Culture container for tissue engineering cartilage scaffold

Abstract

The invention provides a culture container of a tissue engineering cartilage scaffold, comprising: a first container having a first opening and a first chamber; the first cover body is mounted on the first container and used for covering the first opening; the second container is used for being placed in the first cavity, the second container is provided with a second opening and a second cavity, the second cavity is used for placing a tissue engineering cartilage scaffold, and the bottom of the second container has a hydrophobic and breathable function, so that a gas medium can enter the second cavity through the bottom of the second container; the second cover body is mounted on the second container and used for covering the second opening, and the second cover body is connected with the first cover body; the jacking and pressing part is mounted on the second cover body, is positioned on one side, deviating from the first cover body, of the second cover body, and is used for limiting the displacement distance of the tissue engineering cartilage scaffold in the second container towards the second opening direction; the technical problem that the collagen scaffold needs to be repeatedly clamped in an existing culture mode is solved, and the collagen scaffold is prevented from floating or adhering to the wall in the culture and transportation process.

Description

Technical Field

[0001] The present application relates to the technical field of cell culture equipment, and in particular to a culture container for a tissue engineering cartilage scaffold. Background Art

[0002] The "human tissue-engineered regenerative cartilage" treatment technology belongs to the third category of medical technology in the field of tissue engineering medicine. It is currently the cutting-edge clinical application technology used internationally to treat acute and chronic articular cartilage defects. This technology is based on the principle of matrix-induced autologous chondrocyte implantation (MACI), and is a new generation of autologous cell cartilage repair methods. Tens of thousands of patients worldwide have received this type of treatment and achieved good results. Its core treatment process includes: obtaining a small amount of healthy cartilage tissue from the patient, isolating and culturing the chondrocytes therein and amplifying them in vitro, and then compounding these cells with a three-dimensional collagen scaffold to form tissue-engineered cartilage with biological functions. Finally, it is transplanted to the patient's cartilage defect site through surgery to achieve structural repair and functional reconstruction. Among them, the composite construction process of chondrocytes and collagen scaffolds is the key link in the entire technical process, which directly affects the final treatment effect.

[0003] In the current tissue-engineered cartilage construction process, the following steps are typically used to complete the composite of cells and collagen scaffolds: First, a sterile collagen scaffold is placed in the center of a petri dish. Then, a suspension of expanded and cultured chondrocytes is evenly injected or dripped onto the scaffold surface. After inoculation, the petri dish is transferred to a CO2 incubator and incubated for approximately 30 minutes to allow the cells to initially attach to the scaffold structure. The scaffold is then picked up with forceps and rinsed with an appropriate amount of Dulbecco's phosphate buffered saline (DPBS) to remove unattached free cells. The rinse solution is then aspirated from the petri dish to count the cells and calculate the cell attachment rate on the scaffold. After confirming the attachment effect, the scaffold is transferred to a transport bottle, adjusted to its front and back, and a transport protective solution is added. The scaffold is sealed and transported to the hospital. Before surgery, medical staff remove the scaffold from the transport bottle using forceps, place it on a tray, and again distinguish the front and back. It is cut as needed in preparation for implantation.

[0004] Although the above-mentioned construction method has been applied clinically to a certain extent, it still has obvious operational defects. In particular, in key operations such as rinsing, transferring to a transport bottle, adjusting the front and back, and finally taking it out of the transport bottle, the collagen scaffold needs to be repeatedly clamped with tweezers. Since the collagen scaffold itself is soft in texture and delicate in structure, and the chondrocytes are only in a preliminary attachment state and have not yet been firmly bound to the inside of the scaffold, it is very easy to cause cell shedding or damage during frequent mechanical clamping, thereby reducing cell utilization. In addition, due to the physical properties of the collagen scaffold, it is easy to be suspended in the preservation solution. During transportation, it is subjected to the shear force of the preservation solution, which further causes cell damage and shedding. If the product floats to the surface of the preservation solution or adheres to the wall of the container, it will cause the product to dry up locally, reduce cell activity, and affect the treatment effect. In addition, repeated clamping operations not only increase the risk of contamination, but also reduce the consistency and repeatability of the preparation process, limiting the promotion and application of this technology on a larger scale. Therefore, there is an urgent need to develop a new tissue engineering cartilage construction container device and a method for using the container device that can reduce the number of clamping times, avoid cell damage, and improve construction efficiency and stability. Summary of the Invention

[0005] In the embodiments of the present application, a culture container for a tissue engineering cartilage scaffold is provided to avoid the defects of the existing culture method, which requires repeated clamping of the collagen scaffold and the collagen scaffold floating or adhering to the wall during culture and transportation, and to solve the technical problem of damage to chondrocytes caused by such defects. The technical solution is as follows:

[0006] In an embodiment of the present application, a culture container for a tissue engineering cartilage scaffold is provided, comprising: a first container having a first opening and a first chamber; a first cover body installed on the first container, for covering the first opening; a second container for being placed in the first chamber, the second container having a second opening and a second chamber, the second chamber being used to place the tissue engineering cartilage scaffold, the bottom of the second container having a hydrophobic and breathable function, so that the gas medium can enter the second chamber through the bottom of the second container; a second cover body installed on the second container, for covering the second opening, the second cover body being connected to the first cover body; and a pressing component installed on the second cover body, located on the side of the second cover body facing away from the first cover body, for limiting the displacement distance of the tissue engineering cartilage scaffold in the second container toward the second opening.

[0007] In one embodiment, it also includes: a first functional layer having a hydrophobic and breathable function, the first functional layer being arranged at the bottom of the second container and located in the second chamber; a plurality of first air holes being provided at the bottom of the second container, the first functional layer covering the first air holes.

[0008] In one embodiment, the bottom of the second container has a third opening; the culture container of the tissue engineering cartilage scaffold also includes: a third cover body, installed on the second container, used to cover the third opening, and the third cover body is provided with a plurality of first air holes; a first functional layer body, having a hydrophobic and breathable function, the first functional layer body is configured on the third cover body, and is located in the second chamber to cover the first air holes.

[0009] In one embodiment, the pressing component includes: a first connecting member connected to a surface of the second cover body facing away from the first cover body; a pressure plate connected to an end of the first connecting member away from the second cover body;

[0010] When the second cover is closed on the second opening, the first connecting member extends into the second chamber, so that the pressure plate can limit the displacement of the tissue engineering cartilage scaffold in the second container toward the second opening.

[0011] In one embodiment, the second cover has a hydrophobic and breathable function, so that the gas medium enters the second chamber through the second cover.

[0012] In one embodiment, it further includes: a second functional layer having a hydrophobic and breathable function, the second functional layer being disposed on the second cover; a plurality of second air holes being provided on the second cover, the second functional layer covering the second air holes.

[0013] In one embodiment, the pressing component includes: a support plate, which is arranged on the second cover body, the second functional layer body is located between the support plate and the cover body, and the support plate is provided with third air holes corresponding to the second air holes one by one; a first connecting member, which is connected to a side surface of the support plate away from the second functional layer body; and a pressure plate, which is connected to an end of the first connecting member away from the support plate;

[0014] When the second cover is closed on the second opening, the first connecting member extends into the second chamber, so that the pressure plate can limit the displacement of the tissue engineering cartilage scaffold in the second container toward the second opening.

[0015] In one embodiment, the first container and the first cover are connected by a first threaded structure; the second container and the second cover are connected by a second threaded structure.

[0016] In one embodiment, the second connecting member, the first cover body and the second cover body are connected through the second connecting member.

[0017] In one embodiment, when the second container is placed in the first chamber, a gap is formed between the inner wall of the first container and the outer wall of the second container, and the gap is used to accommodate the shock-absorbing sponge.

[0018] Compared with the prior art, the culture container of the tissue engineering cartilage scaffold proposed in the above technical solution, when used, uses sterile tweezers to place the collagen scaffold that needs to be compounded with chondrocytes in the second container, and evenly drips the prepared chondrocyte suspension onto the collagen scaffold. After tightening the second cover on the second container so that the top pressure component presses against the collagen scaffold, the second container is placed face down in a carbon dioxide incubator and incubated for 30 minutes. Subsequently, an appropriate amount of Dulbecco's phosphate buffer (DPBS) is added to the second container for rinsing. The cells in the Dulbecco's phosphate buffer are counted and the attachment rate of the chondrocytes on the collagen scaffold is calculated. A transport protection solution containing 10% autologous serum is added to the second container, and the second container is placed face down in a carbon dioxide incubator for 24 hours. A shockproof sponge is placed in the first chamber of the first container, and the second container is placed in the first container so that the tissue engineering cartilage scaffold can be reliably transported to the hospital for replantation. When used, medical staff use tweezers to transfer the tissue engineering cartilage scaffold from the second container to a tray for cutting and use. Since the second cover is connected to the first cover, and the second cover can be installed on the second container, when operating the tissue engineering cartilage scaffold, the second container can be taken through the first cover to culture the collagen scaffold in the second container. In order to achieve the purpose of incubating and culturing chondrocytes, the bottom of the second container has a hydrophobic and breathable function, so that the gas medium in the second chamber of the second container can be exchanged with the gas medium of the carbon dioxide incubator. When the culture container proposed in this application is used, it is achieved that from the beginning of culturing chondrocytes to the time when medical staff cuts and uses it, only tweezers are used twice to clamp the collagen scaffold for necessary operations, which effectively improves the culture quality of chondrocytes and reduces the risk of chondrocyte damage and falling.

[0019] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0021] Figure 1 This is a schematic diagram of the three-dimensional structure of the culture container of the tissue engineering cartilage scaffold in the embodiment of the present application;

[0022] Figure 2 This is a cross-sectional view of a culture container for a tissue engineering cartilage scaffold in an embodiment of the present application;

[0023] Figure 3 This is an exploded view of a culture container for a tissue engineering cartilage scaffold in an embodiment of the present application;

[0024] Figure 4 This is a schematic diagram of arranging a third opening on the second container in an embodiment of the present application;

[0025] Figure 5 This is a bar graph showing the comparative attachment rates of chondrocytes in the examples of this application;

[0026] Figure 6 This is a bar graph showing the comparative attachment rates of chondrocytes after simulated transport in the examples of this application;

[0027] Figure 7 This is a comparison chart of the chondrocyte migration ability test in the examples of this application;

[0028] Figure 8 This is a photo of the actual growth of chondrocytes observed under an electron microscope in the examples of this application;

[0029] Figure 9 This is a bar chart comparing changes in pH values of chondrocytes in culture medium in the examples of this application;

[0030] Figure 10 This is a photo of the actual state of chondrocytes in the culture medium of the embodiment of the present application observed under an electron microscope.

[0031] Reference numerals:

[0032] 1. First container;

[0033] 11. First opening; 12. First chamber;

[0034] 2. a first cover;

[0035] 3. Second container;

[0036] 31. Second opening; 32. Third opening; 33. Second chamber;

[0037] 4. Second cover;

[0038] 41. Second vent hole;

[0039] 5. Top pressure components;

[0040] 51. First connecting member; 52. Pressure plate; 53. Support plate;

[0041] 531, third vent hole;

[0042] 6. The third cover;

[0043] 61. First vent hole;

[0044] 7. First functional layer;

[0045] 8. Second functional layer;

[0046] 9. Second connecting piece. DETAILED DESCRIPTION

[0047] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0048] First embodiment

[0049] Reference Figures 1 to 4 As shown, in an embodiment of the present application, a culture container for a tissue engineering cartilage scaffold is proposed, and the culture container for the tissue engineering cartilage scaffold may include: a first container 1, having a first opening 11 and a first chamber 12; a first cover 2, installed on the first container 1, for covering the first opening 11; a second container 3, for being placed in the first chamber 12, the second container 3 having a second opening 31 and a second chamber 33, the second chamber 33 being used to place the tissue engineering cartilage scaffold, and the bottom of the second container 3 having a hydrophobic and breathable function, so that the gas medium can enter the second chamber 33 through the bottom of the second container 3; a second cover 4, installed on the second container 3, for covering the second opening 31, the second cover 4 being connected to the first cover 2; and a pressing component 5, installed on the second cover 4, located on the side of the second cover 4 away from the first cover 2, for limiting the displacement distance of the tissue engineering cartilage scaffold in the second container 3 toward the second opening 31.

[0050] Specifically, in the technical solution adopted in the present application, the first chamber 12 in the first container 1 can accommodate the second container 3, the first container 1 is provided with a first cover 2 covering the first opening 11, and the second container 3 is provided with a second cover 4 covering the second opening 31; the first cover 2 is connected to the second cover 4 so that when the first container 1 and the first cover 2 are disassembled, the first cover 2 can drive the second container 3 to detach from the first chamber 12 through the first opening 11 through the second cover 4, thereby achieving the second container 3 being removed from the first chamber 12 without using additional tools and contact, and the second chamber 33 of the second container 3 is used to place the tissue function cartilage scaffold. In order to enable gas exchange in the second chamber 33, the bottom of the second container 3 has a breathable function, specifically a hydrophobic ventilation function. It can be explained that the bottom of the second container 3 can allow the gas medium to pass through, but can block the liquid medium, thereby preventing the liquid medium in the second chamber 33 from flowing out through the bottom of the second container 3. The pressing component 5 is arranged on the side of the second cover body 4 facing away from the first cover body 2, so that when the second cover body 4 is covered on the second opening 31, the pressing component 5 enters the second chamber 33 through the second opening 31, so as to be able to press against the tissue engineering cartilage scaffold in the second chamber 33, so that the tissue functional cartilage scaffold is close to the bottom of the second container 3. When the second container 3 is placed in the first chamber 12 of the first container 1, since the bottom of the second container 3 is facing downward, the pressing component 5 can press against the tissue engineering cartilage scaffold to be completely immersed in the liquid medium in the second chamber 33; and when the tissue engineering cartilage scaffold needs to interact with the gas medium, the first container 1 can be separated from the first cover body 2, and the second container 3 can be separated from the first chamber 12 of the first cover body 2 through the second cover body 4, and the second container 3 can be inverted through the first cover body 2, and the pressing component 5 can support the tissue engineering cartilage scaffold to remain in a position close to the bottom of the second container 3, so as to better interact with the gas medium outside the second container 3.

[0051] Further, refer to Figure 3 As shown, in some embodiments, it also includes: a first functional layer 7, which has a hydrophobic and breathable function, and the first functional layer 7 is arranged at the bottom of the second container 3 and is located in the second chamber 33; a plurality of first air holes 61 are provided at the bottom of the second container 3, and the first functional layer 7 covers the first air holes 61.

[0052] Specifically, in the technical solution adopted in the present application, in order to achieve the hydrophobic and breathable function mentioned in the present application, a first functional layer 7 can be configured at the bottom of the second container 3. The first functional layer 7 can be a breathable silicone membrane. It should be explained that the breathable silicone membrane achieves selective permeability through precisely designed micropores, and the diameter of the micropores is usually 0.2 to 0.3 μm. The diameter of gas molecules such as water vapor, oxygen, and carbon dioxide is much smaller than the micropores (about 0.0004 μm) and can diffuse freely. Liquids are blocked: liquid water molecules form larger molecular clusters (about 20 μm) due to surface tension and cannot pass through the micropores; liquids such as blood and oils are also blocked. For example, a medical silicone membrane contains 12 to 12.5 million micropores per square millimeter, with an average micropore diameter of 0.2 to 0.3 μm, which is both breathable and completely waterproof. A plurality of first air holes 61 are provided on the bottom side wall of the second container 3, and the first functional layer 7 covers each of the first air holes 61. Thus, with the cooperation of each of the first air holes 61 and the first functional layer 7, the bottom of the second container 3 has a hydrophobic and breathable function, so that the second chamber 33 can store liquid media while being able to interact with gas.

[0053] Further, refer to Figure 3 and Figure 4 As shown, in some embodiments, the bottom of the second container 3 has a third opening 32; the culture container of the tissue engineering cartilage scaffold also includes: a third cover body 6, installed on the second container 3, for covering the third opening 32, and a plurality of first air holes 61 are provided on the third cover body 6; a first functional layer body 7, having a hydrophobic and breathable function, the first functional layer body 7 is configured on the third cover body 6, and is located in the second chamber 33 to cover the first air holes 61.

[0054] Specifically, in the technical solution adopted in the present application, in the modified embodiment of the second container 3, the structure of the first functional layer 7 is the same as that in the above embodiment. The difference is that the second container 3 can be set as a bottomless container so that the bottom of the second container 3 has a third opening 32, and the culture container proposed in the present application also includes: a third cover 6 for covering the third opening 32, and the third cover 6 is detachably mounted on the second container 3 so that each first air hole 61 is opened on the third cover 6 to facilitate the disassembly and separation of the third cover 6 from the second container 3, so that the second container 3 can be more thoroughly cleaned and disinfected before use, and the installation of the first functional layer 7 is more accurate and convenient.

[0055] Further, refer to Figure 2 As shown, in some embodiments, the pressing component 5 includes: a first connecting member 51 connected to the surface of the second cover 4 on one side facing away from the first cover 2; a pressure plate 52 connected to the end of the first connecting member 51 away from the second cover 4;

[0056] When the second cover 4 covers the second opening 31 , the first connector 51 extends into the second chamber 33 , so that the pressure plate 52 can limit the displacement of the tissue engineering cartilage scaffold in the second container 3 toward the second opening 31 .

[0057] Specifically, in the technical solution adopted in the present application, the first connecting member 51 is configured as an elongated strip, specifically a straight rod-shaped structure, which is connected to the side of the second cover body 4 facing away from the first cover body 2. In this embodiment, the first connecting member 51 can be fixedly connected to the second cover body 4, or a detachable installation method can be formed by interference fit. The length of the first connecting member 51 can adapt to the depth of the second chamber 33, and the pressure plate 52 is connected to the end of the first connecting member 51 away from the second cover body 4, so that when the second cover body 4 is covered on the second opening 31, the pressure plate 52 can be closer to the bottom of the second container 3 through the length of the first connecting member 51, so as to achieve the purpose of supporting the tissue engineering cartilage scaffold in the second chamber 33. The tissue engineering cartilage scaffold is limited to the bottom of the second container 3 by the pressure plate 52, so that the tissue engineering cartilage scaffold can be immersed in the transport protection liquid throughout the process when necessary, effectively reducing the impact of the transport protection liquid on the tissue engineering cartilage scaffold and avoiding the risk of the transport protection liquid flipping and the float drying up, thereby improving the cell activity of the product. It can be explained that the necessary situation may be during the process of transporting the tissue engineering cartilage scaffold, or the whole process of placing the tissue engineering cartilage scaffold into a carbon dioxide incubator for culturing.

[0058] Second embodiment

[0059] Further, refer to Figure 3 As shown, in some embodiments, the second cover body 4 has a hydrophobic and breathable function, so that the gas medium enters the second chamber 33 through the second cover body 4.

[0060] Specifically, in the technical solution adopted by the present application, the second embodiment differs from the first embodiment in that the second cover 4 also has a hydrophobic and breathable function, allowing the gas medium to exchange gas within the second chamber 33 through the bottom of the second container 3 and the second cover 4, thereby improving the gas flowability of the second chamber 33. After both the second cover 4 and the bottom of the second container 3 have hydrophobic and breathable functions, it can ensure that the chondrocytes on the tissue engineering cartilage scaffold in the second container 3 can exchange gas with the carbon dioxide incubator, thereby reducing the risk of cell hypoxia and acidification of the culture medium. The growth environment of the chondrocytes is better than that of a non-air-permeable culture method.

[0061] Further, refer to Figure 3As shown, in some embodiments, it also includes: a second functional layer 8 with hydrophobic and breathable function, the second functional layer 8 is configured on the second cover 4; the second cover 4 is provided with a plurality of second air holes 41, and the second functional layer 8 covers the second air holes 41.

[0062] Specifically, in the technical solution adopted in the present application, in order to realize the hydrophobic and breathable function of the second cover body 4, the present application proposes an embodiment, specifically, a second functional layer body 8 is configured on the second cover body 4, the second functional layer body 8 can be a breathable silicone mold, and a plurality of second breathable holes 41 are opened on the second cover body 4 to cover the second functional layer body 8 on each second breathable hole 41, so that under the cooperation of the second functional layer body 8 and each second breathable hole 41, the second cover body 4 has a hydrophobic and breathable function, and the gas medium outside the second chamber 33 and the gas medium in the second chamber 33 interact through the second functional layer body 8 and the second breathable holes 41, while the liquid medium in the second chamber 33 is retained in the second chamber 33 through the barrier of the second functional layer body 8, thereby preventing the loss of the liquid medium in the second chamber 33.

[0063] Further, refer to Figure 3 As shown, in some embodiments, the pressing component 5 includes: a support plate 53, which is arranged on the second cover body 4, the second functional layer body 8 is located between the support plate 53 and the cover body, and the support plate 53 is provided with third air holes 531 corresponding to the second air holes 41; a first connecting member 51, which is connected to the side surface of the support plate 53 away from the second functional layer body 8; and a pressure plate 52, which is connected to the end of the first connecting member 51 away from the support plate 53;

[0064] When the second cover 4 covers the second opening 31 , the first connector 51 extends into the second chamber 33 , so that the pressure plate 52 can limit the displacement of the tissue engineering cartilage scaffold in the second container 3 toward the second opening 31 .

[0065] Specifically, in the technical solution adopted in the present application, compared with the first embodiment, a support plate 53 for fixing the second functional layer 8 is added to the top pressure component 5. The support plate 53 is installed in the second cover body 4 and is located on the side of the second cover body 4 facing away from the first cover body 2. The second function is fixed between the inner surface of the second cover body 4 and the support plate 53 in a clamping manner, and third air holes 531 corresponding to each second air hole 41 are opened on the support plate 53; one end of the first connecting member 51 is connected to the side surface of the support plate 53 facing away from the second functional layer 8, and the other end of the second connecting member 9 is connected to the pressure plate member 52. Since when the second cover body 4 covers the second opening 31, the first connecting member 51 and the pressure plate member 52 are both located in the first chamber 12, the diameters of the first connecting member 51 and the pressure plate member 52 are smaller than the diameter of the second chamber 33, the first connecting member 51 is a rod-shaped structure, and the pressure plate member 52 is a disc-shaped structure, so that the diameter of the first connecting member 51 is smaller than the diameter of the pressure plate member 52. Therefore, when the pressure plate member 52 supports the tissue engineering cartilage scaffold, the gap formed between the pressure plate member 52 and the second chamber 33 can allow the liquid medium to flow in the second chamber 33, so that the liquid medium in the second chamber 33 can interact with the gas outside the second container 3 through the second cover body 4.

[0066] Further, refer to Figure 3 As shown, in some embodiments, the first container 1 is connected to the first cover body 2 through a first threaded structure; the second container 3 is connected to the second cover body 4 through a second threaded structure.

[0067] Specifically, in the technical solution adopted in the present application, a first external thread structure can be provided on the radial outer side surface of the first container 1 near the first opening 11, and a first internal thread structure compatible with the first external thread structure can be provided on the inner surface of the first cover body 2, so that when the first cover body 2 is covered on the first opening 11, the first external thread structure is matched with the first internal thread structure by rotating the first cover body 2, thereby realizing the installation of the first cover body 2 on the first container 1 through threaded matching; and a second external thread structure is provided on the radial outer side surface of the second container 3 near the second opening 31, and a second internal thread structure compatible with the second external thread structure is provided on the inner surface of the second cover body 4, so that when the second cover body 4 is covered on the second opening 31, the second external thread structure is matched with the second internal thread structure by rotating the second cover body 4, thereby realizing the second cover body 4 on the second container 3 through threaded matching.

[0068] In some embodiments, the third cover body 6 can also be installed on the bottom of the second container 3 through the same threaded fitting technology principle, and the bottom of the second container 3 can also be embedded in the third cover body 6 through interference fit. Specifically, the outer diameter of the second container 3 is larger than the inner diameter of the third cover body 6, so that when the bottom of the second container 3 enters the third cover body 6, the third cover body 6 is fixed to the bottom of the second container 3 in a snap-fit manner.

[0069] Further, refer to Figure 3 As shown, in some embodiments, the second connecting member 9 , the first cover body 2 and the second cover body 4 are connected via the second connecting member 9 .

[0070] Specifically, in the technical solution adopted in the present application, the second connecting member 9 and the first connecting member 51 adopt the same rod-shaped structure, so that when the first cover body 2 is covered on the first opening 11, the second container 3 can be in the target position of the first chamber 12 through the first connecting member 51. It can be explained that the target position can be the middle of the first chamber 12 or a position close to the middle of the first chamber 12, so that the second chamber 33 can be suspended in the first chamber 12 of the first container 1.

[0071] Furthermore, in some embodiments, when the second container 3 is placed in the first chamber 12 , a gap is formed between the inner wall of the first container 1 and the outer wall of the second container 3 , and the gap is used to accommodate the shock-absorbing sponge.

[0072] Specifically, in the technical solution adopted in this application, the outer diameter of the second container 3 is smaller than the inner diameter of the first container 1, that is, the inner diameter of the first container 1 is the size of the first chamber 12. Therefore, when the second container 3 is placed in the first chamber 12, a gap is formed between the outer wall of the second container 3 and the inner wall of the first container 1, and this gap can be used to accommodate the shock-absorbing sponge. When the tissue engineering cartilage scaffold needs to be transported, the tissue engineering cartilage scaffold can be placed in the second chamber 33, and then the second cover 4 is covered on the second opening 31, and the second cover 4 is rotated to fix it on the second container 3. At this time, the pressing component 5 presses the tissue engineering cartilage scaffold against the bottom of the second container 3, and a shock-proof sponge is placed in the first container 1. Then, the first cover 2 is held and the second container 3 and the second cover 4 are placed in the first chamber 12 through the first opening 11, and the first cover 2 is covered on the first opening 11. Finally, the first cover 2 is rotated to fix it on the first container 1. Since the shock-proof sponge is located between the outer wall of the second container 3 and the inner wall of the first container 1, it can buffer the vibration of the second container 3 in the first container 1 during transportation, so as to avoid unnecessary vibration of the tissue engineering cartilage scaffold during transportation, and effectively improve the integrity of the tissue engineering cartilage scaffold before use.

[0073] This application also proposes a method for using a culture container for a tissue engineering cartilage scaffold, which is performed according to the following steps:

[0074] Take out a set of culture containers, unscrew the first cover 2 in the safety cabinet, so that the second container 3 is separated from the first chamber 12 through the first opening 11, then hold the lower part of the second container 3 and rotate the first cover 2. Since the first cover 2 and the second cover 4 are connected, the second cover 4 can be separated from the second container 3. Turn the first cover 2 upside down on the workbench for later use, so that the second cover 4 and the pressing component 5 are located above the first cover 2;

[0075] Use sterile forceps to place the collagen scaffold to be incorporating chondrocytes into the second chamber 33 of the second container 3 through the second opening 31. Then, drip the chondrocyte suspension onto the collagen scaffold, i.e., the tissue-engineered cartilage scaffold in the above embodiment. It is important to note that during this operation, be careful not to puncture the first functional layer 7 disposed at the bottom of the second container 3.

[0076] Take the first cover 2 and place the second cover 4 over the second opening 31 of the second container 3, so that the pressing member 5 enters the second chamber 33 of the second container 3 through the second opening 31. Hold the second container 3 and the first cover 2 and rotate them relative to each other, so that the first cover 2 drives the second cover 4 to be screwed onto the second container 3. Then, place the second container 3 face downward in a CO2 incubator and incubate for 30 minutes.

[0077] After the incubation is completed, the surface is disinfected and then transferred to a biosafety cabinet. The first cover 2 is rotated by holding the lower part of the second container 3 to separate the second cover 4 from the second container 3 again. The first cover 2 is turned upside down on the workbench for later use, so that the second cover 4 and the pressing component 5 are located above the first cover 2.

[0078] Add an appropriate amount of Dulbecco's Phosphate Buffered Saline (DPBS) to the second container 3 and gently shake it. Aspirate the DPBS solution after rinsing the tissue-engineered cartilage scaffold into a 15ml centrifuge tube. It is important to note that the first functional layer 7 at the bottom of the second container 3 should not be punctured during this operation.

[0079] The adhesion rate of composite chondrocytes on the collagen scaffold was calculated by counting cells in the eluent;

[0080] After adding the transport protection liquid into the second chamber 33 through the second opening 31, hold the second container 3 and the first cover 2, place the pressing component 5 into the second chamber 33 through the second opening 31, and cover the second opening 31 with the second cover 4. Rotate the second container 3 and the first cover 2 relative to each other to install the second cover 4 on the second container 3. The tissue engineering cartilage scaffold in the second chamber 33 is completely immersed in the transport protection by the pressing component 5, and then placed in a carbon dioxide incubator with the second container 3 facing downward for culturing for 24 hours. It should be noted that the transport protection liquid contains 10% autologous serum.

[0081] After the culture is completed, a shock-absorbing sponge is placed in the first chamber 12 of the first container 1 through the first opening 11, and the first cover 2 is closed on the first opening 11, so that the second container 3 and the second cover 4 enter the first chamber 12 through the first opening 11. The first container 1 and the second cover 4 are held and rotated relative to each other to tighten the first cover 2 on the first container 1.

[0082] The culture container equipped with the tissue-engineered cartilage scaffold is packaged, placed in a cold chain transport box, and transported to the hospital for re-implantation;

[0083] On a sterile operating table, medical staff open the first container 1 and the second container 3 by unscrewing the first cover 2 and the second cover 4 in sequence, and use sterile tweezers to clamp the tissue engineering cartilage scaffold from the second chamber 33 of the second container 3 and transfer it to the tray. They can also complete the cutting in the second chamber 33 and directly replant it.

[0084] By using the culture container and method of use proposed in this application, the tissue-engineered cartilage scaffold is grasped twice with forceps from the start of culture until application, significantly reducing the risk of damage and drop of the tissue-engineered cartilage scaffold during handling. Furthermore, washing the tissue-engineered cartilage scaffold instead of grasping it, rinsing it before transferring it to a storage bottle, effectively improves the attachment rate of chondrocytes to the collagen scaffold.

[0085] Reference Figure 5 As shown, the test was carried out for culturing chondrocytes on the collagen scaffold to form the following comparative examples:

[0086] The existing process is to place a collagen scaffold in the center of a plate, add chondrocytes on the collagen scaffold, transfer the plate to a carbon dioxide incubator and incubate for 30 minutes, use tweezers to pick up the tissue-engineered cartilage scaffold and rinse it with Dulbecco's phosphate buffered saline solution, absorb the Dulbecco's phosphate buffered saline solution after rinsing the tissue-engineered cartilage scaffold in the plate, and count the cells to calculate the attachment rate of chondrocytes on the scaffold.

[0087] After adopting the culture container and the method for using the culture container of the present application, you can refer to Figure 1 As shown, after adopting the above-mentioned existing process, the attachment rate of chondrocytes ranges from 92.5% to 99.2%, with an average value of 96.7%; while after adopting the culture container and the use method proposed in the application, the attachment rate of chondrocytes ranges from 98.7% to 99.8%, with an average value of 99.3%. There is a significant difference in the attachment rate data of the two methods. After using the culture container proposed in this application and the corresponding use method, the attachment rate of chondrocytes on the collagen scaffold can be significantly improved.

[0088] Compared with the existing process, a step is added in which chondrocytes and collagen scaffolds are cultured in a transport protective solution containing 10% autologous serum for 24 hours. This step can improve the adhesion of chondrocytes on the collagen scaffold, and the culture conditions are not sufficient to support chondrocyte proliferation and aging, ensuring that chondrocytes can still migrate quickly to the wound surface after reimplantation surgery, reducing the shedding rate of chondrocytes on the collagen scaffold during transportation.

[0089] The following comparative examples were generated for the transport of tissue engineering cartilage scaffolds:

[0090] The existing process involves using tweezers to transfer the tissue engineering cartilage scaffold into a transport bottle, adjusting the front and back of the tissue engineering cartilage scaffold, adding transport protection fluid into the transport bottle, placing a shock-proof sponge inside the outer bottle, and placing the transport bottle inside the outer bottle to simulate a 4-hour transport environment at 15 to 25°C.

[0091] Using the culture container and the method of using the culture container of the present application, add transportation protection liquid to the second container 3, cover the second opening 31 with the second cover 4, rotate the first cover 2 to drive the second cover 4 to rotate together, so that the second cover 4 is installed on the second container 3, and place the second container 3 facing down in a carbon dioxide incubator for culture for 24 hours. Place a shock-proof sponge in the first container 1, hold the first cover 2 and place the second container 3 in the first chamber 12 of the first container 1 to simulate a 4-hour transportation environment at 15 to 25°C.

[0092] Reference Figure 6 As shown, the chondrocyte attachment rate after simulated transport was calculated by collecting the transport protection solution from the transport bottle and the second container 3, centrifuging at 300g for 5 minutes, and removing the supernatant. The chondrocytes on the tissue engineering cartilage scaffold were sampled and counted using 1 ml of Dulbecco's phosphate buffer solution, and the chondrocyte attachment rate was recalculated. Specifically, the cell attachment rate after transport = the number of cells on the collagen scaffold / the number of seeded cells. The results showed that the chondrocyte attachment rate after transport using the existing process decreased to approximately 70%, while the chondrocyte attachment rate remained >95% after using the culture container and the method for using the culture container of the present application.

[0093] Reference Figure 7 As shown, the chondrocyte migration was tested to form the following comparative examples:

[0094] Take the above set of tissue-engineered cartilage scaffolds and cut them into two pieces. One piece is placed face down in a six-well plate and cultured with chondrocyte growth medium for 48 hours. The cells are observed for their growth on the surface of the six-well plate at 24 hours and 48 hours, respectively, and their migration ability is assessed. The comparison results show that the culture method of this application has better migration ability of chondrocytes than the existing process, which can better ensure the repair effect of chondrocytes.

[0095] Reference Figure 8 As shown, the growth of cartilage on the collagen scaffold was observed by electron microscopy.

[0096] Another portion of the cartilage scaffold obtained by cutting can be transferred to a 24-well plate, fixed with 2.5% glutaraldehyde, washed twice with Dulbecco's phosphate buffer solution, dehydrated with a gradient of ethanol at 30%, 50%, 70%, 80%, 90%, and 100%, critical point dried, and subjected to electron microscopy after gold spraying. The results show that chondrocytes obtained by the existing process mostly appear in a semi-adherent and shrunken state on the collagen scaffold. Experiments have shown that this state is prone to falling off during transportation, handling, and cutting. However, chondrocytes cultured using the culture method of the present application appear in an extended, adherent growth state. Experiments have shown that this state is less likely to fall off during transportation, handling, and cutting, thereby ensuring a better therapeutic effect.

[0097] Reference Figure 9 As shown, the chondrocyte culture process was tested to form the following comparative examples:

[0098] After completing the incubation and rinsing steps in the culture method of this application, the collagen scaffold is cut into two parts, one of which is sealed and placed in a 5% carbon dioxide incubator for 24 hours; the other is cultured in a 5% carbon dioxide incubator using the culture container of this application. The pH value of the culture solution is retrieved at 0 hours, 4 hours, 8 hours, 20 hours, and 24 hours during the culture process. After the culture is completed, the collagen scaffold is transferred to a 24-well plate and fixed with 2.5% glutaraldehyde. It is then washed twice with Dulbecco's phosphate buffer solution and dehydrated with an ethanol gradient of 30%, 50%, 70%, 80%, 90%, and 100%. It is critical point dried and scanned with an electron microscope after gold spraying.

[0099] The results showed that the pH value of the culture medium was 7.35 when the preparation was completed, and the pH value of the culture medium rose to 7.86 after the assembly operation was completed; the pH value of the culture medium cultured in a closed manner was adjusted slowly, and there was still no significant change in pH after 8 hours, and the pH only dropped to 7.63 after 24 hours. The pH had dropped to 7.65 after 4 hours of culture using the culture container of the present application, and dropped to 7.42 after 8 hours, and 7.25 after 24 hours. During most of the culture time, the pH of the culture medium was within the optimal range for cell growth. It can be seen that the culture container of the present application can better exchange with carbon dioxide gas, which is conducive to the adjustment of the pH value of the culture medium to an appropriate range, and is more conducive to the growth of chondrocytes.

[0100] Reference Figure 10 As shown, electron microscopic observation shows that some chondrocytes cultured in a closed manner are still in a shrunken state; while the chondrocytes cultured in the culture container of the present application can be stretched and attached to the collagen scaffold more uniformly and consistently.

[0101] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described may be combined in any appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless they are mutually inconsistent.

[0102] 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 being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0103] Any process or method description in a flow chart or otherwise described herein can be understood to represent a module, segment or portion of code comprising one or more executable instructions for implementing the steps of a specific logical function or process. The scope of the preferred embodiments of the present application includes additional implementations in which the functions may be performed in a different order than shown or discussed, including in a substantially simultaneous manner or in a reverse order depending on the functions involved.

[0104] The logic and / or steps represented in the flowchart or otherwise described herein may be considered, for example, as an ordered list of executable instructions for implementing logical functions, and may be embodied in any computer-readable medium for use by, or in conjunction with, an instruction execution system, apparatus, or device (such as a computer-based system, a system including a processor, or other system that can fetch and execute instructions from an instruction execution system, apparatus, or device).

[0105] It should be understood that various parts of the present application can be implemented using hardware, software, firmware, or a combination thereof. In the above embodiments, multiple steps or methods can be implemented using software or firmware stored in a memory and executed by a suitable instruction execution system. All or part of the steps of the above embodiment method can be completed by instructing the relevant hardware through a program, which can be stored in a computer-readable storage medium. When the program is executed, it includes one or a combination of the steps of the method embodiment.

[0106] In addition, the functional units in the various embodiments of the present application may be integrated into a single processing module, or each unit may exist physically separately, or two or more units may be integrated into a single module. The aforementioned integrated modules may be implemented in the form of hardware or in the form of software functional modules. If the aforementioned integrated modules are implemented in the form of software functional modules and sold or used as independent products, they may also be stored in a computer-readable storage medium. The storage medium may be a read-only memory, a magnetic disk, or an optical disk, etc.

[0107] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this application, and such modifications or substitutions should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A culture container for tissue engineering cartilage scaffold, characterized in that: include: a first container having a first opening and a first chamber; a first cover, mounted on the first container and used to cover the first opening; a second container, configured to be placed in the first chamber, the second container having a second opening and a second chamber, the second chamber being configured to hold a tissue engineering cartilage scaffold, the bottom of the second container being hydrophobic and breathable, thereby allowing gaseous media to enter the second chamber through the bottom of the second container; a second cover body, mounted on the second container and used to cover the second opening, the second cover body being connected to the first cover body; as well as, The pressing component is installed on the second cover body and is located on the side of the second cover body away from the first cover body, and is used to limit the displacement distance of the tissue engineering cartilage scaffold in the second container toward the second opening.

2. The culture container for tissue engineering cartilage scaffold according to claim 1, characterized in that: Also includes: a first functional layer having a hydrophobic and breathable function, the first functional layer being disposed at the bottom of the second container and located in the second chamber; The bottom of the second container is provided with a plurality of first air holes, and the first functional layer body covers the first air holes.

3. The culture container for tissue engineering cartilage scaffold according to claim 1, characterized in that: The bottom of the second container has a third opening; The culture container of the tissue engineering cartilage scaffold further comprises: a third cover body, mounted on the second container and used to cover the third opening, the third cover body being provided with a plurality of first air holes; The first functional layer has a hydrophobic and breathable function. The first functional layer is configured on the third cover and located in the second cavity to cover the first breathable hole.

4. The culture container for tissue engineering cartilage scaffold according to claim 1, characterized in that: The pressing component includes: a first connecting member connected to a surface of the second cover body on a side facing away from the first cover body; a pressure plate connected to an end of the first connecting member away from the second cover; When the second cover is closed on the second opening, the first connecting member extends into the second chamber, so that the pressure plate can limit the displacement of the tissue engineering cartilage scaffold in the second container toward the second opening.

5. The culture container for tissue engineering cartilage scaffold according to any one of claims 1 to 3, characterized in that: The second cover has a hydrophobic and breathable function, so that the gas medium enters the second chamber through the second cover.

6. The culture container for tissue engineering cartilage scaffold according to claim 5, characterized in that: Also includes: a second functional layer having a hydrophobic and breathable function, wherein the second functional layer is disposed on the second cover; The second cover is provided with a plurality of second ventilation holes, and the second functional layer covers the second ventilation holes.

7. The culture container for tissue engineering cartilage scaffold according to claim 6, characterized in that: The pressing component includes: a support plate, disposed on the second cover, the second functional layer being located between the support plate and the cover, and the support plate being provided with third ventilation holes corresponding one-to-one to the second ventilation holes; a first connecting member, connecting a surface of the support plate on a side facing away from the second functional layer; a pressure plate connected to an end of the first connecting member away from the support plate; When the second cover is closed on the second opening, the first connecting member extends into the second chamber, so that the pressure plate can limit the displacement of the tissue engineering cartilage scaffold in the second container toward the second opening.

8. The culture container for tissue engineering cartilage scaffold according to claim 1, characterized in that: The first container and the first cover are connected by a first thread structure; The second container and the second cover are cooperatively connected via a second threaded structure.

9. The culture container for tissue engineering cartilage scaffold according to claim 1, characterized in that: A second connecting member, wherein the first cover body and the second cover body are connected via the second connecting member.

10. The culture container for tissue engineering cartilage scaffold according to claim 1, characterized in that: When the second container is placed in the first chamber, a gap is formed between the inner wall of the first container and the outer wall of the second container, and the gap is used to accommodate the shock-absorbing sponge.

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