Colorectal cancer organ-like model and construction method and application thereof

Abstract

The invention discloses a colorectal cancer organoid model and a construction method and application thereof, and relates to the technical field of tissue engineering and organoid chips, the construction method comprises the following steps: designing and manufacturing a micropore array culture substrate, the micropore array culture substrate comprises a plurality of organoid culture structures distributed in an array, a plurality of organoid culture cavities distributed at intervals are arranged on the organoid culture structure, one end of each organoid culture cavity is in an open funnel shape, and the other end of each organoid culture cavity is a closed cavity; and adding an inoculated cell suspension containing inoculated cells and matrigel and a culture medium into the organoid culture chamber, and carrying out in-situ culture of a whole culture process to obtain the colorectal cancer organoid model. The method has the effects of improving the homogeneity of the organoid in the colorectal cancer organoid model, constructing the flux of the organoid, shortening the culture period and reducing the unit dosage of matrigel.

Description

Technical Field

[0001] The present application relates to the field of tissue engineering technology, and in particular to a colorectal cancer organoid model and a construction method and application thereof. Background Art

[0002] The mortality rate of colorectal cancer (CRC) ranks third in the world and has become one of the major public health issues that endanger national health. The conventional treatment of advanced CRC is mainly based on drugs. Due to the different sensitivity of different patients to drugs, the treatment of advanced CRC is prone to drug resistance and recurrence and metastasis after treatment. Therefore, by culturing the patient's tumor tissue in three dimensions in vitro to form micro-tumor tissue, patient-derived tumor organoids (PDOs) are obtained, which highly restore the tissue structure and genetic lineage of human tumors and maintain tumor heterogeneity, thereby facilitating drug sensitivity testing.

[0003] At present, the culture method for constructing colorectal cancer organoids mainly relies on the classical gel droplet method. However, the classical gel droplet culture method is difficult to regulate the tumor microenvironment at the level of a single organoid, resulting in large differences between batches of constructed tumor organoids, low repeatability, and poor uniformity within batches. In addition, the classical gel droplet culture method cannot achieve one-time high-throughput construction of organoids, which in turn leads to a decrease in the accuracy of the interpretation results during drug sensitivity testing. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned related technologies, the present application provides a colorectal cancer organoid model, a construction method thereof, and an application thereof. By constructing an organoid culture structure containing multiple organoid culture chambers of uniform size, and arranging the organoid culture structure array as a microwell array culture substrate, and culturing and inoculating cells in each organoid culture chamber, on the one hand, the uniformity of tumor organoids within the same batch is improved, and on the other hand, the throughput of organoid construction is improved. In addition, the culture cycle is shortened and the unit dosage of matrix gel is reduced.

[0005] In the first aspect, the method for constructing a colorectal cancer organoid model provided in the present application adopts the following technical solution: The method comprises the following steps: designing and manufacturing a microporous array culture substrate, wherein the microporous array culture substrate comprises a plurality of organoid culture structures distributed in an array, wherein the organoid culture structures are provided with a plurality of organoid culture chambers distributed at intervals, wherein one end of the organoid culture chamber is in an open funnel shape and the other end is a closed cavity; The inoculated cell suspension containing inoculated cells and matrix gel and the culture medium are added into the organoid culture chamber, and the in situ culture of the whole culture process is carried out to obtain the colorectal cancer organoid model.

[0006] Preferably, the open funnel end of the organoid culture chamber has an opening diameter of 900-1100 μm, and the other end is hemispherical with a diameter of 500-700 μm.

[0007] Preferably, the open funnel end of the organoid culture chamber has an opening diameter of 1000 μm, and the other end is hemispherical with a diameter of 600 μm.

[0008] Preferably, the depth of the organoid culture chamber is 400-600 μm.

[0009] Preferably, the depth of the organoid culture chamber is 500 μm.

[0010] By adopting the above scheme, the open funnel-shaped design at one end of the organoid culture chamber of the present application is conducive to improving the efficiency of cell collection, and the hemispherical design at the other end helps the inoculated cells to aggregate into balls, thereby improving the success rate of organoid unit preparation.

[0011] Preferably, the number of cells seeded in the organoid culture chamber is 80-300.

[0012] Preferably, the number of cells seeded in the organoid culture chamber is 100.

[0013] By adopting the above scheme, each organoid culture chamber in the present application successfully forms a single uniform organoid at a low inoculation amount, which is conducive to adapting to low sample volume sampling methods such as endoscopic samples and puncture samples.

[0014] Preferably, the volume concentration of the matrix gel in the inoculated cell suspension is 3-8%.

[0015] Preferably, the volume concentration of the matrix gel in the inoculated cell suspension is 5%.

[0016] By adopting the above scheme, the concentration of matrix gel used in constructing the colorectal cancer organoid model in this application is only 5%, which reduces the amount of matrix gel used and reduces the construction cost.

[0017] Preferably, the distance between two adjacent organoid culture chambers is 160-200 μm.

[0018] Preferably, the distance between two adjacent organoid culture chambers is 166.67 μm.

[0019] Preferably, the number of the organoid culture chambers on the organoid culture structure is 19.

[0020] By adopting the above scheme, by controlling the number of organoid culture chambers on the organoid culture structure, controlling the size of the organoid culture chambers, and controlling the distance between adjacent organoid culture chambers, on the one hand, it is beneficial to the uniformity of the distribution of nutrients in the inoculated cell nuclei, and on the other hand, it is beneficial to the aggregation of inoculated cells in the organoid culture chambers and the exchange of nutrients and waste absorption during the inoculation growth process, thereby successfully forming organoids.

[0021] Preferably, the inoculated cells include one or more of human-derived tumor stem cells and animal-derived tumor stem cells.

[0022] Preferably, the inoculated cells include primary source colorectal cancer stem cells and intestinal cancer epithelial cells.

[0023] Preferably, the microwell array culture substrate is obtained by fixing a plurality of the organoid culture structures on a cell culture plate.

[0024] Preferably, the cell culture plate is a 96-well plate.

[0025] By adopting the above scheme, the present application is compatible with cell culture plates, and can perform in situ culture, in situ observation and in situ drug testing of the entire culture process of organoids, thereby improving the universality of the colorectal cancer organoid model, and is compatible with commercially commonly used cell culture plates, thereby improving the convenience of use.

[0026] Preferably, the in situ culture of the full culture process comprises centrifuging the microwell array culture medium to which the inoculated cell suspension is added at 80-200 g for 2-4 minutes.

[0027] Preferably, the in situ culture of the full culture process comprises centrifuging the microwell array culture medium to which the inoculated cell suspension is added at 100 g for 3 minutes.

[0028] Preferably, the steps for preparing the organoid culture structure are as follows: The photosensitive resin is used as a raw material and printed using a surface projection micro-stereoscopic light-curing 3D printing technology to obtain a mold with a micro-column array. A polymer monomer is poured on the mold and cured, and the mold is demolded to obtain the organoid culture structure.

[0029] Preferably, the printing parameters are as follows: The model slice thickness is 5-20μm, the molding temperature is room temperature, the separation speed is 0.5mm / s, the separation distance is 2-3mm / s, the contact speed is 1mm / s, the starting layer thickness range is 20μm, and the starting layer light intensity is 120mW / cm 2 The exposure time of the initial layer is 3-4s, the thickness of the remaining layer is in the range of 5-10μm, and the light intensity of the remaining layer is 120-150mW / cm2 , the exposure time of each remaining layer is 2-3s.

[0030] Preferably, the micro-column array is composed of a plurality of columns with hemispherical tops and truncated cone-like bottoms.

[0031] Preferably, a low adhesion layer is formed on the surface of the mold before pouring the polymer monomer on the mold.

[0032] Preferably, the demoulding layer is formed by the following steps: The mold was first cleaned with oxygen plasma, the surface was acted on for 10 seconds, and then placed under vacuum conditions containing trichloro(1H,1H,2H,2H-perfluorooctyl)silane droplets, the pressure was maintained for 30 minutes, and finally dried in a 90° C. oven for one hour.

[0033] In the second aspect, the colorectal cancer organoid model provided in the present application adopts the following technical solution: A colorectal cancer organoid model is prepared using a colorectal cancer organoid model construction method.

[0034] In a third aspect, the present application provides an application of a colorectal cancer organoid model in drug sensitivity testing.

[0035] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application constructs an organoid culture structure containing multiple organoid culture chambers of uniform size, and arranges the organoid culture structure array as a microwell array culture substrate, and cultures and inoculates cells in each organoid culture chamber, thereby improving the uniformity of tumor organoids within the same batch on the one hand, and improving the throughput of organoid construction on the other hand; 2. The independent distribution of organoid culture chambers in the colorectal cancer organoid model of the present application is conducive to improving the accuracy of controlling the culture environment of each organoid culture chamber, while avoiding the fusion of various organoids as much as possible. The size and morphological variation coefficients of the colorectal cancer organoid model obtained in the present application are lower than those of the colorectal cancer organoid constructed by the classic gel drop method, which improves the uniformity of various organoids in the colorectal cancer organoid model; 3. The expression of cancer stem cell markers CD44 (median increased by 181.66%, P<0.0025) and Lgr5 (median increased by 252.64%, P<0.0001) in the colorectal cancer organoid model of the present application was upregulated, indicating that the colorectal cancer organoid model of the present application provides a suitable physical and chemical microenvironment for the growth of inoculated cells, which is beneficial to improving the activity of inoculated cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1It is a schematic diagram of the structure of the microwell array culture medium of the present application.

[0037] Figure 2 It is a schematic diagram of the preparation process of the microwell array culture medium of the present application.

[0038] Figure 3 This is a time-series bright field image of the colorectal cancer organoid model constructed in Example 1 of the present application.

[0039] Figure 4 This is the homogeneity characterization result of the colorectal cancer organoid model constructed in Example 1 of the present application.

[0040] Figure 5 This is the live or dead staining result of the colorectal cancer organoids in the colorectal cancer organoid model constructed in Example 1 of the present application.

[0041] Figure 6 This is the immunofluorescence identification result of the colorectal cancer organoid model constructed in Example 1 of the present application.

[0042] Figure 7 This is the immunofluorescence semi-quantitative statistical result of the colorectal cancer organoid model constructed in Example 1 of the present application.

[0043] Figure numerals: 1. microwell array culture substrate; 2. organoid culture structure; 3. organoid culture chamber. DETAILED DESCRIPTION

[0044] The present application is further described in detail below in conjunction with examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the recorded content may also be applied to the present invention.

[0045] The raw materials used in the examples are all commercially available.

[0046] Example 1 Example 1 of the present application provides a method for constructing a colorectal cancer organoid model, referring to Figure 1 and Figure 2 , the specific steps are as follows: Preparation of organoid culture structure 2: High temperature resistant resin HTL (Magic Cube Precision, 20230216-1) was used as the raw material, and the surface projection micro-stereoscopic light curing 3D printing technology was used for printing. The printing parameters were as follows: the model slice thickness was 5 μm, the molding temperature was room temperature, the separation speed was 0.5 mm / s, the separation distance was 2 mm / s, the contact speed was 1 mm / s, the starting layer thickness range was 20 μm, and the starting layer light intensity was 120 mW / cm 2 The exposure time of the initial layer is 3s, the thickness of the remaining layer is 5μm, and the light intensity of the remaining layer is 120mW / cm 2 The exposure time of the remaining single layer is 3s; a mold with a micro-pillar array is obtained, referring to Figure 2 The micro-column array is composed of a plurality of columns with hemispherical tops and truncated cone-shaped bottoms.

[0047] The mold was immersed in 95% alcohol for dehydration and cleaning, and then placed in a UV curing device for curing for 2-3 minutes. Next, it was placed in a plasma cleaner and cleaned with oxygen plasma for 10 seconds at a power of 100 W. Finally, it was transferred to a vacuum condition containing trichloro(1H,1H,2H,2H-perfluorooctyl)silane droplets. After maintaining the pressure for 30 minutes, it was dried in a 90°C oven for one hour to form a low adhesion layer on the mold surface.

[0048] A polydimethylsiloxane prepolymer with a ratio of 10:1 between liquid A and liquid B was de-bubbled with a vacuum pump and then cast onto a mold with a low adhesion layer. The bubbles attached to the mold surface were removed with a vacuum pump, and the mold was placed in an 80°C oven for curing and then demolded to obtain polydimethylsiloxane (PDMS). A 6mm punch was used to punch holes on the PDMS to remove the area corresponding to the micro-pillar array, and the organoid culture structure 2 was obtained. The micro-pillar array promoted the formation of the corresponding organoid culture chamber 3 on the organoid culture structure 2. Figure 1 One end of the organoid culture chamber 3 is in the shape of an open funnel with an opening diameter of 1000 μm, and the other end is in the shape of a hemisphere with a diameter of 600 μm; the depth of the organoid culture chamber 3 is 500 μm; the distance between two adjacent organoid culture chambers 3 is 166.67 μm.

[0049] Preparation of the microwell array culture substrate 1: 96 organoid culture structures 2 are fixed on a 96-well plate in an array distribution using PDMS prepolymer to obtain a microwell array culture substrate 1.

[0050] Construction of colorectal cancer organoid model: sterilize the microwell array culture substrate 1 with ultraviolet light for no less than 4 hours, wash the microwell array culture substrate 1 with sterile water and dry it, add 4% F127 solution (Sigma, 9003-11-6) to the microwell array culture substrate 1, centrifuge to remove bubbles, soak it overnight under ultraviolet light, then aspirate the F127 solution, wash the microwell array culture substrate 1 with sterile 1XPBS (phosphate buffered saline), and dry it to make the surface of the organoid culture chamber 3 hydrophobic, avoid the inoculated cells from adhering to the surface of the organoid culture chamber 3 as much as possible, and facilitate the inoculated cells to self-organize into three-dimensional tissues, finally add colorectal cancer organoid culture medium (bioGenous, B213152) to moisten the microwell array culture substrate 1 and precool it at 4°C for later use; 50 μL of the inoculated cell suspension (Matrigel mass concentration 5%) containing matrix gel (Matrigel; Corning, 356231), colorectal cancer organoid culture medium and inoculated cells (Zhongnan Hospital of Wuhan University, Ethics Approval No. 2022018) was added to the organoid culture chamber 3. The initial number of inoculated cells in each organoid culture chamber 3 was 100. The microwell array culture substrate 1 was centrifuged at 4°C and 100g for 3 minutes and then placed in an incubator to support the matrix gel in a non-solidified state and settle to the bottom of the organoid culture chamber 3 without negatively affecting the activity of colorectal cancer stem cells. After the matrix gel at the bottom of the organoid culture chamber 3 solidified, 150 μL of colorectal cancer organoid culture medium was added to the organoid culture chamber 3 for static culture. The medium was changed every 2-3 days to maintain the culture to obtain a colorectal cancer organoid model.

[0051] In this embodiment, the inoculated cells include primary colorectal cancer stem cells and intestinal cancer epithelial cells.

[0052] Comparative Example 1 Comparative Example 1 provides a method for constructing a colorectal cancer organoid model. The difference between Comparative Example 1 and Example 1 is that the initial number of cells seeded in each organoid culture chamber is 50.

[0053] Comparative Example 2 Comparative Example 2 provides a method for constructing a colorectal cancer organoid model. The difference between Comparative Example 2 and Example 1 is that the initial number of cells seeded in each organoid culture chamber is 500.

[0054] Comparative Example 3 Comparative Example 3 provides a method for constructing a colorectal cancer organoid model. The difference between Comparative Example 3 and Example 1 is that the volume concentration of matrix gel in the inoculated cell suspension containing matrix gel, colorectal cancer organoid culture medium and inoculated cells is 2%.

[0055] Comparative Example 4 Comparative Example 4 provides a method for constructing a colorectal cancer organoid model. The difference between Comparative Example 4 and Example 1 is that the volume concentration of matrix gel in the inoculated cell suspension containing matrix gel, colorectal cancer organoid culture medium and inoculated cells is 10%.

[0056] Experimental testing The time-series bright field images of the colorectal cancer organoid model (labeled as CHIP) constructed in Example 1 are compared with the time-series bright field images of the colorectal cancer organoid model (labeled as DOME) constructed by the classic gel drop method at 0-6 days (labeled as D0, D3 and D6, respectively). Figure 3 The homogeneity characterization results of the colorectal cancer organoid model constructed in Example 1 are compared with the homogeneity characterization results of the colorectal cancer organoid model constructed by the classic gel drop method. Figure 4 The live-death staining of the colorectal cancer organoid model constructed in Example 1 is compared with the live-death staining of the colorectal cancer organoid model constructed by the classic gel drop method. Figure 5 The colorectal cancer organoid model constructed in Example 1 was subjected to immunofluorescence identification, and the comparison of the colorectal cancer organoid model constructed by the classic gel drop method was shown in FIG. Figure 6 The immunofluorescence semi-quantitative statistical results of the colorectal cancer organoid model constructed in Example 1 are compared with those of the colorectal cancer organoid model constructed by the classic gel drop method. Figure 7 The culture effects of the colorectal cancer organoid models of Example 1 and Comparative Examples 1-4 were statistically analyzed to determine whether the organoids were formed. The area coefficient of variation was calculated for the colorectal cancer organoid model with normal organoid growth. The calculation method of the area coefficient of variation was: coefficient of variation = (standard deviation / mean value) × 100%. The results are shown in Table 1 below. Table 1:

[0057] Results Analysis The following combination Figure 3-7 And the experimental results provided in Table 1 are used to explain this application in detail.

[0058] Reference Figure 3, the microwell array culture substrate has significant advantages in culturing colorectal cancer organoids. In the colorectal cancer organoid model constructed in Example 1, single cells aggregate and self-assemble to form a three-dimensional structure with budding vacuoles at D3, achieving the effect of high-throughput construction of single organoids in a short period of time (3 days), improving the uniformity and repeatability of organoids, and allowing real-time observation of morphogenesis. The classic gel drop method requires 6 days to construct organoids, and over time, organoids fuse with each other. Cultivating colorectal cancer organoids in a microwell array culture substrate can achieve in situ culture and in situ observation of colorectal cancer organoids throughout the culture cycle. At the same time, the microwell array culture substrate can also track the generation and growth of colorectal cancer organoids from single cells aggregated through the organoid culture chamber.

[0059] Reference Figure 4 The colorectal cancer organoids cultured in the microwell array culture medium were significantly superior to those cultured in the classic gel drop method in terms of size (P=0.0038, CV<10%) and uniformity (P=0.0004). Figure 5 As shown, the live / death staining results showed that there was no significant difference in the live / death between colorectal cancer organoids cultured on the microwell array culture substrate and those cultured on the classic gel drop method (P=0.7416), confirming that the organoids constructed on the microwell array culture substrate were in good activity.

[0060] Reference Figure 6 Immunofluorescence results confirmed that colorectal cancer organoids expressed multiple cell type markers similar to those of colorectal cancer organoids cultured by the classic gel drop method (CD44 and LGR5, colorectal cancer stem cell markers; MUC2, an endocrine cell marker; CK20, a colorectal cancer epithelial marker; CDX2, an intestinal-specific nuclear transcription factor marker), indicating that colorectal cancer organoids cultured on the microwell array culture substrate retained colorectal cancer-related pathological characteristics. Figure 7 As shown, the semi-quantitative statistical results of immunofluorescence confirmed that the expression of cell markers CD44 (median increased by 181.66%, P<0.0025) and Lgr5 (median increased by 252.64%, P<0.0001) in the colorectal cancer organoids cultured in the microwell array culture matrix was upregulated, indicating that the growth of colorectal cancer stem cells in the organoids cultured in the microwell array culture matrix was promoted, which may be related to the low oxygen environment at the bottom of the microwell array culture matrix culture that is closer to the growth environment of colorectal cancer stem cells in vivo, indicating that Example 1 provides a colorectal cancer organoid model that can provide a physical and chemical microenvironment more suitable for the growth of colorectal cancer organoids. That is, the colorectal cancer organoid model of the present application is conducive to retaining the pathological characteristics related to colorectal cancer, and is applied to the field of drug sensitivity testing, which is conducive to improving the accuracy of drug sensitivity testing, and high-throughput and efficient cultivation of multiple organoid units at the same time is conducive to shortening the cycle of drug sensitivity testing.

[0061] Referring to Table 1, Example 1 and Comparative Examples 1-2 show the effect of the initial number of inoculated cells on whether organoids are formed, wherein the organoids in the colorectal cancer organoid model constructed in Example 1 grow normally, while the organoids in the colorectal cancer organoid model constructed in Comparative Examples 1-2 cannot form. The analysis is that if the initial number of inoculated cells in each organoid culture chamber is too small, it is difficult for the cells to self-assemble to form organoids. Excessive initial inoculation of cells in each organoid culture chamber will result in limited cell growth space and lack of nutrients, affecting the normal formation of organoids. Example 1 and Comparative Examples 3-4 show the effect of the volume concentration of matrix gel in the inoculated cell suspension containing matrix gel, colorectal cancer organoid culture medium and inoculated cells on whether organoids are formed and the area coefficient of variation. The organoids in the colorectal cancer organoid model constructed in Example 1 and Comparative Example 4 grow normally, while the organoids in the colorectal cancer organoid model constructed in Comparative Example 3 cannot form. The analysis shows that the concentration of matrix gel is too low to support the extracellular matrix support required for organoid formation, resulting in organoids being difficult to grow normally. Although the organoids in the colorectal cancer organoid model constructed in Comparative Example 4 can grow normally, the area variation coefficient of the organoids reaches 28.77%, which is much higher than the area variation coefficient of 6.20% of the organoids in the colorectal cancer organoid model constructed in Example 1. In summary, it is shown that the colorectal cancer organoid model constructed with the initial inoculation cell inoculation number and matrix gel volume concentration adopted in Example 1 is conducive to the formation of organoids and reduces the area variation coefficient of organoids.

[0062] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for constructing a colorectal cancer organoid model, characterized in that: The method comprises the following steps: designing and manufacturing a microporous array culture substrate, wherein the microporous array culture substrate comprises a plurality of organoid culture structures distributed in an array, wherein the organoid culture structures are provided with a plurality of organoid culture chambers distributed at intervals, wherein one end of the organoid culture chamber is in the shape of an open funnel and the other end is a closed cavity; The inoculated cell suspension containing inoculated cells and matrix gel and the culture medium are added into the organoid culture chamber, and the in situ culture of the whole culture process is carried out to obtain the colorectal cancer organoid model.

2. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The open funnel end of the organoid culture chamber has an opening diameter of 900-1100 μm, and the other end is hemispherical with a diameter of 500-700 μm.

3. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The depth of the organoid culture chamber is 400-600 μm.

4. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The number of cells seeded in the organoid culture chamber is 80-300.

5. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The volume concentration of the matrix gel in the inoculated cell suspension is 3-8%.

6. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The distance between two adjacent organoid culture chambers is 160-200 μm.

7. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The inoculated cells include one or more of human-derived tumor stem cells and animal-derived tumor stem cells.

8. The method for constructing a colorectal cancer organoid model according to claim 1, characterized in that: The in situ culture of the full culture process includes centrifuging the microwell array culture medium added with the inoculated cell suspension at 80-200g for 2-4 minutes.

9. A colorectal cancer organoid model, characterized in that: The method is prepared by the construction method described in any one of claims 1 to 8.

10. Use of a colorectal cancer organoid model as described in claim 9 in drug sensitivity testing.

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