Novel culture method of colorectal cancer organoid

Abstract

The invention discloses a novel colorectal cancer organoid culture method, which obviously improves the activity of primary cells (more than or equal to 90%) and the forming efficiency of the organoid (the success rate is more than 95%) through alcohol gradient cleaning, collagenase-hyaluronidase combined digestion and stent-free culture technologies. The improved culture medium takes R-Spondin1 as a core, nicotinamide and a small-molecule inhibitor are combined, and long-term amplification of organoids (passage is more than or equal to 10 times) is supported. The method is suitable for individualized drug screening, tumor microenvironment simulation and drug resistance mechanism research, has the advantages of high efficiency, stability and low cost, and provides a reliable model for precise medical treatment of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel method for culturing colorectal cancer organoids. Background Art

[0002] Colorectal cancer organoids (CRC organoids), as three-dimensional in vitro models, can closely simulate tumor heterogeneity and microenvironmental characteristics, and are of great value in drug screening and mechanism research. However, existing technologies have the following drawbacks:

[0003] High contamination risk: Traditional digestive fluids rely solely on penicillin-streptomycin for bacteriostasis, with insufficient protection against mycoplasmas and fungi, resulting in a culture failure rate >30%;

[0004] Low digestion efficiency: Single enzymes (such as collagenase or trypsin) can easily damage stem cell activity when dissociating tissues, and the lack of a pH buffer system results in cell recovery rates of less than 50%29;

[0005] Poor culture medium stability: Over-reliance on conditioned medium (such as Wnt3a) leads to batch-to-batch variability, a long organoid formation cycle (>7 days), and a survival rate that drops below 60% after passage.

[0006] Scaffold dependence: Matrix gel embedding requires exogenous material support, which is costly and difficult to apply on a large scale.

[0007] To address the above problems, the present invention significantly improves the stability and practicality of organoids by optimizing the combination of digestive enzymes, introducing new antibacterial agents and scaffold-free culture technology. Summary of the Invention

[0008] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0009] In view of the above problems in the prior art, the inventors proposed the present invention.

[0010] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a novel method for culturing colorectal cancer organoids.

[0011] To solve the above technical problems, the present invention provides the following technical solution: a novel method for culturing colorectal cancer organoids, comprising:

[0012] (1) Tissue pretreatment: Fresh colorectal cancer tissue was obtained, washed with gradient alcohol, and then repeatedly rinsed with PBS buffer containing 1‰ primocin and 1% penicillin-streptomycin to remove impurity cells;

[0013] (2) Multi-enzyme synergistic digestion: Tissue fragments were placed in a digestion solution containing collagenase I (1 mg / mL), hyaluronidase (0.5 mg / mL), DNase I (20 μg / mL), and Y-27632 (10 μM), and digested at 37°C for 30 min with shaking, and crypt clusters were collected multiple times in a short period of time;

[0014] (3) Cell purification: After filtering through a 100 μm filter, centrifuge and discard the supernatant. The cell pellet was mixed with Matrigel at a volume ratio of 2:1 for embedding;

[0015] (4) Scaffold-free culture: The embedded cells were seeded in ultra-low attachment culture plates, added with modified organoid culture medium, and cultured at 37°C and 5% CO2. The culture medium was changed every 48 hours.

[0016] (5) Passaging and expansion: Use a protective agent containing R-Spondin1 (100 ng / mL) and proline (1 mM) to dissolve the matrix gel, repeat the digestion and culture steps, and establish an organoid biobank.

[0017] As a preferred embodiment of the novel colorectal cancer organoid culture method described in the present invention, the digestion solution further comprises HEPES buffer (1×) and GlutaMAX (1×) to maintain the pH stability of the digestion system and cell viability ≥90%.

[0018] As a preferred embodiment of the culture method of a novel colorectal cancer organoid described in the present invention, the improved organoid culture medium is composed of the following components: Advanced DMEM / F12 basal medium, supplemented with 30% VVL-RWN conditioned medium, 100 ng / mL R-Spondin1, 500 nM A83-01, 1 μM SB202190, 10 nM GastrinI, 1 μM PGE2 and 5 μM nicotinamide, and the total antibiotic content is ≤1‰.

[0019] As a preferred embodiment of the novel colorectal cancer organoid culture method described in the present invention, its applications include colorectal cancer drug sensitivity detection, individualized treatment model construction and tumor microenvironment simulation.

[0020] As a preferred embodiment of the novel colorectal cancer organoid culture method described in the present invention, the improved organoid culture medium further contains 1 mM N-acetylcysteine ​​to reduce oxidative stress damage and increase the organoid survival rate to ≥95%.

[0021] As a preferred embodiment of the novel colorectal cancer organoid culture method described in the present invention, the oxygen content in the culture environment in step (4) is 21%, and the carbon dioxide content is 5%, and the physiological microenvironment in the body is simulated by precisely controlling the gas concentration.

[0022] As a preferred embodiment of the method for culturing a novel colorectal cancer organoid described in the present invention, during the passage and amplification in step (5), optogenetics is used to activate oncogenes to achieve a study on the spatiotemporal controllability of organoid tumorigenesis.

[0023] As a preferred embodiment of the novel colorectal cancer organoid culture method described in the present invention, the organoid can be used to construct a co-culture model, including co-culturing with tumor-associated fibroblasts or immune cells to simulate tumor microenvironment interactions.

[0024] As a preferred embodiment of the novel colorectal cancer organoid culture method of the present invention, the organoid culture cycle is 3-5 days, the number of passages is ≥10 times, and the survival rate of each generation is ≥90%.

[0025] Beneficial effects of the present invention:

[0026] 1. Highly effective anti-pollution: Alcohol gradient cleaning combined with primocin (1‰) and broad-spectrum antibiotics reduces the contamination rate from 30% to <5%;

[0027] 2. Multi-enzyme synergistic digestion: Collagenase I and hyaluronidase are used together to increase the dissociation efficiency by 40%, the crypt cluster integrity retention rate is >85%, and the proportion of LGR5+ stem cells reaches 25%;

[0028] 3. Optimization of culture medium stability: Replacing part of the conditioned medium with R-Spondin1 and combining it with nicotinamide (1 mM) to promote stem cell self-renewal, shortening the organoid formation cycle to 3-5 days, and achieving a passage survival rate of ≥90%811;

[0029] 4. Scaffold-free large-scale culture: Ultra-low adsorption plates combined with cell self-aggregation properties avoid dependence on exogenous matrix gel, reducing the cost of a single culture by 60%;

[0030] 5. Precision drug evaluation: The sensitivity test results of the constructed organoid model to drugs such as 5-FU and oxaliplatin are 92% consistent with the clinical response. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it constitute a separate or selective embodiment that is mutually exclusive with other embodiments.

[0034] Example 1

[0035] This example provides a novel method for culturing colorectal cancer organoids.

[0036] Multi-enzyme synergistic digestion and anti-pollution treatment

[0037] Steps and parameters

[0038] (1) Take the primary colorectal cancer tissue (1 cm in diameter) 3 ), placed in a 6 cm Petri dish, and soaked in 10 times the volume of 75% ethanol for 10 seconds to remove surface contaminants;

[0039] (2) Discard the ethanol, wash with PBS buffer containing 1‰ primocin and 1% penicillin-streptomycin three times for 30 seconds each time, and mince into 0.5 mm3 fragments with a scalpel;

[0040] (3) Add 5 times the volume of digestion solution (Advanced DMEM / F12 + collagenase I 1 mg / mL + hyaluronidase 0.5 mg / mL + DNase I 20 μg / mL + Y-27632 10 μM + HEPES 1× + GlutaMAX 1×) and digest at 37°C, 200 rpm, shaking for 30 min;

[0041] (4) Filter through a 100 μm filter, collect the supernatant, centrifuge at 300 × g for 5 minutes at 4°C, and discard the supernatant to obtain the cell pellet.

[0042] Principle analysis:

[0043] Multi-enzyme synergistic digestion mechanism

[0044] Collagenase I: specifically cuts the triple helix structure of collagen (Gly-XY repeating sequence), preferentially decomposes type I and type III collagen fibers in colorectal cancer tissue, destroys the physical barrier of the extracellular matrix (ECM), and releases stem cells (LGR5+) in the crypt structure.

[0045] Hyaluronidase: Degrades hyaluronic acid (HA), one of the main components of tumor stroma. Its degradation can reduce tissue viscosity, promote the penetration of digestive fluid into deep tissues, and reduce the damage of mechanical shear force to cells.

[0046] DNaseⅠ: decomposes free DNA released by cell rupture, prevents DNA adhesion and cell aggregation, and improves the purity of single-cell suspension (single-cell rate >90% by flow cytometry).

[0047] Y-27632: As a Rho kinase (ROCK) inhibitor, it inhibits cell apoptosis (Annexin V positivity is reduced to <5%) and maintains stem cell polarity by blocking ROCK-mediated myosin light chain phosphorylation.

[0048] Anti-pollution design

[0049] Alcohol gradient cleaning: 75% ethanol achieves rapid sterilization by destroying the lipid bilayer of microbial cell membranes (killing >99% of surface bacteria within 10 seconds), while short-term immersion avoids osmotic damage to active cells inside cancer tissues.

[0050] Primocin combined with antibiotics: Primocin contains polymyxin B and macrolide ingredients, which target Gram-negative bacteria and mycoplasmas (coverage rate >95%), while penicillin-streptomycin inhibits Gram-positive bacteria, forming a broad-spectrum protection (contamination rate <5% vs. 30% for traditional methods).

[0051] Effects

[0052] Crypt cluster integrity: Scanning electron microscopy (SEM) showed that the crypt basement membrane structure was intact, and the proportion of LGR5+ stem cells reached 25% (flow cytometry data), an increase of 108% compared with the single collagenase digestion group (12%);

[0053] Cell viability: Trypan blue staining showed a viable cell rate of ≥93% (65% for the traditional trypsin digestion group), and ATP detection showed a 2.1-fold increase in energy metabolism activity;

[0054] Contamination control: Bacterial culture test showed that the fungal and mycoplasma contamination rates were reduced to 0.3% and 0.5%, respectively (n=50 samples).

[0055] Example 2

[0056] This example provides a novel method for culturing colorectal cancer organoids.

[0057] Scaffold-free culture and culture medium optimization

[0058] Steps and parameters

[0059] (1) The cell pellet was mixed with Advanced DMEM / F12 at a ratio of 1:2 and inoculated into a 96-well ultra-low attachment plate with 50 μL per well.

[0060] (2) Add modified culture medium (containing R-spondin 1100 ng / mL, nicotinamide 5 μM, and N-acetylcysteine ​​1 mM) and culture at 37°C and 5% CO2;

[0061] (3) Organoid rudiments can be seen on the 3rd day, and three-dimensional structures with a diameter of >100 μm are formed on the 5th day;

[0062] (4) During subculturing, cells were digested with 0.25% trypsin-EDTA for 5 minutes, centrifuged, and resuspended for inoculation. The survival rate was ≥95%.

[0063] Principle analysis:

[0064] Stentless technology

[0065] Ultra-low attachment plate surface modification: Hydrophilic polymers (such as polyethylene glycol) are used to coat the culture plates, inhibiting cell attachment through steric hindrance (contact angle <10°), forcing cells to self-aggregate to form 3D organoids (100-200μm in diameter), and avoiding batch variability caused by Matrigel dependence (H&E staining showed a 40% increase in organoid structural uniformity).

[0066] Culture medium optimization mechanism

[0067] R-Spondin1: As a Wnt signaling pathway agonist, it binds to the LGR5 receptor to activate β-catenin nuclear translocation (immunofluorescence shows β-catenin nuclear localization rate >80%), maintaining stem cell self-renewal (EdU-labeled proliferating cells account for 35%);

[0068] Nicotinamide: As an NAD+ precursor, it delays stem cell aging (SA-β-gal positive rate <5%) by inhibiting SIRT1 deacetylase activity (Western blot showed SIRT1 expression was downregulated by 60%).

[0069] N-acetylcysteine ​​(NAC): It scavenges reactive oxygen species (ROS detection shows a 70% reduction in levels) by providing sulfhydryl groups (-SH), protects mitochondrial membrane potential (JC-1 staining red / green fluorescence ratio increased by 3 times), and reduces oxidative stress damage.

[0070] Effects

[0071] Organoid formation efficiency: Organoid formation rate on day 5 >95% (compared to 75% for the traditional Matrigel method), with concentrated diameter distribution (85% between 100-150 μm);

[0072] Long-term expansion capacity: survival rate ≥90% after 10 serial passages (survival rate drops to 60% at passage 5 using conventional methods); RT-qPCR shows stable expression of stemness markers LGR5 and ASCL2 (ΔCt value fluctuation <1);

[0073] Cost-effectiveness: The cost per culture is reduced by 60% (ultra-low attachment plates replace 80% of the Matrigel usage).

[0074] Example 3

[0075] This example provides a novel method for culturing colorectal cancer organoids.

[0076] Drug sensitivity testing and microenvironment simulation

[0077] Steps and parameters

[0078] (1) The third-generation organoids were co-cultured with 5-FU (10 μM) and oxaliplatin (50 μM) for 72 h;

[0079] (2) CCK-8 was used to measure the survival rates, which were 35% ± 5% and 42% ± 7%, respectively, consistent with the patients' response to postoperative chemotherapy;

[0080] (3) Immunofluorescence staining showed that the expression of apoptosis marker Caspase-3 was upregulated 3-fold;

[0081] (4) Constructing a co-culture model: Organoids were co-cultured with patient-derived tumor-associated fibroblasts (CAFs) at a ratio of 1:2 to simulate tumor-stroma interactions.

[0082] Principle Analysis

[0083] Mechanisms of drug sensitivity

[0084] Organoid gene fidelity: Whole exome sequencing (WES) showed that the consistency of organoid and primary tumor driver gene mutations (APC, KRAS, TP53) was 98%, and PDX model comparison showed drug response correlation R 2 =0.92;

[0085] 5-FU action pathway: 5-FU interferes with DNA synthesis by inhibiting thymidylate synthase (TS) (BrdU incorporation rate decreased by 60%). The TS expression level in organoids was negatively correlated with the patient's postoperative chemotherapy resistance (Spearman r = -0.85, p < 0.01).

[0086] Microenvironment simulation mechanism

[0087] CAFs co-culture model: Tumor-associated fibroblasts (CAFs) activate the organoid STAT3 pathway (phosphorylated STAT3 levels increased 4-fold) by secreting IL-6 (ELISA concentration >200 pg / mL), inducing epithelial-mesenchymal transition (EMT marker Vimentin expression increased 3-fold, E-cadherin downregulated 80%);

[0088] Immune cell interactions: After adding the patient's autologous CD8+ T cells, the expression of PD-L1 in organoids was upregulated by 2.5 times (flow cytometry), simulating the drug-resistant microenvironment of immune checkpoint inhibitors (such as pembrolizumab).

[0089] Effects

[0090] Drug detection accuracy: Across 30 clinical samples, the organoids' predicted sensitivity to 5-FU / oxaliplatin was 92% consistent with the patients' actual chemotherapy responses (Kappa = 0.88), outperforming traditional 2D cell models (78%).

[0091] Deep microenvironment simulation: The invasive ability of the co-cultured organoids (Transwell assay penetration increased 2.5-fold) was significantly correlated with the patient's lymph node metastasis rate (Pearson r = 0.79, p < 0.001).

[0092] Analysis of drug resistance mechanism: Overexpression of oxaliplatin-resistance-related gene ABCC3 was detected in organoids (ΔCt=-3.2), which was consistent with the patient's plasma ctDNA results.

[0093] The present invention systematically overcomes the technical bottlenecks of high contamination rate, low digestion efficiency, poor culture medium stability and strong scaffold dependence in traditional colorectal cancer organoid culture through three core modules: multi-enzyme synergistic digestion, scaffold-free culture system and precise microenvironment simulation. In the tissue pretreatment and digestion stage (Example 1), an innovative alcohol gradient cleaning method is used in combination with the broad-spectrum antibacterial agent primocin. Through the rapid penetration sterilization of ethanol (killing >99% of surface microorganisms within 10 seconds) and the high inhibition of mycoplasma by primocin (coverage rate >95%), the contamination rate is reduced from more than 30% in traditional methods to <5%, while avoiding the cytotoxicity caused by excessive use of antibiotics. To address the issue of low digestion efficiency, a three-enzyme synergistic system consisting of collagenase I, hyaluronidase, and DNase I was designed: collagenase I specifically decomposes type I / III collagen fibers in the tumor stroma, releasing LGR5+ stem cells; hyaluronidase reduces HA-mediated tissue viscosity and enhances digestive fluid penetration; and DNase I severs free DNA networks, reducing cell aggregation and increasing the purity of single-cell suspensions to over 90% (flow cytometry verification). Combined with the ROCK inhibitor Y-27632 to maintain stem cell activity (Annexin V positivity <5%), the system ultimately achieved breakthroughs in crypt cluster integrity exceeding 85% and a 25% LGR5+ stem cell ratio, representing a 40% increase in efficiency compared to traditional single-enzyme digestion methods. This fundamentally addresses the issues of low primary cell recovery and severe stem cell damage.

[0094] During the organoid construction and expansion stage, the present invention abandons the traditional matrix gel embedding method and adopts ultra-low adsorption culture plates combined with self-aggregation induction technology. It inhibits cell adhesion through surface hydrophilic modification (contact angle <10°), forcing cells to autonomously form 3D structures. It successfully reduces the amount of exogenous matrix gel used by 80%, reduces the cost of single culture by 60%, and improves the uniformity of organoids by 40% (verified by H&E staining). In terms of culture medium formula, R-Spondin1 replaces the traditional Wnt3a conditioned medium to maintain stem cell self-renewal by activating the LGR5 / β-catenin signaling pathway (nuclear localization rate > 80%); nicotinamide inhibits the SIRT1-mediated senescence pathway (SA-β-gal positivity rate < 5%), and cooperates with N-acetylcysteine ​​to clear ROS (level reduced by 70%), shortening the organoid formation cycle to 3-5 days (traditionally 7 days), with a passaging survival rate of ≥95%, and stable expression of stemness markers (LGR5, ASCL2) after 10 generations of continuous amplification (ΔCt fluctuation < 1), completely overcoming the industry problems of large batch differences in culture medium and a sudden drop in activity after passaging.

[0095] During the application validation phase, the organoids constructed in this study demonstrated extremely high clinical concordance: whole-exome sequencing confirmed 98% concordance with primary tumor driver mutations, and comparison with PDX models revealed an R² correlation of drug response of 0.92. Across 30 clinical samples, the organoids' predicted sensitivity to 5-FU / oxaliplatin was concordant with the patient's actual chemotherapy response in 92% of cases (Kappa = 0.88), significantly outperforming the 2D model's 78%. Further, co-culture with CAFs to simulate the tumor microenvironment confirmed that activation of the IL-6 / STAT3 pathway induced EMT (Vimentin ↑3-fold, E-cadherin ↓80%), and enhanced the organoids' invasiveness by 2.5-fold (Transwell assay), highly concordant with patient metastatic lesions (Pearson r = 0.79). Furthermore, this system can be expanded to simulate the immune microenvironment (e.g., PD-L1 regulation) and elucidate drug resistance mechanisms (e.g., ABCC3 overexpression validation), providing a one-stop solution for personalized therapy and translational research.

[0096] In summary, the present invention has increased the success rate of organoid culture to >95%, reduced the cost by 60% through the full-chain innovation of "efficient digestion-stable culture-precise simulation", and deeply coupled with clinical pathological characteristics, successfully breaking through the existing technological barriers and providing a reliable, efficient and scalable in vitro model for precision medicine of colorectal cancer.

[0097] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A novel method for culturing colorectal cancer organoids, characterized by: include (1) Tissue pretreatment: Fresh colorectal cancer tissue was obtained, washed with gradient alcohol, and then repeatedly rinsed with PBS buffer containing 1‰ primocin and 1% penicillin-streptomycin to remove impurity cells; (2) Multi-enzyme synergistic digestion: Tissue fragments were placed in a digestion solution containing collagenase I (1 mg / mL), hyaluronidase (0.5 mg / mL), DNase I (20 μg / mL), and Y-27632 (10 μM), and digested at 37°C for 30 min with shaking, and crypt clusters were collected multiple times in a short period of time; (3) Cell purification: After filtering through a 100 μm filter, centrifuge and discard the supernatant. The cell pellet was mixed with Matrigel at a volume ratio of 2:1 for embedding; (4) Scaffold-free culture: The embedded cells were seeded in ultra-low attachment culture plates, added with modified organoid culture medium, and cultured at 37°C and 5% CO2. The culture medium was changed every 48 hours. (5) Passaging and expansion: Use a protective agent containing R-Spondin1 (100 ng / mL) and proline (1 mM) to dissolve the matrix gel, repeat the digestion and culture steps, and establish an organoid biobank.

2. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: The digestion solution further comprises HEPES buffer (1×) and GlutaMAX (1×) to maintain pH stability of the digestion system and cell viability ≥ 90%.

3. The novel method for culturing colorectal cancer organoids according to claim 2, wherein: The improved organoid culture medium consists of the following components: Advanced DMEM / F12 basal medium, supplemented with 30% VVL-RWN conditioned medium, 100 ng / mL R-Spondin1, 500 nM A83-01, 1 μM SB202190, 10 nM GastrinI, 1 μM PGE2 and 5 μM nicotinamide, and the total antibiotic content is ≤1‰.

4. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: The tissue was pretreated with alcohol immersion for 10 seconds and washed with PBS three times, each time for 30 seconds, to effectively remove the fat layer and microbial contamination.

5. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: Its applications include colorectal cancer drug sensitivity testing, personalized treatment model construction, and tumor microenvironment simulation.

6. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: The improved organoid culture medium further contains 1 mM N-acetylcysteine ​​to reduce oxidative stress damage and increase the organoid survival rate to ≥95%.

7. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: In step (4), the oxygen content of the culture environment is 21%, and the carbon dioxide content is 5%, and the physiological microenvironment in the body is simulated by precisely controlling the gas concentration.

8. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: During the passage and amplification in step (5), optogenetics is used to activate oncogenes to achieve spatiotemporal controllability of organoid tumorigenesis.

9. The method for using the novel colorectal cancer organoid culture method according to claim 1, characterized in that: The organoids can be used to construct co-culture models, including co-culture with tumor-associated fibroblasts or immune cells, to simulate tumor microenvironment interactions.

10. The novel method for culturing colorectal cancer organoids according to claim 1, wherein: The organoid culture cycle is 3-5 days, the number of passages is ≥10 times, and the survival rate of each generation is ≥90%.