EO-CRC source NCIO and culture method and application thereof

Abstract

The invention relates to the technical field of biomedical engineering, and discloses EO-CRC-sourced NCIO and a culture method and application thereof.The method comprises the steps that normal colorectal tissue of a premature colorectal cancer patient with the onset age smaller than 50 years old is obtained; digesting the tissue blocks for 20-60 minutes at 2-6 DEG C by using EDTA (Ethylene Diamine Tetraacetic Acid) digestive juice; stopping the digestion reaction, filtering, and centrifugally collecting a digestion product; mixing the collected digestion product with a basic culture medium and matrigel in proportion, and performing three-dimensional embedding; and after gelling, adding a normal intestinal organ-like culture medium for culturing. According to the method, culture conditions are optimized according to biological characteristics of normal tissues of a patient suffering from premature colorectal cancer, and cell damage is avoided by adopting EDTA low-temperature digestion. The cultured organoid can be stably passed for more than 15 generations, and has important application value in the fields of premature colorectal cancer disease model construction, pathogenesis research, drug screening and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to an EO-CRC source of NCIO, its culture method, and its application. Background Technology

[0002] Colorectal cancer is one of the most common and deadliest malignant tumors worldwide. In recent years, the incidence of early-onset colorectal cancer (usually referring to patients under 50 years of age) has shown a significant upward trend, with clinical characteristics, molecular profiles, and prognosis differing significantly from late-onset colorectal cancer. Early-onset colorectal cancer often exhibits higher microsatellite instability, stronger stem cell characteristics, higher proliferation dependence, and sensitivity to microenvironment signals; these unique biological behaviors present unique challenges for its research and treatment.

[0003] Organoid technology, as an emerging three-dimensional cell culture technique, can reproduce the structural and functional characteristics of tissues and organs in vitro, and has become an important tool for disease modeling, drug screening, and regenerative medicine research. However, existing NCIO (Normal Colorectal Intestinal Organoid) culture methods mainly suffer from the following problems:

[0004] Most existing organoid culture systems are based on advanced or sporadic colorectal cancer tissues, failing to adequately consider the unique biological characteristics of EO-CRC (Early-Onset Colorectal Cancer) patient tissues. Early-onset colorectal cancer often exhibits higher microsatellite instability, stronger stem cell characteristics, greater proliferation dependence, and sensitivity to microenvironment signals. These characteristics make traditional culture methods less suitable for processing early-onset colorectal cancer patient tissues.

[0005] Traditional culture methods have a low success rate in culturing normal colorectal tissue from patients with early-onset colorectal cancer. Existing technologies lack specificity in the selection of digestion methods. Most methods employ a uniform digestion strategy to process different types of tissues, failing to differentiate treatment based on biological differences between normal and tumor tissues in areas such as intercellular connectivity strength and matrix density, thus affecting the efficiency and quality of organoid culture.

[0006] Current culture medium formulations fail to adequately meet the specific nutritional needs of normal intestinal tissue in patients with end-stage renal disease (EO-CRC). The concentration and ratio requirements of signaling pathway factors such as Wnt, Noggin, and R-spondin for normal intestinal epithelial stem cells differ from those for tumor cells, and current formulations often cannot provide the optimal culture environment. Summary of the Invention

[0007] The purpose of this invention is to provide an EO-CRC source NCIO, its culture method, and its application, in order to solve the technical problems of low success rate and insufficient standardization in the culture of organoids from normal tissues of patients with early-onset colorectal cancer in the prior art.

[0008] To achieve the above-mentioned objective, this invention provides a method for cultivating EO-CRC-derived NCIO, comprising:

[0009] Obtain normal colorectal tissue from patients with early-onset colorectal cancer who are under 50 years of age at onset;

[0010] After pretreatment, 1-3mm was obtained 3 The organizational blocks;

[0011] The tissue block was digested with EDTA digestion solution at 2-6°C for 20-60 minutes.

[0012] The digestion reaction is terminated, and the digestion products are collected by filtration and centrifugation.

[0013] The collected digestion products were three-dimensionally embedded with a proportionally mixed basal culture medium and matrix gel.

[0014] After gelation, the culture medium for normal intestinal organoids was added;

[0015] The normal intestinal organoid culture medium comprises: Advanced DMEM / F12 basal medium, N21-MAX medium additive 1x, N-2MAX medium additive 1x, N-acetylcysteine ​​1-1.25mM, Glutamine Plus 1x, HEPES buffer 8-12mM, recombinant human Noggin protein 50-150ng / mL, recombinant human R-Spondin 1 protein 0.5-1.5μg / mL, recombinant human EGF protein 20-100ng / mL and recombinant human Wnt-3a protein 50-200ng / mL.

[0016] Optionally, the digestion process uses an EDTA digestion solution with a concentration of 0.01-0.02M.

[0017] Optionally, during the digestion process, microscopic observation is performed every 10-20 minutes, and digestion is terminated with phosphate buffer containing 0.5-2% BSA when a cell cluster of 5-50 cells is observed.

[0018] Optionally, the centrifugation conditions are 250-350g, 2-6℃, and 3-8 minutes.

[0019] Optionally, the volume ratio of the basal culture medium to the matrix gel is 1:0.8 to 1:1.5.

[0020] Optionally, the three-dimensional embedding culture is carried out at 37°C and 3-7% CO2.

[0021] Optionally, the pretreatment includes: removing blood and tissue debris, and washing the normal colorectal tissue.

[0022] Optional steps include propagation:

[0023] When the diameter of the organoids reaches 200-500 μm, the matrix gel is dissolved, and the organoids are digested and decomposed into cell clusters.

[0024] The organoids were crushed by blowing and pulverizing 2-3 times, collected by centrifugation, and then remixed, embedded, and cultured.

[0025] The present invention also provides an EO-CRC source NCIO, which is prepared using the above-described culture method.

[0026] The present invention also provides the application of the above-mentioned NCIO in constructing disease models of early-onset colorectal cancer, drug screening, or studying the pathogenesis of early-onset colorectal cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The EO-CRC-derived NCIO culture method provided by this invention uses EDTA digestion solution at low temperatures to gently digest normal colorectal tissue, avoiding cell damage that may be caused by collagenase digestion. This method is suitable for the biological characteristics of normal tissue, which has tightly connected intercellular structures but relatively little matrix. This method also establishes a specialized culture system for normal tissue from patients with early-onset colorectal cancer, significantly improving the success rate of culture, producing organoids with typical morphology, and maintaining the physiological characteristics of normal intestinal epithelium and stem cell function. Simultaneously, by using a specially formulated normal intestinal organoid culture medium, the necessary growth factors and nutritional conditions are provided for the stable growth and long-term passage of the organoids.

[0029] Furthermore, the standardized digestion time control, cell cluster formation monitoring, and passage procedures in the method of this invention ensure the reproducibility and stability of organoid culture. Digestion is terminated promptly when cell clusters of appropriate size are observed, avoiding the problem of single-cell formation caused by over-digestion, and significantly improving the initial formation rate and survival rate of organoids. The culture method is simple to operate, easy to standardize and widely apply, and provides a reliable in vitro model platform for mechanistic research, drug screening, and personalized treatment of early-onset colorectal cancer. Attached Figure Description

[0030] Figure 1 This is a time-series diagram of organoid morphological development in experimental groups 1-2 of the comparative experiment in this embodiment of the invention;

[0031] Figure 2 This is a bar chart comparing the ball formation rate of experimental groups 1-2 in the comparative experiment of this invention embodiment;

[0032] Figure 3 This is a line graph comparing the growth curves of experimental groups 1-2 in the comparative experiment of this invention. Detailed Implementation

[0033] The following will describe in more detail, with reference to the schematic diagrams, an EO-CRC source NCIO, its cultivation method, and its application, according to the present invention. Preferred embodiments of the invention are illustrated. It should be understood that those skilled in the art can modify the invention described herein while still achieving its advantageous effects. Therefore, the following description should be understood as being of general knowledge to those skilled in the art and is not intended to limit the invention.

[0034] For clarity, not all features of the actual embodiments are described. In the following description, well-known functions and structures are not detailed in detail, as they would obscure the invention with unnecessary detail. It should be understood that in the development of any actual embodiment, numerous implementation details must be made to achieve the developer's specific objectives, such as changes from one embodiment to another according to limitations related to the system or business. Furthermore, it should be understood that such development work may be complex and time-consuming, but is merely routine work for those skilled in the art.

[0035] Based on the teachings of this specification, those skilled in the art can form new technical solutions through cross-combination of different implementation methods without creating technical contradictions. Such variations should all be considered to fall within the protection scope of this invention.

[0036] The invention is described more specifically by way of example in the following paragraphs with reference to the accompanying drawings. The advantages and features of the invention will become clearer from the following description. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the invention.

[0037] Example 1

[0038] This invention provides a method for culturing EO-CRC-derived NCIO, specifically designed for culturing organoids from normal colorectal tissue of patients with early-onset colorectal cancer under the age of 50. This method can significantly improve the success rate of establishing organoids from normal tissue of patients with early-onset colorectal cancer and maintain the stability of their biological characteristics.

[0039] The cultivation method specifically includes the following steps:

[0040] S1: Obtain normal colorectal tissue from patients with early-onset colorectal cancer who are under 50 years of age at onset.

[0041] Normal colorectal tissue was obtained from patients with early-onset colorectal cancer who were under 50 years of age at diagnosis. This normal colorectal tissue was defined as adjacent normal tissue obtained during surgical resection, located at least 5 cm from the tumor margin to ensure tissue normality.

[0042] Immediately after acquisition, tissues were placed in sampling tubes containing pre-cooled tissue preservation solution, which included DPBS, 2% penicillin-streptomycin antibiotics, and 1% BSA, with a pH of 7.4. The tissues were completely submerged in the preservation solution and transported on ice. Because normal colorectal tissue exhibits better stability than tumor tissue, it can be stored at 4°C for 4-6 hours without significantly affecting subsequent culture results.

[0043] S2: 1-3mm after pretreatment 3 The organizational block.

[0044] After sterilizing the sampling tube with 75% alcohol, place it in a clean bench. Fresh, normal colorectal tissue is removed and placed in a sterile cell culture dish. The tissue morphology is then carefully observed under a stereomicroscope.

[0045] The pretreatment includes removing blood and tissue debris and washing the normal colorectal tissue.

[0046] Use sterile ophthalmic scissors and forceps to remove adhering blood clots, fatty tissue, and obvious necrotic tissue. Normal colorectal tissue is usually pink, homogeneous in texture, and has typical intestinal mucosal structure. Wash the tissue repeatedly 3-5 times with pre-cooled washing solution (DPBS + 2% penicillin-dextrose antibody) until the washing solution is clear and free of blood.

[0047] After cleaning, the tissue is cut into 1-3mm pieces using a sterile blade. 3 The tissue blocks should be kept moist during cutting to prevent drying and cell death. Controlling the size of the tissue blocks is crucial for subsequent digestion; overly large blocks result in incomplete digestion, while overly small blocks can easily cause cell damage.

[0048] In a specific example, a portion of tissue can be retained for pathological examination and molecular biological analysis, such as a 1cm sample. 3 The tissue was added to a general-purpose tissue fixative for histopathological and immunohistochemical analysis, and several small portions were reserved for cryopreservation at -80°C for later use.

[0049] S3: Digest the tissue block with EDTA digestion solution at 2-6°C for 20-60 minutes.

[0050] Prepare an EDTA digestion solution with a concentration of 0.01-0.02M.

[0051] The mechanism of action of the EDTA digestion solution is through chelation of Ca... 2+ and Mg 2+ Divalent cations disrupt calcium-dependent adhesion junctions between cells, particularly E-cadherin-mediated intercellular connections, thereby dissociating intestinal crypt structures.

[0052] The pretreated tissue blocks were transferred to 50 ml centrifuge tubes containing EDTA digestion solution and digested on a shaker at 2-6°C for 20-60 minutes, preferably 25-35 minutes, and more preferably 30 minutes. The low temperature of 2-6°C slows down enzyme activity, preferably 4°C, to avoid cell damage caused by over-digestion, while maintaining the activity of normal stem cells.

[0053] Microscopic observation should be performed every 10-20 minutes during the digestion process. A small drop of digestion solution should be placed on a glass slide and observed under an inverted microscope to assess cell dissociation. When a large cluster of approximately 10 cells is observed, the digestion is considered adequate; at this point, digestion should be terminated using a phosphate-buffered saline solution containing 0.5-2% BSA. Digestion into single cells should be avoided, as intestinal stem cells in a single-cell state are prone to anodic apoptosis, significantly reducing organoid formation rates.

[0054] S4: Terminate the digestion reaction, filter, and collect the digestion products by centrifugation.

[0055] Preferably, 10 ml of phosphate-buffered saline (PBS + 1% BSA) containing BSA is added to the digestion tube to terminate the digestion reaction. The role of BSA is to provide a protective environment for proteins, reducing cell adhesion and aggregation.

[0056] The digested cell suspension was filtered using a filter with a pore size of 80-120 μm, preferably a 100 μm filter, to remove undigested large tissue fragments and cell debris. The filtrate was then collected into a new 50 ml centrifuge tube.

[0057] The centrifugation conditions are 250-350g, 2-6℃, 3-8 minutes.

[0058] Low-temperature centrifugation helps maintain cell viability; moderate centrifugal force can effectively precipitate cell clusters while avoiding excessive mechanical stress that could damage the cells.

[0059] After centrifugation, carefully remove the supernatant, retaining the cell pellet at the bottom.

[0060] S5: Three-dimensional embedding culture.

[0061] In step S5, the collected digestion products are three-dimensionally embedded with a proportionally mixed basal culture medium and matrix gel.

[0062] The volume ratio of matrix gel to culture medium affects the formation efficiency and growth quality of organoids from normal colorectal tissue derived from early-onset colorectal cancer. A 1:1 ratio moderately increases gel thickness, facilitating oxygen and nutrient diffusion, and is suitable for samples with high cell viability. A ratio of 1:1.25 (i.e., 1 volume of culture medium to 1.25 volumes of matrix gel) provides better three-dimensional support, further improving initial organoid survival and colony formation consistency. The volume ratio of basal culture medium to matrix gel is 1:0.8 to 1:1.5, preferably 1:1 or 1:1.25.

[0063] In one specific example, the matrix gel used is a basement membrane extract. Various commercially available basement membrane matrix products can be used, including but not limited to Matrigel, Cultrex BME, and iMatrix. The culture medium base can be Advanced DMEM / F12 or other serum-free media (such as IntestiCult). TM ).

[0064] Specifically, resuspend the cell pellet in an appropriate amount of DMEM / F12 basal medium, and then mix thoroughly with pre-cooled matrix gel. Use half-range pipetting during mixing to avoid generating air bubbles. Seed the mixture at 50 μl / well into preheated 12-well or 24-well plates.

[0065] S6: After gelation, add normal intestinal organoid culture medium for culturing.

[0066] Specifically, place the inoculated culture plates in a 37°C, 3-7% CO2 incubator for 15 minutes to gel, including 5 minutes upright and 10 minutes upside down. Upside-down incubation helps form a uniform gel structure and prevents cells from settling to the bottom of the wells.

[0067] After gelation, the organoids are cultured using an optimized culture medium independently developed by the applicant. This culture medium, through component optimization and formulation improvement, significantly enhances the culture effect of organoids. The specific composition of the normal intestinal organoid culture medium is as follows:

[0068] Advanced DMEM / F12 basal medium: provides a basic nutritional environment; N21-MAX medium additive 1x: contains neurotrophic factors to promote stem cell maintenance; N-2MAX medium additive 1x: provides growth factors such as insulin and transferrin; N-acetylcysteine ​​1-1.25mM: antioxidant to protect cells from oxidative stress; GlutaminePlus 1x: a stable source of glutamine to support cell metabolism; HEPES buffer 8-12mM: maintains pH stability of the medium; Recombinant human Noggin protein 50-150ng / mL: BMP signaling pathway inhibitor to maintain stem cell characteristics; Recombinant human R-Spondin 1 protein 0.5-1.5μg / mL: Wnt signaling pathway enhancer to promote stem cell proliferation; Recombinant human EGF protein 20-100ng / mL: epidermal growth factor to stimulate cell proliferation; Recombinant human Wnt-3a protein 50-200ng / mL: classic Wnt signaling pathway activator to maintain stem cell self-renewal.

[0069] In a specific example, for samples with low stem cell activity, the concentration of Wnt-3a protein can be appropriately increased to 150-200 ng / mL; for organoids with slow proliferation, the concentration of EGF protein can be increased to 80-100 ng / mL; and for organoids that are easy to differentiate, the concentration of Noggin protein can be increased to 120-150 ng / mL to maintain stemness.

[0070] For organoids made from normal intestinal tissue, the concentration and ratio of culture medium components are precisely adjusted to better support the growth and differentiation of normal intestinal stem cells.

[0071] Furthermore, the three-dimensional embedding culture is carried out at 37°C and 3-7% CO2, preferably 5% CO2. The culture medium is changed every 2-3 days, and preheated to 37°C before each change.

[0072] S7: Passage Breeding

[0073] When the organoid diameter reaches 200-500 μm, it is passaged.

[0074] You can choose the appropriate size of culture plates and centrifuge tubes according to the specific conditions of the laboratory. For cell recovery solution, you can use Corning 354253 or a product with similar functions. For TrypLE protease, you should choose a phenol red-free 1× concentration product to avoid interference with subsequent detection.

[0075] The passage steps include: dissolving the matrix gel, digesting and decomposing the organoids into cell clusters; crushing the organoids 2-3 times by pipetting, collecting them by centrifugation, and then remixing them with the matrix gel for embedding and culture.

[0076] Furthermore, the specific steps for generation are as follows:

[0077] S71: Remove the culture medium, add pre-cooled Cell Recovery Solution, and incubate at 4°C for 10 minutes to allow the matrix gel to fully dissolve.

[0078] S72: Add TrypLE enzyme solution and incubate at 37°C for 10 minutes, gently pipetting 1-2 times during the process. When the organoids have decomposed into a suspension mainly composed of cell clusters, terminate the digestion with PBS containing 1% BSA.

[0079] S73: Blow and crush the organoids 2-3 times to break down large organoid structures into smaller cell clusters, but avoid forming single cells.

[0080] S74: 300g, centrifuge at 4℃ for 5 minutes, discard the supernatant and retain the precipitate.

[0081] S75: Re-embedded culture, and perform three-dimensional embedding culture again according to the methods of steps S5-S6 above.

[0082] Specifically, in step S74, centrifugation typically results in a three-layer structure: an upper layer of buffer, a middle layer of matrix gel, and a bottom layer of organoid precipitate. At this point, the supernatant and matrix gel layer are discarded, and the organoid precipitate is retained.

[0083] If the mixture separates into two layers after centrifugation, with the upper layer being buffer and the lower layer being a matrix gel organoid suspension, this may be due to insufficient dissolution of the matrix gel. In this case, discard the supernatant, keeping the matrix gel organoid suspension. Add 4°C PBS and repeat the washing step above. After another cooling and centrifugation, a clear three-layer structure (buffer layer, matrix gel layer, and organoid precipitate layer) should usually appear. Discard the supernatant and matrix gel layer, keeping only the organoid precipitate. If it still consists of two layers (buffer layer and matrix gel organoid suspension layer), discard the buffer and the upper 1 / 3 of the matrix gel organoid suspension, keeping the remaining 2 / 3.

[0084] It should be noted that the conditions that promote the effective separation of matrix gel and organoids include:

[0085] Horizontal centrifuges are more conducive to the separation of matrix gels and organoids than fixed-angle centrifuges; the centrifuge temperature is best at 4°C to avoid matrix gel solidification, the centrifugation speed can be appropriately increased, but should not exceed 500g, and the centrifugation time can be appropriately extended, but should not exceed 10 minutes.

[0086] Furthermore, cryopreservation should be performed during the exponential growth phase of organoids, preferably on the 3rd-4th day after passage, when the diameter of most organoids is 100-200 μm. Gradient cryopreservation or manual cryopreservation methods can be used.

[0087] The organoid cryopreservation solution can be selected from... D10 Cryopreservation Medium without Phenol Red. Gradient cryopreservation involves placing cryovials in a gradient cryopreservation box and storing them at -80°C overnight. The next day, they are removed and placed in a liquid nitrogen tank. Manual cryopreservation involves placing cryovials in a 4°C freezer for 30 minutes, transferring them to -20°C for 1 hour, then transferring them to -80°C overnight. The next day, they are removed and placed in a liquid nitrogen tank.

[0088] The thawing steps include: preheating the water bath to 37°C; removing the cryovials and thawing them quickly, shaking them constantly to allow the liquid in the tubes to melt rapidly; after about 1-2 minutes, when the liquid in the cryovials is completely dissolved, wiping the outer wall of the cryovials with an alcohol swab and then placing them in a clean bench; transferring the organoid cryopreservation solution into centrifuge tubes, resuspending them with washing buffer, centrifuging to collect the organoid precipitate; and re-embedding and culturing the organoids according to steps S5-S6 above.

[0089] Criteria for successful training include:

[0090] Formation rate: Stem cell-derived organoids: single-cell seeding efficiency ≥1%;

[0091] Tissue block-derived organoids: ≥20% of tissue blocks can form structurally complete organoids.

[0092] Growth rate: Typical three-dimensional structures are formed within the system-specific time window.

[0093] Morphological quality: The organoids have clear boundaries, a central lumen and polarized epithelium, and no obvious necrotic area in the center.

[0094] Growth status: During organoid proliferation, the daily volume growth rate is ≥5%.

[0095] The organoids of normal colorectal tissue derived from early-onset colorectal cancer prepared using the above culture method have the following characteristics:

[0096] The organoids exhibit typical intestinal crypt-like structures, including two main morphologies: vesicular and budding. Vesicular organoids have a distinct central chamber. The organoids have clear boundaries, uniform cell distribution, and a diameter ranging from 100 to 800 μm.

[0097] Organoids exhibit stable proliferative capacity, doubling in size every 3-5 days. Under appropriate culture conditions, organoids can be stably passaged for more than 15 generations without losing their basic biological characteristics. After passage, organoids can rapidly resume growth, maintaining a formation rate of over 80%.

[0098] The organoids retained the main functions of normal intestinal epithelium, including barrier function, secretory function, and absorptive function. The FITC-dextran permeability test confirmed that they possessed intact epithelial barrier function.

[0099] Furthermore, the organoids from normal colorectal tissue of patients with early-onset colorectal cancer have significant application value in constructing disease models of early-onset colorectal cancer. Comparative studies with tumor organoids from the same patient can analyze the mechanisms of early-onset colorectal cancer development; identify specific molecular markers of early-onset colorectal cancer; and investigate key molecular events in the transformation of normal tissue into tumors.

[0100] The applications of these organoids in studying the pathogenesis of early-onset colorectal cancer include:

[0101] Applications in constructing disease models of early-onset colorectal cancer, drug screening, or research on the pathogenesis of early-onset colorectal cancer.

[0102] We will introduce pathogenic mutations related to early-onset colorectal cancer into normal organoids using gene editing technology and observe their effects on cell behavior; we will use organoid models to study the effects of environmental factors such as diet, inflammation, and microorganisms on the occurrence of early-onset colorectal cancer; and we will investigate the role and mechanism of key signaling pathways such as Wnt, p53, and PI3K / AKT in the occurrence of early-onset colorectal cancer.

[0103] In this embodiment, the colorectal tissue organoids derived from early-onset colorectal cancer prepared using the above-described culture method can provide patient-specific drug response information, providing a basis for individualized treatment.

[0104] Example 2

[0105] To verify the effectiveness of the culture method provided by this invention, this embodiment provides a comparative experiment to systematically evaluate the impact of different digestion methods on the culture effect of colorectal tissue organoids.

[0106] Experimental group 1: EDTA digestion method was used.

[0107] Experimental Group 2: The only difference from Experimental Group 1 is that it uses 1 mg / mL Dispase digestion method.

[0108] Statistical analysis was used to compare the differences in pelleting efficiency and growth kinetics between the two groups. Please refer to [link / reference]. Figures 1-3 The experimental results showed that the culture effect of experimental group 1 (EDTA digestion method) was significantly better than that of experimental group 2 (Dispase digestion method), mainly reflected in the following two key indicators:

[0109] 1. Significantly improved ball formation efficiency

[0110] Please continue to refer to this. Figure 2 Quantitative analysis using bar charts showed that the spheroidization rate of experimental group 1 reached approximately 78%, which was 20 percentage points higher than the 58% of experimental group 2, representing an increase of 34.5%. Statistical tests confirmed that the difference between the two groups was highly statistically significant (p<0.001), indicating that the EDTA digestion method can significantly improve the success rate of cell aggregation to form organoid structures.

[0111] 2. Significant advantages in growth kinetics

[0112] Please continue to refer to this. Figure 1 and Figure 3 Significant differences between the two groups in growth rate and final size:

[0113] Earlier growth initiation: From the second day of culture, the average diameter of organoids in experimental group 1 showed a significant advantage.

[0114] The growth trend is steeper: the volume growth curve of organoid 1 in experimental group has a steeper slope, showing stronger proliferative activity.

[0115] Larger final size: By day 5 of culture, the average diameter of organoids in experimental group 1 reached approximately 220 μm, which was 29.4% larger than that in experimental group 2 (approximately 170 μm).

[0116] The above comparative experiments fully demonstrate the significant advantages of the EDTA digestion method of this invention in the culture of colorectal tissue organoids. This method not only improves the efficiency of organoid formation but also promotes faster growth and a more ideal final size, providing reliable technical support for the large-scale preparation of high-quality organoids.

[0117] In summary, this invention addresses the unique biological characteristics of normal tissues from patients with early-onset colorectal cancer by employing a low-temperature EDTA digestion strategy, effectively avoiding cell damage and single-cell formation caused by over-digestion. Combined with an optimized culture medium formulation for normal intestinal organoids, it improves the success rate and passage stability of organoids derived from colorectal tissues from early-onset colorectal cancer. The established organoid models not only highly preserve the original tissue characteristics at the morphological and molecular levels but also provide a reliable technical platform for studying the pathogenesis of early-onset colorectal cancer, constructing disease models, screening drugs, and developing personalized treatment plans. The technical solution of this invention has a high degree of standardization and good reproducibility, possessing significant scientific research value and clinical translational significance.

[0118] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A method for culturing NCIO derived from EO-CRC, characterized in that, include: Obtain normal colorectal tissue from patients with early-onset colorectal cancer who are under 50 years of age at onset; After pretreatment, 1-3mm was obtained 3 The organizational blocks; The tissue block was digested with EDTA digestion solution at 2-6°C for 20-60 minutes. The digestion reaction is terminated, and the digestion products are collected by filtration and centrifugation. The collected digestion products were three-dimensionally embedded with a proportionally mixed basal culture medium and matrix gel. After gelation, the culture medium for normal intestinal organoids was added; The normal intestinal organoid culture medium comprises: Advanced DMEM / F12 basal medium, N21-MAX medium additive 1x, N-2MAX medium additive 1x, N-acetylcysteine ​​1-1.25mM, Glutamine Plus 1x, HEPES buffer 8-12mM, recombinant human Noggin protein 50-150ng / mL, recombinant human R-Spondin 1 protein 0.5-1.5μg / mL, recombinant human EGF protein 20-100ng / mL and recombinant human Wnt-3a protein 50-200ng / mL.

2. The cultivation method according to claim 1, characterized in that, The digestion process uses an EDTA digestion solution with a concentration of 0.01-0.02M.

3. The cultivation method according to claim 1, characterized in that, During the digestion process, microscopic observation is performed every 10-20 minutes. When a cell cluster consisting of 5-50 cells is observed, digestion is terminated using a phosphate buffer containing 0.5-2% BSA.

4. The cultivation method according to claim 1, characterized in that, The centrifugation conditions are 250-350g, 2-6℃, 3-8 minutes.

5. The cultivation method according to claim 1, characterized in that, The volume ratio of the basal culture medium to the matrix gel is 1:0.8 to 1:1.

5.

6. The cultivation method according to claim 1, characterized in that, The three-dimensional embedding culture was carried out at 37°C and 3-7% CO2.

7. The cultivation method according to claim 1, characterized in that, The pretreatment includes: removing blood and tissue debris, and washing the normal colorectal tissue.

8. The cultivation method according to claim 1, characterized in that, It also includes the process of propagation: When the diameter of the organoids reaches 200-500 μm, the matrix gel is dissolved, and the organoids are digested and decomposed into cell clusters. The organoids were crushed by blowing and pulverizing 2-3 times, collected by centrifugation, and then remixed, embedded, and cultured.

9. An EO-CRC source NCIO, characterized in that, It was prepared using the culture method described in any one of claims 1-8.

10. The application of the NCIO described in claim 9 in constructing disease models of early-onset colorectal cancer, drug screening, or studying the pathogenesis of early-onset colorectal cancer.

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