A method for training t cells based on a tumor organ chip

Abstract

This invention discloses a T-cell training method based on tumor organ-on-a-chip. The method includes: preparing an organ-on-a-chip with a micropillar array and a biomimetic fishbone structure; introducing primary tumor cells from a patient into the chip, causing them to spontaneously form uniform three-dimensional tumor spheroids; dynamically pumping pre-activated peripheral blood lymphocytes from the same patient into the chip and co-culturing them with the tumor spheroids; collecting the co-cultured lymphocytes and repeating the co-culture process multiple times to obtain a lymphocyte population with enhanced anti-tumor activity. This invention utilizes organ-on-a-chip technology to highly simulate the dynamic tumor immune microenvironment in vivo. Through multiple cycles of "training," it effectively activates and expands tumor-specific T cells, avoiding immune exhaustion, and providing a highly efficient and controllable new platform for personalized immunotherapy of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and immunotherapy technology, specifically relating to a T-cell training method based on tumor organ-on-a-chip and its application. Background Technology

[0002] Neuroblastoma is the most common extracranial solid tumor in children, characterized by high heterogeneity and aggressiveness, and traditional treatments have limited efficacy. T-cell-based immunotherapies, such as CAR-T and TIL therapy, have brought new hope to the treatment of neuroblastoma. However, these therapies still face many challenges in treating solid tumors, including: insufficient numbers of tumor-specific killing T cells in the patient's body; immunosuppression in the tumor microenvironment leading to exhaustion of infiltrating T cell function; and a lack of effective in vitro expansion and functional enhancement methods. Existing two-dimensional co-culture or static three-dimensional culture methods cannot realistically simulate the dynamic, shear-force-dependent tumor immune microenvironment in vivo, thus the obtained T cells often have insufficient in vivo anti-tumor efficacy. Therefore, there is an urgent need for a new method that can efficiently induce, expand, and train autologous T cells with strong anti-tumor activity in vitro. Summary of the Invention

[0003] This invention aims to address the aforementioned problems in the prior art by providing a T-cell training method based on tumor organ-on-a-chip. This method utilizes microfluidic organ-on-a-chip technology to construct a dynamic tumor-immune co-culture microenvironment in vitro that more closely resembles the in vivo environment, thereby achieving high-throughput, repeatable training of autologous T cells to obtain a T-cell population with potent and durable anti-tumor activity.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] A method for training T cells based on tumor organ-on-a-chip, characterized by comprising the following steps:

[0006] (1) Preparation of tumor organ-on-a-chip: an organ-on-a-chip with a special microstructure is prepared, the chip comprising upper and lower layers bonded together; the lower layer is provided with a columnar array composed of multiple micropillars for trapping and culturing tumor cells; the upper layer is provided with a microchannel with a biomimetic fishbone structure for guiding fluid to form turbulence;

[0007] (2) Chip surface smoothing treatment: Pump a surface smoothing agent into the chip prepared in step (1) to modify the surface of the fluid channels inside the chip to prevent non-specific cell adhesion;

[0008] (3) Formation of tumor spheroids: The primary tumor cell suspension derived from the patient is slowly pumped into the chip after step (2) by a microfluidic pump. The tumor cells spontaneously form uniform three-dimensional tumor spheroids between the columnar arrays.

[0009] (4) Dynamic co-culture and training of T cells: Peripheral blood lymphocytes from the same patient were pre-activated in vitro and then pumped into the chip containing tumor spheroids in step (3) by a microfluidic pump to achieve dynamic co-culture of lymphocytes and tumor spheroids; the co-cultured lymphocytes were collected from the chip outlet and the recovered lymphocytes were pumped into a new or the same chip containing tumor spheroids for at least one repeated co-culture to obtain a lymphocyte population with enhanced tumor killing activity.

[0010] This invention differs from existing simple co-culture methods by utilizing the recyclability of the chip to achieve multiple, intermittent contacts between T cells and autoantigens. This simulates the process of repeated antigen exposure by the immune system in vivo, which is fundamentally different from single-stage activation in vitro.

[0011] In one embodiment, in step (1), the columnar array of the lower layer of the chip is a micro-unit composed of multiple micropillars arranged in concentric circles, used to physically trap cells and promote their aggregation into spheres; the central radius of each columnar array is 300±30μm, the diameter and height of the micropillars are both 100±10μm, and the gap between two adjacent micropillars is 100±10μm; the micro-texture spacing of the upper fishbone structure matches the spacing of the lower micropillars, which is 100±10μm, so that the fluid forms a vortex flow in the chip, promoting cell recruitment.

[0012] In this invention, the micropillars not only trap cells but also ensure the uniformity of tumor spheres across multiple training sessions by limiting their size. The fishbone structure not only promotes turbulence but also maximizes the probability of T cell contact with the tumor spheres during multiple perfusions.

[0013] In one embodiment, in step (2), the surface smoothing agent is a 10±5 vol% F-108 solution (a non-ionic polyoxyethylene-polyoxypropylene block copolymer with good interfacial activity and inhibits cell adhesion), which is pumped in and incubated at room temperature for 2 hours.

[0014] In one embodiment, in step (3), the primary tumor cells are derived from pediatric solid malignant tumor tissue, preferably pediatric neuroblastoma tissue. After physical cutting and enzymatic digestion, the tumor tissue is resuspended in tumor stem cell culture medium at 1×10⁻⁶. 6 Pumped into the chip at a density of 1 / mL and a flow rate of 0.05-0.2mL / h.

[0015] In one embodiment, in step (4), the peripheral blood lymphocytes need to be pre-stimulated outside the chip before being co-cultured with tumor spheromeres. The pre-stimulation treatment includes amplification and activation using at least one of CD3 / CD28 co-stimulatory factor and interleukin cytokines IL-2, IL-7, and IL-15.

[0016] In one embodiment, the method further includes a step of functional identification of lymphocyte populations obtained after multiple co-cultures.

[0017] The present invention also provides a population of lymphocytes with tumor-killing activity obtained by training by any of the methods described above.

[0018] The present invention further provides the application of the above method in the preparation of immunomodulatory agents for the treatment of tumors, especially showing good application effects for pediatric solid tumors (neuroblastoma).

[0019] Beneficial effects

[0020] Compared with the prior art, the present invention has the following significant advantages:

[0021] (1) Highly simulates the in vivo microenvironment: Through a unique chip structure and dynamic fluid, namely the specific hydrodynamic effects brought about by the "concentric micropillars + fishbone" structure, this invention constructs a three-dimensional tumor-immune co-culture microenvironment with fluid shear force in vitro that is closer to the real situation in vivo, which can more realistically simulate the interaction process between T cells and tumor cells.

[0022] (2) Enhanced T cell function: Through multiple dynamic co-culture "training" processes, T cells are co-cultured in the chip and then recovered and re-infused into a new chip for multiple co-cultures, ultimately obtaining a toxic lymphocyte population. Unlike the existing one-time co-culture, this can effectively activate and expand tumor-specific T cells, while avoiding immune exhaustion caused by excessive expansion in vitro, thereby obtaining a larger number of T cells with stronger activity and more lasting anti-tumor efficacy.

[0023] (3) Realizing personalized medicine: By using tumor cells and immune cells from the same patient, highly specific autologous T cell products can be "tailor-made" for patients in vitro, providing a new technical platform for personalized immunotherapy.

[0024] (4) High throughput and reproducibility: The microstructure of the chip ensures the high uniformity of tumor spheroids, and the dynamic culture system ensures the controllability and reproducibility of the training process, laying the foundation for the preparation of standardized T cell products. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a schematic diagram of the T-cell training method based on tumor organ-on-a-chip according to the present invention.

[0027] Figure 2 This is a schematic diagram of the structure of the tumor organ-on-a-chip of the present invention. Figure 3 The following diagram shows the simulation results of fluid dynamics within the chip of this invention, where (a) is the flow velocity simulation, (b) is the particle trajectory simulation, and (c) is the turbulence simulation. Figure 4 This is a graph showing the detection results of tumor spheroid formation and activity within the chip of this invention. Figure 5 This is a diagram illustrating the state of peripheral blood lymphocytes during in vitro pre-stimulation and expansion according to the present invention. Figure 6 This is a microscopic image of T cells and tumor spheromeres co-cultured within the chip of this invention.

[0028] Figure 7 This is a real-time fluorescence integrated image of tumor spheroids inside the co-cultured activated T-cell killing chip. Figure 8 This is an analytical diagram illustrating the changes in lymphocyte subsets before and after co-culture detected by flow cytometry according to the present invention.

[0029] Figure 9 It is a flow cytometry method to assess changes in the self-exhaustion state of T cells before and after co-culture. Detailed Implementation

[0030] The present invention can be better understood from the following embodiments.

[0031] Example 1: Preparation and Characterization of Tumor Organ-on-a-Chip

[0032] Chip Design and Mold Fabrication: The chip structure was designed using CAD software. The overall chip dimensions are 2cm (width) × 3cm (length) × 0.4cm (height), and the culture chamber dimensions are approximately 1.5cm × 1.5cm × 0.1cm. The lower layer is a micropillar array, with each micropillar being a cylinder with a diameter and height of 100μm. Six micropillars are arranged concentrically around a central point, forming an array with a central radius of 300±30μm. The gap between adjacent micropillars is 100±10μm. The upper layer is a biomimetic fishbone structure, with the width and spacing of the fishbone stripes both being 100μm. Soft photolithography was used to fabricate a silicon-based positive mold based on the design.

[0033] Chip casting and bonding: Polydimethylsiloxane prepolymer and curing agent (10:1 mass ratio) are mixed evenly and poured onto a silicon-based positive mold. After vacuum degassing, the mixture is placed in a 37°C oven for curing for 24 hours. The cured PDMS upper and lower layers are peeled off the mold, and holes are punched to form the inlet and outlet. The upper and lower PDMS structures are then treated with oxygen plasma, aligned, and bonded to form a complete chip, such as... Figure 2 As shown. Sterile water is introduced into the chip inlet to test airtightness.

[0034] like Figure 1 As shown, when using the aforementioned tumor organ-on-a-chip for T-cell training, a surface smoothing agent (10 vol% F-108 solution) is first pumped into the prepared chip to modify the surface of the fluid channels inside the chip, preventing non-specific cell adhesion. Then, a suspension of primary tumor cells from the patient is slowly pumped into the smoothed chip using a microfluidic pump. The tumor cells spontaneously form uniform three-dimensional tumor spheroids between the columnar arrays. Peripheral blood lymphocytes from the same patient, after in vitro pre-activation, are pumped into the chip containing the tumor spheroids using a microfluidic pump, achieving dynamic co-culture of lymphocytes and tumor spheroids. The co-cultured lymphocytes are collected from the chip outlet, and these recovered lymphocytes are pumped back into a new or identical chip containing tumor spheroids for at least one repeated co-culture, thereby obtaining a lymphocyte population with enhanced tumor-killing activity.

[0035] Fluid Simulation: COMSOL Multiphysics software was used to simulate and analyze the internal fluid dynamics of the chip. Results are as follows... Figure 3 As shown, the special structure inside the chip creates turbulence and eddies between the micropillars, which is beneficial for cell retention and full contact with flowing particles.

[0036] Example 2: Chip pretreatment and formation of tumor spheroids

[0037] Chip sterilization and surface treatment: The chips prepared in Example 1 were sterilized by irradiation with ultraviolet light for 30 minutes. Then, 10 vol% F-108 solution was injected into the chip at a flow rate of 0.1 ml / h using a microfluidic pump until the entire chip was filled. The chip was incubated at room temperature for 2 hours to smooth the surface of the internal channels. Finally, the chip was rinsed with sterile PBS for later use.

[0038] Obtaining primary tumor cells: Tumor tissue from surgically resected pediatric neuroblastoma patients, confirmed pathologically, was collected. Under aseptic conditions, the tumor tissue was physically minced to approximately 1 mm³, then digested with collagenase IV and DNase I at 37°C for 1 hour with intermittent shaking. The digested cell suspension was filtered through a 70 μm cell sieve, the filtrate was collected, centrifuged, and the supernatant was discarded to obtain a single-cell suspension containing tumor cells and stromal cells.

[0039] Tumor spherocyte culture: The obtained cell pellet was resuspended in neuroblastoma stem cell culture medium, and the cell density was adjusted to 1×10⁻⁶. 6 Cells / ml. The stem cell culture medium consisted of DMEM / F12 basal medium supplemented with 1×B27, 1×N2, 1 vol% KnockOut Serum Replacement, 1×Acetylcysteine, 20 ng / ml EGF, 20 ng / ml FGF, 10 μM Y-27632, and 10 μM Noggin. The cell suspension was pumped into the F-108-treated chip at a flow rate of 0.1 ml / h using a microfluidic pump. Under a microscope, once the cells were evenly distributed in the gaps between the bottom microcolumn array, the pumping was stopped, and the chip was placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, uniformly sized three-dimensional tumor spheroids were observed to form between the microcolumns. Figure 4 Then, fresh stem cell culture medium was continuously pumped in at a flow rate of 0.05 ml / h to maintain spheromer growth and activity.

[0040] Example 3: In vitro pre-activation and expansion of peripheral blood lymphocytes

[0041] Lymphocyte isolation: Peripheral blood homologous to that of tumor patients was collected, and peripheral blood mononuclear cells were obtained by Ficoll-Paque density gradient centrifugation.

[0042] Pre-stimulation and expansion: The obtained PBMCs were resuspended in complete T cell culture medium (such as AIM-V or X-VIVO 15) and CD3 / CD28 antibody-conjugated magnetic beads (cells:beads = 1:1), 100 IU / ml IL-2, 10 ng / ml IL-7, and 10 ng / ml IL-15 were added. The cells were placed in 6-well plates and cultured in a 37°C, 5% CO2 incubator. The culture medium was replaced with fresh cytokines every 2-3 days. Cell proliferation and aggregation were observed under a microscope: During the initial proliferation phase, PBMCs could proliferate stably in the culture system and form cell aggregates; subsequently, during the maintenance phase, IL-2 and CD3 / CD28 antibody-conjugated magnetic beads were removed from the culture system, while the cytokines IL-7 and IL-15 were retained to maintain good proliferation efficiency while avoiding immune exhaustion. Figure 5 This transition between the proliferation and maintenance phases effectively maintains the activity of lymphocytes in PBMCs and obtains a sufficient number of lymphocytes for tumor-immune co-culture.

[0043] Example 4: Dynamic training of T cells within a tumor organ-on-a-chip

[0044] Initial co-culture: The pre-activated T cells from Example 3 were centrifuged and resuspended in T cell maintenance medium to a density of 5 × 10⁻⁶ cells / mL. 5 T cell suspension was pumped into the chip of Example 2, which had formed stable tumor spheroids, at a flow rate of 0.1 ml / h using a microfluidic pump. T cells flowed fluidically through the intercolumnar spaces, dynamically contacting the tumor spheroids. Tumor cells and PBMCs from three groups of patients were labeled with green and red tracers, respectively. PBMCs could directly contact the tumor spheroids within the chip and gradually recruited to the periphery of the tumor spheroids. Some active lymphocytes could infiltrate into the tumor spheroids. Ultimately, with increasing co-culture cycles, lymphocyte toxicity and targeted killing effects gradually increased, effectively killing tumor spheroids and disintegrating their structure. Figure 6 and Figure 7 Effluent containing T cells that have interacted with tumor spheroids is continuously collected from the chip outlet.

[0045] Multiple training sessions: The initially collected T cells are centrifuged and concentrated, then resuspended in fresh T cell maintenance medium. They are then pumped back into a new microarray containing the same patient-derived tumor spheroids at the same flow rate for a second co-culture. This "co-culture-recovery-re-co-culture" process is repeated 3-5 times to adequately "train" the T cells.

[0046] T cell function identification: Untrained naive T cells and T cells after multiple training cycles were collected. A portion was used for flow cytometry analysis to detect the expression of cytotoxic T cell subsets (e.g., CD8+CD107a+) and exhaustion markers (e.g., LAG-3, TIM-3). Results showed that the proportion of central memory T cells significantly increased in trained T cells, while the expression levels of exhaustion markers did not significantly increase. Before and after co-culture, the number of CD8+CD107a+ subset cells significantly increased in PBMCs from all three patient groups. Figure 8 and Figure 9 Furthermore, flow cytometry analysis revealed no significant increase in immune exhaustion in PBMCs derived from the three patient groups, indicating that the co-culture system within the chip exhibits stable training efficiency and low immune exhaustion costs.

[0047] Example 5: Validation of the in vivo anti-tumor efficacy of T cells after training

[0048] Construction of a humanized immune mouse model: The trained T cells obtained in Example 4 were transplanted into NCG immunodeficient mice via tail vein injection to construct a humanized immune system.

[0049] Establishment of the orthotopic xenograft model: One week later, 5×10⁵ cells were injected orthotopically into the left adrenal gland of the constructed humanized mice. 5A tumor cell labeled with luciferase, which is homologous to T cells.

[0050] Monitoring of anti-tumor efficacy: Tumor growth was monitored regularly using a small animal in vivo imaging system, and the survival time of mice was recorded. At the end of the experiment, mice were sacrificed, tumor tissue was dissected, and histopathological analysis and immune cell infiltration detection were performed. The results showed that mice treated with trained T cells had significantly slower tumor growth and significantly longer survival than the control group, and a large number of human CD8⁺ T cells infiltrated the tumor tissue.

[0051] This invention provides a concept and method for T-cell training based on tumor organ-on-a-chip. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A T-cell training method based on tumor organ-on-a-chip, characterized in that, The steps include: (1) providing a tumor organ-on-a-chip, the chip having a microstructure for trapping tumor cells and allowing fluid to pass through dynamically; (2) introducing a suspension of primary tumor cells from a patient into the chip to form three-dimensional tumor spheroids within the chip; (3) introducing peripheral blood lymphocytes from the same patient into the chip containing tumor spheroids in step (2) for dynamic co-culture; (4) collecting the lymphocytes after co-culture in step (3) and repeating the operation of step (3) at least once to obtain a lymphocyte population that has been trained multiple times.

2. The method according to claim 1, characterized in that, The tumor organ-on-a-chip described in step (1) comprises two layers bonded together: the lower layer is a columnar array composed of multiple micropillars, used to trap and culture tumor cells; the upper layer is a microchannel with a biomimetic fishbone structure, used to guide fluid to form turbulence.

3. The method according to claim 2, characterized in that, The lower columnar array is composed of multiple micro-column units arranged in concentric circles. The central radius of each columnar array is 300±30μm, the diameter and height of the micro-columns are both 100±10μm, and the gap between two adjacent micro-columns is 100±10μm. The micro-texture spacing of the upper fishbone structure is 100±10μm.

4. The method according to claim 1, characterized in that, After step (1) and before step (2), there is also a step of surface smoothing treatment of the internal channels of the chip, which is to pump the surface smoothing agent F-108 solution into the chip and incubate it.

5. The method according to claim 1, characterized in that, The primary tumor cells mentioned in step (2) are derived from solid malignant tumor tissue. After physical cutting and enzymatic digestion, the tumor tissue is resuspended into a single-cell suspension using tumor stem cell culture medium and then... 6 Cells were pumped into the chip at a density of 1 cell / mL and a flow rate of 0.05-0.2 mL / h.

6. The method according to claim 1, characterized in that, Before the peripheral blood lymphocytes mentioned in step (3) are introduced into the chip, they need to be pre-stimulated outside the chip. The pre-stimulation treatment includes amplification and activation using CD3 / CD28 co-stimulatory molecules and interleukin cytokines.

7. The method according to claim 6, characterized in that, The interleukin cytokines are selected from at least one of IL-2, IL-7, and IL-15.

8. The method according to claim 1, characterized in that, In steps (3) and (4), the introduction and co-culture of lymphocytes are carried out dynamically by a microfluidic pump at a flow rate of 0.05-0.2 mL / h.

9. A population of lymphocytes with tumor-killing activity obtained by training according to any one of claims 1-8.

10. The use of the lymphocyte population of claim 9 in the preparation of a medicament for treating tumors.

Images