Human tumor organoid culture method based on green light volume bioprinting technology

Abstract

This invention proposes a method for culturing human tumor organoids based on green light volumetric bioprinting (GLVBP) technology, belonging to the field of bioculture technology. By optimizing the bio-ink formulation, this invention combines human tumor cells with photocrosslinked biomaterials (such as methacrylated gelatin and hyaluronic acid derivatives) and a matrix adhesive to prepare a photocurable bio-ink suitable for GLVBP. Using GLVBP technology, three-dimensional models of tumor organoids with biomimetic structures can be rapidly constructed, achieving efficient proliferation and functional maintenance of the organoids. This invention solves the problems of poor structural controllability and large batch-to-batch variability in traditional organoid culture. The prepared tumor organoids possess a highly biomimetic tumor microenvironment and stable biological characteristics, and have significant application value in drug screening, personalized medicine, and tumor mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of bioculture technology, and in particular relates to a method for culturing human tumor organoids based on green light volumetric bioprinting technology. Background Technology

[0002] With the rapid development of precision medicine and regenerative medicine, patient-derived organoids (PDOs) have become a core tool for cancer mechanism research, high-throughput drug screening, and personalized clinical medication guidance because they can highly mimic the tissue structure, functional characteristics, and biological responses of tumors in patients.

[0003] Currently, organoid construction mainly relies on the three-dimensional microenvironment provided by natural extracellular matrices such as Matrigel. While traditional culture methods (such as droplet or embedding methods) are simple to operate, they suffer from significant technical bottlenecks: First, the resulting three-dimensional structures are random, failing to accurately simulate the complex spatial geometry and vascular system of tumors; second, limited by manual manipulation, organoids prepared using these methods exhibit significant batch-to-batch variations in morphology and size, leading to poor reproducibility and comparability of experimental results.

[0004] To improve the controllability of structures, 3D bioprinting technology has been introduced into the field of organoid construction. However, mainstream extrusion bioprinting technology still faces severe challenges in application: high shear stress can easily cause mechanical damage to sensitive cells such as primary tumor cells, and the printing efficiency is low and the resolution is insufficient, making it difficult to meet the needs of large-scale applications.

[0005] In recent years, Green Light-based Volumetric Bioprinting (GLVBP), as an emerging non-contact manufacturing technology, has attracted widespread attention due to its advantages such as high-speed light imaging, one-time integral molding, and extremely low cell damage. However, existing volumetric printing solutions still suffer from insufficient material-function compatibility: current GLVBP technologies mostly use synthetic photocrosslinking materials, which, although capable of molding, lack the natural matrix components (such as ECM proteins) required for organoid growth, making it difficult to support the long-term functional maintenance of organoids; while Matrigel, with its excellent bioactivity, cannot be directly photocured due to its physical properties, making it difficult to effectively integrate with efficient volumetric printing technologies.

[0006] Therefore, how to develop a technical solution that can fully utilize the high efficiency and precision of volumetric printing, and provide a highly biomimetic tumor microenvironment to achieve standardized and high-performance construction of human tumor organoids is a key issue that urgently needs to be addressed in the fields of biomedicine and tissue engineering. Summary of the Invention

[0007] The first objective of this invention is to propose a method for culturing human tumor organoids based on green light volumetric bioprinting technology, comprising the following steps:

[0008] S1, Obtain human tumor cell suspension;

[0009] S2, the cell suspension is mixed with a bio-ink containing a photocrosslinking matrix, matrix gel and visible light initiation system to obtain a composite bio-ink;

[0010] S3, Based on the preset three-dimensional model, the composite bio-ink is integrally molded and printed to construct a three-dimensional scaffold structure containing tumor cells and having internal through channels.

[0011] S4. The printed three-dimensional scaffold structure is induced and cultured in vitro to obtain organoids.

[0012] Preferably, in step S1, the isolated human tumor cells are tested and found to have a cell viability rate ≥70% and a live cell concentration ≥1×10⁻⁶. 6 per mL.

[0013] Preferably, in step S2, the components of the composite bio-ink, by mass-volume percentage, include:

[0014] 1.25%-20% photocrosslinking matrix;

[0015] 5%-50% Matrigel;

[0016] And visible light initiation systems;

[0017] The photocrosslinking matrix is ​​selected from at least one of methacrylamide gelatin (GelMA), hyaluronic acid derivative (SF), silk fibroin, and sericin (SS).

[0018] The visible light initiation system is a Ru / SPS system, wherein the working concentration of Ru is 0.1-1.0 mM and the working concentration of SPS is 0.5-20 mM.

[0019] Preferably, in step S3, the sphere is a three-dimensional model with internal intersecting channels, the width of which is 100-500 μm, and is used to provide nutrient exchange and gas diffusion pathways for organoid growth.

[0020] Preferably, in step S3, the overall molding printing uses green visible light with a wavelength of 520nm-535nm as the light source, the printing light intensity is 3-20 mW / cm², and the exposure time is 60-120 seconds.

[0021] Preferably, the human tumor cells are derived from malignant effusion and / or tumor tissue of cancer patients;

[0022] When the source is malignant effusion, the pretreatment includes sequential cell filtration, red blood cell lysis and discontinuous density gradient centrifugation.

[0023] When the source is tumor tissue, pretreatment includes mechanical shearing and complex enzyme digestion.

[0024] The human tumor is lung adenocarcinoma, and the induction culture in step S4 uses a complete culture medium containing EGF, FGF-10, Noggin and R-spondin 1.

[0025] A second objective of this invention is to provide human tumor organoids prepared by the aforementioned method, wherein the organoids are distributed around the channels of a three-dimensional scaffold, have a diameter ≥50μm, and retain the biological characteristics of the primary tumor.

[0026] A third objective of this invention is to provide a human tumor organoid culture system based on the aforementioned method, comprising:

[0027] The GLVBP printing module is used to solidify composite bio-ink into shape in one step according to a preset three-dimensional model;

[0028] The model design module is used to construct a three-dimensional digital model of a structure with an internal through-channel.

[0029] It also includes a culture monitoring module, which provides a constant-temperature culture environment for the printed 3D scaffold and monitors the morphological evolution of the organoid.

[0030] The human tumor organoid culture technology of the present invention is based on the synergistic effect of photochemical crosslinking and hydrodynamic optimization, and its core working mechanism includes the following three aspects:

[0031] In-situ rapid photocuring mechanism:

[0032] Green visible light (525nm) is used to excite the Ru / SPS initiator system to generate free radicals, inducing polymerization at the methacrylation sites in the bio-ink. Compared to traditional layer-by-layer printing, this invention uses a GLVBP optical projection system to project a three-dimensional model onto a rotating ink bottle, achieving synchronous cross-linking of the ink throughout space. This non-contact, instantaneous forming method avoids the mechanical shear forces of extrusion printing, thus preserving the initial activity of primary tumor cells to a great extent.

[0033] The construction principle of biomimetic microenvironments:

[0034] By molecularly combining Matrigel, which possesses excellent bioactivity, with a photocrosslinking matrix (such as GelMA) that provides good mechanical support, a hybrid microenvironment was constructed that meets the requirements for volumetric printing precision while also providing signals from the natural extracellular matrix (ECM). The laminin and collagen in Matrigel provide necessary adhesion sites for tumor cells, inducing cells to evolve from a monodisperse state into a three-dimensional organoid structure.

[0035] Controlled diffusion and nutrient supply principles:

[0036] Leveraging the high precision of volumetric printing, cross-shaped through-channels (300 μm) were pre-formed inside microspheres with a diameter of only 2 mm. According to the laws of fluid diffusion, these channels act as an artificial "vascular system," effectively shortening the distance oxygen and nutrients diffuse to the organoid core by increasing the surface area to volume ratio (S / Vratio). Simultaneously, metabolic waste can be promptly discharged through these channels, overcoming the technical bottleneck of internal necrosis limiting the growth of large-sized organoids.

[0037] Compared with existing traditional matrix gel dispensing and extrusion 3D bioprinting technologies, this invention has the following significant advantages:

[0038] (1) Extremely high construction efficiency and throughput:

[0039] This invention employs volumetric integral molding technology, which reduces the printing time of a single organoid scaffold from several hours using traditional methods to 30-120 seconds, significantly improving the preparation efficiency of human tumor organoids and making large-scale, high-throughput drug screening possible.

[0040] (2) Excellent structural controllability and batch stability:

[0041] By precisely controlling the shape and internal channel structure of organoids using digital models, the randomness of shape and unevenness in size caused by manual operation in traditional drip irrigation methods are eliminated. Experimental data show that the coefficient of variation of organoid diameter prepared by this invention is significantly reduced, ensuring the reproducibility of experimental results.

[0042] (3) Higher cell viability and functional fidelity:

[0043] Utilizing a low-energy, green visible light system combined with a highly biocompatible Ru / SPS initiation system, phototoxicity and chemical toxicity were minimized. The prepared organoids highly expressed specific markers such as TTF-1, NapsinA, and CK7 at the molecular level, enabling precise replication of tumor biological characteristics in patients.

[0044] (4) Enhanced growth performance and shorter culture cycle:

[0045] Thanks to the nutrient exchange advantage provided by the internal perforated channels, organoids exhibit stronger proliferative activity during culture, with a structural integrity rate of over 92.5% within 14 days. They can also grow to larger sizes without core necrosis, providing high-quality samples for long-term drug sensitivity testing and genomics research.

[0046] (5) Wide sample applicability:

[0047] This protocol optimizes the pretreatment process for malignant pleural effusion and tissue samples. In particular, the introduction of discontinuous density gradient centrifugation significantly improves the efficiency of separating and purifying tumor cells from complex clinical samples, stabilizing the initial cell viability at over 70%-80% and expanding the scope of clinical sample utilization. Attached Figure Description

[0048] Figure 1 The diagram shows the processing flow and results of human lung adenocarcinoma samples.

[0049] Figure 2 Human lung adenocarcinoma organoid culture system.

[0050] Figure 3 Morphological characteristics of organoids under different printing parameter groups and culture times.

[0051] Figure 4 Comparison of organoid morphology prepared by the traditional Matrigel drip method and the GLVBP method of this invention.

[0052] Figure 5 This is an RT-qPCR identification image of the human lung adenocarcinoma organoids prepared in this invention.

[0053] Figure 6 A schematic diagram illustrating the entire process of constructing human lung cancer organoids based on green light volumetric bioprinting (GLVBP) technology. Detailed Implementation

[0054] To better understand the present invention, the present invention will be further described below with reference to specific serial numbers. The terminology used in the serial numbers is for describing specific embodiments and does not constitute a limitation on the scope of protection of the present invention.

[0055] In the specific implementation methods, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0056] Unless otherwise specified, percentages, % and so on in the specific implementation method are assumed to be mass percentages.

[0057] In the following examples, all operations were performed in a biosafety cabinet, and cell viability was required to be >80% for subsequent experiments. Matrigel was placed in an ice bath overnight before use at an initial concentration of 100%. Unless otherwise specified, containers and solutions in contact with Matrigel should be pre-cooled to 0-4°C and maintained for at least 30 minutes.

[0058] Figure 2 Human lung adenocarcinoma organoid culture system; Figure 6 This is a schematic diagram illustrating the entire process of constructing human lung cancer organoids based on green light volumetric bioprinting (GLVBP) technology. Both are used to demonstrate the workflow of this invention.

[0059] Example 1: Isolation and purification of human lung adenocarcinoma cells

[0060] For pleural effusion samples: Place a 70 μm cell filter on a sterile 50 mL centrifuge tube, slowly add 20-30 mL of pleural effusion sample for filtration, centrifuge at 440×g for 10 min, discard the supernatant, add 3-5 times the volume of erythrocyte lysis buffer, lyse at 4℃ for 10 min, centrifuge at 400×g at 4℃ for 5 min, discard the supernatant, and wash the precipitate twice with pre-cooled DMEM / F12 medium. Separate cancer cells using Percoll density gradient centrifugation (40%:75% discontinuous density gradient, 780×g, 20 min, 4℃), carefully aspirating the cancer cell layer distributed on the 40% Percoll interface.

[0061] Process and results as follows Figure 1 As shown in (a).

[0062] For tumor tissue samples: Wash 3-5 times with ice-cold physiological saline containing 2% penicillin and antibiotics. Mechanically mince the tissue to approximately 1 mm³ in pre-chilled sterile Petri dishes. Digest with a mixture of collagenase IV (2 mg / mL), hyaluronidase (1 mg / mL), and DNase I (0.1 mg / mL) at 37°C (100 rpm) for 60 min, gently pipetting every 15 min during digestion. After digestion, add 3 volumes of pre-chilled HBSS to terminate the digestion. Filter the solution through a 70 μm cell sieve, centrifuge the filtrate at 300×g for 5 min, and resuspend to obtain a cell suspension.

[0063] Process and results as follows Figure 1 As shown in (b).

[0064] Example 2: Formulation and Optimization of Bio-ink

[0065] This embodiment details the preparation method of the bio-ink: 40% (w / v) methacrylamide gelatin (GelMA), 40% (w / v) silk fibroin (SF) or 40% (w / v) silk fibroin (SS) are dissolved in deionized water at 37°C. An equal volume of pre-cooled 5% Matrigel (v / v) is slowly added and gently mixed to avoid generating bubbles. Finally, the photoinitiator Ru / SPS (final concentration: 0.25 mM Ru, 2.5 mM SPS) and cell suspension (density adjusted to 1500 cells / μL) are added.

[0066] All operations were performed in an ice bath and in the dark.

[0067] Example 3: Volumetric Bioprinting Process Parameters

[0068] This embodiment details the fabrication process for a three-dimensional biological scaffold with internal channel structures based on GLVBP technology, and optimizes and verifies key printing parameters. This invention particularly emphasizes the construction of internal interconnected channels within the three-dimensional structure, a feature crucial for achieving efficient nutrient delivery and gas exchange in organoids.

[0069] (1) Printing process

[0070] The Matrigel-based photocrosslinked composite bio-ink prepared in Example 2 was filled into a pre-cooled dedicated cylindrical glass printing bottle (bottom diameter 1.2 cm, height 2 cm), and after removing air bubbles, it was placed in the center of the printing platform. A 525 nm green visible light source was set up, and a three-dimensional model of a cross-shaped channel sphere was imported into computer-aided design software. The core feature of the three-dimensional model is that it includes an internal through-channel structure; in this example, a channel width of 300 μm is used as a representative example.

[0071] Printing parameters are set as follows: light intensity 3-20 mW / cm², printing time 60-120 seconds.

[0072] After printing, the material was gently rinsed three times with sterile PBS preheated to 37°C to remove uncrosslinked material, and then transferred to an ultra-low adsorption 6-well plate for incubation.

[0073] (2) Parameter verification

[0074] To verify the universality of the printing parameter range (light intensity 3-20 mW / cm², printing time 60-120 seconds) described in the claims, organoid culture verification was performed using the lower limit, upper limit, and median values ​​within this range:

[0075] Lower limit group: light intensity 3 mW / cm², printing time 60 seconds

[0076] Upper limit group: light intensity 20 mW / cm², printing time 120 seconds

[0077] Intermediate group: light intensity 10 mW / cm², printing time 90 seconds

[0078] (3) Parameter optimization

[0079] Based on the characteristics of different photocrosslinking matrix materials, the printing parameters for the three materials were optimized during the printing process, as follows:

[0080] 2.5% GelMA / 20% Matrigel group: Printing light intensity 3.5 mW·cm⁻², printing time 60 seconds;

[0081] 2.5% SF / 20% Matrigel group: Printing intensity 3 mW·cm⁻², printing time 60 seconds;

[0082] 2.5% SS / 20% Matrigel group: Printing intensity 7 mW·cm⁻², printing time 60 seconds.

[0083] It should be noted that the printing process described in this invention is applicable to constructing various three-dimensional structures, and the internal channel feature can be applied to various geometric shapes such as spheres, cylinders, and cubes, and is not limited to the spherical structure described in the embodiments. These channel-containing structures of different shapes can all be successfully fabricated by adjusting the printing parameters described in this invention.

[0084] Furthermore, this embodiment, using a channel width of 300 μm as an example, demonstrates the ability of the printing process of this invention to form internal channel structures. Based on the same technical principles, by adjusting the model design, this invention is also applicable to the fabrication of three-dimensional structures with channel widths of other sizes, and these structures with different channel sizes are all within the protection scope of this invention.

[0085] like Figure 5 The figure shown is an RT-qPCR identification diagram of the human lung adenocarcinoma organoids prepared in this invention. The organoids prepared in this embodiment continuously express lung adenocarcinoma-specific markers TTF-1, Napsin A, and CK7, demonstrating that they highly retain the biological characteristics of the primary tumor at the molecular level.

[0086] like Figure 3 As shown, the GLVBP printing scheme established in this invention exhibits good organoid formation ability under different key printing parameters.

[0087] Figure 3 The results in section a show that, under the conditions of the lower limit, upper limit and middle group of the parameter range, organoids with typical three-dimensional structures can be successfully induced on the fourth day of culture, proving that the printing process of the present invention has a wide window and good robustness.

[0088] Figure 3 Figure b further demonstrates the complete developmental process of organoids over time under optimized parameters: initially, cells successfully aggregate and form initial spheres, then the organoid structure gradually becomes denser, the boundaries become clearer, and finally it develops into a mature, structurally complete three-dimensional organoid, showing a good growth and development trend.

[0089] Example 4: Establishment of an organoid culture system

[0090] This example describes the organoid culture conditions in detail: using DMEM / F12 basal medium, with the following components added:

[0091] 1×B-27 Supplement

[0092] 1×N-2 Supplement

[0093] 10 mM HEPES

[0094] 2 mM GlutaMAX,

[0095] 100 U / mL penicillin-streptomycin

[0096] 1.25 mM N-acetylcysteine

[0097] 5 mM nicotinamide.

[0098] The growth factors added include: 500 ng / mL R-spondin1, 100 ng / mL Noggin, 100 ng / mL FGF-10, 50 ng / mL EGF, and 100 ng / mL Wnt 3a.

[0099] Small molecule inhibitors include: 500 nM SB202190, 10 μM Y-27632, and 500 nM A83-01.

[0100] Cultured at 37℃ and 5% CO2 saturated humidity, with freshly prepared complete culture medium replaced every 3-4 days, for a culture period of 14-21 days.

[0101] Comparative Example 1: Culture using the conventional Matrigel dispensing method

[0102] This comparative example uses the conventional Matrigel droplet method for organoid culture. The specific steps are as follows: cells are mixed with liquid Matrigel under ice bath conditions, and then added to a culture plate at a rate of 30 μL / drop. The mixture is then solidified at 37°C, followed by the addition of complete culture medium for culturing. This comparative example is used to verify the differences between the conventional method and the present invention.

[0103] Comparative Example 2: Printing three-dimensional structures without channels for culture

[0104] This comparative example uses the same bio-ink and printing parameters as the embodiment, but the 3D model is a solid structure without any internal channels. This comparative example aims to verify the crucial role of the internal channel structure in organoid nutrient supply, gas exchange, and growth efficiency, highlighting the technical advantages of this invention in incorporating a channel structure.

[0105] like Figure 4 As shown, by comparing the morphological characteristics of organoids obtained by different preparation methods, the significant advantages of the GLVBP technology system of this invention can be clearly observed.

[0106] Compared to the traditional Matrigel dripping method and printed structures without channels, the channel-containing organoid structures constructed using the GLVBP technology of this invention exhibit significant improvements in morphological regularity, structural integrity, and overall growth status. The organoids are more evenly distributed and exhibit more typical three-dimensional morphology, fully validating that this invention, through integrated printing of channel-containing structures, can provide a superior biomimetic microenvironment for organoids, thereby effectively supporting their high-quality formation and growth.

[0107] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for culturing human tumor organoids based on green light volumetric bioprinting technology, characterized in that, Includes the following steps: S1, Obtain human tumor cell suspension; S2, the cell suspension is mixed with a bio-ink containing a photocrosslinking matrix, matrix gel and visible light initiation system to obtain a composite bio-ink; S3, Based on the preset three-dimensional model, the composite bio-ink is integrally molded and printed to construct a three-dimensional scaffold structure containing tumor cells and having internal through channels. S4. The printed three-dimensional scaffold structure is induced and cultured in vitro to obtain organoids.

2. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 1, characterized in that, In step S1, the isolated human tumor cells are tested, and their cell viability is ≥70% and the concentration of live cells is ≥1×10⁻⁶. 6 per mL.

3. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 1, characterized in that, In step S2, the components of the composite bio-ink, by mass-volume percentage, include: 1.25%-20% photocrosslinking matrix; 5%-50% of the base adhesive; And visible light initiation systems; The photocrosslinking matrix is ​​selected from at least one of methacrylamide gelatin, hyaluronic acid derivatives, silk fibroin, and sericin.

4. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 3, characterized in that, The visible light initiation system is a Ru / SPS system, wherein the working concentration of Ru is 0.1-1.0 mM and the working concentration of SPS is 0.5-20 mM.

5. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 1, characterized in that, In step S3, the sphere is a three-dimensional model with internal intersecting channels. The width of the channels is 100-500 μm, which are used to provide nutrient exchange and gas diffusion pathways for organoid growth.

6. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 1, characterized in that, In step S3, the overall molding printing uses green visible light with a wavelength of 520nm-535nm as the light source, the printing light intensity is 3-20mW / cm², and the exposure time is 60-120 seconds.

7. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 1, characterized in that, The human tumor cells are derived from malignant effusion and / or tumor tissue of cancer patients; When the source is malignant effusion, the pretreatment includes sequential cell filtration, red blood cell lysis and discontinuous density gradient centrifugation. When the source is tumor tissue, pretreatment includes mechanical shearing and complex enzyme digestion.

8. The method for culturing human tumor organoids based on green light volumetric bioprinting technology according to claim 7, characterized in that, The human tumor is lung adenocarcinoma, and the induction culture in step S4 uses a complete culture medium containing EGF, FGF-10, Noggin and R-spondin 1.

9. The human tumor organoids prepared by the method according to any one of claims 1-8, characterized in that: The organoids are distributed around the channels of the three-dimensional scaffold, with a diameter ≥50μm, and retain the biological characteristics of the primary tumor.

10. A human tumor organoid culture system for implementing the method of claim 1, characterized in that, include: The GLVBP printing module is used to solidify composite bio-ink into shape in one step according to a preset three-dimensional model; The model design module is used to construct a three-dimensional digital model of a structure with an internal through-channel. It also includes a culture monitoring module, which provides a constant-temperature culture environment for the printed 3D scaffold and monitors the morphological evolution of the organoid.

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