Patient autologous source cartilage organoid culture medium and modeling method thereof

Abstract

The invention discloses a culture medium for an autologous cartilage organ of a patient and a modeling method of the culture medium, and relates to the field of life sciences, the culture medium is composed of high-glucose DMEM, TGF-beta1, BMP-2, L-ascorbic acid-2-phosphate, insulin-transferrin-selenium (ITS), beta-sodium glycerophosphate, insulin-like growth factor-1 (IGF-1), FGF2, FBS, penicillin-streptomycin (PS) and L-proline, and the pH is 7.0-7.3. The culture medium greatly meets the nutritional requirements of patient autologous source cartilage primary organoid culture, the morphological structure and characteristics of original cartilage tissue can be reproduced in vitro, and the established cartilage organoid can be used for subsequent research and drug sensitive test, and is a good bone research model.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of life science and relates to a patient-derived cartilage organoid culture medium and a modeling method thereof. BACKGROUND

[0002] The movement system supports human movement, provides support and protection, and maintains normal morphology, which can be subdivided into the skeletal system and the muscular system. The skeletal system is mainly composed of bones and joints connecting bones, forming a skeleton with both firmness and mobility; the muscular system includes skeletal muscle (SKM) and its tendon, ligament and other structures, which realize function by driving joint movement. Movement system diseases include degeneration, injury, tumor and other types, among which fractures, osteoporosis, low back pain and osteoarthritis are the most common. Globally, more than 171 million people are affected, of which low back pain (568 million), fractures (436 million) and osteoarthritis (343 million) account for the highest proportion. Population aging exacerbates the disease burden, and endogenous repair fails in late-stage patients, who need to rely on prosthetic replacement or tissue transplantation; while the source of autologous transplantation is limited, and allogeneic transplantation has problems such as immune rejection, which promotes the development of tissue engineering technology. The selection of traditional tissue engineering three elements (seed cells, scaffolds and biological induction factors) often lacks the support of developmental theory, and only through in vitro screening of seed cells and biological induction factors, simulating the morphology of mature tissues, designing tissue scaffolds, and pursuing the "similar" of tissues and organs, the heterogeneity of internal cell composition and the bionics of microstructure are ignored, making it difficult to obtain the correct composition of various cells and matrix components, and it is naturally difficult to reproduce the biomechanical properties consistent with natural tissues.

[0003] Organoids are a technology that uses adult stem cells to culture 3D cell cultures in vitro. Since the construction of organoids is to reproduce the differentiation and development process of stem progenitor cells, the organoids obtained are highly similar to normal tissues in cell composition, matrix composition, tissue microstructure, etc.; at the same time, if stem progenitor cells carrying specific genetic pathogenic genes are used to construct organoids, genetic disease models can also be constructed in vitro. Based on the above two advantages, combined with in vitro large-scale batch preparation technology, organoids have been quickly applied in disease mechanism exploration, large-scale drug screening and other aspects.

[0004] Gradually, the application scenarios of organoids also extend to the field of tissue repair and reconstruction. The biomimetic microenvironment of organoids retains the interaction characteristics between cells and matrix, giving them the potential to promote proliferation, differentiation, and functional recovery. At the same time, organoids also have the ability of self-renewal and self-organization, which can realize sustained tissue repair and regeneration. Compared with traditional biomaterials, organoids can be highly customized to meet the needs of individual patients, and personalized treatment strategies can be implemented by adjusting the type of tissue, structural characteristics, and repair effect. At the same time, with the continuous development of bioengineering technology, various strategies for optimizing organoid construction and culture have emerged: with the help of bioreactors, microfluidic systems, and biological 3D printing technology, not only highly controllable large-scale production platforms are provided, but also the in vivo microenvironment can be accurately simulated, providing ideal support for the development, maturation, and function of organoids.

[0005] A large number of studies have confirmed that organoids have shown significant ability in tissue repair. For example, the relatively simple intestinal organoid has been thoroughly explored, providing a deep understanding of the evolution and regeneration repair process of the intestinal tract. At the same time, with the help of biological 3D printing technology, liver organoids constructed from induced pluripotent stem cells (iPSCs) exhibit superior potential for liver failure treatment compared to primary liver cells. In recent years, the academic community has successfully constructed cartilage organoids, providing a theoretical basis and technical support for the repair and reconstruction of the musculoskeletal system based on organoid technology.

[0006] Cartilage organoids refer to the three-dimensional self-assembly of cartilage-related stem cells induced by various factors and suitable microenvironments in vitro, which can simulate the 3D structure and function, biomechanical properties, and genetic characteristics of natural cartilage tissue. Cartilage tissue is difficult to self-repair. In recent years, through the construction of pluripotent stem cells by three-dimensional culture technology, the highly biomimetic construction of cartilage matrix has been realized, which has attracted widespread attention in the academic community. However, the pluripotent stem cell-derived cartilage organoids have uneven cell differentiation during the induction process, which may result in an undesirable ratio of different cell types in the formed cartilage organoids. At the same time, after transplantation in vivo, the survival rate and integration ability of cartilage organoids need to be improved, and there may be risks such as rejection or abnormal growth. Autologous cartilage organoids are an effective method to solve the above problems. Therefore, it is particularly important to develop a method that can construct autologous cartilage organoids in vitro while reducing the hypertrophy process during the development of cartilage organoids. Autologous cartilage organoids are expected to become a powerful tool for solving bone and joint diseases, and to accumulate valuable experience for developing models for basic research of bone and joint diseases. SUMMARY

[0007] The purpose of the present application is to provide an autologous cartilage organoid culture medium and a modeling method thereof, which solves the problem of difficulty in constructing existing cartilage organoids and easy overgrowth.

[0008] To achieve the above object, the technical scheme adopted by the present application is: An autologous cartilage organoid culture medium, the culture medium is a basic culture medium and specific additives: the specific additives are TGF-β1 and high-sugar DMEM, and the specific formula is as follows: Ingredient Type Ingredient Name 0-7 days 7-14 days 14-28 days Base Medium High glucose DMEM 1x 1x 1x Specificity Additives TGF-β1 10 ng / ml 10 ng / ml 10 ng / ml BMP-2 50 ng / ml 50 ng / ml 50 ng / ml L-Ascorbic Acid-2-Phosphate 150 μM 150 μM 150 μM Insulin-Transferrin-Selenium (ITS) 1x 1x 1x β-Glycerophosphate Sodium / / 5 mM Insulin-like Growth Factor-1 (IGF-1) 50 ng / mL 50 ng / mL 20 ng / mL FGF2 5 ng / mL / / FBS 10% 10% 10% Penicillin-Streptomycin (PS) 1x 1x 1x L-Proline 50 ug / ml 50 ug / ml 50 ug / ml GDF5 10 ng / ml 10 ng / ml 10 ng / ml

[0009] For tissues with older age or poor tissue dryness, the concentration can be adjusted, and the specific formula is as follows: Ingredient Type Ingredient Name 0-14 days 14-28 days Base Medium High glucose DMEM 1x 1x Specificity Additives TGF-β1 10 ng / ml 10 ng / ml L-Ascorbic Acid-2-Phosphate 150 μM 150 μM Insulin-Transferrin-Selenium (ITS) 1x 1x Dexamethasone 0.1 μM 0.1 μM β-Glycerophosphate Sodium / 5 mM Insulin-like Growth Factor-1 (IGF-1) 50 ng / ml 10 ng / ml FGF2 0.2 ng / ml 0.2 ng / ml FBS 10% 10% Penicillin-Streptomycin (PS) 1x 1x L-Proline 50 ug / ml 50 ug / ml GDF5 10 ng / ml 10 ng / ml An application of the culture medium, the culture medium is applied in culturing autologous cartilage organoids.

[0010] 1. Pour the tissue sample into a new sterile 6 cm cell culture dish, use a scissors forceps to cut two pieces of bone tissue the size of a soybean, use a disposable scalpel to scrape off the white mucosa on the inner surface and the material on the outer surface, transfer the tissue to a new sterile 6 cm cell culture dish, use 3-4 mL of 5% tris-antigen-containing ice D-PBS buffer to wash, wash 10 times up, down, left and right; transfer the tissue to a new sterile 6 cm cell culture dish and wash again; repeat this step and wash again 2 times; 2. Transfer the washed tissue to a rinsed 1.5 mL EP tube, use scissors to cut the tissue directly in the tube until the tissue becomes small particles within 2 mm; 3. Prepare 4 mL of 37℃ preheated collagenase II (use a 5 mL centrifuge tube), use a new pair of scissors to cut off the tip of the 1250 μL syringe, resuspend the cut tissue with 1 mL of digestion solution, transfer it to 4 mL of digestion solution, and also wash the scissors in the digestion solution; 4. Digest at 37℃, 100 rpm on a shaker for 2h, take out and observe under a microscope, and terminate digestion when a large number of obvious cells appear; (Note: Rinse a 100 μm cell filter in advance during the digestion process, the rinsing method is to place the filter in a 6 cm cell culture dish, take 1-2 mL of rinsing solution and evenly rinse the filter with a 1250 μL syringe, avoiding the generation of air bubbles) 5. Terminate digestion with 2% ~5% FBS, i.e. add 80 μL FBS to 4 mL tumor tissue digestion solution, invert to mix 3-5 times to terminate digestion; and directly pour into 100 μm cell filter, hold the cell filter to shake slightly until the liquid flows into the cell culture dish, use 1 mL basal medium to wash the centrifuge tube and pour it into the cell filter to filter, 2 mL basal medium to rinse the filter again; then transfer the liquid in the cell culture dish to a new 5 mL centrifuge tube (add gently along the tube wall to avoid impact on the cell mass); 4°C, 300~350 g, centrifuge for 3 min; 6. Use a Pasteur pipette to suck the supernatant, be careful not to suck the precipitate, and retain 50-100 μL liquid; add 1 mL red blood cell lysis solution, gently blow the precipitate to mix, transfer to a 1.5 mL centrifuge tube, immediately 4°C, 300~350 g, centrifuge for 3 min; 7. Suck the supernatant, resuspend with 500 μL basal medium, suck 10 μL to detect cell activity, then re-centrifuge at 4°C, 300~350 g, for 3 min, suck the supernatant, resuspend with 1 mL basal medium and add to a new 6 cm cell culture dish, add 1 mL basal medium for culture.

[0011] 8. Observe the cell state under the microscope every day, the primary cells grow slowly, and it takes about 15-30 days, replace the culture medium every two days, and when the cell density reaches 80%, it can be subcultured; 9. Observe the cell state, when the cell state is good and the density reaches 80-90%, discard the culture medium in the original culture dish, add PBS and shake slowly to wash once, discard the PBS; 10. Add 3 mL TryplE digestion solution for digestion, the digestion time is 3 minutes, and the microscope needs to be observed during the digestion, and the gun head is used for mechanical blowing, when most of the cells are not adherent and separated under the microscope, add Basal to terminate digestion; 11. Transfer the liquid in the culture dish to a 15 mL centrifuge tube, centrifuge at 300 g for 3 min, discard the supernatant after centrifugation, resuspend the precipitate with 1 mL Bascal, take 10 μL for trypan blue staining, count with a cell counter, and according to the plating density of 20,000 cells per well, take the appropriate volume of cell suspension into a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min, discard the supernatant; 12. Add an appropriate amount of Matrigel to resuspend the cell precipitate (the content of Matrigel should not be less than 70%), resuspend and place on ice, and the resuspension time should not exceed 30 s to avoid premature solidification of Matrigel; 13. According to 20 μL per hole, as soon as possible drop to the preheated cell culture plate hole bottom central position, avoid contact with the hole plate side wall, invert in 37℃, 5 % CO2 cell incubator for 15 min~20 min, after Matrigel solidification, slowly add human bone organ complete culture medium along the hole wall, culture in 37℃, 5 % CO2 cell incubator; 14. The bone organ culture uses the foregoing induction culture medium, and the culture time is 28 days, the first stage culture medium is used in 1-14 days, the culture medium is replaced every three days, the second stage culture medium is replaced in 14-28 days, the second stage culture medium is divided into two parts, one is β-glycerophosphate sodium containing, and one is β-glycerophosphate sodium free, the culture medium is replaced every three days, and the state of the organoid is observed under a microscope every day, the β-glycerophosphate sodium containing organoid forms a ball in 16-18 days, and the β-glycerophosphate sodium free organoid forms a ball in 22-25 days.

[0012] In summary, the application has the following beneficial effects: The application provides a culture medium specially suitable for autologous human cartilage organoids, which greatly meets the complex environment required for cartilage cell in-vitro culture, maintains high activity of primary cells, and maximally retains the characteristics of cartilage, so that the success rate of cartilage organoid construction can be improved.

[0013] The application also provides a culture method system of cartilage organoids, which comprises a cartilage organoid culture medium, a method for separating cartilage primary cells, and a method for constructing and culturing cartilage organoids, and has the characteristics of short culture period and high activity. Based on the method system provided by the application, the in-vitro cartilage organoid can be successfully self-assembled in 5-7 days, has a regular shape, uniform size, and highly consistent pathological results with cartilage tissue, and can be used for subsequent mechanism and translational medicine research.

[0014] Based on the cartilage organoid culture medium and the in-vitro culture method system of autologous cartilage organoids provided by the application, the method system is especially suitable for clinical and basic researches on cartilage injury, microtia and other cartilage injury diseases, and the success rate of in-vitro culture of qualified surgical specimens is as high as 95 % or more.

[0015] The cartilage organoids obtained by the application are suitable for individualized chemotherapy drug sensitivity detection, researches on cartilage injury diseases, osteoporosis or bone metabolism related diseases. In particular, the cartilage organoids have a wide application prospect in individualized precise treatment of cartilage injury diseases and osteoporosis, and can provide an efficient in-vitro model for bone disease research. BRIEF DESCRIPTION OF DRAWINGS

[0016] The application will be further described below in combination with the drawings and examples.

[0017] Figure 1 Optical microscope image of the primary culture of the cartilage organoid in Example 1 of the present application at day 21; Figure 2 Alizarin red staining result image of the primary established cartilage organoid in Example 1 of the present application; Figure 3 Safranin O staining image of the primary established cartilage organoid in Example 1 of the present application; Figure 4 Safranin O staining and RUNX2, ACAN, collagen II pathological result image of the primary established cartilage organoid in Example 1 of the present application; DETAILED DESCRIPTION

[0018] In order to better understand the present application, the present application will be further described below in conjunction with examples and drawings. It should be noted that the specific embodiments described herein are only intended to illustrate and explain the present application, and are not limited to the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market.

[0019] Example 1: Primary construction of patient autologous cartilage organoid A patient autologous cartilage organoid culture medium, with a pH of 7.0-7.3, has the following components and concentrations as shown in the table: Ingredient Type Ingredient Name 0-7 days 7-14 days 14-28 days Base Medium High glucose DMEM 1x 1x 1x Specificity Additives TGF-β1 10 ng / ml 10 ng / ml 10 ng / ml BMP-2 50 ng / ml 50 ng / ml 50 ng / ml L-Ascorbic Acid-2-Phosphate 150 μM 150 μM 150 μM Insulin-Transferrin-Selenium (ITS) 1x 1x 1x β-Glycerophosphate Sodium / / 5 mM Insulin-like Growth Factor-1 (IGF-1) 50 ng / mL 50 ng / mL 20 ng / mL FGF2 5 ng / mL / / FBS 10% 10% 10% Penicillin-Streptomycin (PS) 1x 1x 1x L-Proline 50 ug / ml 50 ug / ml 50 ug / ml GDF5 10 ng / ml 10 ng / ml 10 ng / ml The primary construction of the patient autologous cartilage organoid is carried out according to the following specific steps: 1. Pour the tissue sample into a new sterile 6 cm cell culture dish, use a scissors forceps to cut two pieces of bone tissue the size of a soybean, use a disposable scalpel to scrape off the white mucosa on the inner surface and the material on the outer surface, transfer the tissue to a new sterile 6 cm cell culture dish, wash with 3-4 mL of 5% tris-antigen-containing ice D-PBS buffer, wash 10 times up, down, left and right; transfer the tissue to a new sterile 6 cm cell culture dish and wash again; repeat this step and wash again 2 times; 2. Transfer the washed tissue to a rinsed 1.5 mL EP tube, use scissors to cut the tissue directly in the tube until the tissue becomes small particles within 2 mm; 3. Prepare 4 mL of 37℃ preheated collagenase II (use a 5 mL centrifuge tube), use a new pair of scissors to cut off the tip of the 1250 μL gun head, resuspend the cut tissue with 1 mL of the digestion solution, transfer it to 4 mL of the digestion solution, and also wash the scissors in the digestion solution; 4. 37℃, 100 rpm shaker digestion, timing 2h, take out and observe under microscope, a large number of obvious cells appear, then stop digestion; (Note: during the digestion process, rinse 100 μm cell filter in advance, rinse method is to put the filter into a 6 cm cell culture dish, take 1-2 mL rinse solution and evenly rinse the filter with 1250 μL gun head, avoid air bubbles) 5. Terminate digestion with 2% ~5% FBS, i.e. 4 mL tumor tissue digestion solution is added with 80 μL FBS, inverted 3-5 times to terminate digestion; and directly pour into 100 μm cell filter, hold the cell filter and shake slightly until the liquid flows into the cell culture dish, use 1 mL basal medium to wash the centrifuge tube and pour it into the cell filter, rinse the filter with 2 mL basal medium; then transfer the liquid in the cell culture dish to a new rinsed 5 mL centrifuge tube (add along the wall, avoid impact on the cell mass); 4℃, 300~350 g, centrifuge for 3 min; 6. Use a bus suction tube to discard the supernatant, be careful not to suck the precipitate, can reserve 50-100 μL liquid; add 1 mL red blood cell lysis solution, gently blow the precipitate and mix well, transfer to a rinsed 1.5 mL centrifuge tube, immediately 4℃, 300~350 g, centrifuge for 3 min; 7. Suck the supernatant, add 500 μL basal medium and resuspend, suck 10 μL to detect cell activity, then re-4℃, 300~350 g, centrifuge for 3 min, suck the supernatant, add 1 mL basal medium and resuspend, then add to a new 6 cm cell culture dish, add 1 mL basal medium for culture.

[0020] 8. Observe the cell state under the microscope every day, primary cells grow slowly, about 15-30 days, replace the culture medium every two days, when the cell density reaches 80%, it can be subcultured; 9. Observe the cell state, when the cell state is good and the density reaches 80-90%, discard the culture medium in the original culture dish, add PBS and shake slowly to wash once, discard the PBS; 10. Add 3 mL TryplE digestion solution for digestion, digestion time is 3 minutes, during which the microscope is observed and the gun head is mechanically blown, when most of the cells are not adherent and separated under the microscope, add Basal to terminate digestion; 11. Transfer the liquid in the culture dish to a 15 mL centrifuge tube, centrifuge at 300g for 3 min, discard the supernatant after centrifugation, add 1 mL Bascal to resuspend the precipitate, take 10 μL for staining with trypan blue, count the cells using a cell counter, count the cells according to the plating density of 20,000 cells per well, take the appropriate volume of cell suspension into a rinsed 1.5 mL centrifuge tube, centrifuge at 300g for 3 min, discard the supernatant; 12. Add an appropriate amount of Matrigel to resuspend the cell precipitate (the content of Matrigel should not be less than 70%), after resuspension, place on ice, and the resuspension time should not exceed 30 s to avoid premature solidification of Matrigel; 13. According to 20 μL per well, drop as soon as possible to the bottom center of the preheated cell culture plate, avoid contact with the side wall of the plate, invert in a 37℃, 5 % CO2 cell incubator for 15 min~20 min, after Matrigel solidification, slowly add human bone organ complete culture medium along the wall of the hole, cultivate in a 37℃, 5 % CO2 cell incubator; 14. The bone organ culture uses the aforementioned induction medium, the culture time is 28 days, the first stage medium is used from 1-14 days, the medium is replaced every three days, the second stage medium is replaced from 14-28 days, the second stage medium is divided into two parts, one contains β-glycerophosphate sodium, and the other does not contain β-glycerophosphate sodium, the medium is replaced every three days, the state of the organoid is observed under a microscope every day, the organoid containing β-glycerophosphate sodium forms a ball in 16-18 days, and the organoid without β-glycerophosphate sodium forms a ball in 22-25 days.

Claims

1. A culture medium for cartilage organoid culture, the culture medium being a basal medium and specific additives: the specific additives being high glucose DMEM, TGF-β1, with the following specific formulation: Concentration I: Concentration II:

2. The method for primary construction of a primary cartilage organoid according to claim 1, characterized in that: Cartilage tissue is derived from surgical resection or biopsy specimens from clinical patients, and blood clots, necrotic tissue, fat and connective tissue on the tissue specimen are removed and placed in a tissue preservation solution, and transported back to the laboratory within 24 h under a low temperature environment of 0-4 ℃ for primary cartilage cell separation.

3. Use of the medium according to claim 1, characterized in that: Application in primary construction of cartilage organoids.

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