MULTILAYERED MICROFLUID CHIP-ON-IMMUNE-TUMOR SYSTEM AND ITS USAGE METHOD

Abstract

This invention relates to a multilayer microfluidic chip system (1) and a method (100) for evaluating the paracrine effects of macrophages on a tumor spheroid using system (1), which allows the study of immune-tumor interactions in a controlled microenvironment.

Description

1 TARIFF MULTILAYERED MICROFLUID CHIP-ON-IMMUNE TUMOR THE SYSTEM AND ITS USAGE INSTRUCTIONS Technical Area 5 This invention allows for the study of immune-tumor interactions in a controlled microenvironment. using a multilayer microfluidic chip system that enables A method for evaluating the paracrine effects of macrophages on a tumor spheroid. It is related to. 10 Previous Technique Tumor-associated macrophages (TAMs) are involved in the tumor microenvironment (TME). by increasing growth, promoting angiogenesis and an immunosuppressive niche 15 It plays a critical role by creating various therapeutic approaches to retraining TAMs. Although strategies have been developed, due to the complex structure of the TMJ, these cells... Effectively reversing tumor-promoting behaviors remains a challenge. That is the issue. Malignant tumors have a highly sensitive tumor microenvironment known as the tumor microenvironment (TME). It forms, grows, and acquires metastatic capabilities in specialized niches. This is complex and Heterogeneous environment, cancerous and non-cancerous cells, and various soluble and environmental factors. It arises as a result of intense interactions between factors and in the tissue It supports the development of malignancy. The temporal and spatial composition of the TMJ supports tumor 25 It significantly affects its behavior; especially altered immune cells, Macrophages, in particular, play an effective role in almost every situation. Macrophages constitute the dominant immune cell population within the TME, and transition between both pro-inflammatory (M1) and anti-inflammatory (M2) states They have the capacity to do so; this means they can interact with cancerous and normal cells for 30 years. It shapes their interactions. This repolarization ability is key to neoplastic development. It is effective from the early stages of TMJ formation; during these stages, macrophages 2 It interacts closely with cancer cells and polarizes into a tumor-associated state. They become; these cells, which need nutrients and oxygen in a hypoxic environment, are "under stress". They perceive these tumor-associated macrophages (TAMs) as "healthy cells". enhancing angiogenesis, clearing cellular debris, and regulating the extracellular matrix They exhibit M2-like characteristics, restructuring TME through activities such as these. 5 TAMs not only promote tumor growth through angiogenesis, but also They also contribute to immunosuppression within the TMJ at times. These are their critical roles. Therefore, TAMs have become the target of various therapeutic strategies; these gene editing with the CRISPR / Cas9 system, monoclonal antibodies, metabolic manipulations and toll-like receptors (TLRs) and STING receptors 10 There are approaches that aim to achieve this. However, TAMs are more like M2-like situations. Returning to the pro-inflammatory M1 state, both TAMs and complex processes It is a challenging process both because of its biology and the difficult-to-target nature of TMJ. This continues. Therefore, these therapeutic approaches are generally carried out in the laboratory and It yields inconsistent or inadequate results in clinical settings. 15 Traditional in vitro cell culture systems do not fully mimic the complexity of TME. It has limitations in terms of implementation. In response, microfluidic technologies... They have emerged as promising tools; these systems measure flow rate, oxygen level, 20 on microscopic-scale fluid properties such as chemical gradients and pressure It provides precise control. These technologies are used in two-dimensional (2D) and three-dimensional (3D) cultures. They can mimic physiological conditions and, thanks to their highly customizable structures, are very versatile. Microfluidic platforms allow for the addressing of various research questions. Simultaneous co-culture of different cell types in compartmentalized chambers It provides dynamic culture conditions that support the development of intercellular 25%. Juxacrine and paracrine interactions, a transparent device that can be equipped with various sensors. It can be observed within it. In recent years, microfluidic models have been used to study the TME and the immune cells within it. In its analysis, it has demonstrated superiority compared to traditional models. For example, a 30 breast cancer microfluidic model, T cells and macrophages in tumor Other studies have revealed their opposing roles in its progression. Other studies have focused on the endothelium, immune system, etc. 3 and by enabling the co-culture of cancer cells, the relationships between these cell types This has made it possible to observe interactions in real time. Furthermore, Microfluidic technology is also valuable in immunotherapy research. It has been proven that these systems reduce the TMJ that occurs after drug administration. It enables real-time monitoring of changes. 5 Therefore, simultaneous culturing of macrophages and cancer spheroids is recommended. facilitating effective paracrine communication and enabling 3D cancer detection by immune cells. a microfluidic chip system that allows culturing along with its models It is needed. 10 United States patent number US2021062129, which is included in the prior art. The document describes the structure and at least one function of specific areas of the epithelial system in living organisms. An in vitro microfluidic "organ-on-chip" device that mimics a microfluidic device is described. Brief Description of the Invention The aim of this invention is to study immune-tumor interactions in a controlled microenvironment. a multilayer microfluidic chip system and system that allows for its examination using 20 paracrine effects of macrophages on a tumor spheroid The evaluation method is to carry out the assessment. Another aim of this invention is to study immune cells and three-dimensional (3D) tumor spheroids. used to study only paracrine-only interactions between them The goal is to realize a microfluidic chip system. 25 Detailed Description of the Invention To achieve the purpose of this invention, "Multilayer Microfluidic Chip-on-Immune- The "Tumor System and Method of System Use" are shown in the attached figures, and these 30 from shapes; 4 Figure 1. a) General view of the system subject to the invention, b) View of the lower macrophage layer. a) a view of the upper chamber layer and d) spheroid micro-well insert This is a view belonging to the platform. Figure 2. Flowchart of the method described in the invention. The parts shown in the figures are individually numbered, and each number corresponds to the following: given below: 1. Microfluidic chip system 2. Lower macrophage layer 10 3. Upper chamber layer 4. Spheroid micro-well insert platform The only paracrine pathway between immune cells and three-dimensional tumor spheroids The invention is a microfluidic chip system (1) used to study the interactions; 15 - for culturing, stimulating, and phenotypic reprogramming macrophages a single microfluidic channel structured to allow controlled movement of immune cells used to examine the channel's stimulation and interaction with therapeutic agents. By continuously supplying cell culture medium through an inlet, a stable environment for the cells is created. 20 that make it possible to provide a microenvironment and to deliver nutrients and medicines. at least one sub-macrophage layer in the form of a plate (2), -to house a suspension of cancer cells forming a 3D tumor spheroid at least one large well in the form of a structured opening to prevent dehydration containing a small surrounding medium reservoir and acting as a nutrient medium reservoir at least one small 25 that performs its function, preventing the culture medium from evaporating and drying out. At least one plate-shaped well placed on the lower macrophage layer (2) containing wells upper chamber layer (3) and -sized to fit into the opening in the large well of the upper reservoir layer (3) and here At least one micro-well to create and maintain a 3D tumor spheroid. containing and enabling cancer cells to form three-dimensional structures, al 30 having a single micropore on its surface and through this pore while preventing direct physical contact between macrophages in the lower macrophage layer (2) and the upper A controlled diffusional paracrine between (3) 3D tumor spheroids in the reservoir layer. It includes at least one spheroid micro-well insert platform (4) that forms a communication pathway. The subject of the invention is a microfluidic chip system (1), lower macrophage layer (2), upper chamber layer (3) and spheroid micro-well insert platform (4) reversible (detachable) or 5 It is obtained by irreversibly joining the components in a microfluidic chip system. (1) The single micro-pore located there maintains the physical separation of cells while allowing cytokines, selective diffusion of chemokines, ROS (reactive oxygen species) and small molecules It has a diameter between 5–100 μm. In the microfluidic chip system (1) only Paracrine configuration, single pore usage, porous membranes 10 the absence of a vertical gap of 0.2–2 mm between the micro-pore and the spheroid center It is obtained by finding the distance. In the microfluidic chip system (1) The spheroid microwell allows for the formation of a single, homogeneous 3D tumor spheroid. The recognizable macrophage is 200–800 μm in diameter, hemispherical (half-spherical) or conical. layer (2), controlled macrophage stimulation, polarization or re-15 To enable programming, it will provide continuous perfusion at a rate of 1–100 μL / min. It is structured as follows. Macrophages in the lower macrophage layer (2) from M2 to M1 Resiquimod is used to induce (trigger) phenotypic reprogramming. It is exposed to a liposomal formulation (LipRes). The lower macrophage layer (2) and upper reservoir layer (3); polydimethylsiloxane (PDMS), cyclic olefin copolymer, 20 polylactic acid (PLA), polymethyl methacrylate (PMMA) or their It is produced from combinations of spheroid micro-well insert platforms (4), is separable and the upper chamber layer of preformed spheroids (3) It allows for placement. The single micro-pore size, geometry, and its location, with diffusion gradients directed from macrophages towards the tumor spheroid 25 This results in: Microfluidic chip system (1), immunotherapeutic A structured multiplex for screening (immunotherapy) agents. It contains numerous spheroidal micro-well inserts. 6 Method 100 The invention also involves the paracrine activity of macrophages on a tumor spheroid. It is related to a method for evaluating immunomodulatory effects (100); 101) culturing of macrophages in the (1) lower macrophage layer of the system (2); 102) 5 retraining or stimulating macrophages with a liposomal TLR agonist 103) a 3D tumor spheroid in the micro-well of the upper chamber layer (3) 104) formation of paracrine diffusion only through a single micro-pore 105) allowing it to occur; and the spheroid morphology of the tumor over time, The steps for measuring its size, viability, or secretome changes are 10. Method (100) includes liposomal TLR agonist, liposomal resiquimod (LipRes) It also contains IL-6, TNF-α, ROS, or other substances that can diffuse through micropores. Quantification of macrophage-derived cytokines It includes. The invention system (1) targets macrophage-mediated tumor modulation. It is used for screening immunotherapeutic agents. Also, M2 to M1. Effects of macrophage reprogramming on cancer spheroid growth and behavior It is used for modeling and quantification on the surface. In the microfluidic chip system (1), the lower macrophage layer (2) and the upper chamber layer (3) It was produced using the standard soft lithography method. First The chip design is created using computer-aided design (CAD) software, and transferred onto a photomask. To define microchannel and chamber geometries. Molds were prepared on silicon wafers using SU-8 photoresist. 25 Polydimethylsiloxane (PDMS) and curing agent are mixed in a 10:2 ratio, in air. The bubbles were removed and it was cured at 80 °C for 1.5 hours. The cured PDMS layers... Biopsy punch with removed mold, 1.5 mm entry holes and 8 mm well openings. It was created using [method name]. The layers were cleaned with ethanol and oxygenated for 2 minutes. The hydrophilicity of the surfaces was increased by applying plasma treatment, thus creating a strong surface 30 The chip has been secured. It has been sterilized in an autoclave and left overnight at 60°C. It has been dried throughout the process and made ready for use. 7 While preparing the spheroid micro-well insert platform (4), cancer cell spheres To create them, each containing 24 micro-wells was formed using a special mold. Platforms were produced. Each of these platforms is a standard cell culture platform with 96 wells. It fits perfectly into a well in the plates. Platforms for cell culture 5 It was previously sterilized under UV (ultraviolet) light. HT-29 colorectal cancer The cells are seeded into these micro-wells, resulting in the formation of uniform three-dimensional spheres. has been provided. Industrial Application of the Invention 10 In the microfluidic chip system that is the subject of the invention (1), macrophages in the lower macrophage layer (2) were seeded into the canal and cultured under static conditions for 3 days. This period Cell culture medium was regularly supplied from the inlet throughout, in the upper chamber layer (3) The reservoir tank is also kept full to prevent evaporation. In the first stage 15 macrophages, conditioned medium collected from cancer cells (medium) by treatment with tumor-associated macrophage (TAM) phenotype They were transformed. Then, the TAMs were retrained with liposomal resiquimod, Reprogrammed macrophages with tumor suppressor phenotype (ReTAM) were obtained. HT-29 cells were inserted into the spheroid micro-well insert platform (4) 20 outside the chip. It has taken on a spherical form by being seeded onto it. During this process, it has direct contact with macrophages. no cancer cells were found. By the end of the third day, the previously formed cancer cells The platform containing the spheres is placed in the large chamber of the upper layer and macrophages It is aligned on the channel. After the layers are combined, the system has (1) indirect A shared culture (paracrine interactive co-culture) has been established. 25 The aim of this co-culture system is to facilitate the interaction between macrophages and cancer cells. It is the physiological simulation of interactions in a microfluidic environment. This system (1), different phenotypes of macrophages (TAM and ReTAM) tumor growth, Investigation of its effects on invasion and epithelial-mesenchymal transition (EMT) 30 provides. 8 During the four-day co-culture period, the size, morphology, and migration of cancer spheres were analyzed. Their abilities have been monitored. Additionally, immunostaining methods have been used. vimentin (mesenchymal marker) and E-cadherin (epithelial marker) proteins The biological consequences of macrophage effect are analyzed by evaluating expression levels. It has been done. 5 The subject of the invention is “Multilayer Microfluidic Chip-On-Immune-Tumor System (1) and Developing a wide variety of applications for the System (1) Usage Method (100)” It is possible, and the invention is not limited to the examples described here, but is essentially As stated in the requests. 10 20 30

Claims

9 REQUESTS 1. Only paracrine between immune cells and three-dimensional tumor spheroids. a microfluidic chip system (1) used to study interactions Feature; 5 - culturing, stimulating, and phenotypic reconstruction of macrophages an immune system containing a single microfluidic channel structured for programming controlled stimulation of cells and their interaction with therapeutic agents used for examination, continuous cell culture through an inlet into the channel by providing the medium, a stable microenvironment is created for the cells and nutrients are supplied. at least one plate-shaped substrate that makes it possible to administer drug supplements macrophage layer (2), -a suspension of cancer cells forming a 3D tumor spheroid with at least one large well in the form of an opening structured to accommodate 15 containing at least one surrounding medium reservoir that prevents dehydration and acting as a culture medium reservoir, preventing the evaporation of the culture medium. and the lower macrophage layer (2) which contains at least one small well that prevents it from drying out. at least one upper reservoir layer in the form of a plate (3) placed on it and -sized to fit into the opening in the large well of the upper reservoir layer (3) and At least one 20-inch probe is needed to create and maintain the 3D tumor spheroid placed here. containing micro-wells and allowing cancer cells to form three-dimensional structures enabling, having a single micropore on its surface, and this while preventing direct physical contact through the pore, sub-macrophage macrophages in the (2) upper chamber layer and (3) 3D tumor spheroid at least one 25 that forms a controlled diffusional paracrine communication pathway between them It includes a spheroid micro-well insert platform (4).

2. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; sub-macrophage layer (2), upper chamber layer (3) and spheroid micro-well insert platform (4) obtained by combining in a reversible (separable) or irreversible manner 30 It is done.

3. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; single micro- the pore, while maintaining the physical separation of cells, allows cytokines, chemokines, Selective diffusion of ROS (reactive oxygen species) and small molecules This requires having a diameter between 5–100 μm.

4. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; only The use of a single pore in a paracrine configuration is porous. absence of membranes and between micro-pore and spheroid center This is achieved by maintaining a vertical distance of 0.2–2 mm.

5. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; spheroid The micro-well allows for the formation of a single, homogeneous 3D tumor spheroid. The defining characteristic is that it has a diameter of 200–800 μm and is either hemispherical or conical.

6. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; sub-macrophage 15 Controlled macrophage stimulation, polarization or re-stimulation of layer (2) To enable programming, continuous perfusion at a rate of 1–100 μL / min It is structured in a way that will provide this.

7. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; sub-macrophage 20 phenotypic remodeling of macrophages (2) in the layer from M2 to M1 to trigger programming a liposomal formulation of resiquimod (LipRes) exposure.

8. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; sub-macrophage 25 layer (2) and upper reservoir layer (3); polydimethylsiloxane (PDMS), cyclic olefin copolymer, polylactic acid (PLA), polymethyl methacrylate (PMMA) or It is produced from combinations of these. 11 9. The microfluidic chip system mentioned in Claim 1 is (1) and its characteristic is; Spheroid micro-well insert platform (4), detachable and pre-formed It allows the spheroids to be placed in the upper chamber layer (3).

10. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; single micro-5 Pore ​​size, geometry, and location, from macrophages to tumor spheroid This results in directional diffusion gradients.

11. The microfluidic chip system mentioned in Claim 1 is (1) and its feature is; A structured system for multiple screening of immunotherapeutic agents, number 10 It contains a spheroidal micro-well insert.

12. Paracrine immunomodulatory activity of macrophages on a tumor spheroid. to evaluate the effects of the system defined by the above requirements (1) a method (100) which uses and its feature is; 101) system (1) lower macrophage 15 (2) culturing of macrophages in the layer; 102) macrophages in one Retraining or stimulation with liposomal TLR agonist; 103) upper Creating a 3D tumor spheroid in the micro-well of the reservoir layer (3); 104) Paracrine diffusion through only a single micropore allowing it to happen; and 105) the tumor spheroid 20 in time morphology, size, viability or secretome changes It includes the steps for measurement.

13. The method mentioned in claim 12 is (100) and its characteristic is that of a liposomal TLR agonist, It contains liposomal resiquimod (LipRes). 25 14. The method mentioned in claim 12 is (100) and its feature is that it is micro-pore. diffusion-borne IL-6, TNF-α, ROS, or macrophage-derived cytokines It involves determining (quantifying) its amount.

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