A system and standardized method for constructing organ-on-a-chip for colorectal cancer

Abstract

This invention relates to the fields of biomedical engineering, precision oncology, and in vitro model construction, providing a system and standardized method for constructing organ-on-a-chip for colorectal cancer. It aims to address the problems of lack of unified standards in existing tissue processing and microarray implantation procedures, and poor reproducibility of experimental data. The method comprises six steps: standardized tissue pretreatment and quality control, differentiated segmentation, controlled enzymatic digestion, cell collection and processing, precise preparation of implantation suspension, and standardized microarray implantation and culture. Key parameters are quantified and quality control nodes are established. Experimental validation shows that this method can significantly improve cell viability, sample utilization, and organoid formation efficiency. The inter-batch coefficient of variation for each key indicator is less than 10%, ensuring consistency of drug sensitivity test data. This provides reliable technical support for personalized drug screening for colorectal cancer and has clear prospects for clinical translation.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering, precision oncology and in vitro model construction technology, and specifically to a system and standardized method for constructing organ-on-a-chip for colorectal cancer. Background Technology

[0002] Colorectal cancer is a prevalent malignant tumor worldwide, and its treatment heavily relies on personalized approaches. The combination of patient-derived organoids and organ-on-a-chip (OOC) technology provides a revolutionary platform for simulating the patient-specific tumor microenvironment in vitro and conducting high-throughput drug sensitivity testing. This platform holds the promise of directly guiding clinical medication and achieving precision medicine.

[0003] However, a key bottleneck currently exists in the translation of this technology from patient tissue to microarray culture: the lack of standardized tissue processing and inoculation procedures leads to significant arbitrariness in operation. This directly results in substantial differences in the growth status, morphology, and cell activity of organoids constructed by different batches and operators on the microarray, leading to poor reproducibility and low data comparability in drug testing results. This instability severely wastes valuable patient samples and greatly hinders the clinical reliability and widespread application of organoid microarray drug sensitivity testing results.

[0004] Furthermore, existing tissue processing methods are mostly based on laboratory experience, and standardized procedures have not been developed to match specific organ-on-a-chip platforms in terms of tissue cutting size, determination of digestion endpoints, cell seeding density, and matrix gel mixing ratio. Therefore, there is an urgent need to develop a standardized operating procedure to ensure high success rate, high fidelity, and high reproducibility of experimental data in the construction of colorectal cancer organoids on microarrays. Summary of the Invention

[0005] The purpose of this invention is to provide a system and standardized method for constructing organ-on-a-chip for colorectal cancer, in order to solve the technical problems of non-standardization of existing colorectal cancer organ-on-a-chip construction processes and poor repeatability of experimental data.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A standardized method for constructing organ-on-a-chip for colorectal cancer includes the following steps:

[0008] (1) Pre-treatment of tissues: Cleaning, recording morphology, weighing and archiving information of fresh colorectal cancer surgical specimens;

[0009] (2) Controllable enzymatic digestion: The processed specimen is digested with enzymes, and the morphology of the digestion products is monitored in real time by microscope to control the digestion endpoint. The criteria for judging the digestion endpoint are: the tissue block basically disappears and the digestion products are mainly composed of cell clusters with a size of 20-100μm.

[0010] (3) Cell collection and processing: Filter and centrifuge the digestion products to obtain cell precipitate, and count the live cells in the precipitate;

[0011] (4) Precision inoculation suspension preparation: Based on the preset target number of inoculation chips, the preset target cell density in the chip and the structural parameters of the organ chip used, calculate and prepare a cell-matrix suspension containing a specific number of cells and a matrix gel volume;

[0012] (5) Standardized chip seeding and culture: The cell-matrix gel suspension is injected into the gel channel of the organ chip, solidified, culture medium is added, and a dynamic perfusion culture system is started for culture to obtain colorectal cancer organ chips.

[0013] In a preferred embodiment, after step (1), a differential tissue segmentation step is further included: the pre-treated tissue is segmented into three parts for in vitro culture, cryopreservation backup and pathological analysis, and the volume relationship of the three parts of the tissue satisfies: the volume of the part for in vitro culture > the volume of the part for cryopreservation backup > the volume of the part for pathological analysis.

[0014] In a preferred embodiment, the criteria for determining the digestion endpoint further include: the scarcity of discrete single cells in the microscope field of view.

[0015] In a preferred embodiment, step (3) further includes an inoculation decision based on cell viability:

[0016] a. If the cell viability is ≥70%, the obtained primary cells will be directly used to prepare cell-matrix suspension and seeded on organ-on-a-chip.

[0017] b. If the cell viability is <70%, the obtained primary cells will be cultured and expanded in well plates. After they form organoids and are passaged, they will be used to prepare cell-matrix gel suspensions and seeded on organ-on-a-chip.

[0018] In a preferred embodiment, in step (4), the matrix gel volume and the required total number of cells are calculated using the following model:

[0019] matrix gel volume

[0020] Where N represents the number of organ-on-a-chip targets for implantation, and C represents the total number of gel channels in the individual chips contained in a single device. The standard inoculation volume for each gel channel, k is the coefficient for reserving operational losses, and the value of k ranges from 1.25 to 1.3;

[0021] Total number of cells required

[0022]

[0023] Where D is the preset target cell density within the chip.

[0024] In a preferred embodiment, in step (4), the cell-matrix gel suspension is prepared by mixing the counted cell precipitate with the matrix gel taken according to the calculated volume at a low temperature of 4°C.

[0025] In a preferred embodiment, in step (5), the cell-matrix gel suspension is slowly injected from the gel channel inlet of the organ-on-a-chip; the culture medium is added from the perfusion channel inlet of the organ-on-a-chip; and the dynamic perfusion culture system is a microfluidic peristaltic pump or a rocking incubator that can generate liquid flow.

[0026] The present invention also provides a colorectal cancer organ-on-a-chip construction system for implementing any of the above methods, comprising:

[0027] The tissue pretreatment unit is used to clean, record the morphology of, weigh, and archive information of fresh colorectal cancer surgical specimens.

[0028] The controllable enzyme digestion unit is used to perform enzyme digestion on the processed specimen and to control the digestion endpoint by observing the cell cluster state under a microscope. The digestion endpoint is the formation of digestion products mainly consisting of cell clusters with a size of 20-100 μm.

[0029] The cell processing unit is used to filter and centrifuge the digestion products to obtain cell precipitate, and to perform live cell counting and cell viability assessment.

[0030] The precision inoculation suspension preparation unit includes a calculation module, which is used to calculate and prepare a cell-matrix suspension containing a specific number of cells and a matrix gel volume based on the number of organ-on-a-chip to be inoculated, the preset target cell density within the chip, and the structural parameters of the organ-on-a-chip.

[0031] A standardized chip seeding and culture unit is used to inject the cell-matrix gel suspension into the gel channel of the organ-on-a-chip, add culture medium after solidification, and start the dynamic perfusion culture system for culture.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. This invention significantly reduces human error through standardized operation throughout the entire process and quantitative control of key parameters, enabling colorectal cancer organoid chips constructed in different batches to have high uniformity in growth status, structural morphology and cell composition. This significantly improves the repeatability, stability and comparability of experimental data such as drug sensitivity screening and efficacy evaluation, and provides a reliable model basis for in vitro high-throughput screening.

[0034] 2. This invention, through a clearly defined digestion endpoint judgment standard and a differentiated tissue segmentation strategy, achieves a rational allocation of multiple uses for in vitro culture, cryopreservation backup, and pathological detection while ensuring the viability and yield of primary cells. This significantly improves organoid formation rate and chip construction success rate, maximizes the use of precious clinical tumor tissue samples, and reduces sample waste.

[0035] 3. Based on the structural parameters of organ-on-a-chip, such as the number of channels, the volume of a single channel, and the perfusion system, this invention establishes a standardized seeding volume calculation model, which can accurately control the cell seeding density and the amount of matrix gel used, ensuring a high degree of consistency in seeding conditions between different chips and different channels. This solves the problem that traditional manual seeding is difficult to match with micro-chip systems, and realizes the standardized and large-scale construction of organ-on-a-chip from tissue samples.

[0036] 4. The colorectal cancer organ-on-a-chip structure obtained by the method of this invention is stable and has high physiological relevance. It can better preserve the tissue morphology and biological characteristics of the primary tumor. It can be directly used for personalized drug sensitivity testing, drug screening and mechanism research, providing objective and reliable in vitro evaluation basis for the formulation of clinical personalized treatment plans. It has good application prospects and clinical translation potential in precision medicine. Attached Figure Description

[0037] Figure 1 This is a flowchart of the standardized method of the present invention;

[0038] Figure 2 A diagram illustrating organizational differentiation;

[0039] Figure 3 A schematic diagram of the end-stage microscopic examination criteria for digestion;

[0040] Figure 4 This is a schematic diagram of the structure of an organ-on-a-chip used in one embodiment of the present invention;

[0041] Figure 5 This image shows the healthy growth state of colorectal cancer organoids constructed using the method of this invention on a microarray. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0043] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0044] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] Example 1

[0047] The sample used in this embodiment was rectal adenocarcinoma tissue. After ex vivo, the sample was placed in a tissue preservation solution containing 5% penicillin and streptomycin at 4°C for 30 minutes before being transported to the laboratory. The main reagents included: basement membrane matrix, tumor tissue digestion solution, erythrocyte lysis buffer, and human colorectal cancer organoid culture medium. The experiment employed a colorectal cancer organ-on-a-chip construction system to complete the entire process. This system includes a tissue pretreatment unit, a controlled enzyme digestion unit, a cell processing unit, a precise seeding suspension preparation unit, and a standardized chip seeding and culture unit. These units work together to complete the standardized operation from tissue sample processing to organoid culture.

[0048] A standardized method for constructing organ-on-a-chip for colorectal cancer includes the following steps (such as...) Figure 1 ):

[0049] 1. Pretreatment and quality control of tissue samples

[0050] In the biosafety cabinet, the received samples are transferred to pre-cooled PBS buffer through the tissue pretreatment unit and washed repeatedly 3-5 times until the liquid is clear. The tissue is then placed in a sterile culture dish, observed and recorded under a stereomicroscope, and all information is archived in the sample information database after the tissue quality inspection is completed.

[0051] 2. Differentiated organizational segmentation

[0052] After quality inspection, the tissue was placed on a 4°C constant-temperature operating table and cut using a sterile scalpel, with differential segmentation based on volume ratios (e.g., Figure 2 (As shown): The portion for in vitro culture > the portion for cryopreservation and backup > the portion for pathological analysis. Specifically:

[0053] For in vitro culture: approximately 250 mg, placed in pre-cooled PBS for later use.

[0054] For cryopreservation backup: approximately 80 mg, transferred to cryovials, subjected to programmed cooling, and then stored in a liquid nitrogen tank.

[0055] The portion used for pathological fixation and embedding: approximately 40 mg, fixed in 4% paraformaldehyde for subsequent pathological control analysis.

[0056] 3. Controlled enzymatic digestion and cell collection

[0057] Finely chop 250 mg of tissue blocks for culture into a paste approximately 1 mm³ in a culture dish. Transfer this paste to a digestion tube pretreated with 1% BSA, add 8 mL of tissue digestion solution, and digest in a 37°C shaker for 5–10 minutes. During digestion, gently mix every 2 minutes. After approximately 10 minutes of digestion, control the endpoint using the microscopic examination module of the controlled enzyme digestion unit: aspirate a small amount of the digestion suspension and observe under an inverted microscope. When the tissue block has largely disappeared from the field of view, forming mainly relatively uniform cell clusters (approximately 20–100 μm) with relatively few discrete individual cells (e.g., ...), the digestion is complete. Figure 3 As shown in the figure, immediately add an equal volume of pre-cooled culture medium to terminate the digestion.

[0058] The digestion products were processed using a cell processing unit: the digestion products were filtered through a 100 μm cell filter, the filtrate was collected, and centrifuged at 300g for 5 minutes to obtain a cell pellet. Observation of the pellet revealed minimal red blood cell contamination, so the red blood cell lysis step was omitted. The cell pellet was resuspended in 1 mL PBS, and 10 μL was mixed with trypan blue for viable cell counting. The total cell count was calculated to be 8 × 10⁻⁶. 6The cell viability was 65% (<70%). According to the technical solution of the present invention, when the cell viability is lower than the threshold, it is not advisable to directly seed the primary cells into the chip. Therefore, plate culture and expansion were performed first.

[0059] After amplification in well plates, organoids are collected and incubated at 4°C for 1 hour using organoid recovery solution (e.g., cold medium containing dispersing enzymes) to dissolve the matrix gel. After washing with PBS, the cells are digested with TrypLE for 5-10 minutes, gently pipetting them into single cells or small cell clusters. After terminating digestion, the cells are centrifuged and resuspended in medium for cell counting, preparing for subsequent microarray seeding.

[0060] 4. Precision inoculation suspension preparation based on chip specifications

[0061] The organ-on-a-chip used in this embodiment is the OCplex-8 / 32 chip (part number: 100-032-00) manufactured by Shenzhen Yaosu Technology Co., Ltd., and its specific structure is as follows. Figure 4 As shown, this chip device contains 8 independent gel channels (C=8), numbered A1 to A4 (upward) and B1 to B4 (downward), corresponding to 8 independent perfusion channels; the volume of a single gel channel is 1.4 μL, and the volume of a single perfusion channel is 1.25 μL. According to the product manual, to ensure adequate filling and allow for overflow, the standard inoculation volume for each gel channel is ( The concentration was 1.5 μL.

[0062] This experiment plans to implant cells into two organ-on-a-chip devices (N=2), with a pre-set target cell density (D) of 2×10⁻⁶ cells per chip. 6 The cells / mL was set with a pre-set operational loss factor (k) of 1.30. The calculation was performed using the calculation module of the precise inoculation suspension preparation unit, substituting the values ​​into the formula:

[0063] Total volume of matrix adhesive required: = 2 × 8 × 1.5 μL × 1.30= 31.2 μL;

[0064] Total number of cells required: = 2×10 6 cells / mL×31.2 μL / 1000= 6.24×10 4 cells;

[0065] Based on the calculation results, take the corresponding number of cells from the cell precipitate obtained above, mix them thoroughly with 31.2 μL of matrix gel in an ice bath to prepare a cell-matrix gel suspension.

[0066] 5. Standardized chip seeding and culture

[0067] Subsequent operations were performed using a standardized microarray seeding and culture unit: A precision pipette was used to aspirate the mixed suspension and slowly inject it into the gel channel inlet of the organ-on-a-chip, ensuring the matrix gel filled the entire gel channel without air bubbles. The seeded chip was carefully placed in a 37°C, 5% CO2 incubator and allowed to cure for 60 minutes. After the matrix gel had completely polymerized, 200 μL of preheated human colorectal cancer organoid culture medium was added through the perfusion channel inlet, and the chip was placed on a shaking table to initiate dynamic perfusion culture at a flow rate of 8 μL / min. Thereafter, the culture medium in the perfusion channel was replaced every 2 days.

[0068] On day 5 of culture, observation under an inverted microscope revealed the formation of numerous colorectal cancer organoids with typical three-dimensional structures within the microarray gel channels. These organoids were spherical or cystic, with clear margins and translucent cytoplasm (e.g., ...). Figure 5 This signifies that the organoid has successfully grown and entered a rapid growth phase, meaning the organoid chip culture is complete and will be used for subsequent downstream experiments such as drug sensitivity testing.

[0069] Examples of drug susceptibility testing applications:

[0070] Three days after the completion of culture and drug treatment, the organoids in each gel channel were carefully recovered. Following the instructions of the CellTiter-Glo (CTG) luminescence assay kit, the ATP content of each sample was measured. The luminescence value of the negative control group was normalized to 100%, and the relative viability of the treated cells was calculated. The results showed that the viability of the organoids in the 30 μM 5-FU treatment group was significantly lower than that in the control group (relative viability approximately 42.3% ± 5.1%, p < 0.01), indicating that the colorectal cancer organoids have good drug sensitivity to 5-FU. This example demonstrates that the organoid microarray constructed using the method of this invention can be used for the efficacy evaluation of antitumor drugs.

[0071] Example 2: Verification of Technical Effect

[0072] To verify the technical effectiveness of the standardized construction method for colorectal cancer organ-on-a-chip proposed in this invention, this embodiment compares and verifies the method of the embodiment (hereinafter referred to as the experimental group) with the traditional method (hereinafter referred to as the control group) from three dimensions: cell viability and sample utilization rate, organoid formation efficiency, and batch-to-batch reproducibility.

[0073] The control group used conventional tissue processing methods, specifically: the tissue was minced and directly added to digestion solution for digestion. The digestion time was fixed at 60 minutes based on experience, without strict control of microscopic examination points during digestion, and the endpoint determination relied entirely on the operator's experience; the tissue was divided only by volume, without implementing a differentiated segmentation strategy; after digestion and centrifugation, cell pellets were obtained, resuspended, and directly seeded onto the microarray. The seeding density was estimated based on the operator's experience, without performing precise calculations based on microarray channel parameters and target density; the remaining operation steps were consistent with the example.

[0074] 1. Verification of cell viability and sample utilization

[0075] Five clinical colorectal cancer tissue samples (Case 1-Case 5) were digested according to the experimental and control groups. Immediately after digestion, the cell suspension was tested for viability, and the number of viable cells obtained per unit tissue weight (viable cell yield) was calculated. The results are shown in Tables 1 to 4.

[0076] Table 1 Cell viability after digestion

[0077]

[0078] Table 2. Statistical results of cell viability after digestion

[0079]

[0080] Table 3. Yield of viable cells after digestion

[0081]

[0082] Table 4 Statistical results of live cell yield

[0083]

[0084] As shown in Tables 1 to 4, the experimental group using the method of this invention achieved an average cell viability of 91.0%, significantly higher than the 75.0% of the control group; the average number of viable cells obtained per unit tissue reached 2.50 × 10⁻⁶. 5 The number of cells / g was also significantly higher than that of the control group (1.76 × 10⁻⁶). 5 The cell / g ratio was also significantly lower in the experimental group than in the control group, indicating that the method of this invention can effectively improve cell viability, enhance sample utilization efficiency, and provide more stable results.

[0085] 2. Verification of organoid formation efficiency

[0086] The cells from the above 5 samples were digested and collected and then seeded into chips for culture. On the 9th day of culture, the number of organoids with a diameter ≥25μm in the gel channels of each chip was counted. The results are shown in Tables 5 and 6.

[0087] Table 5 Number of organoids formed

[0088]

[0089] Table 6. Statistical results of organoid formation.

[0090]

[0091] As shown in Tables 5 and 6, the average number of organoids formed in the experimental group was 99.8, which was significantly higher than the 80.2 in the control group. Furthermore, the coefficient of variation in the experimental group (3.84%) was much lower than that in the control group (14.34%), indicating that the method of the present invention can significantly improve the success rate of organoid culture in the chip and reduce individual differences among samples.

[0092] 3. Inter-batch repeatability verification

[0093] Five independent batch experiments (Batch 1-Batch 5) were conducted using the same colorectal cancer organoid sample and the method of this invention. Cell viability (after digestion), organoid formation number (day 9), and inhibition rate after 4 days of treatment with 30 μM 5FU were measured in each batch. The results are shown in Tables 7 and 8.

[0094] Table 7 Results of inter-batch repeatability experiments

[0095]

[0096] Table 8 Statistical results of inter-batch repeatability experiments

[0097]

[0098] As shown in Tables 7 and 8, in the five independent replicate experiments, the coefficients of variation for cell viability, organoid formation number, and drug inhibition rate were 3.93%, 6.92%, and 7.55%, respectively, all at low levels (<10%). The results indicate that the method of this invention exhibits good stability and repeatability in different batches of experiments, providing reliable data consistency assurance for subsequent downstream applications such as drug sensitivity testing.

[0099] 4. Conclusion

[0100] In summary, multi-dimensional verification experiments have confirmed that the standardized construction method for colorectal cancer organoids on-chip proposed in this invention has significant technical advantages: This method, through differentiated segmentation and controllable enzymatic digestion, can significantly reduce cell damage, improve cell viability and sample utilization, and obtain higher quality cell clusters, thereby improving the efficiency and speed of organoid formation on the chip; In addition, the key indicators of this method have low coefficients of variation, good batch-to-batch reproducibility, and the system is robust and reliable, providing a stable culture environment for drug sensitivity experiments and significantly improving the accuracy and consistency of drug sensitivity detection data.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. A standardized method for constructing organ-on-a-chip for colorectal cancer, characterized in that, Includes the following steps: (1) Pre-treatment of tissues: Cleaning, recording morphology, weighing and archiving information of fresh colorectal cancer surgical specimens; (2) Controlled enzymatic digestion: The processed specimen is digested with enzymes to obtain digestion products. The digestion endpoint is controlled by observing the cell cluster state under a microscope. The digestion endpoint is determined by the formation of relatively uniform cell clusters with a size of 20-100 μm. (3) Cell collection and processing: Filter and centrifuge the digestion products to obtain cell precipitate, and perform live cell counting; (4) Precision inoculation suspension preparation: Based on the number of target organ-on-a-chip, the preset target cell density in the chip and the structural parameters of the organ-on-a-chip, calculate and prepare a cell-matrix suspension containing a specific number of cells and a matrix gel volume; (5) Standardized chip seeding and culture: The cell-matrix gel suspension is injected into the gel channel of the organ chip, solidified, culture medium is added, and a dynamic perfusion culture system is started for culture to obtain colorectal cancer organ chips.

2. The standardized method for constructing organ-on-a-chip for colorectal cancer according to claim 1, characterized in that, Following step (1), a differential tissue segmentation step is also included: the pre-treated tissue is segmented into three parts for in vitro culture, cryopreservation backup, and pathological analysis, and the volume relationship of the three parts satisfies: volume of the part for in vitro culture > volume of the part for cryopreservation backup > volume of the part for pathological analysis.

3. A standardized method for constructing organ-on-a-chip for colorectal cancer according to claim 1, characterized in that, The criteria for determining the digestion endpoint also include: the tissue block has basically disappeared in the microscope field of view, and there are few individual discrete cells.

4. A standardized method for constructing organ-on-a-chip for colorectal cancer according to claim 1, characterized in that, Step (3) also includes an inoculation decision based on cell viability: a. If the cell viability is ≥70%, the obtained primary cells will be directly used to prepare cell-matrix suspension and seeded on organ-on-a-chip. b. If the cell viability is <70%, the obtained primary cells will be cultured and expanded in well plates. After they form organoids and are passaged, they will be used to prepare cell-matrix gel suspensions and seeded on organ-on-a-chip.

5. A standardized method for constructing organ-on-a-chip for colorectal cancer according to claim 1, characterized in that, In step (4), the required matrix gel volume and the required total number of cells are calculated using the following model: Required matrix gel volume ； Where N represents the number of organ-on-a-chip targets for implantation, and C represents the total number of gel channels in the individual chips contained in a single device. The standard inoculation volume for each gel channel, k is the coefficient for reserving operational losses, and the value of k ranges from 1.25 to 1.3; Total number of cells required ； Where D is the preset target cell density within the chip.

6. A standardized method for constructing organ-on-a-chip for colorectal cancer according to claim 1, characterized in that, In step (4), the cell-matrix gel suspension is prepared by mixing the counted cell precipitate with the matrix gel taken according to the calculated volume at a low temperature of 4°C to prepare the cell-matrix gel suspension.

7. A standardized method for constructing organ-on-a-chip for colorectal cancer according to claim 1, characterized in that, In step (5), the cell-matrix gel suspension is slowly injected from the gel channel inlet of the organ-on-a-chip; the culture medium is added from the perfusion channel inlet of the organ-on-a-chip; and the dynamic perfusion culture system is a microfluidic peristaltic pump or a rocking incubator that can generate liquid flow.

8. A system for constructing colorectal cancer organ-on-a-chip for implementing the method of any one of claims 1-7, characterized in that, include: The tissue pretreatment unit is used to clean, record the morphology of, weigh, and archive information of fresh colorectal cancer surgical specimens. The controllable enzyme digestion unit is used to perform enzyme digestion on the processed specimen and to control the digestion endpoint by observing the cell cluster state under a microscope. The digestion endpoint is the formation of digestion products mainly consisting of cell clusters with a size of 20-100 μm. The cell processing unit is used to filter and centrifuge the digestion products to obtain cell precipitate, and to perform live cell counting and cell viability assessment. The precision inoculation suspension preparation unit includes a calculation module, which is used to calculate and prepare a cell-matrix suspension containing a specific number of cells and a matrix gel volume based on the number of organ-on-a-chip to be inoculated, the preset target cell density within the chip, and the structural parameters of the organ-on-a-chip. A standardized chip seeding and culture unit is used to inject the cell-matrix gel suspension into the gel channel of the organ-on-a-chip, add culture medium after solidification, and start the dynamic perfusion culture system for culture.

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