Culture vessel for tissue engineered cartilage scaffolds
By designing a tissue-engineered cartilage scaffold culture container with hydrophobic and breathable functions, the number of times the cells are handled is reduced, solving the problems of cartilage cell damage and shedding in existing technologies, achieving efficient cell culture and transportation, and improving treatment efficacy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- BEIHAO STEM CELL & REGENERATIVE MEDICINE RES INST CO LTD
- Filing Date
- 2025-06-30
- Publication Date
- 2026-07-14
AI Technical Summary
In the current process of constructing tissue-engineered cartilage, frequent clamping of collagen scaffolds leads to chondrocyte damage and shedding, affecting cell utilization and therapeutic effect, and also carries a high risk of inconsistency and contamination.
Design a tissue-engineered cartilage scaffold culture container, comprising a first container and a second container. The second container has hydrophobic and breathable functions, a top pressure component restricts scaffold displacement to reduce the number of clamping operations, and the hydrophobic and breathable functions ensure gas exchange to avoid cell damage.
It significantly improved the culture quality of chondrocytes, reduced the risk of cell damage and shedding, enhanced the stability and consistency of the procedure, increased cell adhesion and migration ability, and ensured therapeutic efficacy.
Smart Images
Figure CN224494224U_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of cell culture equipment, and more particularly to a culture container for a tissue-engineered cartilage scaffold. Background Technology
[0002] "Human-derived tissue-engineered regenerated cartilage" is a Class III medical technology in the field of tissue engineering medicine and is currently a cutting-edge clinical application technology used internationally to treat acute and chronic articular cartilage defects. Based on the principle of matrix-induced autologous chondrocyte transplantation (MACI), this technology is a new generation of autologous cell cartilage repair methods, and tens of thousands of patients worldwide have received this treatment with good results. Its core treatment process includes: obtaining a small amount of healthy cartilage tissue from the patient, isolating and culturing the chondrocytes within it, expanding them in vitro, and then combining these cells with a three-dimensional collagen scaffold to construct biologically functional tissue-engineered cartilage. Finally, it is surgically transplanted to the patient's cartilage defect site to achieve structural repair and functional reconstruction. The composite construction process of chondrocytes and the collagen scaffold is the key step in the entire process and directly affects the final treatment outcome.
[0003] In current tissue-engineered cartilage construction processes, the following steps are typically used to integrate cells with a collagen scaffold: First, a sterile collagen scaffold is placed in the center of a petri dish. Then, a suspension of expanded and cultured chondrocytes is uniformly injected or dropped onto the scaffold surface. After seeding, the petri dish is transferred to a CO2 incubator and incubated for approximately 30 minutes to allow the cells to initially adhere to the scaffold structure. Next, the scaffold is picked up with forceps and rinsed with an appropriate amount of Duchenne phosphate-buffered saline (DPBS) to remove any unattached free cells. The rinse solution is then aspirated from the petri dish for cell counting, and the cell adhesion rate on the scaffold is calculated. After confirming the adhesion effect, the scaffold is transferred to a transport bottle, its orientation adjusted, transport protection solution added, and the bottle sealed before transport to the hospital. Before surgery, medical staff use forceps to remove the scaffold from the transport bottle, place it on a tray, distinguish the front and back sides again, and trim it as needed, preparing for implantation.
[0004] Although the aforementioned construction method has been applied clinically to some extent, significant operational drawbacks remain. Particularly in critical steps such as rinsing, transfer to transport bottles, adjusting the orientation, and final removal from the transport bottle, repeated handling of the collagen scaffold with forceps is required. Because the collagen scaffold itself is soft and intricately structured, and chondrocytes are only initially attached and not yet firmly integrated into the scaffold, frequent mechanical handling easily leads to cell detachment or damage, reducing cell utilization. Furthermore, due to the physical properties of the collagen scaffold, it tends to remain suspended in the preservation solution. During transport, the shear force from the vibration of the preservation solution further damages and causes cell detachment. If the product floats to the surface of the preservation solution or adheres to the container wall, it can lead to localized drying, reduced cell activity, and compromised therapeutic efficacy. In addition, repeated handling increases the risk of contamination and reduces the consistency and reproducibility of the preparation process, limiting the wider application of this technology. Therefore, there is an urgent need to develop a novel tissue-engineered cartilage construction container device and a method for using the container device that can reduce the number of clamping operations, avoid cell damage, and improve construction efficiency and stability. Utility Model Content
[0005] This application provides a culture container for a tissue-engineered cartilage scaffold to avoid the shortcomings of existing culture methods, such as the need for repeated clamping and unclamping of the collagen scaffold and the floating or adherence of the collagen scaffold during culture and transportation. This solves the technical problem of chondrocyte damage caused by these shortcomings. The technical solution is as follows:
[0006] This application provides a culture container for a tissue-engineered cartilage scaffold, comprising: a first container having a first opening and a first chamber; a first cover mounted on the first container for covering the first opening; a second container for placement in the first chamber, the second container having a second opening and a second chamber, the second chamber for placing the tissue-engineered cartilage scaffold, the bottom of the second container having a hydrophobic and breathable function, thereby allowing a gaseous medium to enter the second chamber through the bottom of the second container; a second cover mounted on the second container for covering the second opening, the second cover being connected to the first cover; and a pressing member mounted on the second cover, located on the side of the second cover away from the first cover, for limiting the displacement distance of the tissue-engineered cartilage scaffold in the second container toward the second opening.
[0007] In one embodiment, it further includes: a first functional layer having hydrophobic and breathable functions, 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 vent holes, and the first functional layer covering the first vent holes.
[0008] In one embodiment, the bottom of the second container has a third opening; the culture container for the tissue-engineered cartilage scaffold further includes: a third cover, mounted on the second container for covering the third opening, the third cover having a plurality of first vent holes; and a first functional layer having hydrophobic and breathable functions, the first functional layer being disposed on the third cover and located in the second chamber to cover the first vent holes.
[0009] In one embodiment, the pressing component includes: a first connector connected to a side surface of the second cover away from the first cover; and a pressure plate connected to the end of the first connector away from the second cover.
[0010] When the second cover is closed over the second opening, the first connector extends into the second chamber, so that the pressure plate can restrict the displacement of the tissue-engineered cartilage scaffold in the second container toward the second opening.
[0011] In one embodiment, the second cover has a hydrophobic and breathable function, thereby allowing the gaseous medium to enter the second chamber through the second cover.
[0012] In one embodiment, it further includes: a second functional layer having hydrophobic and breathable functions, the second functional layer being disposed on the second cover; the second cover having a plurality of second vent holes, the second functional layer covering the second vent holes.
[0013] In one embodiment, the top-pressing component includes: a support plate disposed on a second cover, a second functional layer located between the support plate and the cover, and a third vent hole on the support plate corresponding to the second vent hole; a first connector connected to the side surface of the support plate away from the second functional layer; and a pressure plate connected to the end of the first connector away from the support plate.
[0014] When the second cover is closed over the second opening, the first connector extends into the second chamber, so that the pressure plate can restrict the displacement of the tissue-engineered 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 connector connects the first cover to the second cover.
[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, the gap being used to accommodate the shock-absorbing sponge.
[0018] Compared to existing technologies, the tissue-engineered cartilage scaffold culture container proposed in the above technical solution involves placing the collagen scaffold requiring composite chondrocytes into a second container using sterile forceps. The prepared chondrocyte suspension is then evenly added dropwise onto the collagen scaffold. The second cap is tightened onto the second container so that the pressure-pressing component presses against the collagen scaffold. The second container is then placed face down in a CO2 incubator for 30 minutes. Subsequently, an appropriate amount of DuPont phosphate-buffered saline (DPBS) is added to the second container for rinsing. Cells in the DPBS are counted, and the adhesion rate of chondrocytes to the collagen scaffold is calculated. A transport protection solution containing 10% autologous serum is added to the second container, and the container is placed face down in a CO2 incubator for 24 hours. A shock-absorbing sponge is placed in the first chamber of the first container, and the second container is placed inside the first container to reliably transport the tissue-engineered cartilage scaffold to the hospital for reimplantation. During application, medical staff use forceps to transfer the tissue-engineered cartilage scaffold from the second container to a tray for cutting and use. Because the second cover is connected to the first cover, and the second cover can be installed on the second container, the second container can be accessed through the first cover to culture the collagen scaffold within it during the manipulation of the tissue-engineered cartilage scaffold. To achieve the purpose of incubating and culturing chondrocytes, the bottom of the second container has a hydrophobic and breathable function, allowing the gas medium in the second chamber of the second container to exchange with the gas medium in the carbon dioxide incubator. When using the culture container proposed in this application, only two operations are required—using tweezers to grasp the collagen scaffold—from the start of chondrocyte culture until the medical staff cuts and applies it. This effectively improves the culture quality of chondrocytes and reduces the risk of chondrocyte damage and loss.
[0019] The above overview 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 this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description
[0020] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.
[0021] Figure 1 This is a three-dimensional structural diagram of the culture container for the tissue-engineered cartilage scaffold in the embodiments of this application;
[0022] Figure 2 This is a cross-sectional view of the culture container for the tissue-engineered cartilage scaffold in an embodiment of this application;
[0023] Figure 3 This is an exploded view of the culture container for the tissue-engineered cartilage scaffold in the embodiments of this application;
[0024] Figure 4 This is a schematic diagram showing the arrangement of a third opening on the second container in an embodiment of this application;
[0025] Figure 5 This is a bar graph showing the comparative adhesion rate of chondrocytes in the embodiments of this application;
[0026] Figure 6 This is a bar graph showing the comparative adhesion rate of chondrocytes after simulated transport in the embodiments of this application.
[0027] Figure 7 This is a comparative bar graph showing the pH changes of chondrocytes in the culture medium in the embodiments of this application.
[0028] Figure label:
[0029] 1. First container;
[0030] 11. First opening; 12. First chamber;
[0031] 2. First cover;
[0032] 3. Second container;
[0033] 31. Second opening; 32. Third opening; 33. Second chamber;
[0034] 4. Second cover;
[0035] 41. Second vent;
[0036] 5. Top pressure components;
[0037] 51. First connecting component; 52. Pressure plate component; 53. Support plate;
[0038] 531. Third vent hole;
[0039] 6. The third cover;
[0040] 61. First vent hole;
[0041] 7. First functional layer;
[0042] 8. Second functional layer;
[0043] 9. Second connector. Detailed Implementation
[0044] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.
[0045] First Embodiment
[0046] Reference Figures 1 to 4 As shown, an embodiment of this application proposes a culture container for a tissue-engineered cartilage scaffold. This culture container may include: a first container 1 having a first opening 11 and a first chamber 12; a first cover 2, mounted on the first container 1 to cover the first opening 11; a second container 3 for placement within the first chamber 12, the second container 3 having a second opening 31 and a second chamber 33, the second chamber 33 for placing the tissue-engineered cartilage scaffold, the bottom of the second container 3 having a hydrophobic and breathable function, allowing gaseous media to enter the second chamber 33 through the bottom of the second container 3; a second cover 4, mounted on the second container 3 to cover the second opening 31, the second cover 4 being connected to the first cover 2; and a pressing member 5, mounted on the second cover 4, located on the side of the second cover 4 opposite to the first cover 2, for limiting the displacement distance of the tissue-engineered cartilage scaffold in the second container 3 towards the second opening 31.
[0047] Specifically, in the technical solution adopted in this application, the first chamber 12 of the first container 1 can accommodate the second container 3. The first container 1 is equipped with a first cover 2 that closes the first opening 11, and the second container 3 is equipped with a second cover 4 that closes the second opening 31. The first cover 2 and the second cover 4 are connected so that when the first container 1 is disassembled from the first cover 2, the first cover 2 can drive the second container 3 out of the first chamber 12 through the first opening 11 via the second cover 4. This allows the second container 3 to be removed from the first chamber 12 without the use of additional tools and without contact. The second chamber 33 of the second container 3 is used to place a tissue functional 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 and breathable function. This can be explained as the bottom of the second container 3 allowing gaseous media to pass through, but blocking liquid media, thereby preventing liquid media in the second chamber 33 from flowing out through the bottom of the second container 3. The pressure-reducing component 5 is disposed on the side of the second cover 4 away from the first cover 2. When the second cover 4 is closed on the second opening 31, the pressure-reducing component 5 enters the second chamber 33 through the second opening 31 to press against the tissue-engineered cartilage scaffold in the second chamber 33, so that the tissue-engineered 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 down, the pressure-reducing component 5 can press against the tissue-engineered cartilage scaffold so that it is completely immersed in the liquid medium in the second chamber 33. When the tissue-engineered cartilage scaffold needs to interact with the gas medium, the first container 1 can be separated from the first cover 2, and the second container 3 can be separated from the first cover 2 through the second cover 4. The second container 3 can be inverted through the first cover 2, and the pressure-reducing component 5 can support the tissue-engineered cartilage scaffold to be held 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.
[0048] Furthermore, refer to Figure 3 As shown, in some embodiments, it further includes: a first functional layer 7 having hydrophobic and breathable functions, the first functional layer 7 being disposed at the bottom of the second container 3 and located in the second chamber 33; the bottom of the second container 3 is provided with a plurality of first vent holes 61, and the first functional layer 7 covering the first vent holes 61.
[0049] Specifically, in the technical solution adopted in this application, to achieve the hydrophobic and breathable function mentioned in this application, a first functional layer 7 can be disposed 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 permeation through precisely designed micropores, with micropore diameters typically ranging from 0.2 to 0.3 μm. Water vapor, oxygen, carbon dioxide, and other gas molecules have diameters much smaller than the micropores (approximately 0.0004 μm) and can diffuse freely. Liquids are blocked: liquid water molecules form larger molecular clusters (approximately 20 μm) due to surface tension and cannot pass through the micropores; blood, oils, and other liquids are also blocked. For example, medical silicone membranes contain 12 to 12.5 million micropores per square millimeter, with an average micropore diameter of 0.2 to 0.3 μm, making them both breathable and completely waterproof. A plurality of first vent holes 61 are provided on the bottom side wall of the second container 3, and the first functional layer 7 is covered on each of the first vent holes 61. Thus, with the cooperation of each of the first vent 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 not only carry out gas interaction, but also store liquid media in the second chamber 33.
[0050] Furthermore, 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 for the tissue-engineered cartilage scaffold also includes: a third cover 6, which is installed on the second container 3 to cover the third opening 32, and the third cover 6 is provided with a plurality of first vent holes 61; a first functional layer 7, which has hydrophobic and breathable functions, and the first functional layer 7 is disposed on the third cover 6 and located in the second chamber 33 to cover the first vent holes 61.
[0051] Specifically, in the technical solution adopted in this 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. The culture container proposed in this application also includes a third cover 6 for covering the third opening 32. The third cover 6 is detachably installed on the second container 3 so that each first vent hole 61 is opened on the third cover 6, so as to facilitate the disassembly and separation of the third cover 6 from the second container 3. This allows the second container 3 to be more thoroughly cleaned and disinfected before use, and also makes the installation of the first functional layer 7 more precise and convenient.
[0052] Furthermore, refer to Figure 2 As shown, in some embodiments, the pressing component 5 includes: a first connector 51 connected to the surface of the second cover 4 facing away from the first cover 2; and a pressing plate 52 connected to the end of the first connector 51 away from the second cover 4.
[0053] When the second cover 4 is closed over the second opening 31, the first connector 51 extends into the second chamber 33, so that the pressure plate 52 can restrict the tissue-engineered cartilage scaffold in the second container 3 from displacing toward the second opening 31.
[0054] Specifically, in the technical solution adopted in this application, the first connecting member 51 is configured as a long strip, specifically a straight rod structure, connected to the side of the second cover 4 away from the first cover 2. In this embodiment, the first connecting member 51 can be fixedly connected to the second cover 4, or it can be configured as a detachable installation by means of an interference fit. The length of the first connecting member 51 can be adapted to the depth of the second chamber 33. The pressure plate member 52 is connected to the end of the first connecting member 51 away from the second cover 4, so that when the second cover 4 is closed on the second opening 31, the pressure plate member 52 can be closer to the bottom of the second container 3 by the length of the first connecting member 51, so as to achieve the purpose of pressing against the tissue-engineered cartilage scaffold in the second chamber 33. By limiting the tissue-engineered cartilage scaffold to the bottom of the second container 3 by the pressure plate member 52, the tissue-engineered cartilage scaffold can be fully immersed in the transport protective fluid when necessary, effectively reducing the impact of the transport protective fluid on the tissue-engineered cartilage scaffold, avoiding the risk of the transport protective fluid turning over, floating and drying out, thereby improving the cell activity of the product. It can be explained that, if necessary, this could be during the transportation of tissue-engineered cartilage scaffolds, or throughout the entire process of culturing tissue-engineered cartilage scaffolds in a carbon dioxide incubator.
[0055] Second Embodiment
[0056] Furthermore, refer to Figure 3 As shown, in some embodiments, the second cover 4 has a hydrophobic and breathable function, thereby allowing the gas medium to enter the second chamber 33 through the second cover 4.
[0057] Specifically, in the technical solution adopted in this application, the difference between the second embodiment and the first embodiment lies in that the second cover 4 also has hydrophobic and breathable functions, thereby allowing the gas medium to exchange gases in the second chamber 33 through the bottom of the second container 3 and the second cover 4, thus improving the gas flow of the second chamber 33. With both the second cover 4 and the bottom of the second container 3 having hydrophobic and breathable functions, it can be ensured that the chondrocytes on the tissue-engineered cartilage scaffold in the second container 3 can have good gas exchange with the carbon dioxide incubator, thereby reducing the risk of cell hypoxia and acidic culture medium, and providing a better growth environment for chondrocytes than with airtight culture methods.
[0058] Furthermore, refer to Figure 3As shown, in some embodiments, it further includes: a second functional layer 8 having hydrophobic and breathable functions, the second functional layer 8 being disposed on the second cover 4; the second cover 4 having a plurality of second vent holes 41, the second functional layer 8 covering the second vent holes 41.
[0059] Specifically, in the technical solution adopted in this application, in order to achieve the hydrophobic and breathable function of the second cover 4, this application proposes an embodiment in which a second functional layer 8 is disposed on the second cover 4. The second functional layer 8 can be a breathable silicone mold, and a plurality of second vent holes 41 are opened on the second cover 4 so that the second functional layer 8 covers each of the second vent holes 41. Thus, with the cooperation of the second functional layer 8 and each of the second vent holes 41, the second cover 4 has a hydrophobic and breathable function. The gas medium outside the second chamber 33 and the gas medium inside the second chamber 33 interact through the second functional layer 8 and the second vent holes 41, while the liquid medium in the second chamber 33 is retained in the second chamber 33 by the barrier of the second functional layer 8, so as to prevent the liquid medium in the second chamber 33 from being lost.
[0060] Furthermore, refer to Figure 3 As shown, in some embodiments, the top pressing component 5 includes: a support plate 53 disposed on the second cover 4, the second functional layer 8 located between the support plate 53 and the cover, the support plate 53 having third vent holes 531 corresponding one-to-one with the second vent holes 41; a first connector 51 connected to the side surface of the support plate 53 away from the second functional layer 8; and a pressure plate 52 connected to the end of the first connector 51 away from the support plate 53.
[0061] When the second cover 4 is closed over the second opening 31, the first connector 51 extends into the second chamber 33, so that the pressure plate 52 can restrict the tissue-engineered cartilage scaffold in the second container 3 from displacing toward the second opening 31.
[0062] Specifically, in the technical solution adopted in this application, compared with the first embodiment, a support plate 53 for fixing the second functional layer 8 is added to the top pressing component 5. The support plate 53 is installed in the second cover 4 and is located on the side of the second cover 4 away from the first cover 2. The second functional layer is fixed between the inner surface of the second cover 4 and the support plate 53 by clamping. A third vent 531 corresponding to each of the second vent 41 is 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 away from the second functional layer 8, and the other end of the second connecting member 9 is connected to the pressure plate component 52. When the second cover 4 is closed on the second opening 31, the first connector 51 and the pressure plate 52 are both located in the first chamber 12. The diameters of the first connector 51 and the pressure plate 52 are smaller than the diameter of the second chamber 33. The first connector 51 has a rod-like structure, while the pressure plate 52 has a disc-like structure. Therefore, the diameter of the first connector 51 is smaller than the diameter of the pressure plate 52. Thus, when the pressure plate 52 supports the tissue-engineered cartilage scaffold, the gap formed between the pressure plate 52 and the second chamber 33 allows 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 4.
[0063] Furthermore, refer to Figure 3 As shown, in some embodiments, the first container 1 and the first cover 2 are connected by a first threaded structure; the second container 3 and the second cover 4 are connected by a second threaded structure.
[0064] Specifically, in the technical solution adopted in this application, a first external thread structure can be provided on the radially outer side of the first container 1 near the first opening 11, and a first internal thread structure adapted to the first external thread structure can be provided on the inner surface of the first cover 2. When the first cover 2 is covered on the first opening 11, the first external thread structure and the first internal thread structure can be engaged by rotating the first cover 2, thereby realizing the installation of the first cover 2 on the first container 1 through thread engagement. Similarly, a second external thread structure is provided on the radially outer side of the second container 3 near the second opening 31, and a second internal thread structure adapted to the second external thread structure can be provided on the inner surface of the second cover 4. When the second cover 4 is covered on the second opening 31, the second external thread structure and the second internal thread structure can be engaged by rotating the second cover 4, thereby realizing the installation of the second cover 4 on the second container 3 through thread engagement.
[0065] In some embodiments, the third cover 6 can also be installed at the bottom of the second container 3 using the same threaded fit technology principle. Alternatively, the bottom of the second container 3 can be embedded in the third cover 6 by interference fit. Specifically, the outer diameter of the second container 3 is larger than the inner diameter of the third cover 6, so that when the bottom of the second container 3 enters the third cover 6, the third cover 6 is fixed to the bottom of the second container 3 by snap-fit.
[0066] Furthermore, refer to Figure 3 As shown, in some embodiments, the second connector 9 connects the first cover 2 and the second cover 4.
[0067] Specifically, in the technical solution adopted in this application, the second connector 9 and the first connector 51 adopt the same rod-shaped structure so that when the first cover 2 is closed on the first opening 11, the second container 3 can be positioned at the target position of the first chamber 12 through the first connector 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.
[0068] 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, the gap being used to accommodate the shock-absorbing sponge.
[0069] 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. Thus, 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 transporting tissue-engineered cartilage scaffolds, the scaffolds can be placed in the second chamber 33, and the second cover 4 can be placed over the second opening 31. The second cover 4 can be rotated to fix it onto the second container 3. At this time, the pressing component 5 presses the tissue-engineered cartilage scaffold against the bottom of the second container 3. A shock-absorbing sponge is placed in the first container 1. Then, the first cover 2 is held and placed into the first chamber 12 through the first opening 11, and the first cover 2 is placed over the first opening 11. Finally, the first cover 2 is rotated to fix it onto the first container 1. Since the shock-absorbing 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-engineered cartilage scaffold during transportation and effectively improve the integrity of the tissue-engineered cartilage scaffold before use.
[0070] This application also proposes a method for using the culture container of tissue-engineered cartilage scaffold, which is operated according to the following steps:
[0071] 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, and then hold the lower part of the second container 3 and rotate the first cover 2. Since the first cover 2 is connected to the second cover 4, the second cover 4 can be separated from the second container 3. Place the first cover 2 upside down on the workbench for later use, so that the second cover 4 and the top pressing component 5 are located above the first cover 2.
[0072] Using sterile forceps, the collagen scaffold requiring composite chondrocytes is placed through the second opening 31 into the second chamber 33 of the second container 3. Then, a chondrocyte suspension is dripped onto the collagen scaffold, resulting in the tissue-engineered cartilage scaffold described in the above embodiment. It is important to note that care must be taken not to puncture the first functional layer 7 located at the bottom of the second container 3 during the procedure.
[0073] Take the first cover 2 and cover the second cover 4 onto the second opening 31 of the second container 3, so that the top pressing component 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 second cover 4 is screwed and fixed onto the second container 3 by the first cover 2. Then place the second container 3 face down in the carbon dioxide incubator for incubation for 30 minutes.
[0074] After incubation, the surface is disinfected and then transferred to a biosafety cabinet. The lower part of the second container 3 is held and the first cover 2 is rotated to separate the second cover 4 from the second container 3 again. The first cover 2 is then placed upside down on the workbench for later use, so that the second cover 4 and the top pressing component 5 are located above the first cover 2.
[0075] Add an appropriate amount of DuPont phosphate-buffered saline (DPBS) to the second container 3, gently agitate, and aspirate the remaining DPBS solution after rinsing the tissue-engineered cartilage scaffold into a 15 ml centrifuge tube. Note that this procedure must not puncture the first functional layer 7 at the bottom of the second container 3.
[0076] The adhesion rate of composite chondrocytes to the collagen scaffold was calculated by cell counting in the eluent.
[0077] After adding the transport protective solution into the second chamber 33 through the second opening 31, hold the second container 3 and the first cover 2, and place the top pressing component 5 into the second chamber 33 through the second opening 31. Cover the second opening 31 with the second cover 4, and rotate the second container 3 relative to the first cover 2 to install the second cover 4 on the second container 3. The tissue-engineered cartilage scaffold in the second chamber 33 is completely immersed in the transport protective solution by the pressure of the top pressing component 5. Then, place it in a carbon dioxide incubator with the second container 3 facing down for 24 hours. It should be noted that the transport protective solution contains 10% autologous serum.
[0078] 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 placed on the first opening 11 so that the second container 3 and the second cover 4 enter the first chamber 12 together through the first opening 11. The first container 1 and the second cover 4 are held and rotated relative to each other to tighten and fix the first cover 2 on the first container 1.
[0079] The culture container equipped with the tissue-engineered cartilage scaffold was packaged and then placed in a cold chain transport box for transport to the hospital for reimplantation.
[0080] 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. They then use sterile forceps to pick up the tissue-engineered cartilage scaffold from the second chamber 33 of the second container 3 and transfer it to a tray. Alternatively, the scaffold can be cut in the second chamber 33 and then directly reimplanted.
[0081] Using the culture container and its usage method proposed in this application, the tissue-engineered cartilage scaffold is handled twice with forceps from the start of culture until its application, significantly reducing the risk of damage and dislodgement during the operation. Furthermore, replacing the handling of the tissue-engineered cartilage scaffold with washing before transferring it to a preservation bottle effectively improves the adhesion rate of chondrocytes to the collagen scaffold.
[0082] Reference Figure 5 As shown, tests were conducted on chondrocytes cultured on collagen scaffolds, resulting in the following comparative examples:
[0083] The existing process involves placing a collagen scaffold in the center of a petri dish, adding chondrocytes to the scaffold, transferring the dish to a CO2 incubator for 30 minutes, using forceps to remove the tissue-engineered cartilage scaffold and rinsing it with DuPont phosphate-buffered saline solution, aspirating the DuPont phosphate-buffered saline solution from the petri dish after rinsing the tissue-engineered cartilage scaffold, and performing cell counting to calculate the adhesion rate of chondrocytes on the scaffold.
[0084] After adopting the culture container and its usage method as described in this application, please refer to... Figure 1 As shown, the adhesion rate of chondrocytes using the existing process ranges from 92.5% to 99.2%, with an average of 96.7%; while the adhesion rate using the culture container and its usage method proposed in this application ranges from 98.7% to 99.8%, with an average of 99.3%. The adhesion rate data of the two methods show a significant difference. Using the culture container and its corresponding usage method proposed in this application can significantly improve the adhesion rate of chondrocytes on the collagen scaffold.
[0085] Compared to existing processes, this method adds a step of culturing chondrocytes and collagen scaffolds in a transport protective solution containing 10% autologous serum for 24 hours. This step can improve the adhesion of chondrocytes to the collagen scaffold, and the culture conditions are insufficient to support chondrocyte proliferation and aging, ensuring that chondrocytes can still quickly migrate to the wound after reimplantation surgery, reducing the rate of chondrocyte detachment from the collagen scaffold during transportation.
[0086] Tests were conducted on cartilage scaffolds used in transport tissue engineering, resulting in the following comparative examples:
[0087] The existing process involves using tweezers to transfer the tissue-engineered cartilage scaffold into a transport bottle, adjusting the front and back of the scaffold, adding a transport protective solution to the transport bottle, placing a shock-absorbing sponge inside the outer bottle, and then placing the transport bottle inside the outer bottle to simulate a 4-hour transport environment at 15 to 25°C.
[0088] Using the culture container and its usage method of this application, a transport protective liquid is added to the second container 3, the second cover 4 is placed on the second opening 31, the first cover 2 is rotated to drive the second cover 4 to rotate together, so that the second cover 4 is installed on the second container 3, the second container 3 is placed face down in a carbon dioxide incubator for 24 hours, a shock-absorbing sponge is placed in the first container 1, and the second container 3 is placed in the first chamber 12 of the first container 1 by holding the first cover 2, simulating a 4-hour transport environment of 15 to 25°C.
[0089] Reference Figure 6 As shown, the chondrocyte adhesion rate after simulated transport was calculated. Transport preservation solutions were collected from the transport bottle and the second container (3), centrifuged at 300g for 5 minutes, and the supernatant was removed. Chondrocytes on the tissue-engineered cartilage scaffold were sampled and counted using 1 mL of DuPont phosphate buffer solution. The chondrocyte adhesion rate was then recalculated, specifically: post-transport cell adhesion rate = number of cells on the collagen scaffold / number of seeded cells. The results showed that the chondrocyte adhesion rate decreased to approximately 70% after transport using existing processes, while the chondrocyte adhesion rate remained >95% after using the culture container and its usage method as described in this application.
[0090] Tests were conducted to investigate chondrocyte migration, resulting in the following comparative examples:
[0091] A set of tissue-engineered cartilage scaffolds was taken and cut into two parts. One part was placed face down in a six-well plate and cultured for 48 hours with chondrocyte growth medium. The growth of cells reaching the surface of the six-well plate was observed at 24 and 48 hours, and the migration ability of chondrocytes was assessed. The comparative results show that, compared with existing processes, the culture method of this application has better chondrocyte migration ability and can better ensure the repair effect of chondrocytes.
[0092] The growth of cartilage on a collagen scaffold was observed using electron microscopy.
[0093] Another cartilage scaffold obtained through the above-mentioned trimming process can be transferred to a 24-well plate, fixed with 2.5% glutaraldehyde, washed twice with Duchenne phosphate buffer, and dehydrated using an ethanol gradient of 30%, 50%, 70%, 80%, 90%, and 100%. Critical-point drying and gold sputtering are then performed followed by electron microscopy. Results show that chondrocytes grown on the collagen scaffold using existing methods are mostly in a semi-adherent, rounded state, which is prone to detachment during transportation, handling, and trimming. In contrast, chondrocytes grown using the culture method described in this application exhibit an extended, adherent growth state, which is less prone to detachment during transportation, handling, and trimming, thus ensuring better therapeutic effects.
[0094] Reference Figure 7 As shown, tests were conducted on the chondrocyte culture process to form the following comparative examples:
[0095] After completing the incubation and rinsing steps in the culture method of this application, the collagen scaffold was cut into two parts. One part was placed in a closed environment and cultured in a 5% carbon dioxide incubator for 24 hours; the other part was placed in the culture container of this application and cultured in a 5% carbon dioxide incubator for 24 hours. The pH value of the culture medium was checked at 0, 4, 8, 20, and 24 hours during the culture process. After the culture was completed, the collagen scaffold was transferred to a 24-well plate, fixed with 2.5% glutaraldehyde, washed twice with Duchenne phosphate buffer, and dehydrated with ethanol gradients of 30%, 50%, 70%, 80%, 90%, and 100%. Critical point drying was used, followed by gold sputtering and scanning electron microscopy.
[0096] The results showed that the pH of the culture medium was 7.35 when it was first prepared, and rose to 7.86 after assembly. The pH of the medium cultured in the closed method adjusted more slowly; there was no significant change in pH after 8 hours, and it only dropped to 7.63 after 24 hours. Using the culture container of this application, the pH dropped to 7.65 after 4 hours, 7.42 after 8 hours, and 7.25 after 24 hours. For most of the culture period, the pH of the culture medium remained within the optimal range for cell growth. Therefore, the culture container of this application allows for better exchange with carbon dioxide gas, which is beneficial for adjusting the pH of the culture medium to a suitable range and promotes chondrocyte growth.
[0097] Electron microscopy revealed that some chondrocytes cultured in a closed manner remained in a shrunken state, while chondrocytes cultured in the container described in this application were able to extend and adhere to the collagen scaffold more uniformly.
[0098] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.
[0099] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.
[0100] Any process or method description in the flowchart or otherwise herein can be understood as representing a module, segment, or portion of code comprising one or more executable instructions for implementing a particular logical function or process. Furthermore, the scope of the preferred embodiments of this application includes additional implementations in which functions may be performed not in the order shown or discussed, including substantially simultaneously or in reverse order depending on the functionality involved.
[0101] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a sequenced list of executable instructions for implementing logical functions, and can 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 processor-included system or other system that can fetch and execute instructions from, an instruction execution system, apparatus or device).
[0102] It should be understood that various parts of this 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 memory and executed by a suitable instruction execution system. All or part of the steps of the methods in the above embodiments can be implemented by a program instructing related hardware, the program being stored in a computer-readable storage medium, which, when executed, includes one or a combination of the steps of the method embodiments.
[0103] Furthermore, the functional units in the various embodiments of this application can be integrated into a processing module, or each unit can exist physically separately, or two or more units can be integrated into a module. The integrated module can be implemented in hardware or as a software functional module. If the integrated module is implemented as a software functional module and sold or used as an independent product, it can also be stored in a computer-readable storage medium. This storage medium can be a read-only memory, a disk, or an optical disk, etc.
[0104] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.
Claims
1. A culture container for a tissue-engineered cartilage scaffold, characterized in that, include: A first container having a first opening and a first chamber; A first cover is installed on the first container to cover the first opening; A second container is placed in the first chamber. The second container has a second opening and a second chamber. The second chamber is used to place a tissue-engineered cartilage scaffold. The bottom of the second container has a hydrophobic and breathable function, so that a gaseous medium can enter the second chamber through the bottom of the second container. A second cover is installed on the second container to cover the second opening; the second cover is connected to the first cover. as well as, A pressure-reducing component, mounted on the second cover and located on the side of the second cover opposite to the first cover, is used to limit the displacement distance of the tissue-engineered cartilage scaffold in the second container toward the second opening.
2. The culture container for the tissue-engineered cartilage scaffold according to claim 1, characterized in that, Also includes: The first functional layer has hydrophobic and breathable functions, and is disposed at the bottom of the second container and located in the second chamber; The bottom of the second container is provided with a number of first vent holes, and the first functional layer covers the first vent holes.
3. The culture container for the tissue-engineered cartilage scaffold according to claim 1, characterized in that, The bottom of the second container has a third opening; The culture vessel for the tissue-engineered cartilage scaffold also includes: A third cover is installed on the second container to cover the third opening, and the third cover is provided with a plurality of first vent holes; The first functional layer has hydrophobic and breathable functions. The first functional layer is disposed on the third cover and located in the second chamber to cover the first vent.
4. The culture container for the tissue-engineered cartilage scaffold according to claim 1, characterized in that, The pressure-reducing component includes: The first connector is attached to the surface of the second cover opposite to the first cover. A pressure plate component is connected to the end of the first connector that is furthest from the second cover. When the second cover is closed over the second opening, the first connector extends into the second chamber, so that the pressure plate can restrict the displacement of the tissue-engineered cartilage scaffold in the second container toward the second opening.
5. The culture container for the tissue-engineered 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 can enter the second chamber through the second cover.
6. The culture container for the tissue-engineered cartilage scaffold according to claim 5, characterized in that, Also includes: The second functional layer has hydrophobic and breathable functions, and the second functional layer is disposed on the second cover. The second cover has a plurality of second vent holes, and the second functional layer covers the second vent holes.
7. The culture container for the tissue-engineered cartilage scaffold according to claim 6, characterized in that, The pressure-reducing component includes: A support plate is disposed on the second cover, the second functional layer is located between the support plate and the cover, and the support plate is provided with third vent holes that correspond one-to-one with the second vent holes; The first connector connects to the side surface of the support plate that is away from the second functional layer. A pressure plate component is connected to the end of the first connector that is furthest from the support plate. When the second cover is closed over the second opening, the first connector extends into the second chamber, so that the pressure plate can restrict the displacement of the tissue-engineered cartilage scaffold in the second container toward the second opening.
8. The culture container for the tissue-engineered cartilage scaffold according to claim 1, characterized in that, 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.
9. The culture container for the tissue-engineered cartilage scaffold according to claim 1, characterized in that, The second connector connects the first cover to the second cover.
10. The culture container for the tissue-engineered 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.