Composite oral squamous cell carcinoma organoid model as well as preparation method and application thereof
By co-culturing cancer-related fibroblasts, endothelial cells and oral squamous cell epithelial cells, combined with matrix gel, a complex oral squamous cell carcinoma organoid model was solved, and the problem of lack of vascular system support in traditional organoid culture was achieved better tumor microenvironment simulation and personalized drug screening.
Patent Information
- Application Number
- CN202311306036.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-08
- Publication Date
- 2024-12-20
AI Technical Summary
Traditional in vitro cultured tumor organoids lack the vascular system support in the tumor microenvironment, resulting in cell apoptosis and tissue necrosis, making it difficult to fully simulate the morphology and physiological functions of parent tumor tissue.
Coculture method of stable GFP-expressing cancer-related fibroblasts and stable m-Cherry-expressing endothelial cells and oral squamous cell epithelial cells was used to construct a complex oral squamous cell carcinoma organoid model to simulate the tumor microenvironment Cell interaction.
A contact co-culture system for tumor cells, cancer-related fibroblasts and endothelial cells was successfully constructed, real-time observation and integration of the tumor microenvironment was realized, and a more superior tumor research model and personalized drug screening platform were provided.
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Figure CN119162084A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical fields of stem cells, organoids, organoid chips and tissue engineering, and in particular to a composite oral squamous cell carcinoma organoid model and a preparation method and application thereof. Background Art
[0002] Tumor organoids are three-dimensional structures formed by self-assembly of stem cells in vitro. They are a new type of tumor research model that can simulate the personalized characteristics of patients. A large number of studies have extracted human colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, oral cancer and other tissues to establish organoids, and confirmed that tumor-derived organoids have strong similarities with primary tumors in both genotype and phenotype. The progress made in organoid research in recent years has greatly promoted the progress of advanced medical treatment.
[0003] The tumor microenvironment (TME) plays an extremely important role in tumor evolution, and cancer associated fibroblasts (CAFs) are the most important type of stromal cells in the tumor microenvironment, playing an important role in tumor extracellular matrix remodeling. However, traditional in vitro cultured organoids lack such cell support.
[0004] Blood vessels play a vital role in tumor growth, but organoids cultured in vitro lack the support of the vascular system. As the volume of organoids increases, hypoxia and accumulation of metabolic waste lead to apoptosis of cells inside the organoids, ultimately causing necrosis within the organoid tissue.
[0005] In summary, the existing traditional organoid culture technology lacks the integration and support of tumor stromal cells, resulting in difficulty in completely simulating the parental tumor tissue in terms of morphology, size and physiological function. This problem has also become a bottleneck in the development of organoid culture technology.
[0006] Therefore, it is necessary to develop a composite oral squamous cell carcinoma organoid model that can better simulate the parental tumor tissue. Summary of the invention
[0007] The purpose of the present invention is to provide a composite oral squamous cell carcinoma organoid model and its preparation method and application, which can better simulate the parental tumor tissue and can observe and integrate a variety of stromal cells in real time.
[0008] In order to achieve the above object, the present invention adopts the following technical solution:
[0009] In a first aspect of the present invention, a method for preparing a composite oral squamous cell carcinoma organoid model is provided, the method comprising:
[0010] Cancer-associated fibroblasts stably expressing GFP, endothelial cells stably expressing m-Cherry, and oral squamous cell carcinoma epithelial cells were co-cultured to obtain co-culture cell clusters;
[0011] The co-cultured cell clusters were centrifuged and the supernatant was discarded to obtain cell spheres; the cell spheres were then mixed with matrix gel and inoculated into a well plate and cultured in an incubator; after solidification, culture medium was added for culture to obtain a composite oral squamous cell carcinoma organoid model.
[0012] Furthermore, the method for preparing cancer-associated fibroblasts that stably express GFP includes: inoculating cancer-associated fibroblasts in a six-well plate, and when the cells grow to a density of 30%, infecting them with a lentiviral vector expressing GFP green fluorescent protein, and when the cells converge to a density of 80%, adding puromycin to screen successfully infected cells.
[0013] Furthermore, the method for preparing endothelial cells stably expressing m-Cherry includes: inoculating umbilical vein endothelial cells in a six-well plate, and when the cells grow to a density of 30%, infecting them with a lentiviral vector expressing m-Cherry red fluorescent protein, and when the cells converge to a density of 80%, adding puromycin to screen successfully infected cells.
[0014] Furthermore, the addition ratio of the cancer-associated fibroblasts stably expressing GFP, the endothelial cells stably expressing m-Cherry, and the oral squamous cell carcinoma epithelial cells is (4-6):1:(4-6) according to the number of cells.
[0015] In a second aspect of the present invention, a composite oral squamous cell carcinoma organoid model obtained by the method is provided.
[0016] In the third aspect of the present invention, the application of the composite oral squamous cell carcinoma organoid model in disease model, drug development and toxicity analysis is provided.
[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
[0018] 1. The present invention provides a composite oral squamous cell carcinoma organoid model and its preparation method and application, which successfully optimizes the composite oral squamous cell carcinoma organoid culture technology integrating vascular endothelial cells and cancer-associated fibroblasts, and stably constructs a contact co-culture system of tumor cells, cancer-associated fibroblasts and umbilical vein endothelial cells (human umbilical vein endothelial cell, HUVEC). The composite oral squamous cell carcinoma organoid obtained by the present invention can observe and integrate a variety of stromal cells in real time, providing new ideas for studying the interaction mechanism between tumor and matrix in the tumor microenvironment and developing treatment methods for the tumor microenvironment. At the same time, it provides a more superior organoid culture model for personalized drug screening and treatment of tumor patients.
[0019] 2. The preparation method of a composite oral squamous cell carcinoma organoid model provided by the present invention is easy to construct and promote. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0021] Figure 1 This is a comparative identification diagram of HE staining of primary tumor organoids and parental tumor tissues in the example (scale bar = 50 μm);
[0022] Figure 2 This is a cell immunofluorescence identification image of surface markers of primary tumor epithelial cells in the example (scale bar = 50 μm);
[0023] Figure 3 It is a fluorescence identification image of the surface markers of cancer-associated fibroblasts in the example (scale bar = 50 μm);
[0024] Figure 4 Figure 1 shows cancer-associated fibroblasts stably expressing GFP green fluorescent protein for the purpose of implementing the method (scale bar = 50 μm);
[0025] Figure 5 Figure 1 shows HUVEC stably expressing m-Cherry red fluorescent protein for the purpose of implementation (scale bar = 50 μm);
[0026] Figure 6 It is a pattern diagram of the present invention in the embodiment;
[0027] Figure 7Bright field and fluorescence images of CAF-HUVEC-OSCC co-culture cells constructed in the example (scale bar = 50 μm);
[0028] Figure 8 Bright field and fluorescence images of composite oral squamous cell carcinoma organoids constructed in the example (scale bar = 50 μm);
[0029] Fig. 9 This is a HE staining image of a composite oral squamous cell carcinoma organoid section in the example (scale bar = 50 μm). DETAILED DESCRIPTION
[0030] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.
[0031] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.
[0032] It should be understood that the terms "length", "width", "up", "down", "front", "back", "first", "second", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.
[0033] In addition, in the description of the present application, “multiple” and “several” mean two or more, unless otherwise clearly and specifically defined.
[0034] The overall idea of the technical solution of this application is as follows:
[0035] According to a typical embodiment of the present invention, a method for preparing a composite oral squamous cell carcinoma organoid model is provided, the method comprising:
[0036] Step S1, co-culturing cancer-associated fibroblasts stably expressing GFP, endothelial cells stably expressing m-Cherry, and oral squamous cell carcinoma epithelial cells to obtain co-cultured cell clusters;
[0037] In the step S1,
[0038] The method for preparing cancer-associated fibroblasts stably expressing GFP comprises:
[0039] Cancer-associated fibroblasts were seeded in six-well plates. When the cells grew to a density of 30%, they were infected with a lentiviral vector expressing GFP green fluorescent protein (Gene Gene, China, Catalog No. GV077). When the cells converged to a density of 80%, puromycin (White Shark, China, Catalog No. BL528A) was added to screen for successfully infected cells.
[0040] The method for preparing endothelial cells stably expressing m-Cherry comprises:
[0041] Umbilical vein endothelial cells were inoculated in six-well plates. When the cells grew to a density of 30%, they were infected with a lentiviral vector expressing m-Cherry red fluorescent protein (Gene Gene, China, Catalog No. GV549). When the cells converged to a density of 80%, puromycin (White Shark, China, Catalog No. BL528A) was added to screen for successfully infected cells.
[0042] In the co-culture, the added ratio of the cancer-associated fibroblasts stably expressing GFP, the endothelial cells stably expressing m-Cherry, and the oral squamous cell carcinoma epithelial cells is (4-6):1:(4-6) according to the number of cells;
[0043] The purpose of co-culturing according to the above ratio is to better simulate the ratio of various cells in the patient's oral squamous cell carcinoma tissue. If the ratio of endothelial cells is too high or too low, it will affect the construction of the model.
[0044] The culture medium used was an organoid culture medium (DMEM / F12 culture medium containing 1×B27, 1×N2, 50 ng / ml hEGF, 500 nM A83-01 (Sciencecell, China, catalog number SH30023.01)) and an ECM culture medium (Sciencecell, USA, catalog number 1001) in a volume ratio of (4-6):1.
[0045] Step S2, centrifuging the co-cultured cell cluster and discarding the supernatant to obtain a cell sphere;
[0046] The cell spheres were then mixed with matrix gel and inoculated into a well plate and cultured in an incubator. After solidification, culture medium was added for differentiation culture to obtain a composite oral squamous cell carcinoma organoid model.
[0047] In the step S2,
[0048] The volume ratio of the cell sphere to the matrix gel is 1:100±10.
[0049] The culture medium used for the differentiation culture is an organoid culture medium and an ECM culture medium in a volume ratio of (4-6):1.
[0050] According to another typical embodiment of the present invention, a composite oral squamous cell carcinoma organoid model obtained by the method is provided.
[0051] The present invention stably constructs a contact co-culture system of tumor cells, cancer-associated fibroblasts and umbilical vein endothelial cells for the first time. This model can better simulate the characteristics of parental tumor tissues and provide a better in vitro model for tumor research.
[0052] The following will describe in detail a composite oral squamous cell carcinoma organoid model of the present application and its preparation method and application in combination with the embodiments and drawings.
[0053] Example 1: Method for constructing a composite human oral squamous cell carcinoma organoid model
[0054] 1. Extraction of primary human oral squamous cell carcinoma epithelial cells:
[0055] Take fresh oral squamous cell carcinoma tissue and cut it into pieces less than 1 mm 3 , add DMEM / F12 medium containing digestive enzymes (trypsin, 200U / mL type IV collagenase, 200μg / mL hyaluronidase), and incubate at 37°C on a shaker for 1 hour. After lysing the red blood cells and removing necrotic debris, filter with a 400-mesh cell sieve to obtain a single-cell suspension. Mix the single-cell suspension with the matrix gel evenly, and then inoculate the matrix gel in a preheated 24-well plate. After organoid culture, select epithelial cell clones for expansion;
[0056] 2. Identification of primary human oral squamous cell carcinoma epithelial cells and organoids:
[0057] (1) After the organoids mature, transfer the matrix gel-organoid mixture to OCT gel for coating as completely as possible and prepare frozen sections;
[0058] (2) HE staining identification: Add PBS to the frozen sections, rinse three times, and shake off the liquid. Add an appropriate amount of hematoxylin stain to the frozen sections, shake for 10 seconds, and rinse with running water. Add an appropriate amount of eosin stain to the frozen sections, shake for 1 minute, and rinse with running water. Observe the staining results under a microscope and seal the sections with neutral gum, such as Figure 1 As shown, take pictures under a microscope and record the results.
[0059] (3) Identification of organoid surface markers: Rinse the frozen sections three times with PBS and fix with 4% paraformaldehyde. Add 1:200 diluted cytokeratin, CD44, and β-catenin primary antibody dilutions and incubate overnight at 4 degrees. The next day, add fluorescent secondary antibodies of the same species as the primary antibodies and incubate at room temperature for one hour. After washing three times with PBS, add DAPI staining solution and anti-fluorescence quenching sealing solution, and seal with a coverslip. Figure 2 As shown, the staining results were observed under a fluorescence microscope and images were collected and recorded;
[0060] 3. Extraction of human primary oral squamous cell carcinoma-associated fibroblasts:
[0061] Place the cell tissue blocks filtered during the extraction of the above primary human oral squamous cell carcinoma epithelial cells on the bottom of a T25 culture dish to allow them to adhere to the wall. Add DMEM / F12 culture medium containing 10% serum, but do not touch the tissue. Flip the T25 culture dish over and leave it overnight. After the tumor tissue blocks adhere to the wall, flip it back to the normal culture state. After 7-10 days, cancer-associated fibroblasts can be seen crawling out of the tumor tissue blocks;
[0062] 4. Identification of human primary oral squamous cell carcinoma-associated fibroblasts:
[0063] The cultured cancer-associated fibroblasts were subjected to cell immunofluorescence identification to detect the expression of cancer-associated fibroblast surface markers α-SMA and FAP. The cancer-associated fibroblasts were digested and seeded into a 24-well plate one day in advance. The next day, the cells were washed 2-3 times with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% TritonX-100 at room temperature for 20 minutes. Blocked with 10% serum homologous to the secondary antibody, added the primary antibody, and incubated overnight at 4 degrees. The next day, the secondary antibody homologous to the primary antibody was added, incubated at 37 degrees for 1 hour, the secondary antibody was washed off, the DAPI staining solution was added, and the plates were sealed with an anti-fluorescence quenching sealing agent containing DAPI. Figure 3 As shown, the images were observed and recorded under a fluorescence microscope;
[0064] 5. Cell transfection:
[0065] Using the lentiviral transfection method, cancer-associated fibroblasts expressing GFP green fluorescent protein and HUVEC expressing m-Cherry red fluorescent protein were established. Stably transfected cells were obtained by puromycin selection, as shown in the attached Figure 4-5 As shown;
[0066] 6. Preparation of human oral squamous cell carcinoma CAFs-HUVECs-OSCC co-culture cell clusters:
[0067] like Figure 6-7As shown, cancer-associated fibroblasts stably expressing GFP, endothelial cells stably expressing m-Cherry, and oral squamous cell carcinoma epithelial cells were cultured in ultra-low attachment plates at a ratio of 5:1:5. Organoid culture medium and ECM culture medium were added at the same ratio;
[0068] 7. Construction of composite oral squamous cell carcinoma organoids:
[0069] Centrifuge the cell clusters created in step 6 and discard the supernatant. Mix 3 μl of cell spheres with 300 μl of Matrigel, inoculate in a preheated 24-well plate, and immediately place in a 37°C incubator to solidify. Add organoid culture medium and ECM culture medium in a ratio of 5:1. Figure 8 As shown, the morphology and size of organoids were recorded every day, the culture medium was replaced every 3 days, and the organoids were passaged when they reached their growth limit;
[0070] 8. Identification of compound oral squamous cell carcinoma organoids:
[0071] After the organoids mature, transfer the matrix gel-organoid mixture to OCT gel for coating as completely as possible and prepare frozen sections.
[0072] Rinse the frozen sections with PBS for 3 times and shake off the excess liquid. Take an appropriate amount of hematoxylin staining solution and add it to the frozen sections, shake evenly for 10 seconds, and rinse with running water. Take an appropriate amount of eosin staining solution and add it to the frozen sections, shake evenly for 1 minute, and rinse with running water. Observe the staining results under a microscope, seal the sections with neutral gum, take pictures under a microscope, and record the experimental results.
[0073] The results are as follows Fig. 9 As shown, we successfully constructed a symbiotic system of cancer-associated fibroblasts, endothelial cells, and oral squamous cell carcinoma epithelial cells.
[0074] Example 2: Method for constructing a composite human oral squamous cell carcinoma organoid model
[0075] In this example, the ratio of the cancer-associated fibroblasts stably expressing GFP, the endothelial cells stably expressing m-Cherry, and the oral squamous cell carcinoma epithelial cells added is 4:1:6 according to the number of cells. Other operations are the same as in Example 1.
[0076] Example 3: Method for constructing a composite human oral squamous cell carcinoma organoid model
[0077] In this example, the ratio of the cancer-associated fibroblasts stably expressing GFP, the endothelial cells stably expressing m-Cherry, and the oral squamous cell carcinoma epithelial cells added is 6:1:4 according to the number of cells. Other operations are the same as in Example 1.
[0078] Comparative Example 1
[0079] In this comparative example, the ratio of the cancer-associated fibroblasts stably expressing GFP, the endothelial cells stably expressing m-Cherry, and the oral squamous cell carcinoma epithelial cells added is 1:1:1 according to the number of cells. Other operations are the same as those in Example 1.
[0080] Experimental Example 1: Performance Measurement
[0081] The endothelial cell extension rates of the oral squamous cell carcinoma organoid models of Examples 1 to 3 were measured, as shown in the following table.
[0082] Table 1
[0083] Group Endothelial cell stretch rate Build success rate Example 1 98％ 100％ Example 2 97.5％ 99％ Example 3 97.2％ 97％ Comparative Example 1 40％ Unable to build successfully
[0084] From Table 1, we can see that:
[0085] In Comparative Example 1, the proportion of endothelial cells was too high, and a composite oral squamous cell carcinoma organoid model could not be prepared.
[0086] The composite oral squamous cell carcinoma organoid models of Examples 1 to 3 of the present invention can better simulate the characteristics of parental tumor tissues and cell-cell interactions, provide a better in vitro model for tumor research, and have a construction success rate of up to 100%.
[0087] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.
[0088] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A method for preparing a composite oral squamous cell carcinoma organoid model, characterized in that: The method comprises: Cancer-associated fibroblasts stably expressing GFP, endothelial cells stably expressing m-Cherry, and oral squamous cell carcinoma epithelial cells were co-cultured to obtain co-culture cell clusters; The co-cultured cell clusters were centrifuged and the supernatant was discarded to obtain cell spheres; the cell spheres were then mixed with matrix gel and inoculated into a well plate and cultured in an incubator; after solidification, culture medium was added for differentiation culture to obtain a composite oral squamous cell carcinoma organoid model.
2. The method for preparing a composite oral squamous cell carcinoma organoid model according to claim 1, characterized in that: The method for preparing cancer-associated fibroblasts stably expressing GFP comprises: Cancer-associated fibroblasts were seeded in six-well plates. When the cells grew to a density of 30%, they were infected with a lentiviral vector expressing GFP green fluorescent protein. When the cells converged to a density of 80%, puromycin was added to screen for successfully infected cells.
3. The method for preparing a composite oral squamous cell carcinoma organoid model according to claim 1, characterized in that: The method for preparing endothelial cells stably expressing m-Cherry comprises: Umbilical vein endothelial cells were seeded in six-well plates. When the cells grew to a density of 30%, they were infected with a lentiviral vector expressing m-Cherry red fluorescent protein. When the cells converged to a density of 80%, puromycin was added to screen for successfully infected cells.
4. The method for preparing a composite oral squamous cell carcinoma organoid model according to claim 1, characterized in that: The added ratio of the cancer-associated fibroblasts stably expressing GFP, the endothelial cells stably expressing m-Cherry, and the oral squamous cell carcinoma epithelial cells is (4-6):1:(4-6) according to the number of cells.
5. The method for preparing a composite oral squamous cell carcinoma organoid model according to claim 1, characterized in that: The culture medium used for the co-culture is an organoid culture medium and an ECM culture medium in a volume ratio of (4-6):
1.
6. The method for preparing a composite oral squamous cell carcinoma organoid model according to claim 1, characterized in that: The volume ratio of the cell sphere to the matrix gel is 1:100±10.
7. The method for preparing a composite oral squamous cell carcinoma organoid model according to claim 1, characterized in that: The culture medium used for the differentiation culture is an organoid culture medium and an ECM culture medium in a volume ratio of (4-6):
1.
8. A composite oral squamous cell carcinoma organoid model obtained by the method described in any one of claims 1 to 7.
9. An application of the composite oral squamous cell carcinoma organoid model according to claim 8 in disease models, drug development and toxicity analysis.