Method for preparing cartilage micro-tissue preparation and cartilage cell preparation by adopting three-dimensional microcarrier and application of cartilage micro-tissue preparation and cartilage cell preparation

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a method for preparing a cartilage micro-tissue preparation and a cartilage cell preparation by adopting a three-dimensional microcarrier and application of the cartilage micro-tissue preparation and the cartilage cell preparation. According to the preparation method, cartilage cells are prepared in a three-dimensional microcarrier environment, interaction between cells and interaction between cells and extracellular matrixes are promoted, and the actual microenvironment of the cells in tissue is simulated more accurately. According to the invention, the microcarrier is matched with the stirring bioreactor, so that large-scale amplification of cartilage cells can be realized in in-vitro culture. The invention further provides the cartilage micro-tissue preparation and the cartilage cell preparation which are prepared by the method and application of the cartilage micro-tissue preparation and the cartilage cell preparation in preparation of products for repairing cartilage injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for preparing cartilage microtissue preparations and chondrocyte preparations using three-dimensional microcarriers and its applications. Background Art

[0002] As an important human tissue, cartilage mainly plays roles such as load-bearing, lubrication, and anti-wear in the body. Articular cartilage lacks the nutritional support of blood vessels and nerves, and it is difficult to self-repair after injury. In daily life and sports, when the cartilage tissue is slightly damaged, it can repair the damaged part through its weak self-repair ability. When the cartilage defect exceeds a certain area and continues to develop, it is likely to cause joint pain, movement disorders, and even osteoarthritis, seriously affecting the quality of life of patients.

[0003] Cartilage injury repair is an international problem in the field of tissue regeneration and repair. Currently, commonly used clinical treatment options such as microfracture surgery, autologous cartilage tissue transplantation, autologous chondrocyte transplantation (ACI), and allogeneic tissue transplantation all have their own limitations. Microfracture surgery is prone to generating fibrocartilage with poor mechanical strength; autologous cartilage tissue transplantation has limited autologous tissue for larger defects; autologous chondrocyte tissue transplantation uses 2D culture of chondrocytes, which is likely to cause chondrocyte dedifferentiation and is costly; while allogeneic tissue transplantation has limited donors. Therefore, there is an urgent need to find a solution to achieve cartilage injury repair.

[0004] The proliferative capacity of existing in vitro two-dimensional cultured chondrocytes is limited, and they are prone to dedifferentiation, which affects the repair effect. Since body cells grow in a complex microenvironment, conventional in vitro two-dimensional cell culture cannot provide the environmental conditions required for the normal growth and development of tissues. Deviations in the growth environment will block the transmission of growth signals, chemical signals, and stress signals, leading to cell apoptosis and the loss of normal cell morphology or physiological functions (see Ruoslahti E. Stretching is good for a cell [J]. Science 1997, 276 (5317): 1345-1346). During the in vitro culture of chondrocytes, due to the differences between the extracellular environment and the in vivo environment, their biological properties gradually become unstable after long-term monolayer culture or repeated passage. A series of changes will occur in the cell morphology, differentiation, and proliferation. However, with the increase in the number of passages, their dedifferentiation characteristics gradually increase, and the overall expression of functional proteins decreases. Generally, chondrocytes within three generations have high activity and maintain the cell phenotype, making them ideal cells for experimental research. After three generations, the cells gradually undergo dedifferentiation, with an increase in cell volume, the appearance of many finger-like protrusions, an increase in the cell nucleus, a significant slowdown in the cell proliferation rate, and a decrease in the ability to secrete the matrix (see Ye Zhengzhi, Li Xihai, Liang Wenna, et al. Experimental study on the isolation, culture, and identification of chondrocytes in vitro [J]. Journal of Fujian University of Traditional Chinese Medicine, 2008, 18 (6): 32-33, 37).

[0005] However, research has shown that the in vitro three-dimensional culture environment is more conducive to the maintenance and exertion of cell functions than two-dimensional culture (see Shea LD, Wang D, Franceschi RT, et al. Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds [J]. Tissue Eng 2000, 6(6): 605-617). Compared with two-dimensional culture, three-dimensional culture greatly increases the surface area convenient for cell adhesion, effectively increases the diffusion of signaling factors and the synthesis of the extracellular matrix, and can provide a good microenvironment for cell adhesion, growth, and function exertion, facilitating cell migration, differentiation, and massive proliferation in multiple directions (see Shea LD, Wang D, Franceschi RT, et al. Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds [J]. Tissue Eng 2000, 6 (6): 605-61).

[0006] Three-dimensional cell culture is divided into static culture and dynamic culture. Static culture is further divided into three-dimensional microcarrier static culture and matrix gel culture. When Holy et al. inoculated cells into three-dimensional carriers for static culture, they found that the maximum number of cells inside the carrier accounted for 25% of the initial number of cells inoculated, and the remaining 75% of the cells were at the bottom of the carrier (see, Holy CE Shoichet MS Davies JE. Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell seeding density and culture period[J].J Biomed Mater Res 2000 51 (3): 376-382). Ishaug-Riley et al. cultured bone marrow cells statically on a three-dimensional carrier, producing mineralized tissue on the outer layer, but with little or no bone tissue formed inside the carrier. In addition, due to the effect of gravity, three-dimensional static culture has the disadvantages of insufficient number of cells inside the carrier and uneven distribution. Therefore, static three-dimensional culture still has many problems (see, Ishaug-Riley SL Crane-Kruger GM Yaszemski MJ et al. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers[J].Biomaterials 1998 19 (15): 1405-1412).

[0007] The matrix gel products used in matrix gel culture mainly rely on imports from abroad, and the use of matrix gel in the culture process is limited by many factors. During the culture process, temperature is the biggest factor affecting the matrix gel, and the items in contact with the matrix gel need to be pre-cooled. If the temperature exceeds 10°C during the operation, gelation begins, and the gelation speed is faster when it exceeds 22°C. There is a large difference between batches of matrix gel. The three-dimensional porous microcarriers used in the present invention have clear weight labels and are divided into 10 mg / tablet, 20 mg / tablet and powder. The sterile packaged microcarriers will disperse when exposed to water, without any pretreatment, and can be used at any time. They have obtained FDA-DMF / MF and CDE pharmaceutical excipient qualifications, and the high porosity makes the effective growth area of ​​the microcarriers reach 9000 cm 2 / g, can carry 10 8 ~10 9 At the same time, the bionic elasticity of the microcarrier has the advantages of withstanding shear force and effectively protecting cells, which can significantly reduce the preparation work during the culture process, is simple to operate, and can obtain large-scale cells.

[0008] Chondrocytes cultured with three-dimensional porous microcarriers can effectively improve production efficiency and reduce costs. When the number of P0 cells is 2.5×10 6 cells, the P0 cells can be inoculated into a 125 mL-scale three-dimensional microcarrier dynamic culture. When the P1 cells are harvested to reach 2.5×10 7 cells, the culture can be expanded to a 500 mL-scale three-dimensional microcarrier dynamic culture. When the cells in the 500 mL spinner flask proliferate at least 10-fold, at least 2.5×10 8 cells can be harvested, and then they can be inoculated into a 5 L large-scale process. After harvesting from 5 L, the number of harvested cells is at least 1×10 9 cells. In this process flow, only one dynamic system needs to be inoculated each time, and sufficient, uniform, and stable target cells can be harvested from a small amount of primary cells without the need for cumbersome large numbers of cell culture flasks or cell factories.

[0009] In addition, stem cells have strong proliferation ability and can divide rapidly in vivo. However, chondrocytes themselves have extremely low proliferation activity and need to proliferate under special circumstances. Moreover, during the culture process, chondrocytes are prone to dedifferentiation, changing from a normal morphology to a fibroblast-like shape and losing the ability to secrete cartilage matrix. It can be seen that the methods suitable for culturing stem cells in the prior art are not necessarily suitable for culturing chondrocytes.

[0010] Therefore, to solve the problem of cartilage injury repair, it is necessary to first find a culture method with a short culture time that can achieve a large amount of uniform and stable production of chondrocytes in a short time. Summary of the Invention

[0011] In order to overcome the defects and deficiencies in the prior art, the present invention provides a new method for large-scale dynamic culture of chondrocytes using three-dimensional microcarriers and its application.

[0012] In order to achieve the above invention object, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a method for preparing cartilage microtissue preparations and chondrocyte preparations using three-dimensional microcarriers, and the method includes the following steps: S1. Cartilage tissue extraction: S1.1. Cartilage tissue collection: The excised cartilage tissue is placed in a tissue preservation solution for storage and transportation; S1.2. Cartilage tissue extraction: The cartilage tissue is washed, divided into appropriate tissue blocks, soaked in an appropriate amount of culture medium, washed, centrifuged, and then an extraction enzyme is added, and it is placed in a shaker at 36 - 38°C and 100 - 200 rpm; S1.3. Inoculation of tissue suspension: After there are no obvious cartilage tissue masses in the bottle, take out the cartilage suspension, centrifuge it, resuspend the precipitate with an appropriate amount of culture medium, and inoculate the suspension into a culture bottle for culture; S2. Two-dimensional culture of chondrocytes: S2.1. Tissue medium change: Change the medium within 3 - 5 days after primary culture; S2.2. Cell digestion: When the cell confluence reaches 40 - 90% after medium change, digest the chondrocytes with cell digestive enzymes, centrifuge, and harvest the P0 generation of chondrocytes; S2.3. Cell passage: Inoculate the P0 generation of chondrocytes into a culture bottle for culture. When the cell confluence reaches 70 - 90%, harvest the P1 generation of chondrocytes; Repeat the above steps to harvest the P2 generation of chondrocytes; S3. Three-dimensional inoculation and culture of chondrocytes: S3.1. Preparation of chondrocytes: Passage the chondrocytes until harvest, resuspend the chondrocytes with an appropriate amount of culture medium, mix well, and set aside; S3.2. Preparation of microcarriers: Add microcarriers to an appropriate amount of culture medium, mix well, and soak the microcarriers completely, then set aside; S3.3. Inoculation of chondrocytes: Mix the microcarrier suspension and the chondrocyte suspension, and transfer them to a three-dimensional culture system; S3.4. Cell medium supplementation: After culturing in the three-dimensional culture system for 1 - 2 days, supplement with fresh culture medium; S4. Preparation of cartilage microtissue preparation: S4.1. After culturing chondrocytes on microcarriers for 3 - 5 days, harvest the cartilage microtissue (containing chondrocytes and microcarriers); S4.2. Collection of microtissue: Take out / pump out the microtissue suspension, transfer it to a centrifuge tube, centrifuge, and discard the supernatant; S4.3. Preparation of cartilage microtissue preparation: Resuspend the microtissue with an injectable buffer to prepare a cartilage microtissue preparation for transportation and storage, and set aside; S5. Preparation of chondrocyte preparation: S5.1. After culturing chondrocytes on microcarriers for 3 - 5 days, harvest the cartilage microtissue (containing chondrocytes and microcarriers); S5.2. Collection of microtissue: Take out / pump out the microtissue suspension, transfer it to a centrifuge tube, centrifuge, and discard the supernatant; S5.3. Microtissue lysis: Add an appropriate amount of microcarrier lysis solution to the microtissue precipitate to lyse the microcarriers. After the microcarriers are completely lysed, centrifuge and discard the supernatant; S5.4. Washing of chondrocytes: Add an appropriate amount of injectable buffer to the chondrocyte precipitate to wash the cells, centrifuge, and discard the supernatant; S5.5. Preparation of chondrocyte preparation: Add an appropriate amount of injectable buffer to the chondrocyte precipitate, resuspend the cells to prepare a chondrocyte preparation, and store it for transportation and later use; S6. Identification of the cartilage microtissue preparation or chondrocyte preparation.

[0013] As an alternative, in the above method, in S1.2, the extraction enzyme is a type II collagenase working solution with a mass percentage concentration of 0.01 - 0.5%.

[0014] As an alternative, in the above method, in S2.2, during the cell digestion process, the extraction condition is to place the cartilage tissue in a shaker at 36 - 38 °C and 100 - 200 rpm for treatment.

[0015] As an alternative, in the above method, in S3.3, the culture system is selected from: roller bottle, shake flask, culture bag or bioreactor.

[0016] As an alternative, in the above method, the microcarrier is 3D TableTrix ® microcarrier.

[0017] As an alternative, in the above method, in S5.3, the microcarrier lysate is 3D FloTrix ® Digest lysate.

[0018] As an alternative, in the above method, in S6, the identification method includes tissue staining, qPCR identification or flow cytometry detection.

[0019] In the second aspect, the present invention provides a cartilage microtissue preparation or chondrocyte preparation prepared by the method described in the first aspect above.

[0020] In the third aspect, the present invention provides the use of the cartilage microtissue preparation or chondrocyte preparation described in the second aspect above in the preparation of products for cartilage injury repair.

[0021] In a more specific embodiment, the present invention provides a method for preparing a cartilage microtissue preparation and a chondrocyte preparation using a three-dimensional microcarrier. The method specifically includes the following steps: (1) Cartilage tissue extraction: Step 1: Tissue collection: After the tissue sample is collected and tested negative for infectious disease items, the tissue is placed in a tissue collection bottle containing tissue preservation solution and stored and transported at 2 - 8 °C; Step 2: Tissue extraction: Take out the tissue in the tissue collection bottle and place it in a 90-cm culture dish. Wash it 3 times with the cartilage tissue washing solution. Use an 8-mm corneal trephine to divide the cartilage into cartilage blocks with a diameter of 8 mm. Use instruments such as scissors and forceps to cut the cartilage tissue into 1-mm 3 small pieces. During this period, soak it with 3D FloTrix ® mesenchymal stem cell serum-free medium to prevent tissue drying. Transfer the tissue pieces to a 50-mL sterile centrifuge tube, add saline to wash once, centrifuge at 500 g for 5 min. After centrifugation, aspirate and discard the supernatant. Add at least 3 times the volume of extraction enzyme to the tissue pieces in the 50-mL centrifuge tube, and place it at 37°C for extraction, and observe during this period; Step 3: First enzyme digestion and inoculation of tissue: After extraction, use a pipette to blow and mix the suspension. After mixing, let it stand. Aspirate the supernatant containing the tissue, centrifuge the supernatant to collect the cells, and resuspend the cells with 5 mL of 3D FloTrix ® mesenchymal stem cell serum-free medium. After inoculating the cells, place them in an incubator at 37°C and 5% CO2 for culture.

[0022] The following further describes the cartilage tissue extraction in terms of specific implementation methods: In Step 1, the tissue preservation solution is prepared by adding 2 mL of gentamicin sulfate to 200 mL of 3D FloTrix ® mesenchymal stem cell serum-free medium to prepare a tissue preservation solution with a volume ratio of 1%; In Step 2, the cartilage tissue washing solution is prepared by adding 1 mL of gentamicin sulfate to 500 mL of 0.9% sodium chloride injection to prepare a tissue washing solution containing 0.2% antibiotic; the extraction enzyme is a type II collagenase working solution with a mass percentage concentration of 0.01 - 0.5%; extraction at 36 - 38°C is carried out in a water bath, a carbon dioxide incubator or a shaker. If a shaker is used, the rotation speed is set at 100 - 200 rpm for 3 - 4 hours; In Step 3, the method of aspirating the supernatant containing the tissue includes aspirating the supernatant containing the tissue after the tissue pieces settle or are filtered. When inoculating the cells, the cells extracted from a cartilage block with a diameter of 8.00 mm are used as the cell inoculation amount for one T25 culture flask.

[0023] (2) Two-dimensional culture of chondrocytes: Step 1: Tissue medium change: Perform medium change treatment within 3 - 5 days after primary tissue culture; Step 2: Cell digestion: When the cell confluence reaches 40 - 90% after medium change, take a photo to record the cell state (for example, see Figure 1), add an appropriate amount of cleaning solution and wash twice. Use digestive enzymes to digest the cells. Shake the culture flask to evenly contact the digestive enzymes with the cells. Place the culture flask in a 5% CO2, 37 °C cell incubator and incubate for 2 - 6 min. Observe under a microscope until the cells retract and become round, then stop digestion. Add an equal amount of 3D FloTrix ® mesenchymal stem cell serum-free medium to terminate digestion, and centrifuge to collect the cells.

[0024] The following further describes the two-dimensional culture of chondrocytes in specific embodiments: During the cell digestion process in step two, use 1X TRYPLE TM SELECT digestive enzyme to digest the cells; During the cell washing process in step two, use sodium chloride injection (T25, 2 mL; T75, 5 mL; T175, 10 mL) to wash the cells; During the centrifugation process in step two, centrifuge at 500 g for 5 min to collect chondrocytes; After cell harvesting, inoculate the cells at a concentration of 6000 - 8000 cells / cm 2 again to inoculate and culture P1 - P2 cells.

[0025] (3) Three-dimensional microcarrier inoculation and culture: Step one: Microcarrier preparation: Take out the microcarriers according to the 3D TableTrix ® microcarrier W01 specification and place them in a 50 mL centrifuge tube. Use a pipette to take 10 mL of 3D FloTrix ® mesenchymal stem cell serum-free medium into the 50 mL centrifuge tube containing the microcarriers and soak the microcarriers completely; Step two: Reagent and consumable preparation: Start, clean, and disinfect the biosafety cabinet. Wipe the outer surfaces of the 3D FloTrix ® mesenchymal stem cell serum-free medium, microcarrier suspension, and 125 mL spinner flask with 75% sterile alcohol from the bottle cap downwards and immediately place them in the biosafety cabinet. Open the cap of the 125 mL spinner flask (non-breathable cap), suck the microcarrier suspension, place the pipette against the bottom of the culture flask and then eject the microcarrier suspension. After adding the microcarrier suspension to the culture flask, minimize the movement of the culture flask to prevent the microcarrier suspension from sticking to the side wall of the culture flask; Step three: Cell preparation: Passage the chondrocytes to P2 and then harvest the cells. Use a 5 / 10 mL pipette to aspirate and blow the cells 10 times to mix the cell suspension evenly. Immediately take out the mixed cell suspension and add it to a 40 mL 3D FloTrix ®In a 50 mL centrifuge tube of mesenchymal stem cell serum-free medium, the cells were mixed with 3D FloTrix ® and thoroughly mixed with mesenchymal stem cell serum-free medium, then added to a 125 mL spinner flask, and the cells were cultured in an intermittent culture mode; at the same time, two-dimensional T75 cells were inoculated and cultured for 3 days, and the cell status was observed at harvest; Step 4: Rotation speed adjustment: After 1-2 days of intermittent culture, turn on the constant speed mode, and the parameter is set to 40 rpm; Step 5: Cell fluid supplementation: Take out the prepared 3D FloTrix ® mesenchymal stem cell serum-free medium, equilibrate to room temperature, prepare the reagents and materials required for the experiment, wipe the outer surface with 75% sterile alcohol from the bottle cap downwards and immediately place it in the biosafety cabinet. Adjust the 3D TableTrix ® microcarrier combined with a stirred bioreactor to a constant speed of 50 rpm for standby; open the cap of the 125 mL spinner flask (non-breathable cap), supplement 25 mL of complete culture medium, tighten the cap; place it on the reactor console and mix for 5 min; Step 6: Sampling: Take 3 sterile EP tubes, label the sampling tubes, open the cap of the 125 mL spinner flask (non-breathable cap), and evenly take 1.1 mL of microtissue into the EP tubes, taking a total of 2 samples (the stirring of the turntable is not turned off during this operation process); then take 0.5 mL into another EP tube for cell staining; Step 7: Centrifuge the three taken samples at 500 g for 5 min. After centrifugation, gently discard the supernatant. Add 1 mL of 1× lysis working solution to each sample tube, gently pipette and mix well, incubate at 37°C for 20 min with constant temperature, pipette and mix well, and then incubate at 37°C for 10 min with constant temperature; gently suck out the precipitate in the sampling tube with a sterile Pasteur pipette and transfer it to a 96-well plate, and use a 100 μL micropipette to aspirate and discard the excess supernatant; add 100 μL of staining working solution to each well, cover the lid of the 96-well plate, and stain in the dark for 30 min; after staining, place it under an inverted fluorescence microscope and take pictures to record the cell attachment status of D1, and save the pictures (see Figure 2 , green fluorescence labels live cells, and red fluorescence labels dead cells).

[0026] The three-dimensional culture of chondrocytes is further described below with specific examples: The weight of the microcarrier in Step 1 is 50-100 mg; The 125 mL spinner flask in Step 2 is a disposable 125 mL breathable in-built impeller culture flask, abbreviated as 125 mL spinner flask; The number of cells inoculated in Step 3 is 2.5~5×10 6 cells; In Step 3, the medium-speed culture mode: 40 rpm for 5 min, 0 rpm for 25 min, 48 cycles; Before harvesting the two-dimensional cells inoculated in Step 3, take a comparison photo (see Figure 3 ).

[0027] In Step 4, after culturing the cells for 1 - 2 days, exit the medium-speed culture mode and start the constant-speed culture mode to culture the cells; In Step 6, during the cell sampling process, when using a 125 mL spinner flask for culture, sampling can be done at any time, which is simple and convenient. By sampling and counting at different culture durations, more effective monitoring of the cell growth state can be achieved. Taking the existing pictures as an example, the cell attachment per day (see Figure 4 , daily sampling and staining pictures during the 3-day culture in a 125 mL spinner flask), proliferation status, and glucose consumption can be observed (see Figure 5 , cell daily, proliferation, glucose consumption).

[0028] In Step 7, for cell staining, a live / dead cell viability / toxicity detection kit is used for microtissue staining.

[0029] (4) Three-dimensional microtissue preparation: Step 1: After culturing the cells for 3 - 5 days, open the cap of the 125 mL bottle, take 0.5 mL of microtissue into a sampling EP tube for staining and save the pictures, and then harvest the cells.

[0030] Step 2: Microtissue collection: Take out / pump out the microtissue suspension, transfer it to a centrifuge tube, place the centrifuge tube in a bench-top refrigerated centrifuge for centrifugation, set the centrifuge parameters to 500 g for 5 min, after centrifugation, discard the supernatant; Step 3: Preparation of cartilage microtissue preparation: Add a resuspension solution to resuspend the microtissue to make a cartilage cell preparation.

[0031] The following further explains the three-dimensional microtissue preparation in terms of specific implementation manners: In Step 3, the resuspension solution is 20 - 40 mL of injectable buffer.

[0032] (5) Three-dimensional microtissue cell identification: After harvesting the microtissue, send it for inspection and stain the microtissue (Alcian blue staining) (see Figure 6 ).

[0033] After harvesting the microtissue, send it for inspection and perform qPCR identification on the harvested cells (see Figure 7), two items, COL II and SOX9, were detected. COL II is the main collagen synthesized by chondrocytes. In the COL II detection project, it can be seen that the content of this item expressed by chondrocytes cultured in three dimensions is higher than that expressed by chondrocytes cultured in two dimensions; SOX9, as a transcription factor that plays a key role in chondrocyte differentiation and bone development, shows no significant difference in the expression levels in two-dimensional culture and three-dimensional culture in the SOX9 project.

[0034] After harvesting the microtissues, they were sent for inspection, and flow cytometry was performed on the harvested cells (see Figure 8 ). The flow cytometry detection items included: CD73, CD90, CD105, CD34, CD45, CD44, among which the positive indicators all exceeded 95% and the negative ones were all lower than 2%.

[0035] Therefore, compared with Matrigel culture and static three-dimensional cell culture, dynamic three-dimensional cell culture has obvious advantages: (1) A large number of cells can be effectively and evenly inoculated in dynamic three-dimensional cell culture; (2) Dynamic three-dimensional culture can effectively transport oxygen, nutrients, etc. to the inside of the carrier, providing a good environment for cell growth and metabolism; (3) A large number of uniform and stable cells can be obtained in a short time.

[0036] The present invention has the following beneficial effects compared with the prior art: (1) The preparation method of the present invention prepares chondrocytes in a three-dimensional stereoscopic microcarrier environment, increasing the interaction between cells and between cells and the extracellular matrix, more accurately simulating the actual microenvironment of cells in tissues, and the cell behavior characteristics are closer to the survival state in vivo.

[0037] (2) The advantage of the present invention is that a small amount of cartilage tissue is used, and after separation, extraction and large-scale in vitro culture, a large number of uniform and stable chondrocytes are obtained. The cell growth cycle is short, the operation method is simple, and the repeatability is strong.

[0038] (3) The present invention adopts the 3D TableTrix ® microcarrier of Sinogene Biotechnology Co., Ltd. combined with a stirred bioreactor can achieve the formation of chondrocyte microtissues (chondrocytes + microcarriers) in in vitro culture, jointly regulating the proliferation and differentiation of cells in terms of time and space, enabling the tissue structure and function to be retained to a large extent, and the chondrocytes in the microtissues maintain a high viability within a certain culture period.

[0039] (4) The present invention adopts the 3D TableTrix ® microcarrier of Sinogene Biotechnology Co., Ltd. combined with a stirred bioreactor can achieve real-time monitoring during the cell culture process in in vitro culture.

[0040] (5) The chondrocyte preparation obtained by the present invention can be made into a product for repairing cartilage damage, and the experimental results show that it has better clinical efficacy than the traditional microfracture (MF). Description of the Drawings

[0041] The present invention will be further described below in conjunction with the drawings.

[0042] Figure 1 : Passage 0 chondrocytes.

[0043] Figure 2 : Fluorescent staining map of chondrocytes cultured on 3D microcarriers for 1 day. Among them, Figure 2 A is a live cell staining picture, and the live cells are labeled with green fluorescence; Figure 2 B is a dead cell staining picture, and the dead cells are labeled with red fluorescence.

[0044] Figure 3 : Passage 2 chondrocytes cultured in 2D.

[0045] Figure 4 : Fluorescent staining map of chondrocytes on 3D microcarriers. Among them, Figure 4 A is the live cell staining map of chondrocytes cultured on 3D microcarriers for 1 day, Figure 4 B is the live cell staining map of chondrocytes cultured on 3D microcarriers for 2 days, Figure 4 C is the live cell staining map of chondrocytes cultured on 3D microcarriers for 3 days.

[0046] Figure 5 : Amplification and glucose consumption of chondrocytes on 3D microcarriers in 3 batches. Among them, Figure 5 A is the amplification curve of chondrocytes on 3D microcarriers; Figure 5 B is the glucose consumption curve of chondrocytes on 3D microcarriers.

[0047] Figure 6 : Staining map of chondrocytes cultured on 3D microcarriers.

[0048] Figure 7 : Expression of type II collagen (COL II) and key early chondrogenic transcription factor (SOX9) genes in 2D and 3D chondrocytes. Among them, Figure 7 A is the expression of COL II in 2D and 3D chondrocytes, Figure 7 B is the expression of SOX9 in 2D and 3D chondrocytes.

[0049] Figure 8 : Detection of cell surface markers of chondrocytes cultured on 3D microcarriers. Among them, Figure 8 A is the detection result of CD73, Figure 8B is the detection result of CD90, Figure 8 C is the detection result of CD105, Figure 8 D is the detection result of CD34, Figure 8 E is the detection result of CD45, Figure 8 F is the detection result of CD44. Detailed implementation manners

[0050] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0051] For those technical or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0052] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all commercially available products unless otherwise specified.

[0053] Preparation example: After flushing the tissue 3 times with 0.9% sodium chloride injection medium (containing 0.2% gentamicin sulfate), cut the cartilage tissue into pieces with a size of 1.0 mm 3 Transfer the tissue fragments to a 50 mL centrifuge tube, add 0.9% sodium chloride injection medium containing 0.2% gentamicin sulfate to wash once, and centrifuge at 500 g for 5 min.

[0054] After centrifugation, discard the supernatant, add 3 mL of 0.2% type II collagenase to the 50 mL centrifuge tube, and place it in a 37°C water bath to extract tissue cells.

[0055] After extraction, aspirate the supernatant, centrifuge at 500 g for 5 min, collect the cells, and inoculate and culture the cells two-dimensionally at 6000 cells / cm 2 Perform two-dimensional cell inoculation and culture.

[0056] When the cell confluence reaches 80% during two-dimensional culture, add 1X TRYPLE TM SELECT digestive enzyme (without phenol red) to digest the cells. After terminating the digestion, pipette the cells and collect them into a 50 mL centrifuge tube. Centrifuge the cells at 500 g for 5 min, collect the cells and inoculate and culture the cells two-dimensionally at 6000 cells / cm 2 Perform two-dimensional cell inoculation and culture, harvest the P2 generation chondrocytes, and adjust the cell concentration to 2.5×10 6 cells / mL for standby.

[0057] Add 50 mg of 3D TableTrix to a 50 mL centrifuge tube ® microcarrier W01. Pipette 10 mL of 3DFloTrix ® mesenchymal stem cell serum-free medium into the 50 mL centrifuge tube containing 50 mg of microcarriers and soak the microcarriers completely.

[0058] Wipe the outer surface of the microcarrier suspension and the 125 mL spinner flask downwards from the bottle cap with 75% sterile alcohol and immediately place it in the biosafety cabinet. Open the cap of the 125 mL spinner flask (non-breathable cap), aspirate the pre-soaked 10 mL microcarrier suspension, place the pipette against the bottom of the culture flask and then dispense the microcarrier suspension. After adding the microcarrier suspension to the culture flask, minimize the movement of the culture flask to prevent the microcarrier suspension from sticking to the side wall of the culture flask.

[0059] Add 40 mL of 3D FloTrix ® mesenchymal stem cell serum-free medium to a 50 mL centrifuge tube. Pipette the harvested passage 2 cells up and down 10 times with a 10 mL pipette to mix the cell suspension. Take out 2 mL of the mixed cell suspension (5×10 6 cells) and add it to the 50 mL centrifuge tube containing 40 mL of 3D FloTrix ® mesenchymal stem cell serum-free medium. Mix the cells and 3D FloTrix ® mesenchymal stem cell serum-free medium well and transfer it to a 125 mL spinner flask for three-dimensional cell culture. The three-dimensional culture is carried out using an intermittent speed culture mode. The parameters of the intermittent speed culture mode are set as follows: 40 rpm for 5 min; 0 rpm for 25 min; 48 cycles.

[0060] On the first day of three-dimensional culture: ① Rotation speed adjustment: After culturing in the intermittent speed mode for 16 - 18 h, turn on the constant speed mode, and the parameters of the constant speed mode are set to 40 rpm; ② Cell rehydration: Take out the prepared 3D FloTrix ® mesenchymal stem cell serum-free medium, equilibrate to room temperature, prepare the reagents and materials required for the experiment, wipe the outer surface downwards from the bottle cap with 75% sterile alcohol and immediately place it in the biosafety cabinet. Adjust the 3D TableTrix ® microcarrier combined with a stirred bioreactor to a constant speed of 50 rpm for standby; open the cap of the 125 mL spinner flask (non-breathable cap), add 25 mL of 3D FloTrix ® mesenchymal stem cell serum-free medium, tighten the cap; place it on the reactor console and mix for 5 min; ③ Sampling: Take 3 sterile EP tubes, label the sampling tubes, open the lid of the 125 mL spinner flask (non-breathable lid), evenly take 1.1 mL of microtissue into the EP tubes, and take a total of 2 samples (the stirring of the turntable is not turned off during this operation process); then take 0.5 mL into another EP tube for cell staining.

[0061] ④ Sample counting: Centrifuge the three taken samples at 500 g for 5 min. After centrifugation, gently discard the supernatant. Add 1 mL of 1× lysis working solution to each sample tube, gently pipette and mix well. After incubating at 37°C for 20 min with gentle pipetting and mixing, then incubate at 37°C for another 10 min with gentle pipetting and mixing. ⑤ Sample staining: Gently aspirate the microtissue precipitate in the sampling tube with a sterile Pasteur pipette, transfer it to a 96-well plate, and use a 100 μL micropipette to aspirate and discard the excess supernatant; add 100 μL of staining working solution to each well, cover the lid of the 96-well plate, and stain in the dark for 30 min; after staining, place it under an inverted fluorescence microscope to take pictures and record the attachment state of D1 cells, and save the pictures.

[0062] After culturing for 3 days in the three-dimensional culture method, perform cell sampling treatment. Gently aspirate the microtissue precipitate in the sampling tube with a sterile Pasteur pipette, transfer it to a 96-well plate, and use a 100 μL micropipette to aspirate and discard the excess supernatant; add 100 μL of staining working solution to each well, cover the lid of the 96-well plate, and stain in the dark for 30 min; after staining, place it under an inverted fluorescence microscope to take pictures and record the growth state of D4 cells, and save the pictures.

[0063] Transfer the microtissue suspension in the 125 mL spinner flask to a centrifuge tube, centrifuge at 500 g for 5 min, discard the supernatant, add an injectable buffer, resuspend the microtissue to prepare a cartilage microtissue preparation, and transport and store it for later use.

[0064] Effect implementation example: In the operation, the examples were used for the extraction and culture of cartilage, and the following harvested data were obtained: (1) Method of collecting cells after enzymatic digestion of tissue during tissue extraction: During the experiment, after enzymatic digestion of the tissue, one group collected cells by filtering the tissue suspension, and the other group collected cells by the sedimentation method. During the experiment, it was found that there was no significant difference between the two groups. Considering comprehensively, the tissue was collected by sedimentation to collect cells.

[0065] (2) Screening of cell seeding density in two-dimensional cell seeding: The preliminary culture of chondrocytes adopts the two-dimensional amplification method, but there is a phenomenon of chondrocyte dedifferentiation in two-dimensional culture. Therefore, it is determined that the cell proliferation in the preliminary two-dimensional culture needs to be controlled within a suitable range to avoid chondrocyte dedifferentiation in order to better culture chondrocytes in good condition.

[0066] After multiple experimental tests, it is shown in all two-dimensional data that when the seeding density is adjusted to 5000 - 1000 / cm 2 , the cell proliferation multiple of the seeded cells is relatively high, and the cell state is good. Discard other densities within this range. Considering comprehensively, the density of 5000 - 1000 / cm 2 is used as the two-dimensional cell seeding density.

[0067] (3) Confirmation of cell passage number: The cells are continuously passaged for several generations, and the cell proliferation multiple gradually decreases as the passage number increases.

[0068] Experimental group 1: Table 1 As shown in Table 1, the proliferation of C001 cells after 3 consecutive passages is more than 8 times, and the dedifferentiation phenomenon of chondrocytes after passage P3 is obvious. Therefore, cells within 3 passages are used as the preparation cells within the limited passage number of cells, which meets the final cell harvest requirements.

[0069] Taking experimental group 1 as an example, when the primary cell number is 2.54×10 6 cells, about 9.76×10 9 uniform and stable cells can be finally obtained, and the operation is convenient.

[0070] Experimental group 2: Table 2 As shown in Table 2, the proliferation of C002 cells after 3 consecutive passages is more than 8 times, and the dedifferentiation phenomenon of chondrocytes is obvious as the passage number increases. Cells within 3 passages are all used as the preparation cells, which meets the final cell harvest requirements.

[0071] Taking experimental group 2 as an example, when the primary cell number is 8.18×10 6 cells, about 2.13×10 10 uniform and stable cells can be finally obtained, and the operation is convenient.

[0072] (4) Screening of cell seeding density in three-dimensional cell seeding: During the three-dimensional culture process, if the cell seeding is too few, it will lead to slow cell growth and poor cell state; if the cell seeding is too much, the number of added microcarriers needs to be verified, and it is necessary to consider whether the volume of the microcarriers is suitable for the final preparation requirements. Therefore, it is necessary to verify the appropriate cell seeding density and the appropriate number of microcarriers to meet the final preparation requirements.

[0073] After multiple experimental demonstrations, the experiments were finally divided into 3 groups: ① Take 5 tablets (100 mg) of microcarriers and inoculate 2.5×10 6 cells, culture for 4 days and calculate the cell attachment and growth status; ② Take 2.5 tablets (50 mg) of microcarriers and inoculate 5×10 6 cells, culture for 3 - 4 days and calculate the cell attachment and growth status; ③ Take 2.5 tablets (50 mg) of microcarriers and inoculate 2.5×10 7 cells, culture for 1 day and calculate the cell attachment status.

[0074] Table 3 The results are shown in Table 3. According to the results of the three experimental groups, all three experimental groups meet the final preparation requirements. In order to better meet the final preparation requirements, we adopt the cell inoculation method of group ②, and both the cell volume and the total number of harvested cells meet the requirements for final harvest.

[0075] (5) Re - confirmation of the cell inoculation quantity during three - dimensional culture: After the normal harvest of the second - generation two - dimensional cells (P3), inoculate them into a 125 mL spinner flask at a concentration of 2.50×10 6 cells / 0.1 g of carrier, and obtain the growth curve, growth status and sugar consumption of the cells by sampling and counting daily, so as to more accurately monitor the growth status of the cells at different times. The results are shown in Table 4.

[0076] Table 4 (6) Confirmation of the cell inoculation method in three - dimensional cell inoculation: In three - dimensional culture, the cell attachment rate of static culture is relatively low. The chondrocytes are cultured by the method of three - dimensional dynamic culture, but the rotation speed setting, the duration setting of different rotation speeds, etc. in dynamic culture need to be verified multiple times before being finally determined.

[0077] Multiple dynamic rotation speeds were tested, and the rotation speed with the best proliferation state and the best cell state was selected from them. The best rotation speed setting was obtained as 40 rpm for 5 min; 0 rpm for 25 min, and the rotation speed was adjusted to a constant speed after 16 - 18 h, so as to meet the final preparation requirements of the cells.

[0078] (7) Confirmation of cell viability: Aliquot the completed micro - tissue suspension of the preparation into 15 mL centrifuge tubes, keep the environmental temperature at 2 - 8℃, simulate the preparation requirements, and perform lysis and counting at 0 h, 2 h, 4 h, and 6 h respectively.

[0079] Table 5 The results are shown in Table 5. When the microtissue preparation was stored for 6 hours at 2 - 8°C, the cell viability could still be maintained above 95%, proving that the cell viability in the microtissue was relatively high.

[0080] For the cartilage microtissue preparation and chondrocyte preparation prepared by the method of the present invention, the present invention carried out corresponding clinical studies. After the microtissue injection, according to the law of rehabilitation after cartilage injury repair and referring to the opinions of clinical experts, the subjects were followed up at 1.5 months, 3 months, 6 months, 1 year, and 2 years after the operation, and the effects were evaluated by the following methods to assess the tolerance, vital signs, physical examination, and occurrence of adverse events of the subjects. The abnormal values of laboratory tests were used as safety evaluation indicators. Plain magnetic resonance imaging (MRI) was used for imaging examination of articular cartilage injury. Secondly, VAS pain score, ICRS score, AOFAS score, FAAM score for measuring ankle flexibility, Tegner score, and Lysholm score were also added as efficacy evaluation indicators. For example, AOFAS score: Pain, walking ability, gait, range of motion of the lower leg joint, ankle stability, and joint alignment were evaluated respectively, with a full score of 100 points, and a score of 75 or above was considered excellent.

[0081] The test results at each evaluation time point showed that the patients treated with autologous chondrocyte microcarrier implantation (ACMI) using the cartilage microtissue preparation and chondrocyte preparation of the present invention for ankle talar cartilage injury had good tolerance, and no abnormal results were found in terms of their vital signs, physical examination, and laboratory test results. In addition, the imaging examination results of articular cartilage injury and the results of each score showed that the clinical efficacy of autologous chondrocyte microcarrier implantation was superior to that of traditional microfracture (MF).

[0082] In summary, arthroscopic autologous chondrocyte microcarrier implantation (ACMI) using the chondrocyte preparation prepared by the present invention for the treatment of ankle talar cartilage injury is a safe method and has good clinical application prospects.

[0083] 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 its equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A method for preparing cartilage microtissue preparations and chondrocyte preparations using three-dimensional microcarriers, characterized in that: The method includes the following steps: S1. Cartilage tissue extraction; S2. Two-dimensional culture of chondrocytes; S3. Three-dimensional culture of chondrocytes: S3.

1. Preparation of chondrocytes; S3.

2. Preparation of microcarrier suspension; S3.

3. Seeding of chondrocytes: Mix the microcarrier suspension and chondrocyte suspension, and transfer them to a three-dimensional culture system for culture; S4. Preparation of cartilage microtissue preparation: After three-dimensional culture of chondrocytes for 3-5 days, harvest the cartilage microtissue containing chondrocytes and microcarriers, and prepare it into a cartilage microtissue preparation; S5. Preparation of chondrocyte preparation: After three-dimensional culture of chondrocytes for 3-5 days, harvest the cartilage microtissue containing chondrocytes and microcarriers, lyse the microtissue to obtain chondrocytes, and prepare it into a chondrocyte preparation; S6. Identification of cartilage microtissue preparation or chondrocyte preparation.

2. The method according to claim 1, wherein: In S1, it includes a cartilage tissue treatment step: wash the cartilage tissue, divide the cartilage tissue into appropriate tissue blocks, soak it in an appropriate amount of culture medium, wash, centrifuge, then add extraction enzyme, and place it in a shaker at 36-38 °C and 100-200 rpm for treatment.

3. The method according to claim 2, wherein: The extraction enzyme is a type II collagenase working solution with a mass percentage concentration of 0.01-0.5%.

4. The method according to claim 1, characterized in that: The microcarrier is a three-dimensional porous microcarrier.

5. The method according to claim 1 or claim 4, characterized in that: The microcarrier is 3D TableTrix ® microcarrier 6. The method according to claim 1, characterized in that: In S3.3, the culture system is selected from: spinner flask, shake flask, culture bag or bioreactor.

7. The method according to claim 1, characterized in that: In S6, the identification method includes tissue staining, qPCR identification or flow cytometry detection.

8. A cartilage microtissue preparation or chondrocyte preparation prepared by the method according to any one of claims 1 to 7.

9. Use of the cartilage microtissue preparation or chondrocyte preparation according to claim 8 in the preparation of a product for cartilage injury repair.

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