in the dark for 30 min. After staining, the cells were washed 1-2 times with PBS or flow cytometry staining buffer to remove unbound antibodies, and the cells were resuspended before analysis. The results of CD25 expression in mouse T cells after 48 h under different activation conditions are shown below. Figure 7 As shown.

[0114] The above results indicate that the expression of CD69 and CD25 in mouse T cells under different activation conditions shows that the adaptive liquid-liquid interface group can induce an early activation level of CD69 comparable to that of the anti-CD3 / CD28 magnetic bead group and the culture plate coated with antibody group, while further increasing the proportion of CD25 positive cells. This suggests that the adaptive liquid-liquid interface described in this invention can enhance the response of T cells to IL-2-related proliferation signals while ensuring effective activation of T cells, and is beneficial to subsequent T cell expansion and functional maintenance.

[0115] 4. Detection of mouse T cell expansion capacity under different activation conditions The proliferation capacity of T cells under different activation conditions was detected using the CFSE fluorescence tracer dilution method. The isolated mouse spleen T cells were washed twice with PBS or serum-free medium to remove residual serum components, and the cell concentration was adjusted to 1×10⁻⁶. 6 The cells / mL were then added with CFSE dye (Beyotime, C0051) to a final concentration of 0.5-5 μM, preferably 1 μM, and incubated at 37°C in the dark for 10 min to allow CFSE to enter the cells and bind to intracellular proteins.

[0116] Immediately after staining, add 5-10 volumes of serum-containing complete T-cell culture medium to stop the staining reaction and incubate at room temperature or on ice in the dark for 5 minutes. Then, centrifuge to collect the cells and wash 2-3 times with complete culture medium or PBS to remove free CFSE dye. After washing, resuspend the CFSE-labeled T cells in complete T-cell culture medium for later use.

[0117] CFSE-labeled T cells were seeded into different activation conditions, including an unstimulated blank group, an anti-CD3 / CD28 magnetic bead group, a culture plate coated with anti-CD3 / CD28 antibody group, and an adaptive liquid-liquid interface group. The unstimulated blank group did not contain anti-CD3 antibody, anti-CD28 antibody, or other activation materials; the anti-CD3 / CD28 magnetic bead group was processed according to the methods described in the corresponding comparative examples; the culture plate coated with anti-CD3 / CD28 antibody group was processed according to the methods described in the corresponding comparative examples; and the adaptive liquid-liquid interface group was processed according to the method described in Example 1. The seeding density of T cells in each group was 1 × 10⁻⁶. 6 The concentration of cells / mL was increased, and recombinant mouse IL-2 was added to the culture system to a final concentration of 30 IU / mL. All groups were cultured at 37℃ and 5% CO2.

[0118] During culture, cells can be collected on day 3 and washed 1-2 times with PBS or flow cytometry staining buffer. For the anti-CD3 / CD28 magnetic bead group, magnetic separation is used to remove the magnetic beads before collection. For the culture plate coated with antibody group, suspension and weakly attached T cells are collected by gentle pipetting. For the adaptive liquid-liquid interface group, T cells are collected by gentle pipetting, low-speed centrifugation or other gentle methods and the interface material is removed.

[0119] Using unstimulated cells or initially CFSE-labeled cells as a high-fluorescence, non-dividing control, the proportion of CFSE-low fluorescent cells, the proportion of dividing cells, and the division index or proliferation index were calculated based on the degree of decrease in CFSE fluorescence intensity. CFSE fluorescence was diluted with each cell division; an increase in the proportion of CFSE-low fluorescent cells indicated T cell proliferation. By comparing the CFSE dilution levels under different activation conditions, the promoting effect of the adaptive liquid-liquid interface of this invention on T cell proliferation and expansion could be evaluated.

[0120] Comparison of CFSE proliferation efficiency of mouse T cells on day 3 under different activation conditions: Figure 8 As shown, the CFSE fluorescence of T cells in the unstimulated blank group remained mainly in the high fluorescence region, with no obvious generation-wise dilution peaks, indicating that T cells did not proliferate significantly. The anti-CD3 / CD28 magnetic bead group, the culture plate coated with anti-CD3 / CD28 antibody group, and the adaptive liquid-liquid interface group all showed decreased CFSE fluorescence intensity and multi-generational peaks, indicating that the above activation systems can all induce T cell proliferation. Compared with the traditional culture plate coated with antibody group, the adaptive liquid-liquid interface group showed more significant CFSE fluorescence dilution and a significantly higher proportion of proliferating cells; at the same time, the proliferation rate of the adaptive liquid-liquid interface group was also higher than that of the anti-CD3 / CD28 magnetic bead group. These results indicate that the adaptive liquid-liquid interface described in this invention can effectively induce T cell proliferation and is superior to the traditional rigid planar coated antibody system in promoting T cell expansion, and is no less than or superior to commercial magnetic bead activation systems.

[0121] At the start of culture, the initial number of T cells inoculated in each group was recorded as the cell inoculation number on day 0. On day 5, cells from each group were collected. For the anti-CD3 / CD28 magnetic bead group, the magnetic beads were removed using a magnetic separation device; for the antibody-coated culture plate group, suspended and weakly attached T cells were collected by gentle pipetting; for the adaptive liquid-liquid interface group, T cells were collected and interface material was removed using gentle pipetting, low-speed centrifugation, or other gentle methods. The collected cells were washed 1-2 times with PBS or complete T cell culture medium.

[0122] The number of viable cells in each group was detected using a cell counting chamber. The cell expansion fold was calculated based on the total number of viable T cells obtained on day 5 and the initial number of T cells inoculated on day 0. The formula was: Expansion fold = Total number of viable T cells on day 5 / Initial number of T cells inoculated on day 0. A comparison of the expansion fold of mouse T cells on day 5 under different activation conditions is shown in the figure below. Figure 9 As shown in the figure. These results were used to evaluate the effect of the adaptive liquid-liquid interface of this invention on the in vitro expansion capacity of T cells.

[0123] The graph shows a comparison of the fold expansion of mouse T cells on day 5 under different activation conditions. Figure 9As shown, after culturing under different activation conditions for 5 days, the unstimulated blank group showed a lower fold increase in T cell proliferation, indicating that T cells are unlikely to proliferate significantly in the absence of effective activation signals. The anti-CD3 / CD28 magnetic bead group, the culture plate coated with anti-CD3 / CD28 antibody group, and the adaptive liquid-liquid interface group all induced significant T cell proliferation in mice. The fold increase in the adaptive liquid-liquid interface group was similar to that of the anti-CD3 / CD28 magnetic bead group and the culture plate coated with antibody group, and statistical analysis showed no significant difference. These results indicate that the adaptive liquid-liquid interface described in this invention can achieve T cell proliferation effects comparable to the traditional CD3 / CD28 activation system. Combined with the aforementioned TCM-like cell ratio detection results, this indicates that this platform, while maintaining effective proliferation capacity, is more conducive to improving the memory-like phenotype and cell quality of proliferated T cells.

[0124] 5. Detection of immunosuppressive and exhaustion-like phenotypes of mouse T cells under different activation conditions using flow cytometry. Mouse T cells were cultured according to the grouping method described above. After 7 days of culture, T cells were collected from the unstimulated control group, the anti-CD3 / anti-CD28 magnetic bead group, the culture plate coated with anti-CD3 / CD28 antibody group, and the adaptive liquid-liquid interface group. The collection method was the same as above, avoiding vigorous pipetting or excessive centrifugation to prevent affecting cell state. After collection, an appropriate amount of cells was washed 1-2 times with PBS or flow cytometry staining buffer, followed by Treg phenotype detection. An appropriate amount of cells was added to a flow cytometry tube, and flow cytometry antibodies for surface markers were added for staining. The flow cytometry antibodies were CD4-FITC (Biolegend, 100405) and CD25-APC (Biolegend, 102007). After thorough mixing, the cells were incubated at 4°C in the dark for 30 min. After staining, the cells were washed 1-2 times with flow cytometry staining buffer. Subsequently, the cells were fixed and permeabilized using Foxp3 / transcription factor flow cytometry fixation and permeabilization buffer (Invitrogen, 00-5523-00). After fixation and perforation, Foxp3-PE flow cytometry antibody (Biolegend, 126403) was added, and the cells were incubated at 4°C in the dark for 60 min. After incubation, the cells were washed with perforation buffer or flow cytometry staining buffer, resuspended, and then analyzed by flow cytometry. The proportion of Treg cells in each group was determined by flow cytometry.

[0125] The figure shows a comparison of the proportion of Treg cells in mouse T cells on day 7 under different activation conditions. Figure 10 As shown, after culturing under different activation conditions for 7 days, CD25 in T cells of mice in each group... + FOXP3 +The proportion of Treg-like cells differed. The proportion of Treg cells in the unstimulated control group was low, suggesting it was at a basal level. The proportion of Treg cells was significantly increased in the anti-CD3 / CD28 magnetic bead group and the culture plate coated with anti-CD3 / CD28 antibody group, indicating that the traditional CD3 / CD28 stimulation system, while inducing T cell activation and expansion, may be accompanied by a certain degree of increase in Treg-like immunosuppressive phenotypes. In contrast, the proportion of Treg cells in the adaptive liquid-liquid interface group was lower than that in the anti-CD3 / CD28 magnetic bead group and the culture plate coated with antibody group, especially significantly lower. These results indicate that the adaptive liquid-liquid interface described in this invention can effectively activate and expand T cells while reducing the formation of Treg-like immunosuppressive phenotypes, which is beneficial for improving the functional quality of expanded T cells.

[0126] Mouse T cells were cultured according to the grouping method described above. After 10 days of culture, T cells were collected from the unstimulated control group, the anti-CD3 / anti-CD28 magnetic bead group, the culture plate coated with anti-CD3 / CD28 antibody group, and the adaptive liquid-liquid interface group. Collection methods were the same as above, minimizing the impact on cell state caused by vigorous pipetting or excessive centrifugation. For the anti-CD3 / anti-CD28 magnetic bead group, magnetic beads were removed before collection using magnetic separation; for the culture plate coated with antibody group, suspended and weakly attached T cells were collected by gentle pipetting; for the adaptive liquid-liquid interface group, T cells were collected and interface material was removed using gentle pipetting, low-speed centrifugation, or other gentle methods.

[0127] After collection, take an appropriate amount of cells and wash them 1-2 times with PBS or flow cytometry staining buffer, followed by T cell exhaustion-like phenotype detection. Add an appropriate amount of cells to a flow cytometry tube, and add flow cytometry antibodies containing surface markers and exhaustion-related markers for staining. The flow cytometry antibodies include PD1-FITC flow cytometry antibody (Biolegend, 135213) and Tim3-PE flow cytometry antibody (Biolegend, 119715); after thorough mixing, incubate at 4°C in the dark for 30 min. After staining, wash 1-2 times with PBS or flow cytometry staining buffer, resuspend the cells, and then perform flow cytometry analysis.

[0128] The figure shows a comparison of the proportion of exhausted-like cells in mouse T cells on day 10 under different activation conditions. Figure 11As shown in the figure, the proportion of exhausted cells in the unstimulated control group was low, suggesting that the exhaustion-like phenotype in unstimulated T cells was at a basal level. The proportion of exhausted cells in the anti-CD3 / CD28 magnetic bead group was significantly increased, indicating that traditional strong magnetic bead stimulation may more easily induce an exhaustion-like phenotype in T cells during long-term culture. The proportion of exhausted cells in the culture plate coated with anti-CD3 / CD28 antibody group was also higher than that in the control group, suggesting that traditional rigid planar antibody stimulation can also be accompanied by a certain degree of exhaustion-like phenotype formation. In contrast, the proportion of exhausted cells in the adaptive liquid-liquid interface group was lower than that in the anti-CD3 / CD28 magnetic bead group and the culture plate coated with antibody group, especially significantly lower than the magnetic bead group and the culture plate coated with antibody group. These results indicate that the adaptive liquid-liquid interface described in this invention can reduce the formation of exhaustion-like T cells during long-term culture while maintaining effective T cell activation and expansion, which is beneficial for improving the functional state and cell quality of expanded T cells.

[0129] 6. In vitro tumor-killing function detection of T cells: target cell lysis analysis based on LDH release method The in vitro killing function of T cells obtained from different activation systems against B16-OVA melanoma cells was detected using the LDH release assay. The B16-OVA cells were self-constructed; the original B16 cells were obtained from the National Experimental Cell Resource Sharing Platform. Based on this, B16 cells were transfected and screened using a lentiviral system carrying the OVA (ovalbumin) gene, ultimately preparing a stable B16-OVA cell line expressing OVA protein. Detailed construction methods are referenced in "Eisel D, DasK, Dickes E, König R, Osen W, Eichmüller SB. Cognate Interaction With CD4+ TCells Instructs Tumor-Associated Macrophages to Acquire M1-Like Phenotype. Front Immunol. 2019;10:219. Published 2019 Feb 22." The study (doi:10.3389 / fimmu.2019.00219) used a stable cell line obtained by overexpressing the OVA gene in B16 mouse melanoma cells via lentivirus and then screening it. This cell line can be used as an OVA-positive target cell to detect the killing activity of OVA-specific OT-1 T cells (isolated from the spleen of 8-week-old OT-1 transgenic female mice, which were purchased from Guangdong Nanmo Biotechnology Co., Ltd.).

[0130] The day before the experiment, B16-OVA cells were digested, counted, and seeded into 96-well cell culture plates, with 1×10⁶ cells per well. 4Cells were cultured overnight at 37°C with 5% CO2, and an appropriate amount of complete culture medium was added to allow the target cells to adhere. On the day of detection, the culture medium in the wells was gently aspirated, and the cells were gently washed once with PBS or fresh test medium to remove dead cells and background LDH. Then, fresh test medium was added for later use.

[0131] OT-I T cells activated and expanded using anti-CD3 / CD28 magnetic beads and adaptive liquid-liquid interface groups were used as effector cells. Effector T cells were collected before detection. For the magnetic bead activation group, magnetic beads were removed using magnetic separation; for the adaptive liquid-liquid interface group, interface material was removed using a gentle method. Cells were then washed 2-3 times with PBS or detection medium and resuspended in fresh T cell culture medium or LDH-compatible detection medium for cell counting.

[0132] Effector T cells were added to 96-well plates containing B16-OVA target cells according to different effector-to-target ratios. The effector-to-target ratio could be set to 0.5:1, 1:1, 2:1, 5:1, and 10:1, with at least 3 replicates per group. After adding the effector T cells, the effector T cells and target cells were co-cultured at 37°C and 5% CO2 for 8 h.

[0133] The experiment also included the following control wells: spontaneous release wells for target cells (containing only B16-OVA target cells and culture medium); spontaneous release wells for effector cells (containing only effector T cells and culture medium, with the number of effector cells set according to different effector-to-target ratios); maximum release wells for target cells (containing B16-OVA target cells and the lysis buffer provided in the kit); background wells for culture medium (containing only culture medium); and experimental wells (containing both B16-OVA target cells and effector T cells). These controls were used to correct for spontaneous LDH release from target and effector cells and for background absorbance from the culture medium.

[0134] After co-culture, the 96-well plate was centrifuged at low speed to allow the cells to settle. The culture supernatant from each well was then carefully aspirated and transferred to a new 96-well assay plate. LDH reaction solution was added according to the LDH assay kit (Dojindo, CK12-05) instructions. The plate was incubated in the dark for approximately 30 minutes, followed by the addition of stop solution. The absorbance at 490 nm was then measured using a microplate reader. After subtracting the OD values ​​of the culture medium background wells from the OD values ​​of each well, the target cell killing rate is calculated using the following formula: Kill rate (%) = [OD value of experimental wells - OD value of spontaneous release from effector cells - OD value of spontaneous release from target cells] / [OD value of maximum release from target cells - OD value of spontaneous release from target cells] × 100%.

[0135] If, when the above method is used for detection, the target cell lysis rate gradually increases as the effector-to-target ratio increases, it indicates that the obtained T cells have in vitro anti-tumor killing activity.

[0136] Test results as follows Figure 12 As shown, the in vitro killing ability of OT-I T cells activated and expanded by the anti-CD3 / CD28 magnetic bead group and the adaptive liquid-liquid interface group against B16-OVA target cells was detected using the LDH release method. The results showed that as the effector-to-target ratio gradually increased, the lysis rate of B16-OVA cells by both groups of T cells gradually increased, exhibiting a significant effector-to-target ratio dependence, indicating that both activation and expansion methods can obtain T cells with antigen-specific tumor-killing activity. Further comparison showed that the adaptive liquid-liquid interface group generally exhibited tumor-killing ability comparable to or better than the anti-CD3 / CD28 magnetic bead group at different effector-to-target ratios, especially maintaining a good target cell lysis level under higher effector-to-target ratio conditions. These results indicate that the adaptive liquid-liquid interface described in this invention can not only achieve functional activation of T cells comparable to the traditional magnetic bead system, but also obtain effector T cells with good in vitro anti-tumor killing activity.

[0137] 7. Evaluation of in vivo antitumor activity of T cells based on the B16-OVA subcutaneous xenograft model To evaluate the in vivo antitumor function of T cells obtained through different activation systems, a B16-OVA subcutaneous xenograft mouse model was used. The B16-OVA cells were a stable cell line obtained by overexpressing the OVA gene in B16 mouse melanoma cells via lentivirus and then selecting them. This line could be used as OVA-positive tumor target cells to evaluate the in vivo antitumor activity of OVA-specific OT-I T cells.

[0138] Specifically, on day 0, B16-OVA melanoma cells in the logarithmic growth phase were digested, counted, and resuspended in sterile PBS or other suitable injection buffer. Subsequently, B16-OVA cells were subcutaneously inoculated into the right back or axillary region of C57BL / 6 mice, with each mouse receiving 1 × 102 cells. 6 One B16-OVA cell.

[0139] On day 5 post-tumor inoculation, once the tumors had formed and become palpable, tumor-bearing mice were randomly divided into different treatment groups, with 10 mice in each group. The treatment groups included a PBS blank control group, an anti-CD3 / CD28 magnetic bead-activated OT-1 T cell group, and an OT-1 T cell group adapted to liquid-liquid interface activation according to the present invention. The anti-CD3 / CD28 magnetic bead-activated OT-1 T cells were prepared according to the method described in Comparative Example 1; the adaptive liquid-liquid interface-activated OT-1 T cells were prepared according to the method described in Example 1.

[0140] Before T cell reinfusion, activated and expanded OT-IT T cells were collected from each group. For the anti-CD3 / CD28 magnetic bead activation group, residual magnetic beads were removed using magnetic separation; for the adaptive liquid-liquid interface activation group, interface materials were removed using gentle pipetting, low-speed centrifugation, or other suitable methods. Cells were then washed with sterile PBS and viable cell counts were performed. The resulting OT-IT cells were resuspended in sterile PBS, and the cell concentration was adjusted to 1 × 10⁶ cells per 100 μL of PBS. 6 One live T cell.

[0141] On day 5 post-tumor inoculation, each treatment group received a tail vein injection of 100 μL PBS or 1×10⁻⁶ PBS. 6 One anti-CD3 / CD28 magnetic bead activated OT-I T cell or 1×10 6 OT-I T cells were activated by an adaptive liquid-liquid interface. On day 10 post-tumor inoculation, a second PBS or T cell reinfusion was performed using the same dose and administration method.

[0142] During the experiment, the mice's general condition, weight changes, and tumor growth were observed regularly, and the long and short diameters of the tumors were measured using calipers. Tumor volume was calculated using the formula V = length × width. 2 The formula is 2 / 2, where length represents the major diameter of the tumor and width represents the minor diameter. According to the experimental design, tumor volume can be measured every two days, and tumor growth curves for each group can be plotted. When the tumor volume exceeds 1500 mm²... 3 If a mouse exhibits significant emaciation, reduced activity, tumor ulceration, weight loss exceeding ethical limits, or other animal ethical endpoints, the mouse shall be euthanized.

[0143] The antitumor effects of OT-I T cells obtained by different activation systems were evaluated by comparing tumor growth curves of the PBS blank control group, the anti-CD3 / CD28 magnetic bead activated OT-I T cell group, and the adaptive liquid-liquid interface activated OT-I T cell group.

[0144] Experimental results are as follows Figure 13As shown in the figure, compared with the PBS blank control group, both the anti-CD3 / CD28 magnetic bead activated OT-I T cell group and the adaptive liquid-liquid interface activated OT-I T cell group could delay the growth of B16-OVA tumors to a certain extent, indicating that the OT-I T cells obtained by in vitro activation and expansion have in vivo anti-tumor activity. Further comparison showed that the tumor growth rate of the adaptive liquid-liquid interface activated OT-I T cell group was lower than that of the PBS blank control group, and it showed a better tumor growth delay effect compared with the anti-CD3 / CD28 magnetic bead activated group. These results indicate that the adaptive liquid-liquid interface described in this invention can obtain OT-I T cells with in vivo anti-tumor function and is beneficial to improve the in vivo functional performance of expanded T cells.

[0145] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of a liquid-liquid interface culture system in T cell activation and expansion; wherein the liquid-liquid interface comprises a two-dimensional liquid-liquid interface or liquid-liquid interface microspheres; in, The two-dimensional liquid-liquid interface is constructed from an upper aqueous phase and a lower oil phase. A protein nanofilm is formed at the interface of the two-dimensional liquid-liquid interface. The protein nanofilm is loaded with molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulation signals. The liquid-liquid interface microspheres are formed by dispersing an oil phase in an aqueous phase, and a protein nanofilm is formed at the oil-water interface of the liquid-liquid interface microspheres. The protein nanofilm is loaded with molecules that provide T cell receptor activation signals and / or molecules that provide T cell co-stimulation signals.

2. The application according to claim 1, characterized in that, The molecules providing T-cell receptor activation signals include at least one of anti-CD3 antibodies, anti-TCR antibodies, peptide-MHC complexes, CD3-binding molecules, TCR-binding molecules, or functional fragments thereof; and / or, the molecules providing T-cell co-stimulation signals include at least one of anti-CD28 antibodies, CD80, CD86, or 4-1BBL.

3. The application according to claim 1, characterized in that, The liquid-liquid interface culture system also includes auxiliary interface functional molecules; Preferably, the auxiliary interface functional molecule includes at least one of anti-CD2 antibody, anti-LFA-1 antibody, ICAM-1, cell adhesion molecule, extracellular matrix protein or serum albumin; Preferably, the cell adhesion molecule or extracellular matrix protein includes a protein, polypeptide or derivative thereof containing an RGD sequence, and more preferably includes at least one of fibronectin, hydrin, osteopontin, fibrinogen, laminin, collagen or gelatin. Preferably, the serum albumin includes at least one of bovine serum albumin, human serum albumin, or recombinant serum albumin.

4. The application according to any one of claims 1-3, characterized in that, The oil phase includes a fluorinated liquid that is immiscible or poorly miscible with the aqueous phase and is suitable for cell culture or biological interface construction; and / or, the aqueous phase is an aqueous system. Preferably, the fluorinated liquid is at least one of FC-40, FC70, HFE-7500 or HFE-7100; Preferably, the aqueous system includes at least one of water, PBS buffer, physiological saline, cell culture medium, protein-containing aqueous solution, antibody-containing aqueous solution, or cytokine-containing aqueous solution.

5. The application according to claim 4, characterized in that, The method for forming the microspheres includes at least one of printing, microfluidics, dropwise addition, emulsification, oscillation, stirring, membrane emulsification, or ultrasonic emulsification; and / or, the particle size or equivalent diameter of the microspheres is 5-200 μm.

6. A method for T cell activation and expansion, comprising contacting T cells with a liquid-liquid interface of any one of claims 1-5 loaded with molecules providing T cell receptor activation signals and / or molecules providing T cell co-stimulatory signals, and culturing them in a culture medium containing cytokines.

7. The method according to claim 6, characterized in that, When the liquid-liquid interface is a two-dimensional liquid-liquid interface, the method includes the following steps: partially or completely replacing the aqueous phase with a culture medium containing cytokines, seeding T cells into the aqueous phase of the culture medium containing cytokines, so that the T cells come into contact with the two-dimensional liquid-liquid interface and are cultured; or When the liquid-liquid interface is a liquid-liquid interface microsphere, the method includes the following steps: adding the liquid-liquid interface microsphere to a culture medium containing cytokines, seeding T cells into the culture medium containing the liquid-liquid interface microsphere, and bringing the T cells into contact with the liquid-liquid interface microsphere and culturing them.

8. The method according to claim 7, characterized in that, The cytokines include interleukin-type cytokines; Preferably, the cytokines include at least one of IL-2, IL-7, IL-15 or IL-21; more preferably, they include IL-2, or a combination of IL-7 and IL-15, or a combination of IL-7, IL-15 and IL-21. Preferably, the final concentration of the cytokine in the culture medium is 10-300 IU / mL, or 0.1-100 ng / mL by mass concentration.

9. The method according to claim 7, characterized in that, The seeding density of the T cells was (0.1-100) × 10⁻¹⁰. 6 cells / mL; And / or, the culture time is 2-14 days; And / or, during the culture process, at least one of the following is performed: replenishment of medium containing cytokines, half-volume replacement of medium, replacement of medium, separation of cells into flasks, resuspension of cells, addition of cytokines, or adjustment of cell density.

10. The method according to any one of claims 6-9, characterized in that, The T cells include peripheral blood T cells, umbilical cord blood T cells, tumor-infiltrating T cells, αβ T cells, γδ T cells, and CD4+ T cells. + T cells, CD8 + T cells, natural T cells and / or genetically modified T cells; Preferably, the gene-modified T cell contains an introduced exogenous gene; Preferably, the genetically modified T cell comprises any one or more of the following: a gene segment encoding a chimeric antigen receptor (CAR), a gene segment encoding a T cell receptor that specifically recognizes tumor antigens, and a gene segment encoding a fusion protein of the T cell receptor.

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