Colorectal tumor model based on 3D printing and construction method and application thereof

Abstract

The invention discloses a construction method of a colorectal tumor model based on 3D printing, which comprises the following steps: dissolving methacrylated gelatin and phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate together to obtain a mixed solution, adding colorectal cancer cells HCT-116 and human skin fibroblasts BJ into the mixed solution, and uniformly mixing to obtain bio-ink; and loading the bio-ink into a charging barrel of an extrusion type biological 3D printer for 3D printing to obtain the tumor cell-carrying gel microspheres, and culturing in vitro to obtain the tumor cell-carrying gel microspheres. According to the method, the upper limit of cell density of liquid drop biological 3D printing is broken through, stable printing of biological ink with the cell density as high as 1 * 10 < 8 > cells / mL is successfully achieved through optimization of a rheological window, the density is close to the physiological level of solid tumors, the printed tumor model can truly simulate the high compactness of the tumors and the nutrition / oxygen gradient effect caused by the high compactness of the tumors, and the tumor model has good application prospects. And an experimental platform closer to the in vivo is provided for researching the microenvironment heterogeneity in the tumor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a 3D-printed colorectal tumor model, its construction method, and its application. Background Technology

[0002] A core challenge in current tumor biology research lies in accurately simulating the complex pathological features of the tumor microenvironment (TME) to construct in vitro tumor models. Solid tumors not only contain high-density tumor cell clusters but also are rich in fibrotic stroma composed of cancer-associated fibroblasts (CAFs), abnormal extracellular matrix (ECM) deposition, and internal hypoxic necrosis zones due to insufficient vascularization. These factors collectively shape the unique spatial heterogeneity and drug resistance of tumors. However, traditional two-dimensional (2D) cell culture models lack three-dimensional structure and dynamic intercellular interactions, failing to reflect real tumor biological behavior. Although three-dimensional tumor spheroid models compensate for the lack of dimension to some extent, their cell density is typically limited to 1×10⁻⁶. 7 Tumors with a cell / mL count of less than 100 cells have small size and lack controllable size uniformity, resulting in short survival time. Furthermore, they are difficult to reproduce the pathologically significant topological structure of "tumor core-fibrotic encapsulation," which further restricts the reproducibility and clinical relevance of drug screening.

[0003] The rise of bioprinting technology has provided a new pathway for precisely biomimetic tumor microenvironments, but it still faces multiple technical bottlenecks in constructing highly physiologically relevant tumor models. In extrusion printing, nozzle clogging and cell damage are limiting factors for achieving high cell densities (>1×10⁻⁶). 7The primary obstacle to printing cells / mL is the excessive shear stress, which not only significantly reduces cell viability but also easily causes nozzle malfunction. Therefore, there are limitations to using bioprinting technology to construct in vitro models with cell densities reaching organ and tissue levels. Meanwhile, droplet bioprinting methods based on inkjet printing and other technologies, while showing promise due to their high-throughput forming capabilities, have revealed significant limitations when applied to tumor model construction. Traditional droplet printing technology is only suitable for inks with extremely low viscosity (typically <10 mPa·s). For commonly used medium-viscosity bioinks, such as methacrylamide gelatin (GelMA), it is difficult to stably form droplets with controllable sizes. Regarding temperature control strategies, existing methods mostly employ low-temperature rapid freezing and solidification, which not only requires complex refrigeration systems but also directly damages cell membrane integrity during the freeze-thaw process, making it unsuitable for long-term live cell culture. Room-temperature printing, on the other hand, suffers from droplet tailing and size inhomogeneity due to the high viscosity of the bioink. These technical limitations restrict the application of existing droplet bioprinting technology in constructing tumor models with cell density levels matching solid tumors. In summary, existing technologies have failed to effectively integrate the rheological properties of temperature-responsive bio-inks with droplet printing processes to achieve the construction of tumor models with high cell density, multicellular heterogeneity, and rapid self-support within a mild physiologically relevant temperature range. There is an urgent need for a new method that can precisely regulate the rheology of bio-inks to maintain a "weak gel" state within the inherent gel-sol transition temperature window, combining shear-thinning printability with rapid structural recovery after extrusion, thereby constructing a biomimetic topological structure of "tumor core - fibroblast encapsulation" in one step. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a colorectal tumor model based on 3D printing, which addresses the shortcomings of the prior art.

[0005] Another technical problem to be solved by the present invention is to provide a colorectal tumor model constructed by the aforementioned construction method.

[0006] The final technical problem to be solved by this invention is to provide the application of the aforementioned colorectal tumor model in the study of the occurrence and development of colorectal cancer, the development of clinical therapies, the screening and evaluation of antitumor drugs, the detection of antitumor drug sensitivity, and the study of drug resistance mechanisms.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0008] The first aspect of this invention provides a method for constructing a colorectal tumor model based on 3D printing, comprising the following steps:

[0009] Step 1: Dissolve methacrylamide gelatin (GelMA) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate together to obtain a mixed solution. Add colorectal cancer cells HCT-116 and human skin fibroblasts BJ to the solution and mix well to obtain bio-ink.

[0010] Step 2: Load the bio-ink obtained in Step 1 into the barrel of the extrusion-type bio-3D printer for 3D printing to obtain tumor cell-loaded gel microspheres, which are then cultured in vitro.

[0011] In step 1, the total cell density of HCT-116 colorectal cancer cells and BJ human skin fibroblasts in the bio-ink is 1 × 10⁻⁶. 6 ~ 1×10 8 cells / mL, preferably 8 × 10⁻⁶ 7 ~ 1×10 8 cells / mL.

[0012] In step 2, during 3D printing, the temperature of the bio-ink is controlled at 25~28℃. This temperature range corresponds to the gel-sol transition temperature window of GelMA. Within this window, the ratio tanδ of the storage modulus G' to the loss modulus G'' of the bio-ink is adjusted to 0.3~0.5, ensuring that the bio-ink is in a weak gel state with shear-thinning properties, while simultaneously preventing cell sedimentation in the barrel, which could lead to uneven cell density.

[0013] In step 1, the concentration of methacrylamide gelatin in the bio-ink is 0.03 ~ 0.08 g / mL, preferably 0.05 g / mL, and the concentration of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate is 0.5 ~ 5 g / L, preferably 1 g / L.

[0014] Preferably, in step 1, the solvent for dissolving the methacrylamide gelatin and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is a phosphate buffer solution with a pH of 7.4 and a concentration of 0.01 M.

[0015] Preferably, in step 1, the mixed solution is filtered through a 0.22 μm filter to remove bacteria, and then colorectal cancer cells HCT-116 and human skin fibroblasts BJ are added to it.

[0016] In step 1, the ratio of the cell density of colorectal cancer cells HCT-116 and human skin fibroblasts BJ in the bio-ink is (1 ~ 3): (1 ~ 3), preferably 3:1.

[0017] In step 2, during 3D printing, a pneumatic pressure of 0.5 to 2 bar is applied to the bio-ink, causing the bio-ink to be extruded from the printing needle of the extrusion bio-3D printer under shear force to form droplets, which then settle on the culture dish or culture well plate of the receiving platform of the extrusion bio-3D printer, are exposed to solidify, and tumor cell-loaded gel microspheres are obtained.

[0018] Preferably, the pneumatic pressure applied to the bio-ink during 3D printing is 1 bar.

[0019] Droplet printing typically uses inkjet bioprinters, but this method can only use ultra-low viscosity inks (<20 mPa·s), and the available bioinks have low cell densities (<10). 6 The method for constructing a colorectal tumor model based on 3D printing provided by this invention uses an extrusion-type bio-3D printer (controlled by a pneumatic piston). Rheological regulation is used to keep the bio-ink in a "weak gel" state. Unlike the lines typically printed by conventional extrusion-type bioprinters, this "weak gel" state allows the cell-loaded bio-ink to be extruded in droplet form and deposited onto the receiving platform. This also avoids the problems of ink selection and cell density inherent in traditional droplet printing such as inkjet bioprinting.

[0020] Preferably, the temperature of the culture dish or culture plate is controlled at 4~20℃; the wavelength of the exposed light is 340~405 nm, and the light power density is 0.5~3 W / cm². 2 The exposure time is 10 to 120 seconds.

[0021] More preferably, the temperature of the culture dish or culture plate is controlled at 10°C; the wavelength of the exposed light is 405 nm, and the light power density is 1.6 W / cm². 2 The exposure time is 30 seconds.

[0022] After the droplets are deposited on a culture dish or culture plate on a receiving platform with a low temperature, they achieve self-support and solidification by relying on the viscoelasticity and temperature sensitivity of GelMA. Subsequently, they are cured by exposure to complete secondary photocrosslinking and form stable tumor cell-loaded gel microspheres.

[0023] Preferably, the inner diameter of the printing needle is 240 μm.

[0024] Preferably, the extrusion time of the droplet is 0.5 to 2.5 s, and the diameter of the droplet is controlled to be 1000 to 3000 μm; the roundness of the droplet is controlled by controlling the temperature of the bio-ink during 3D printing to be >0.9.

[0025] Preferably, the in vitro culture is carried out in a cell culture incubator at 37°C and a CO2 concentration of 5% for 3 to 7 days.

[0026] During in vitro culture, colorectal cancer cells HCT-116 proliferated and aggregated to form a high-density cell cluster core, while human skin fibroblasts (BJ) formed a fibrotic encapsulation layer around the core. At the same time, a central necrotic zone naturally formed inside the core due to the nutrient and oxygen gradient. Finally, a tumor model with a biomimetic tumor fibrotic encapsulation microenvironment was obtained through culture.

[0027] The second aspect of the present invention provides a colorectal tumor model constructed by the aforementioned construction method.

[0028] The colorectal tumor model described herein has a diameter of 1000-3000 μm, a roundness greater than 0.9, and a cell density of 1×10⁻⁶. 6 ~ 1×10 8 cells / mL.

[0029] The colorectal tumor model described is microspherical, with a diameter of 1000-3000 μm and a sphericity greater than 0.9. It exhibits a distinct nucleus-shell structure, and the cell density can reach 1×10⁻⁶ at the level of solid tumors. 8 The cell / mL core area is a central necrotic zone formed due to limited nutrient and oxygen diffusion, and the outer layer is a fibroblast encapsulation layer.

[0030] The third aspect of this invention provides the application of the aforementioned colorectal tumor model in the study of the occurrence and development of colorectal cancer, the development of clinical therapies, the screening and evaluation of antitumor drugs, the detection of antitumor drug sensitivity, and the study of drug resistance mechanisms.

[0031] This invention provides a droplet-based bio-3D printing method based on the controlled gel-sol transition temperature of GelMA, enabling the bio-3D printing of colorectal tumor models. This method achieves ultra-high cell density (up to 1×10⁻⁶) by precisely controlling the rheological properties of the bio-ink. 8 The stable extrusion and rapid self-curing of bio-ink (cells / mL) enable one-step construction of a tumor microenvironment model with a "tumor core-fibroblast encapsulation" topology and pathological features such as fibrosis and internal necrosis. This model highly simulates solid tumors in terms of cell density, spatial heterogeneity, and pathological features, solving the technical bottlenecks of existing technologies in high-cell-density printing, multi-cell spatial heterogeneity construction, and biomimetic pathological features.

[0032] Beneficial effects:

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] (1) The method of the present invention utilizes the gel-sol transition characteristics of GelMA at physiologically relevant temperatures (25 ~ 28℃) for the first time. By precisely controlling the temperature, the rheological parameters of the bio-ink are adjusted to the optimal printing window, realizing droplet self-curing and molding without the need for low-temperature freezing or chemical cross-linking support. This avoids cell damage caused by low temperature and overcomes the defect of uncontrolled viscosity during room temperature printing. The process is mild and significantly improves cell survival rate (>80%).

[0035] (2) The method of this invention breaks through the upper limit of cell density in droplet bioprinting. Through the optimization of the rheological window, it successfully achieves a cell density of up to 1×10⁻⁶ cells / cm². 8 Stable printing with bio-ink at a cell density of cells / mL, close to the physiological level of solid tumors, allows the printed models to realistically simulate the high density of tumors and the resulting nutrient / oxygen gradient effect, providing a more in vivo experimental platform for studying the heterogeneity of the tumor microenvironment.

[0036] (3) The method of the present invention uses single-step printing and single-nozzle extrusion of bio-ink containing HCT-116 and BJ cells. By utilizing the self-organizing behavior and differential proliferation of cells within the droplet, a biomimetic topological structure of "tumor core-fibrotic encapsulation" is constructed in one step. This structure spontaneously forms a central necrotic area and a fibrotic encapsulation layer. Compared with the traditional multi-nozzle asynchronous printing and subsequent assembly method, it greatly simplifies the process flow, improves the structural fidelity and batch-to-batch consistency, and is suitable for the rapid construction of high-throughput biomimetic tumor models.

[0037] (4) The tumor model printed by this invention highly simulates the clinical tumor pathological features in terms of morphology and function: the droplet size is uniform, the roundness is high, the core-shell heterogeneous structure is present, and there is an internal necrotic area. It can be applied to high-throughput screening of anti-tumor drugs, research on tumor-matrix interaction mechanism and evaluation of personalized medical plans, and has significant potential for clinical application transformation.

[0038] (5) The method of the present invention has good material and equipment compatibility, does not require complex modification of existing extrusion-type bio 3D printers, reduces technical threshold and equipment cost, and is conducive to the rapid promotion and industrial application of the technology. Attached Figure Description

[0039] 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.

[0040] Figure 1 The viscosity of the bio-ink prepared in Example 1 varies with temperature.

[0041] Figure 2The curve showing the change of tanδ, the ratio of storage modulus G' to loss modulus G'', of the bio-ink prepared in Example 1 as a function of temperature.

[0042] Figure 3 These are microscope images of droplets printed at different temperatures and extrusion times in Example 1; where Figure a is a microscope image of droplets printed at different temperatures, and Figure b is a microscope image of droplets printed at different extrusion times.

[0043] Figure 4 The figures show the roundness of droplets printed at different temperatures and the diameter of droplets printed at different extrusion times in Example 1.

[0044] Figure 5 The figures show the cell survival in tumor cell-loaded gel microspheres printed under different cell densities at a bio-ink temperature of 26°C in Example 2. Figure a shows the live / dead cell staining images of HCT-116 cells and BJ cells, Figure b shows the cell survival rate of HCT-116 cells, and Figure c shows the cell survival rate of BJ cells.

[0045] Figure 6 The image shows the survival of HCT-116 cells in tumor cell-loaded gel microspheres printed at a bio-ink temperature of 23°C and under different cell densities in Example 2. Figure a shows the staining images of live and dead HCT-116 cells, and Figure b shows the cell survival rate of HCT-116 cells.

[0046] Figure 7 Figure 3 shows the cell viability statistics of tumor cell-loaded gel microspheres obtained by 3D printing with bio-ink at different temperatures in Example 3. Figure a shows the cell viability per well of the tumor cell-loaded gel microspheres printed with bio-ink at 23°C after 24 h of culture in a 96-well plate; Figure b shows the cell viability per well of the tumor cell-loaded gel microspheres printed with bio-ink at 26°C after 24 h of culture in a 96-well plate; Figure c shows the cell viability per well of the tumor cell-loaded gel microspheres printed with bio-ink at 29°C after 24 h of culture in a 96-well plate; Figure d shows the 450 nm optical density (OD) of cells in the tumor cell-loaded gel microspheres printed with bio-ink at the three temperatures. 450 Figure 1 shows the distribution map; Figure 2 shows the coefficient of variation (CV) of the 450 nm optical density of cells in the tumor cell-loaded gel microspheres printed with bio-ink at three different temperatures.

[0047] Figure 8 Images showing cell viability and mortality staining of multicellular colorectal tumor spheres prepared on ultra-low adhesion surfaces in Example 4 and 3D-printed multicellular colorectal tumor models after in vitro culture for different time periods.

[0048] Figure 9Images showing the staining of cell nuclei (DAPI), α-smooth muscle actin (α-SMA), and cytokeratin 18 (CK18) in the 3D-printed multicellular colorectal tumor model in Example 4.

[0049] Figure 10 The image shows the cell viability statistics of the tumor models DBB CRC, DBB CRC / FB, ULA CRC, and ULA CRC / FB in Example 5 under the action of different drugs alone.

[0050] Figure 11 The images show statistical diagrams of cell viability of tumor models DBB CRC, DBB CRC / FB, ULA CRC, and ULA CRC / FB in Example 5 under the effects of irinotecan and 5-fluorouracil alone and in combination, as well as cell liveness / death staining images of tumor model DBB CRC / FB under the combined effects of irinotecan and 5-fluorouracil.

[0051] Figure 12 The images show statistical diagrams of cell viability of tumor models DBB CRC, DBB CRC / FB, ULA CRC, and ULA CRC / FB in Example 5 under the effects of tumor-penetrating peptide and 5-fluorouracil alone and in combination, as well as cell liveness / death staining images of tumor model DBB CRC / FB under the combined effects of tumor-penetrating peptide and 5-fluorouracil. Detailed Implementation

[0052] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0053] The extrusion-type bio-3D printer used in the following examples was purchased from EnvisionTec, product number 3D-Bioplotter production model.

[0054] Example 1

[0055] (1) Appearance of droplets obtained by printing with bio-ink at different temperatures during droplet-type bio-3D printing

[0056] ①Preparation of bio-ink

[0057] Methacrylamide gelatin (GelMA) and the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were dissolved together in phosphate buffer (0.01 M, pH=7.4) to prepare a homogeneous solution with a GelMA concentration of 0.05 g / mL and a LAP concentration of 1 g / L, thus obtaining the cell-free bioink.

[0058] The viscosity of the above-mentioned bio-ink was tested at different temperatures, and viscosity-temperature curves were plotted, as shown below. Figure 1As shown. In addition, the storage modulus G' and loss modulus G'' of the bio-ink were measured at different temperatures, and their ratio tanδ was calculated. A curve showing the change of tanδ with temperature was plotted, as shown below. Figure 2 As shown. Figure 1 As shown, the viscosity transition temperature range of 0.05 g / mL GelMA in bio-ink is 25 ~ 30℃, and its tanδ increases with increasing temperature within this temperature range, indicating that GelMA has undergone a sol-to-gel transition. Therefore, this temperature range can be confirmed as the sol-gel transition of GelMA bio-ink. In particular, at 26 ~ 27℃, the tanδ is 0.3 ~ 0.5, indicating that the GelMA bio-ink is in a "weak gel" state at this time.

[0059] ② Droplet-based bio-3D printing

[0060] The bio-ink prepared in step ① was loaded into the barrel of an extrusion-type bio-3D printer, and its temperature was controlled by a temperature control system connected to the barrel. A pneumatic pressure of 1 bar was applied to the bio-ink, causing it to be extruded from the printing nozzle under shear force to form droplets. These droplets settled on a culture dish on a 10°C receiving platform, where they self-supported and solidified due to the viscoelasticity and thermosensitivity of GelMA. The extrusion time of the bio-ink from the printing nozzle was 1 second.

[0061] To compare the appearance of droplets obtained by droplet-based bio-3D printing using bio-inks at different temperatures, the bio-ink temperatures were controlled at 24℃, 25℃, 26℃, 27℃, and 28℃. Self-supporting, solidified droplets were obtained, and their appearance was observed under a microscope. Figure 3 As shown in Figure a. The roundness of the printed droplets at various temperatures was measured using ImageJ software, and the results are as follows. Figure 4 As shown in Figure a, the results indicate that the average sphericity of droplets printed at bio-ink temperatures of 24℃ and 25℃ were 0.79 and 0.84, respectively, significantly lower than the sphericity of droplets printed at bio-ink temperatures of 26℃, 27℃, and 28℃ (average sphericity of 0.96, 0.97, and 0.98, respectively). Furthermore, the uniformity of sphericity of droplets printed at 24℃ and 25℃ was much lower than that of droplets printed at 26~28℃. Therefore, controlling the temperature of the bio-ink during 3D printing to 26~28℃ is crucial for obtaining spherical microspheres with uniform and high sphericity loaded with tumor cells.

[0062] (2) Appearance of droplets obtained at different extrusion times during droplet-type bioprinting

[0063] The preparation of the bio-ink and the droplet-type bio-3D printing method are the same as in this embodiment (1), except that during 3D printing, the temperature of the bio-ink is controlled at 26℃, and the droplet extrusion time is controlled at 0.5 s, 1 s, 1.5 s, 2 s, and 2.5 s, respectively. After obtaining the self-supported and solidified droplets, they are immersed in PBS buffer (pH 7.4, concentration 0.01 M) and observed under a microscope. The results are as follows. Figure 3 As shown in b, the droplet diameter was measured, as follows. Figure 4 As shown in b, the droplet diameter analysis results show that the average diameters of the droplets obtained at extrusion times of 0.5 s, 1 s, 1.5 s, 2 s and 2.5 s are 1000, 1500, 2000, 2500 and 3000 μm, respectively, indicating that controlling the extrusion time of the droplets is the key to controlling the diameter of the tumor cell-loaded gel microspheres.

[0064] Example 2

[0065] Cell survival rate in tumor cell-loaded gel microspheres obtained by 3D printing with bio-inks of different cell densities.

[0066] (1) Preparation of bio-ink

[0067] Methacrylamide gelatin (GelMA) and the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were dissolved together in phosphate buffer (0.01 M, pH=7.4) to prepare a homogeneous solution with a GelMA concentration of 0.05 g / mL and a LAP concentration of 1 g / L. The solution was then filtered through a 0.22 μm filter for sterilization to obtain a mixed solution. Colorectal cancer cells HCT-116 were digested, centrifuged, and resuspended in the above mixed solution. The density of the HCT-116 cell suspension was counted to be 1 × 10⁻⁶. 8 The HCT-116 cell suspension was mixed with the above mixed solution to obtain a bio-ink containing only HCT-116 cells. The final cell density in the bio-ink was controlled to be 1×10⁻⁶ cells / mL. 6 5×10 6 1×10 7 2×10 7 5×10 7 and 1×10 8 cells / mL.

[0068] In addition, a bio-ink containing only BJ cells was prepared using the same method as the bio-ink containing only HCT-116 cells, the difference being that the final cell density was not prepared to be 1×10⁻⁶. 8 Bio-ink containing only BJ cells per mL.

[0069] (2) Droplet-based bio-3D printing

[0070] The bio-inks prepared in step (1) were loaded into the barrel of an extrusion-type bio-3D printer, and the temperature of the bio-inks was controlled by a temperature control system connected to the barrel. A pneumatic pressure of 1 bar was applied to the bio-inks, causing them to be extruded from the printing needle under shear force to form droplets. These droplets settled in a 96-well plate on a 10°C receiving platform. The droplets achieved self-support and solidification due to the viscoelasticity and thermosensitivity of GelMA. Subsequently, light was passed through the printer at a wavelength of 405 nm and an intensity of 1.6 W / cm². 2 Irradiation with light for 30 seconds completes secondary photocrosslinking, forming stable tumor cell-loaded gel microspheres. The bio-ink is extruded from the printing needle in 1.5 seconds.

[0071] To compare the cell viability of tumor cell-loaded gel microspheres obtained by 3D printing with bio-ink at different temperatures, the bio-ink temperatures were controlled at 23℃ and 26℃, respectively.

[0072] The cell viability of tumor cell-loaded gel microspheres obtained by 3D printing with bio-ink at different cell densities was assessed using the Calcein-AM / PI staining method. Results are as follows: Figure 5 and Figure 6 As shown, when the temperature of the bio-ink is controlled at 26℃, the cell density is 1×10⁻⁶. 6 ~ 1×10 8 The cell viability of tumor cell-loaded gel microspheres obtained by 3D printing with bio-ink at a density of cells / mL remained above 80%. Figure 5 ); while when the temperature of the bio-ink is controlled at 23℃, even if the HCT-116 cell density is 1×10 7 The cell count was low (cells / mL), indicating a high number of dead cells and a cell viability rate below 60%. Figure 6 This confirms the crucial role of the temperature control strategy of this invention in high cell density printing.

[0073] Example 3

[0074] Cell viability of tumor cell-loaded gel microspheres obtained by 3D printing with bio-ink at different temperatures.

[0075] (1) Preparation of bio-ink

[0076] Methacrylamide gelatin (GelMA) and the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were dissolved together in phosphate buffer (0.01 M, pH=7.4) to prepare a homogeneous solution with a GelMA concentration of 0.05 g / mL and a LAP concentration of 1 g / L. The solution was then filtered through a 0.22 μm filter for sterilization to obtain a mixed solution. Colorectal cancer cells HCT-116 and human neonatal prepuce dermal fibroblasts (BJ) were digested, centrifuged, and resuspended in the above mixed solution. The densities of the HCT-116 cell suspension and the BJ cell suspension were counted to be 1×10⁻⁶ and 1×10⁻⁶, respectively. 8 cells / mL and 5×10 7 The HCT-116 cell suspension and the BJ cell suspension were mixed at a volume ratio of 3:2 to obtain the bio-ink, resulting in an HCT-116 cell density of 6 × 10⁻⁶ cells / mL. 7 cells / mL, BJ cell density is 2×10⁻⁶ 7 cells / mL.

[0077] (2) Droplet-based bio-3D printing

[0078] The bio-inks prepared in step (1) were loaded into the barrel of an extrusion-type bio-3D printer, and the temperature of the bio-inks was controlled by a temperature control system connected to the barrel. A pneumatic pressure of 1 bar was applied to the bio-inks, causing them to be extruded from the printing needle under shear force to form droplets. These droplets settled in a 96-well plate on a 10°C receiving platform. The droplets achieved self-support and solidification due to the viscoelasticity and thermosensitivity of GelMA. Subsequently, light was passed through the printer at a wavelength of 405 nm and an intensity of 1.6 W / cm². 2 Irradiation with light for 30 seconds completes secondary photocrosslinking, forming stable tumor cell-loaded gel microspheres. The bio-ink is extruded from the printing needle in 1.5 seconds.

[0079] To compare the cell viability of tumor cell-loaded gel microspheres obtained by 3D printing with bio-ink at different temperatures, the bio-ink temperatures were controlled at 23℃, 26℃, and 29℃, respectively.

[0080] (3) In vitro culture

[0081] DMEM medium (containing 10% v / v fetal bovine serum and 1% v / v penicillin-streptomycin) was added to 96-well plates containing tumor cell-loaded gel microspheres printed at different temperatures in step (2), and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. After culture, cell viability in the tumor cell-loaded gel microspheres was detected using a cell counting kit-8 (CCK-8).

[0082] Cell viability test results as follows Figure 7 As shown, when the bio-ink temperature is 26℃, the cell viability of the printed tumor cell-loaded gel microspheres is the most uniform, and the stability is the best. OD 450 The coefficient of variation (CV) was 0.44 ± 0.03, with the lowest CV (6.0%), which was significantly better than the cell viability of tumor cell-loaded gel microspheres printed at bio-ink temperatures of 23℃ and 29℃, indicating that high consistency and reproducibility of high-throughput printing can be achieved at this temperature.

[0083] Example 4

[0084] Verification of biomimetic topological structure formation in tumor model.

[0085] The difference between the tumor model construction method in this embodiment and that in embodiment 3 is only that in step (2), the temperature of the bio-ink is controlled at 26°C, the bio-ink is squeezed out from the printing needle at 0.5 s, 1 s and 1.5 s respectively, and the in vitro culture time in step (3) is 7 days, and finally a mature colorectal tumor model is obtained.

[0086] Tumor models obtained on days 1, 3, 5, and 7 of in vitro culture were stained with Calcein AM / Propidium iodide (Calcein-AM / PI) to observe cell viability and mortality. Additionally, conventional ultra-low adhesion surface tumor spheres were prepared, and calcein AM / Propidium iodide (Calcein-AM / PI) staining was also performed on tumor models obtained on days 1, 3, 5, and 7 of in vitro culture. The staining results are shown below. Figure 8 As shown.

[0087] In addition, the mature tumor model (extrusion time 1.5 s) constructed by 3D printing in this embodiment and cultured in vitro for 7 days was fixed and permeable. Then, it was stained sequentially with DAPI (nucleus), α-smooth muscle actin (α-SMA, labeling tumor-associated fibroblasts), and cytokeratin 18 (CK18, labeling epithelial cancer cells) antibodies. Immunofluorescence staining was used to verify the formation of multicellular spatial heterogeneity in the tumor model constructed in this embodiment. The staining results are as follows: Figure 9 As shown.

[0088] The results showed that the mature tumor model constructed based on 3D printing in this embodiment exhibited a distinct core-shell structure: the outer layer was an α-SMA-positive fibroblast encapsulation layer, and the inner layer was a cluster of CK18-positive cancer cells. Figure 9 A clear necrotic area appeared in the central region. Figure 8 b). Compared to tumor spheres prepared on ultra-low adhesion surfaces, the tumor model obtained by 3D printing in this embodiment has a larger and more uniform diameter, and can maintain structural integrity for more than 7 days. Figure 8 ).

[0089] Ultra-low adhesion surface tumor spheroids were prepared using Corning ultra-low adhesion U-shaped bottom 96-well plates: HCT-116 cells and BJ cells were mixed at a fixed ratio of 3:1 and seeded into U-shaped bottom plates at densities of 1000, 2500, and 5000 cells per well, respectively. The cells were then statically cultured in a 37°C, 5% CO2 incubator to encourage spontaneous aggregation and formation of dense three-dimensional spherical structures.

[0090] Example 5

[0091] Pharmacodynamics were validated using standard antitumor drugs to compare differences in drug sensitivity among different tumor models.

[0092] A 3D-printed single-cell tumor model (DBB CRC) was constructed according to the method in Example 3. When constructing the DBBCRC, the bio-ink contained only cells with a density of 8 × 10⁸ cells. 7 HCT-116 cells / mL were bio-inked using droplet-based bio-3D printing, with the bio-ink temperature controlled at 26°C, extrusion time at 1.5 s, and in vitro culture time at 3 days. DBBCRC / FB is the colorectal tumor model constructed in Example 4 under the conditions of 1.5 s bio-ink extrusion time and 3 days in vitro culture time.

[0093] In addition, single-cell tumor spheres (ULACRC) and multi-cell tumor spheres (ULA CRC / FB) were prepared according to the ultra-low adhesion surface tumor sphere preparation method given in Example 4. Specifically, for the preparation of ULA CRC, HCT-116 cells were seeded into U-shaped bottom-well plates at a density of 5000 cells per well; for the preparation of ULA CRC / FB, HCT-116 and BJ cells were mixed at a fixed ratio of 3:1 and seeded into U-shaped bottom-well plates at a density of 5000 cells per well, with an in vitro culture time of 3 days.

[0094] DBB CRC, DBB CRC / FB, ULA CRC, and ULA CRC / FB were treated with different concentrations (3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μM) of standard antitumor drugs for 72 hours. Cell viability was then assessed using the CCK-8 assay, and IC50 values ​​were calculated. The standard antitumor drugs used were 5-fluorouracil (5-FU), irinotecan (IRI), and oxaliplatin (OXA), each applied individually to the tumor model. Results are as follows: Figure 10 As shown in Table 1, the DBB CRC / FB model exhibited the strongest resistance to single-drug chemotherapy, significantly higher than other models, indicating that the fibrotic encapsulation structure constructed in this invention better reflects common drug resistance in clinical tumors compared to other models. Figure 10 ).

[0095] Further evaluation of combined drug use was conducted. Irinotecan and 5-fluorouracil (IRI / 5-FU) were first used alone and in combination for the four tumor models mentioned above. The combined effect experiment of irinotecan and 5-fluorouracil (IRI / 5-FU) was conducted in three groups (I, II, III), with three subgroups (1, 2, 3) under each group. Different concentrations of the drug were used to treat the four tumor models for 72 h in each group. The specific experimental design is shown in Table 2. The combined use of tumor-penetrating peptide (CEND-1) and 5-fluorouracil (5-FU) was evaluated using the same method, also in three groups (I, II, III), with two subgroups (1, 2) under each group. The specific experimental design is shown in Table 3. After treatment, cell viability of each tumor model was detected by CCK-8 assay, and Calcein-AM / PI staining was performed to observe cell survival in the tumor models.

[0096] Experimental results are as follows Figure 11 and Figure 12 As shown, in the DBB CRC / FB model, the combination therapy of irinotecan and 5-fluorouracil (IRI / 5-FU) exhibited the lowest toxicity at different doses. Figure 11 The results indicate that the DBB CRC / FB model exhibits some resistance to combined chemotherapy, which can avoid the problem of conventional tumor models exaggerating the effects of chemotherapy drugs during drug screening. Furthermore, the DBB CRC / FB model has been used to evaluate novel combination therapies. Matrix remodeling combination therapies (such as CEND-1 tumor-penetrating peptide combination therapy) have entered clinical trials in several solid tumors (such as colorectal cancer, pancreatic cancer, and osteosarcoma) as a promising combination strategy. Figure 12 As shown, the combination of CEND-1 tumor-penetrating peptide and 5-FU can greatly enhance the efficacy of the drug and exhibit a significant synergistic effect, which is not obvious in other models, proving that this model can more accurately predict the clinical combined treatment effect.

[0097] Table 1. Dose-response parameters of different drugs in tumor models

[0098]

[0099] Table 2. Experimental design for efficacy evaluation of irinotecan and 5-fluorouracil (IRI / 5-FU) combination therapy.

[0100]

[0101] Table 3. Experimental design for efficacy evaluation of the combination of tumor-penetrating peptide (CEND-1) and 5-fluorouracil (5-FU)

[0102]

[0103] This invention provides a 3D-printed colorectal tumor model, its construction method, and its application. 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 method for constructing a colorectal tumor model based on 3D printing, characterized in that, Includes the following steps: Step 1: Dissolve methacrylamide gelatin and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate together to obtain a mixed solution. Add colorectal cancer cells HCT-116 and human skin fibroblasts BJ to the solution, mix well, and obtain bio-ink. Step 2: Load the bio-ink obtained in Step 1 into the barrel of an extrusion-type bio-3D printer for 3D printing to obtain tumor cell-loaded gel microspheres, which are then cultured in vitro to obtain the final product. In step 1, the concentration of methacrylamide gelatin in the bio-ink is 0.03 ~ 0.08 g / mL; In step 2, during 3D printing, the temperature of the bio-ink is controlled at 25 ~ 28℃.

2. The construction method according to claim 1, characterized in that, In step 1, the total cell density of HCT-116 colorectal cancer cells and BJ human skin fibroblasts in the bio-ink is 1 × 10⁻⁶. 6 ~ 1×10 8 cells / mL; the concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate is 0.5 ~ 5 g / L.

3. The construction method according to claim 1, characterized in that, In step 1, the ratio of the cell densities of colorectal cancer cells HCT-116 and human skin fibroblasts BJ in the bio-ink is (1 ~ 3): (1 ~ 3).

4. The construction method according to claim 1, characterized in that, In step 2, during 3D printing, a pneumatic pressure of 0.5 to 2 bar is applied to the bio-ink, causing the bio-ink to be extruded from the printing needle of the extrusion bio-3D printer to form droplets, which then settle on the culture dish or culture well plate on the receiving platform of the extrusion bio-3D printer, are exposed to solidify, and tumor cell-loaded gel microspheres are obtained.

5. The construction method according to claim 4, characterized in that, The temperature of the culture dish or culture plate is controlled at 4 ~ 20℃; the wavelength of the exposed light is 340 ~ 405 nm, and the light power density is 0.5 ~ 3 W / cm². 2 The exposure time is 10 to 120 seconds.

6. The construction method according to claim 4, characterized in that, The inner diameter of the printing needle is 240 μm.

7. The construction method according to claim 6, characterized in that, The extrusion time of the droplets is 0.5 to 2.5 seconds.

8. A colorectal tumor model constructed by the construction method according to any one of claims 1 to 7.

9. The colorectal tumor model according to claim 8, characterized in that, The colorectal tumor model described herein has a diameter of 1000-3000 μm, a roundness greater than 0.9, and a cell density of 1×10⁻⁶. 6 ~ 1×10 8 cells / mL.

10. The application of the colorectal tumor model as described in claim 8 in the study of the occurrence and development of colorectal cancer, the development of clinical therapies, the screening and evaluation of antitumor drugs, the detection of antitumor drug sensitivity, and the study of drug resistance mechanisms.

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