A method for preparing collagen fibers on a medium scale and a method for culturing organoids

Abstract

The present application relates to the field of collagen fiber preparation, and discloses a medium-scale collagen fiber preparation method and an organoid culture method, the medium-scale collagen fiber preparation method comprising the following steps: preparing a gelatin solution; preparing a transglutaminase solution; medium-scale collagen fiber preparation; adding the prepared medium-scale collagen fiber into the prepared gelatin solution; and adding the prepared transglutaminase solution into the obtained new matrix gel solution. The method is convenient to use, and the material is derived from a tissue block homologous to an organoid or digestion residues of extracted primary cells. The medium-scale collagen fiber effectively simulates the medium-scale morphology of the in-vivo microenvironment, can effectively enrich cells, and promote the formation of organoids. The new matrix gel culture formed by adding the material into the commonly used matrix gel can effectively improve the success probability of organoid culture, and the growth speed and the generated quantity of organoids can be greatly improved.

Description

Technical Field

[0001] This invention relates to the technical field of collagen fiber preparation, and particularly to a medium-scale collagen fiber preparation method and an organoid culture method. Background Technology

[0002] Patient-specific tumor organoid models are revolutionizing our understanding of cancer heterogeneity and its impact on personalized medicine. These advances are attributed to the ability of organoid models to stably preserve the genetic, proteomic, morphological, and pharmacological characteristics of in vivo tumors in vitro, while also providing unprecedented opportunities for genomic and environmental manipulation. The rise of tumor organoids has provided a completely new technological platform for translational medicine. From the initial successful construction of organoids from single tumor samples to the establishment of large-scale tumor organoid libraries, tumor organoid research has become an important tool in basic and clinical cancer research, especially when combined with gene modification technologies, which is of great significance for elucidating the mechanisms of tumor development and progression and rapidly evaluating the therapeutic effects of cancer drugs and immune cells.

[0003] Currently, it is common practice to use matrix gels (MGEs) made from basic materials such as Matrigel, gelatin, and hydrogels to culture tumor organoids. However, in practical applications, problems such as colloid instability, inconvenience in use, and low organoid culture success rates lead to culture failures. There are many reasons for this, one of which is the neglect of the morphology of in vivo collagen fibers. Studies have shown that medium-scale morphology plays a crucial role in 3D cell culture and tumor cell migration. Morphology plays a central role in cancer progression and treatment response, independent of the solid stress, fluid forces, and stiffness of the microenvironment. The morphology and arrangement of collagen fibers dynamically change during tumor progression. Under normal conditions, ECM fibers are randomly arranged isotropically, while during tumor growth, they exhibit organized anisotropic arrangement with global millimeter- or mesoscale characteristics, significantly influencing cell adhesion, cell mechanotransduction, long-distance intercellular communication, and migration. Current research on in vitro remodeling of tumor ECMs mainly focuses on local properties, including microstructure, pore size, fiber diameter, collagen arrangement, and stiffness. These models typically focus on submicron morphology and mechanics, but lack similarity to the mesoscale morphology of in vivo ECM structures. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing medium-sized collagen fibers and an organoid culture method. This invention aims to develop a method for preparing collagen fibers from the digestion residue of a patient's autologous tissue, forming medium-sized fibers (approximately 100 μm in length and 1 μm in diameter). These medium-sized collagen fibers have a similar morphology to mesoscale collagen fibers in vivo, promoting cell spheroidization and organoid formation. These medium-sized collagen fibers significantly increase the probability of cell aggregation and the success rate of organoid culture.

[0005] In a first aspect, this application provides a method for preparing collagen fibers on a medium scale, the method comprising:

[0006] a. Preparation of gelatin solution: Dissolve gelatin in deionized water to a concentration of 0.05-0.1 w / v (g / ml) and autoclave.

[0007] b. Preparation of transglutaminase solution: Transglutaminase powder is dissolved in phosphate solution to prepare a transglutaminase solution with an activity concentration higher than 100 U / ml, which is then filtered and sterilized using a 0.22 μm needle filter;

[0008] c. Preparation of medium-sized collagen fibers: During the extraction of primary cells, tissue blocks will have residual tissue fibers that are difficult to digest in the short term after being digested and filtered by collagenase I. These fibers are placed at -80℃ and thawed repeatedly 3-5 times to remove the cells from the residue. These fibers are then ground at high speed using a tissue grinder to obtain medium-sized collagen fibers. The size of medium-sized collagen fibers is 50μm-150μm. The medium-sized collagen fibers are sterilized and inactivated by soaking in 75% alcohol for 1 hour, and then washed 4-5 times with sterile deionized water before use.

[0009] d. Add the prepared medium-sized collagen fibers at a concentration of 0.001 g / ml to the gelatin solution prepared in step a;

[0010] e. Add the transglutaminase solution prepared in step b to the novel matrix gel solution obtained in step d, with a volume ratio of matrix gel solution: transglutaminase solution 1000:50;

[0011] f. Place the mixed solution from step e in a 37°C incubator and let it stand for about 1 hour until it gels.

[0012] Secondly, this application provides a method for culturing organoids, using the aforementioned medium-sized collagen fibers for organoid culture, the method specifically comprising:

[0013] S1. After obtaining normal / tumor tissue, cut it into small pieces of about 1 cubic millimeter, add 10ml of collagen hydrolase and place it in a 37℃ cell culture incubator for about 30 minutes to digest.

[0014] S2. Organoid culture medium contains the following reagents: The basal medium contains 10 μM Y-27632-HCl (Selleckchem, S1049), 5% fetal bovine serum (Gibco#10270-106, LOT 42G7277K), 1×B-27 supplement (Thermo Fisher Scientific, 17504044), 10 mM Nicotinamide (Sigma, n0636100g), 500 ng / ml Rspondin (Peprotech, 120-38), 1.25 mM n-acetylcysteine ​​(Sigma, A9165), 10 μM MSB202190 (Selleckchem, S1077), 100 ng / ml Noggin (Peprotech, 250-38), 500 nM A83-01 (Tocris, 2939), and 10 nM DHT (Fluka). Chemica (10300), 10 ng / ml Wnt3a (Peprotech, 315-20), 50 ng / ml HGF (Peprotech, 100-39), 50 ng / ml EGF (Peprotech, AF-100-15), 10 ng / ml FGF10 (Peprotech, 100-26), 1 ng / ml FGF2 (Peprotech, 100-18B), 1 μM PGE2 (Tocris, 2296); the culture medium was prepared and stored at 4°C for no more than 7 days.

[0015] S3. Add 10 ml of complete culture medium to stop digestion, and filter the tissue digested in step S1 through a 100-mesh cell sieve to obtain a cell suspension.

[0016] S4. Centrifuge the cell suspension from step S3 and remove the supernatant. Wash the cells twice with phosphate buffer. After the last centrifugation, remove as much residual buffer as possible.

[0017] S5. Count the cells and resuspend them using the matrix gel obtained in step a of claim 1 at a concentration of 1 million to 2 million cells per milliliter of matrix gel volume.

[0018] S6. Add transglutaminase solution to the cell suspension according to the proportion in step e of claim 1, and seed the mixed liquid into a culture dish as needed, and transfer it to a cell culture incubator at 37°C.

[0019] S7. After standing for about 1 hour, the matrix gel will be fully gelled, and then the appropriate cell culture medium can be added for culture and observation.

[0020] Thirdly, this application also provides an application of the aforementioned medium-sized collagen fibers for the culture of normal tissues and tumor organoids.

[0021] Specifically, the tumors include lung cancer, esophageal cancer, thyroid cancer, pancreatic cancer, tongue cancer, intestinal cancer, breast cancer, ovarian cancer, liver cancer, laryngeal cancer, bladder cancer, and cervical cancer.

[0022] This application presents a method for producing medium-sized collagen fibers. The method utilizes readily available materials derived from organoid homologous tissue blocks or digestive residues extracted from primary cells. These medium-sized collagen fibers effectively mimic the medium-scale morphology of the in vivo microenvironment, enabling efficient cell enrichment and promoting organoid formation. Adding this material to commonly used matrix gels to create a novel matrix gel culture significantly improves the success rate of organoid culture, substantially increasing both organoid growth rate and the number of organoids generated. The novel matrix gel formed by incorporating this material can be applied to the culture of organoids from various tumors and normal tissues. Attached Figure Description

[0023] Figure 1 This is a microscopic comparison image of a blank gelatin matrix and a gelatin colloid with a medium-sized amount of collagen fibers added.

[0024] Figure 2 This is a comparison image of lung adenocarcinoma organoids growing in a blank gelatin matrix and a gelatin matrix with added collagen fibers of moderate scale.

[0025] Figure 3 This is a statistical comparison chart of the growth of lung adenocarcinoma organoids in blank gelatin matrix and gelatin matrix with added collagen fibers of moderate scale.

[0026] Figure 4 This is a comparison image of lung squamous cell carcinoma organoids growing in a blank gelatin matrix and a gelatin matrix with added collagen fibers of moderate scale.

[0027] Figure 5 This is a comparison image of thyroid cancer organoids growing in a blank gelatin matrix and a gelatin matrix with added collagen fibers of moderate scale.

[0028] Figure 6 This is a comparison image of the growth of renal cell carcinoma organoids in blank gelatin matrix gel and gelatin matrix gel with added collagen fibers of medium scale.

[0029] Figure 7 This is a comparison image of colorectal cancer organoids growing in a blank gelatin matrix and a gelatin matrix with added collagen fibers of moderate scale. Detailed Implementation

[0030] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0032] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0033] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0034] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0035] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0036] Reference Figures 1-7 In a first aspect, this application provides a method for preparing collagen fibers on a medium scale, the method comprising:

[0037] a. Preparation of gelatin solution: Dissolve gelatin in deionized water to a concentration of 0.05-0.1 w / v (g / ml) and autoclave.

[0038] b. Preparation of transglutaminase solution: Transglutaminase powder is dissolved in phosphate solution to prepare a transglutaminase solution with an activity concentration higher than 100 U / ml, which is then filtered and sterilized using a 0.22 μm needle filter;

[0039] c. Preparation of medium-sized collagen fibers: During the extraction of primary cells, tissue blocks will have residual tissue fibers that are difficult to digest in the short term after being digested and filtered by collagenase I. These fibers are placed at -80℃ and thawed repeatedly 3-5 times to remove the cells from the residue. These fibers are then ground at high speed using a tissue grinder to obtain medium-sized collagen fibers. The size of medium-sized collagen fibers is 50μm-150μm. The medium-sized collagen fibers are sterilized and inactivated by soaking in 75% alcohol for 1 hour, and then washed 4-5 times with sterile deionized water before use.

[0040] d. Add the prepared medium-sized collagen fibers at a concentration of 0.001 g / ml to the gelatin solution prepared in step a;

[0041] e. Add the transglutaminase solution prepared in step b to the novel matrix gel solution obtained in step d, with a volume ratio of matrix gel solution: transglutaminase solution 1000:50;

[0042] f. Place the mixed solution from step e in a 37°C incubator and let it stand for about 1 hour until it gels.

[0043] Secondly, this application provides a method for culturing organoids, using the aforementioned medium-sized collagen fibers for organoid culture, the method specifically comprising:

[0044] S1. After obtaining normal / tumor tissue, cut it into small pieces of about 1 cubic millimeter, add 10ml of collagen hydrolase and place it in a 37℃ cell culture incubator for about 30 minutes to digest.

[0045] S2. Organoid culture medium contains the following reagents: The basal medium contains 10 μM Y-27632-HCl (Selleckchem, S1049), 5% fetal bovine serum (Gibco#10270-106, LOT 42G7277K), 1×B-27 supplement (Thermo Fisher Scientific, 17504044), 10 mM Nicotinamide (Sigma, n0636100g), 500 ng / ml Rspondin (Peprotech, 120-38), 1.25 mM n-acetylcysteine ​​(Sigma, A9165), 10 μM SB202190 (Selleckchem, S1077), 100 ng / ml Noggin (Peprotech, 250-38), 500 nM A83-01 (Tocris, 2939), and 10 nM DHT (Fluka). Chemica (10300), 10 ng / ml Wnt3a (Peprotech, 315-20), 50 ng / ml HGF (Peprotech, 100-39), 50 ng / ml EGF (Peprotech, AF-100-15), 10 ng / ml FGF10 (Peprotech, 100-26), 1 ng / ml FGF2 (Peprotech, 100-18B), 1 μM PGE2 (Tocris, 2296); the culture medium was prepared and stored at 4°C for no more than 7 days.

[0046] S3. Add 10 ml of complete culture medium to stop digestion, and filter the tissue digested in step S1 through a 100-mesh cell sieve to obtain a cell suspension.

[0047] S4. Centrifuge the cell suspension from step S3 (1000 rpm, 10 min) and remove the supernatant. Wash the cells twice with phosphate buffer. After the last centrifugation, remove as much residual buffer as possible.

[0048] S5. Count the cells and resuspend them using the matrix gel obtained in step a of claim 1 at a concentration of 1 million to 2 million cells per milliliter of matrix gel volume.

[0049] S6. Add transglutaminase solution to the cell suspension according to the proportion in step e of claim 1, and seed the mixed liquid into a culture dish as needed, and transfer it to a cell culture incubator at 37°C.

[0050] S7. After standing for about 1 hour, the matrix gel will be fully gelled, and then the appropriate cell culture medium can be added for culture and observation.

[0051] Thirdly, this application also provides an application of the aforementioned medium-sized collagen fibers for the culture of normal tissues and tumor organoids.

[0052] Specifically, the tumors include lung cancer, esophageal cancer, thyroid cancer, pancreatic cancer, tongue cancer, intestinal cancer, breast cancer, ovarian cancer, liver cancer, laryngeal cancer, bladder cancer, and cervical cancer.

[0053] Experimental example:

[0054] A method for preparing collagen fibers on a medium scale, the method comprising:

[0055] a. Preparation of gelatin solution: Dissolve gelatin in deionized water to a concentration of 0.085 w / v (g / ml) and autoclave.

[0056] b. Preparation of transglutaminase solution: Transglutaminase powder is dissolved in phosphate solution to prepare a transglutaminase solution with an activity concentration higher than 100 U / mL, which is then filtered and sterilized using a 0.22 μm needle filter;

[0057] c. Preparation of medium-sized collagen fibers: During the extraction of primary cells, tissue blocks will have residual tissue fibers that are difficult to digest in the short term after being digested and filtered by collagenase I. These fibers are placed at -80℃ and thawed repeatedly 4 times to remove the cells from the residue. These fibers are then ground at high speed using a tissue grinder to obtain medium-sized collagen fibers with a size of 100μm. The medium-sized collagen fibers are sterilized and inactivated by soaking in 75% alcohol for 1 hour, and then washed 5 times with sterile deionized water before use.

[0058] d. Add the prepared medium-sized collagen fibers at a concentration of 0.001 g / ml to the gelatin solution prepared in step a;

[0059] e. Add the transglutaminase solution prepared in step b to the novel matrix gel solution obtained in step d, with a volume ratio of matrix gel solution: transglutaminase solution 1000:50;

[0060] f. Place the mixed solution from step e in a 37°C incubator and let it stand for 1 hour to gel.

[0061] Comparative example:

[0062] a. Preparation of gelatin solution: Dissolve gelatin in deionized water to a concentration of 0.085 w / v (g / ml) and autoclave.

[0063] b. Place the solution from step a in a 37°C incubator and let it stand for 1 hour to gel.

[0064] Figure 1The figures show the control group (blank gelatin colloid for comparison) and the ECM group (gelatin colloid with added medium-sized collagen fibers in the experimental example). According to the measurements, the size of medium-sized collagen fibers is between 30um and 200um, with an average value of about 90um.

[0065] Figure 2 The figures show the effects of the Control group (blank gelatin colloid for comparison) and the ECM group (gelatin matrix colloid with added collagen fibers for experimental examples) on organoid formation. Primary tumor cells from lung adenocarcinoma patients were used. It can be clearly seen that, compared with the blank gelatin matrix colloid, the lung adenocarcinoma organoids with added collagen fibers formed and grew better.

[0066] Figure 3 This is a statistical graph showing the growth of lung adenocarcinoma organoids in the Control group (blank gelatin colloid for comparison) and the ECM group (gelatin matrix colloid with added collagen fibers for experimental examples). Specifically, it shows the distribution of organoid diameters formed under the two matrix colloid conditions. This result indicates that, compared to the blank gelatin matrix colloid, the matrix colloid with added collagen fibers exhibits better lung adenocarcinoma organoid formation and growth.

[0067] Figure 4 The figures show the effects of the Control group (blank gelatin colloid) and the ECM group (gelatin matrix colloid with added collagen fibers in the experimental cases) on organoid formation. The primary tumor cells used were from patients with squamous cell carcinoma of the lung. It can be clearly seen that compared with the blank gelatin matrix colloid, the lung squamous cell carcinoma organoids with added collagen fibers formed and grew better.

[0068] Figure 5 The figures show the effects of the Control group (blank gelatin colloid for comparison) and the ECM group (gelatin matrix colloid with added medium-sized collagen fibers for experimental examples) on organoid formation. Primary tumor cells from thyroid cancer patients were used. It can be clearly seen that, compared with the blank gelatin matrix colloid, the matrix colloid with added medium-sized collagen fibers showed better organoid formation and growth in thyroid cancer patients.

[0069] Figure 6 The figures show the effects of the Control group (blank gelatin colloid for comparison) and the ECM group (gelatin matrix colloid with added collagen fibers for experimental examples) on organoid formation. The primary tumor cells used were from renal cell carcinoma patients. It can be clearly seen that the renal cell carcinoma organoids formed and grew better in the matrix colloid with added collagen fibers compared to the blank gelatin matrix colloid.

[0070] Figure 7 The figures show the effects of the Control group (blank gelatin colloid for comparison) and the ECM group (gelatin matrix colloid with added medium-sized collagen fibers in the experimental cases) on organoid formation. Primary tumor cells from colorectal cancer patients were used. It can be clearly seen that compared with the blank gelatin matrix colloid, the matrix colloid with added medium-sized collagen fibers showed better organoid formation and growth in colorectal cancer.

[0071] This invention establishes a method for producing medium-sized collagen fibers. The method utilizes readily available materials derived from organoid homologous tissue blocks or digestive residues extracted from primary cells. These medium-sized collagen fibers effectively mimic the medium-scale morphology of the in vivo microenvironment, enabling efficient cell enrichment and promoting organoid formation. Adding this material to commonly used matrix gels to create a novel matrix gel culture significantly improves the success rate of organoid culture, substantially increasing both organoid growth rate and the number of organoids generated. The novel matrix gel formed by incorporating this material can be applied to the culture of organoids from various tumors and normal tissues.

[0072] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0073] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing collagen fibers on a medium scale, characterized in that, The preparation method includes: a. Preparation of gelatin solution: Dissolve gelatin in deionized water to a concentration of 0.05-0.1 g / mL, and autoclave. b. Preparation of transglutaminase solution: Transglutaminase powder is dissolved in phosphate solution to prepare a transglutaminase solution with an activity concentration higher than 100 U / mL, which is then filtered and sterilized using a 0.22-micron needle filter; c. Preparation of medium-sized collagen fibers: During the extraction of primary cells, tissue blocks will have residual tissue fibers that are difficult to digest in the short term after being digested and filtered by collagenase I. These fibers are placed at -80℃ and thawed repeatedly 3-5 times to remove the cells from the residue. These fibers are then ground at high speed using a tissue grinder to obtain medium-sized collagen fibers. The size of medium-sized collagen fibers is 50µm-150µm. The medium-sized collagen fibers are sterilized and inactivated by soaking in 75% alcohol for 1 hour, and then washed 4-5 times with sterile deionized water before use. d. Add the prepared medium-sized collagen fibers at a concentration of 0.001 g / mL to the gelatin solution prepared in step a; e. Add the transglutaminase solution prepared in step b to the matrix gel solution obtained in step d, with a volume ratio of matrix gel solution: transglutaminase solution 1000:50; f. Place the mixed solution from step e in a 37°C incubator and let it stand for 1 hour to gel.

2. A method for culturing organoids, characterized in that, Organoid culture is performed using the medium-sized collagen fibers obtained according to claim 1, wherein the culture method specifically includes: S1. After obtaining normal / tumor tissue, cut it into small pieces of about 1 cubic millimeter, add 10 mL of collagen hydrolase and place it in a 37°C cell culture incubator for 30 minutes to digest. S2. Organoid culture medium contains the following reagents: basal medium contains 10 µM Y-27632-HCl, 5% fetal bovine serum, 1× B-27 supplement, 10 mM Nicotinamide, 500 ng / mL Rspondin, 1.25 mM n-acetylcysteine, 10 μM SB202190, 100 ng / mL Noggin, 500 nM A83-01, 10 nM DHT, 10 ng / mL Wnt3a, 50 ng / mL HGF, 50 ng / mL EGF, 10 ng / mL FGF10, 1 ng / mL FGF2, and 1 μM PGE2; the culture medium should be prepared and stored at 4°C for no more than 7 days. S3. Add 10 mL of complete culture medium to stop digestion, and filter the tissue digested in step S1 through a 100-mesh cell sieve to obtain a cell suspension. S4. Centrifuge the cell suspension from step S3 and remove the supernatant. Wash the cells twice with phosphate buffer. After the last centrifugation, remove as much residual buffer as possible. S5. Count the cells and resuspend them using the matrix gel obtained in step a of claim 1 at a concentration of 1 million to 2 million cells per milliliter of matrix gel volume. S6. Add transglutaminase solution to the cell suspension according to the proportion of step e in claim 1, and seed the mixed liquid into a culture dish as needed, and transfer it to a 37°C cell culture incubator. S7. After standing for 1 hour, the matrix gel will be fully gelled, and then the appropriate cell culture medium can be added for culture and observation.

3. The application of medium-scale collagen fibers prepared by the method of preparing medium-scale collagen fibers according to claim 1 for the culture of normal tissues and tumor organoids.

4. The application according to claim 3, characterized in that, The tumors include lung cancer, esophageal cancer, thyroid cancer, pancreatic cancer, tongue cancer, intestinal cancer, breast cancer, ovarian cancer, liver cancer, laryngeal cancer, bladder cancer, and cervical cancer.

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