A microstent structure-based in-vitro tumor microenvironment dynamic construction method

Abstract

The application discloses a micro-scaffold structure-based dynamic construction method of an in-vitro tumor microenvironment, and belongs to the technical field of biomedical engineering and tumor model construction. The method comprises the following steps: firstly, a 3D micro-scaffold with specific pore structure is prepared through a two-photon polymerization printing technology; then, key cell components in the tumor microenvironment are inoculated on the scaffold through a cell-scaffold co-culture technology to obtain a cell-micro-scaffold composite loaded with different cell types; finally, a bubble microrobot generated based on a photothermal bubble microrobot control system is used to pick up, move and spatially arrange the cell-micro-scaffold composite loaded with different cell types, so that a dynamic construction process of the in-vitro tumor microenvironment is realized. The method can construct a tumor microenvironment with high-precision spatial configuration, multi-cell component integration and real-time dynamic observation capability, and can be used for real-time monitoring of tumor cell migration, invasion, cell-cell interaction and high-throughput drug sensitivity screening.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering and tumor model construction technology, and more specifically, to a method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure and a photothermal-driven bubble micromanipulation robot. Background Technology

[0002] The tumor microenvironment is a complex ecosystem composed of tumor cells, various stromal cells, extracellular matrix, and soluble factors. Its dynamic interactions play a crucial role in tumor progression, invasion, metastasis, and treatment resistance. Glioblastoma, for example, is the most aggressive malignant tumor of the central nervous system and has a high postoperative recurrence rate. Studies have shown that the self-renewal, multi-lineage differentiation, and treatment resistance of glioma stem cells (GSCs) are core drivers of glioblastoma (GBM) recurrence and heterogeneity. However, the mechanisms driving GSC invasion and their interactions with microenvironment cells (such as vascular endothelial cells and macrophages) remain unclear, necessitating the construction of physiologically relevant in vitro models for further research.

[0003] Traditional two-dimensional cell culture models (such as scratch assays and Transwell chambers) cannot simulate the three-dimensional complexity of the tumor microenvironment in vivo. To overcome this deficiency, 3D culture models have emerged. Existing 3D models can be divided into scaffold-free models (such as tumor spheroids and organoids) and scaffold-based models. Scaffold-free models rely on cell self-assembly, which can partially preserve cell-cell interactions and tumor heterogeneity, but lacks the precise structural control required to simulate the microenvironment. Scaffold-based methods, such as extrusion bioprinting, have insufficient resolution (hundreds of micrometers), while photolithography bioprinting (such as stereolithography and digital light processing) has higher resolution, but ultraviolet exposure may affect cell viability and it is difficult to achieve heterogeneous co-culture. At the same time, the spatial configuration of many models is oversimplified, failing to reveal the key regulatory role of the relative spatial arrangement of cells in tumor invasion and other behaviors. Therefore, developing an in vitro model platform that can highly simulate the complexity of the tumor microenvironment, has precise spatial configuration control capabilities, supports dynamic assembly of multiple cell components, and is standardized and reproducible is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure. Specifically, it is a method for dynamically constructing an in vitro tumor microenvironment based on two-photon polymerization (2PP) microscaffolds and bubble manipulation technology. This method can construct a tumor microenvironment with high-precision spatial configuration, multi-cellular component integration, and real-time dynamic observation capabilities, which can be used for real-time monitoring of tumor cell migration, invasion, intercellular interactions, and high-throughput drug sensitivity screening.

[0005] The technical solution adopted by this invention to achieve the above objectives is as follows: First, 3D microscaffolds with specific pore structures are mass-produced using two-photon polymerization (2PP) printing technology. Then, key cell components within the tumor microenvironment are seeded onto the scaffolds using cell-scaffold co-culture technology to obtain cell-microscaffold complexes loaded with different cell types. Finally, a bubble microrobot, generated by a photothermal bubble microrobot control system, picks up, moves, and spatially arranges the cell-microscaffold complexes loaded with different cell types, thereby realizing the dynamic construction process of the in vitro tumor microenvironment.

[0006] The 3D microscaffold structure is 3D microprinted on an indium tin oxide coated glass substrate using a focusing objective and IP-S photoresist. The laser source is a 760 nm-800 nm femtosecond laser with a pulse width of 80 fs-100 fs and a repetition frequency of 60 MHz-100 MHz. The printing parameters are as follows: laser power of 30 mW-60 mW, scanning speed of 80000 μm / s-120000 μm / s, slicing distance of 1 μm-2 μm, and filling distance of 0.5 μm-0.8 μm.

[0007] The 3D microscaffolds are fabricated using biocompatible IP-S photoresist and two-photon polymerization (2PP) technology, based on a pre-defined three-dimensional microenvironment configuration and cell type, to produce cell microscaffolds with specific geometric shapes and pore sizes.

[0008] The cell-scaffold co-culture technique involves sterilizing the microscaffold, treating it with adhesion proteins for 2-4 hours, washing the microscaffold, and then seeding the corresponding cells.

[0009] In the cell-scaffold co-culture technology, cell adhesion is promoted by functionalizing the surface of the microscaffold with adhesion proteins. At the same time, different colors of fluorescence are used to pre-label different cell types in the microenvironment so as to track the movement of different cell populations and analyze cell migration and intercellular interaction characteristics during subsequent co-culture.

[0010] The photothermal bubble microrobot control system consists of a laser, a reflector, a focusing objective lens, a petri dish, a chip, a microscope, a signal generator, and a displacement platform. The chip is made of amorphous silicon, indium tin oxide, and glass.

[0011] The method for constructing the photothermal bubble microrobot control system is to place a laser on a displacement platform, and the laser beam first passes through a reflector, then through a focusing lens, and then irradiates the chip to generate a bubble microrobot.

[0012] The method for dynamically constructing a microenvironment based on bubble microrobot manipulation is achieved through the following approach: The bubble microrobot precisely controls the position and movement of bubbles, sequentially picking up cell-microscaffold complexes loaded with different cell types and dynamically assembling them according to a pre-defined spatial configuration. This method uses a bubble microrobot based on the photothermal effect as a surface manipulation tool. First, the generation and movement mechanism of microbubbles were studied by precisely generating and controlling them through the photothermal effect, verifying their good biocompatibility and flexibility in manipulating cell-microscaffold complexes. Based on this, according to the experimental design, the bubble microrobot precisely picks up, pushes, or rotates the cell-microscaffold complexes loaded with different cell types, performing high-precision, low-damage spatial arrangement and assembly, ultimately constructing an in vitro tumor microenvironment model with a pre-defined heterogeneous spatial configuration.

[0013] The in vitro tumor microenvironment includes the in vitro microenvironment for glioma, breast cancer, pancreatic cancer, lung cancer, liver cancer, and melanoma.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention combines 2PP ultra-high precision machining with bubble-manipulated flexible assembly technology to achieve standardized and reproducible fabrication of microscaffolds, as well as precise, programmable, and reconfigurable arrangement of the microenvironment spatial configuration. This provides an unprecedented tool for studying the impact of cell spatial relationships on tumor behavior. Taking the construction of an in vitro glioma microenvironment as an example, this method can achieve comprehensive cell integration within the microenvironment, integrating the three core components of the glioblastoma (GBM) microenvironment—glioma stem cells, cerebral vascular endothelial cells, and macrophages—on a single high-precision three-dimensional platform, more realistically simulating the cellular diversity and heterogeneity of the tumor microenvironment. After the tumor microenvironment is constructed, the platform can maintain the activity and phenotypic stability of each cellular component for a long period, supporting real-time tracking of tumor cell migration, invasion, and intercellular dynamic interactions. Attached Figure Description

[0016] Figure 1 The preparation method and design configuration of the microscaffold of the present invention are shown below; 1 is a macrophage scaffold, 2 is a glioma stem cell scaffold, and 3 is a cerebral vascular endothelial cell scaffold.

[0017] Figure 2 This describes the preparation process of cell-microscaffold complexes based on cell-scaffold co-culture technology.

[0018] Figure 3 Fluorescence images of the prepared cell-microscaffold complex;

[0019] Figure 4This is a schematic diagram of the assembly of the photothermal bubble microrobot control system in this invention;

[0020] Figure 5 The process of dynamically constructing an in vitro microenvironment using the method of this invention is shown in (a), (b), (c), and (d), which sequentially illustrate the process of bubble pickup, movement, spatial arrangement, and assembly. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0022] Example

[0023] A method for dynamically constructing an in vitro tumor microenvironment based on microscaffold structures includes the following steps:

[0024] (1) 3D microscaffolds with specific pore structures were prepared by 2PP printing technology. The specific operation is as follows: Figure 1 As shown. In this embodiment, the in vitro tumor microenvironment is the glioma in vitro microenvironment. The 3D microscaffold is prepared using biocompatible IP-S photoresist and 2PP technology, based on the glioma in vitro microenvironment configuration and cell type, to create a cell microscaffold with a specific geometry and pore size. Figure 1This invention describes the fabrication method and design configuration of the microscaffold. The specific fabrication method involves printing using two-photon polymerization technology and employing biocompatible and high-resolution IP-S photoresist. A 3D microscaffold structure with regularly arranged micropillars, interconnected pores, and internal bridges is designed based on the key cell types within the microenvironment. The microscaffold structure is 3D microprinted on an indium tin oxide (ITO) coated glass substrate using a 25× objective lens (numerical aperture NA=0.8) and IP-S photoresist. The laser source is a 780 nm femtosecond laser with a pulse width of approximately 80 fs-100 fs and a repetition rate of 80 MHz. The printing parameters are as follows: laser power set to 100% (approximately 50 mW), scanning speed of 100,000 μm / s, slicing distance of 1 μm, and hatching distance of 0.5 μm. Taking the construction of an in vitro microenvironment for gliomas as an example, in the figure, 1 represents a macrophage scaffold, 2 represents a glioma stem cell scaffold, and 3 represents a brain vascular endothelial cell scaffold. Specifically, regarding pore design, “×”-shaped bridges, hollow bridges, and “+”-shaped bridges were designed to support the dispersed growth characteristics of macrophages, the clustered growth of glioma stem cells (GSCs), and the adherent growth characteristics of human brain microvascular endothelial cells (hCMECs), respectively. These designs aimed to facilitate the enrichment of macrophages within the scaffold, the clustered growth of GSCs, and the adhesion and guidance of hCMECs. Based on the differences in growth characteristics among the three cell types (macrophages, GSCs, and hCMECs), a pore size selection range was first set according to cell size. Since the diameter of a single cell is approximately 10 μm-20 μm, the minimum pore size was set at 15 μm; considering that cells may aggregate during culture, the upper limit was further extended to 55 μm. The three cell types were seeded on scaffolds with pore sizes of 15 μm, 35 μm, and 55 μm and different internal structures (“×”-shaped bridges, hollow bridges, and “+”-shaped bridges), and their growth and filling were observed. After comprehensive evaluation, the optimal pore size was determined to be 50 μm for the following reasons: At a pore size of 50 μm, all three cell types can effectively enter the scaffold, and the scaffold can be filled within approximately 48 hours, ensuring cell viability while meeting the experimental time window requirements. Therefore, the optimal pore size was set at 50 μm. Each pore unit adopts a cubic design with precise dimensional control: the outer frame width is 5.7 μm, and the internal bridge structure width is 2.9 μm. Regarding the external contour, a semi-circular scaffold was designed for macrophages and hCMECs, and a circular scaffold was designed for GSCs; the macrophage scaffold has an outer diameter of 396 μm and an inner diameter of 223 μm; the glioma stem cell scaffold has a diameter of 219 μm; and the cerebral vascular endothelial cell scaffold has an outer diameter of 396 μm and an inner diameter of 223 μm; the scaffold height for all three is 61.5 μm.

[0025] (2) Using cell-scaffold co-culture technology, key cell components in the tumor microenvironment were seeded onto scaffolds to obtain cell-microscaffold complexes loaded with different cell types. The specific operation is as follows: Figure 2 , Figure 3 As shown. The cell-scaffold co-culture technique involves sterilizing the microscaffolds, treating them with adhesion proteins for 2 hours, washing the microscaffolds, and then seeding the corresponding cells. In this cell-scaffold co-culture technique, adhesion proteins are functionalized on the surface of the microscaffolds to promote cell adhesion; simultaneously, different colors of fluorescence are used to pre-label different cell types within the microenvironment to track the movement of different cell populations during subsequent co-culture and analyze cell migration and intercellular interaction characteristics.

[0026] Figure 2 To illustrate the preparation process of cell-microscaffold complexes based on cell-scaffold co-culture technology, the microscaffolds were sterilized, treated with adhesion proteins for 2 hours, and then washed with phosphate-buffered saline (PBS) before being seeded with appropriate cells. Typically, a high-concentration cell suspension of 100,000 cells per milliliter was added to the scaffold area in 75 μL droplets. The cells were co-cultured with the scaffolds at 37°C and 5% CO2 for 4 hours. Then, 5 mL of cell culture medium was gently added to the culture dish, and the dish was incubated for 48 hours to allow for sufficient cell colonization and scaffold filling, resulting in the cell-microscaffold complex.

[0027] Figure 3 The image shows a fluorescence image of the prepared cell-microscaffold complex, with a scale bar of 100 μm. Different cell types were stained with fluorescence. Taking the glioma in vitro microenvironment as an example, scaffold complexes loaded with macrophages, GSCs, and hCMECs were incubated for 30 minutes in 3 mL of red membrane fluorescent probes (DiI; Beyotime), green membrane fluorescent probes (DiO; Beyotime), and far-infrared membrane fluorescent probes (DiD; Beyotime), respectively. These staining methods generally do not affect cell viability and can be used to detect cell migration. After entering the cell membrane, DiI-stained macrophages emitted red fluorescence under 555 nm excitation, DiO-stained GSCs emitted green fluorescence under 484 nm excitation, and DiD-stained hCMECs emitted far-infrared fluorescence under 640 nm excitation, appearing purple in the image. Subsequently, the cells were gently washed twice with PBS buffer and added to the appropriate culture medium for subsequent construction of the in vitro microenvironment.

[0028] (3) Based on the bubble microrobot control system, the bubble microrobot picks up, moves, and spatially arranges the cell-microscaffold complex loaded with different cell types, thereby realizing the dynamic construction process of the in vitro microenvironment of glioma. The specific operation is as follows: Figure 4 , Figure 5 As shown, the photothermal bubble microrobot control system consists of a laser, a reflector, a focusing objective lens, a petri dish, a chip, a microscope, a signal generator, and a displacement platform. The chip is made of amorphous silicon, indium tin oxide, and glass. The system is constructed by placing the laser on the displacement platform; the laser beam first passes through the reflector, then through the focusing objective lens, and finally irradiates the chip, generating a bubble microrobot.

[0029] The method for dynamically constructing a microenvironment based on bubble microrobot manipulation is achieved through the following approach: The bubble microrobot precisely controls the position and movement of bubbles, sequentially picking up cell-microscaffold complexes loaded with different cell types and dynamically assembling them according to the spatial configuration of the glioma in vitro microenvironment. This method uses a bubble microrobot based on the photothermal effect as a surface manipulation tool. First, microbubbles are precisely generated and controlled through the photothermal effect, and the generation and movement mechanisms of the bubbles are studied, verifying their good biocompatibility and flexibility in manipulating the cell-microscaffold complexes. Based on this, according to the experimental design, the bubble microrobot precisely picks up, pushes, or rotates the cell-microscaffold complexes loaded with different cell types, performing high-precision, low-damage spatial arrangement and assembly, ultimately constructing a glioma in vitro microenvironment model.

[0030] Figure 4This is a schematic diagram of the assembly of the photothermal bubble microrobot control system of this invention. A 980nm wavelength laser is placed horizontally on a displacement platform. The laser beam is first redirected by a mirror (BB1-E03, Thorlabs, Newton, USA), then focused by a 25× focusing objective, and irradiates a chip in a culture dish to generate bubble microrobots at the solid-liquid interface. The chip has an amorphous silicon (α-Si) absorber layer with a thickness of 1 µm, and the substrate is a 1 mm thick glass plate coated with indium tin oxide. Furthermore, a square wave signal is generated using a signal generator to modulate the laser, which can change the bubble growth rate, thereby precisely controlling the bubble size. In PBS solution, typical experimental parameters are set as follows: laser power 30 mW, square wave frequency 100 Hz, duty cycle 50%. Under these conditions, the bubble grows rapidly and reaches a steady state within 10 s after laser irradiation, with a diameter stabilizing in the range of 40 μm-50 μm. In practice, the bubble size can be flexibly controlled by adjusting the laser power (20 mW-50 mW) or duty cycle (25%–75%). For the various cell-scaffold composite structures used in this study, bubbles with a diameter in the range of 20 μm-50 μm can effectively drive the scaffold to complete the operation—this size range can generate sufficient driving force without affecting cell viability due to local overheating. Based on this, we determined the optimal parameter window for bubble manipulation and uniformly adopted the above conditions in subsequent experiments.

[0031] Figure 5 This invention describes the process of dynamically constructing an in vitro microenvironment using the method described in this invention. Cell-microscaffold complexes are scraped from a substrate and transferred onto a chip using a pipette tip. A bubble-microrobot precisely controls the position and movement of bubbles to sequentially pick up one macrophage-scaffold complex, one glioma stem cell-scaffold complex, and one cerebral vascular endothelial cell-scaffold complex. These are then precisely assembled and dynamically joined together according to a pre-defined spatial configuration (in this case, a circular microenvironment).

Claims

1. A method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure, characterized in that, Includes the following steps: First, 3D microscaffolds with specific pore structures were fabricated using two-photon polymerization printing technology. Subsequently, key cellular components within the tumor microenvironment were seeded onto scaffolds using cell-scaffold co-culture technology to obtain cell-microscaffold complexes loaded with different cell types. Finally, based on the bubble microrobot control system, the bubble microrobot is used to pick up, move, and spatially arrange the cell-microscaffold complex loaded with different cell types, thereby realizing the dynamic construction process of the in vitro tumor microenvironment.

2. The method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure according to claim 1, characterized in that, The 3D microscaffold structure is 3D microprinted on an indium tin oxide coated glass substrate using a focusing objective and IP-S photoresist. The laser source is a 760 nm-800 nm femtosecond laser with a pulse width of 80 fs-100 fs and a repetition frequency of 60 MHz-100 MHz. The printing parameters are as follows: laser power of 30 mW-60 mW, scanning speed of 80000 μm / s-120000 μm / s, slicing distance of 1 μm-2 μm, and filling distance of 0.5 μm-0.8 μm.

3. The method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure according to claim 1, characterized in that, The cell-scaffold co-culture technique involves sterilizing the microscaffold, treating it with adhesion proteins for 2-4 hours, washing the microscaffold, and then seeding the corresponding cells.

4. The method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure according to claim 1, characterized in that, The photothermal bubble microrobot control system consists of a laser, a reflector, a focusing objective, a petri dish, a chip, a microscope, a signal generator, and a displacement platform; wherein the chip is composed of amorphous silicon, indium tin oxide, and glass.

5. The method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure according to claim 1, characterized in that, The method for constructing the photothermal bubble microrobot control system is to place a laser on a displacement platform, and the laser beam first passes through a reflector, then through a focusing lens, and then irradiates the chip to generate a bubble microrobot.

6. The method for dynamically constructing an in vitro tumor microenvironment based on a microscaffold structure according to claim 1, characterized in that, The in vitro tumor microenvironment includes the in vitro microenvironment for glioma, breast cancer, pancreatic cancer, lung cancer, liver cancer, and melanoma.

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