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CN-121991894-A - Colorectal tumor model based on 3D printing and construction method and application thereof

CN121991894ACN 121991894 ACN121991894 ACN 121991894ACN-121991894-A

Abstract

The invention discloses a construction method of a colorectal tumor model based on 3D printing, which comprises the following steps of dissolving methacryloylated gelatin and phenyl (2, 4, 6-trimethyl benzoyl) lithium phosphate together to obtain a mixed solution, adding colorectal cancer cells HCT-116 and human skin fibroblasts BJ into the mixed solution, uniformly mixing to obtain biological ink, loading the biological ink into a feed cylinder of an extrusion type biological 3D printer for 3D printing to obtain tumor cell carrying gel microspheres, and culturing in vitro. The method breaks through the upper limit of the cell density of the liquid drop biological 3D printing, successfully realizes the stable printing of the biological ink containing the cell density as high as 1 multiplied by 10 8 cells/mL through the optimization of a rheological window, and the density is close to the physiological level of solid tumors, so that a printed tumor model can truly simulate the high compactness of the tumor and the nutrition/oxygen gradient effect caused by the tumor, and provides a more in-vivo experimental platform for researching the heterogeneity of the microenvironment inside the tumor.

Inventors

  • MAO HONGLI
  • WU LIHUANG
  • GU ZHONGWEI
  • CUI YUWEN

Assignees

  • 南京工业大学

Dates

Publication Date
20260508
Application Date
20260209

Claims (10)

  1. 1. The method for constructing the colorectal tumor model based on 3D printing is characterized by comprising the following steps of: Step 1, dissolving methacryloyl gelatin and phenyl (2, 4, 6-trimethyl benzoyl) 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 the biological ink; step 2, loading the biological ink prepared in the step 1 into a charging barrel of an extrusion type biological 3D printer for 3D printing to obtain tumor cell-carrying gel microspheres, and culturing in vitro to obtain the tumor cell-carrying gel microspheres; in the step 1, the concentration of the methacryloylated gelatin in the biological ink is 0.03-0.08 g/mL; In the step 2, when 3D printing is performed, the temperature of the bio-ink is controlled to be 25-28 ℃.
  2. 2. The method according to claim 1, wherein in step 1, the total cell density of colorectal cancer cells HCT-116 and human skin fibroblasts BJ is 1X 10 6 ~ 1×10 8 cells/mL, and the concentration of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate is 0.5-5 g/L.
  3. 3. The method according to claim 1, wherein 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. 4. The construction method according to claim 1, wherein in step 2, when 3D printing is performed, pneumatic pressure of 0.5-2 bar is applied to the bio-ink, so that the bio-ink is extruded from a printing needle of the extrusion type bio-3D printer to form liquid drops, and the liquid drops are settled on a culture dish or a culture pore plate on a receiving platform of the extrusion type bio-3D printer, and are exposed and cured, so as to obtain the tumor cell gel-loaded microspheres.
  5. 5. The method according to claim 4, wherein the temperature of the culture dish or the culture well plate is controlled to be 4-20 ℃, the wavelength of the light for exposure is 340-405 nm, the optical power density is 0.5-3W/cm 2 , and the exposure time is 10-120 seconds.
  6. 6. The method of claim 4, wherein the inner diameter of the printing tip is 240 μm.
  7. 7. The method of claim 6, wherein the droplet is extruded for a time period of 0.5 to 2.5 s.
  8. 8. The colorectal tumor model constructed by the construction method of any one of claims 1to 7.
  9. 9. The colorectal tumor model of claim 8, wherein the colorectal tumor model has a diameter of 1000-3000 μm, a roundness of greater than 0.9, and a cell density of 1 x10 6 ~ 1×10 8 cells/mL.
  10. 10. The colorectal tumor model of claim 8 for use in research of occurrence and development of colorectal cancer, development of clinical therapy, screening and evaluation of anti-tumor drugs, detection of sensitivity of anti-tumor drugs and research of drug resistance mechanism.

Description

Colorectal tumor model based on 3D printing and construction method and application thereof Technical Field The invention belongs to the technical field of biomedical science, and particularly relates to a colorectal tumor model based on 3D printing and a construction method and application thereof. Background One of the core challenges facing current tumor biology research is how to accurately simulate the complex pathological features of Tumor Microenvironment (TME) to construct in vitro tumor models. Solid tumors contain not only a high density of tumor cell mass, but also a high concentration of fibrotic matrix composed of cancer-associated fibroblasts (CAFs), abnormal extracellular matrix (ECM) deposition, and internal hypoxic necrosis zones due to insufficient vascularization, which together shape the tumor's unique spatial heterogeneity and drug tolerance. However, the conventional two-dimensional (2D) cell culture model lacks a three-dimensional structure and dynamic interactions between cells, and cannot reflect the actual biological behavior of tumors. Although the three-dimensional tumor sphere model compensates for the dimensional deficiency to a certain extent, the cell density is generally limited to below 1×10 7 cells/mL, the size is small, the controllable size uniformity is lacked, the survival period is short, and meanwhile, the topology structure with pathological significance, namely tumor core-fibrosis package, is difficult to reproduce, so that the repeatability and clinical relevance of drug screening are more severely restricted. The rise of biological 3D printing technology provides a brand new path for the accurate bionic tumor microenvironment, but the biological 3D printing technology still faces multiple technical bottlenecks when constructing a tumor model with high physiological relevance. In extrusion printing, nozzle clogging and cell damage are the primary obstacles restricting high cell density (> 1 x 10 7 cells/mL) printing, and too high shear stress not only results in significant decrease in cell activity, but also is very prone to nozzle failure. Therefore, there are limitations in constructing in vitro models that reach organ and tissue level cell densities using biological 3D printing techniques. Meanwhile, although the liquid drop biological 3D printing method based on the technologies of ink-jet printing and the like is brand-new in terms of the high-flux forming characteristics, the method has obvious limitations when being applied to tumor model construction. Conventional drop printing techniques are only suitable for very low viscosity inks (typically <10 mPa s), and for common medium viscosity bioinks such as methacryloylated gelatin (GelMA) and the like, it is difficult to stably form drops of controllable size. In the aspect of temperature control strategies, the existing method mostly adopts low-temperature quick freezing and solidification, which not only needs a complex refrigerating system, but also directly damages the integrity of cell membranes in the freezing and thawing process, and is not suitable for long-term culture of living cells, and the tail of liquid drops and nonuniform size are caused by overhigh viscosity of biological ink in normal-temperature printing. The technologies restrict the application of the existing liquid drop biological 3D printing technology in constructing a tumor model matched with the solid tumor cell density level. In summary, the prior art fails to effectively integrate the rheological property and the droplet printing process of the temperature-responsive bio-ink, and realizes the establishment of a tumor model with high cell density, multi-cell heterogeneity and rapid self-support in a mild physiological related temperature range, and a new method is needed to enable the bio-ink to be in a 'weak gel' state through precise rheological control in a gel-sol transition temperature window inherent in the bio-ink, and has both shear thinning printability and rapid structure recovery capability after extrusion, so that a bionic topological structure of 'tumor core-fibroblast package' is established by one-step molding. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a method for constructing a colorectal tumor model based on 3D printing. The invention also solves the technical problem of providing the colorectal tumor model constructed by the construction method. The invention finally solves the technical problem of providing the application of the colorectal tumor model in colorectal cancer occurrence and development research, clinical therapy development, anti-tumor drug screening evaluation, anti-tumor drug sensitivity detection and drug resistance mechanism research. In order to solve the technical problems, the technical scheme provided by the invention is as follows: The invention provides a method for constructing a colorectal tumor model based on 3D printing, which comprises th