Cell culture carrier capable of recycling cells
The cell culture carrier with alternating roll membrane structure and rhomboid pore design solves the problems of uneven cell distribution and low exosome production, realizes unlimited cell recovery and increased exosome production, simplifies the cell expansion process and maintains high cell viability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- TONGTENG INNOVATION (SUZHOU) CO LTD
- Filing Date
- 2025-03-27
- Publication Date
- 2026-05-15
AI Technical Summary
Existing cell culture vectors result in uneven cell distribution during large-scale culture, making cell recovery impossible. They also produce low exosome yields and are difficult to monitor and regulate cell state, leading to resource waste and shortened culture cycles.
The membrane structure consists of an alternating layer of cell growth layer, a thin film, and a thick film. Cell recovery is achieved through different sizes of rhomboid pores and centrifugal rotation. The alternating stacking of thin and thick films promotes exosome secretion, thereby improving cell viability and exosome production.
It enables unlimited cell recycling and continuous proliferation, simplifies the expansion process, increases exosome secretion, solves the problems of cell contact inhibition and metabolic waste accumulation, and maintains continuous expansion with high cell viability.
Smart Images
Figure CN224243106U_ABST
Abstract
Description
Technical Field
[0001] This application relates to the fields of exosome production and cell recycling technology, and in particular to a cell culture vector for recyclable cells. Background Technology
[0002] Contact inhibition of adherent cells is a key biological phenomenon in cell culture, which has a significant impact on the efficiency and scale of cell expansion in bioreactors.
[0003] Existing fixed-bed bioreactors, such as those based on randomly compacted paper-type PET membranes into donut-shaped cell culture media, allow for large-scale cell culture production. However, they suffer from poor cell distribution uniformity and the inability to recover cells. While roll-up PET membrane cell culture carriers offer uniform cell distribution and allow for cell density sampling and detection during the culture process, accurately reflecting the cell density within the reactor, their alternating double-layer PET membrane and single-layer PP mesh roll-up structure prevents cell recovery. Furthermore, the structure limits further increases in exosome production. Therefore, these bioreactors still exhibit the following shortcomings in practical applications:
[0004] When the cell culture carriers of the aforementioned patents are used in practice, the existing adherent cell culture scale is limited at one time, and the processing is cumbersome, requiring a lot of time and labor. The processing process has a high risk of bacterial contamination, and it is impossible to monitor and precisely control DO, pH and biochemical indicators during the culture process. Therefore, it is impossible to guarantee the optimal cell state and the quality of the final harvested product. After a single culture in a large-scale bioreactor, the cells in the tank cannot be recovered for continuous culture and reuse, which wastes cell resources and greatly increases the cell expansion culture time. In the existing rolled membrane structure, when the cells reach the plateau phase, non-tumor adherent cells will stop expanding due to contact inhibition. For larger cell clusters, this will cause insufficient dissolved oxygen and nutrient supply to the cells in the center of the cell cluster, as well as the accumulation of metabolic waste, which will cause cell senescence, apoptosis and ultimately cell death, shortening the cell expansion and culture product harvesting cycle. Utility Model Content
[0005] To address the challenges of unsustainable cell proliferation, cell recovery, seed cell preparation, and low exosome production efficiency, this application provides a cell culture vector capable of cell recovery.
[0006] The cell culture vector for recyclable cells provided in this application adopts the following technical solution:
[0007] A cell culture carrier for recyclable cells includes a cell growth layer for cell growth, wherein a thin film is disposed on the outer wall of the cell growth layer, and a thick film is disposed on the outer wall of the thin film.
[0008] Preferably, the cell growth layer material can be selected from materials such as polybutylene terephthalate, polypropylene terephthalate fiber, or polyethylene terephthalate.
[0009] By adopting the above technical solution, the basis weight of the cell growth layer is between 50 g / m³. 2 -200g / m 2 This allows for better cell culture. The different sizes of rhomboid pores one and two work together to allow centrifugation to detach most of the cells, thus achieving cell recovery. Meanwhile, the remaining cells continue to proliferate, resulting in an unlimited harvest of cells. The combination of a thin film and a thick film can also promote the exocytosis effect within the carrier, thereby increasing the amount of exosomes secreted.
[0010] Preferably, the sidewall of the thin film has a plurality of rhomboid holes I, and the sidewall of the thick film has a plurality of rhomboid holes II.
[0011] By adopting the above technical solution, the rhomboid holes are arranged in a completely equidistant manner, thereby maximizing the function of the thin film.
[0012] Preferably, the thickness of the film is set in the range of 0.1-2 mm.
[0013] By adopting the above technical solution, the film has certain stretching and bending properties, thereby improving the adaptability of cell attachment.
[0014] Preferably, the size of the diamond-shaped hole is set within the range of 1mm*1mm to 5mm*5mm.
[0015] By adopting the above technical solution, the flow of cells can be satisfied within this size range, and there is a reasonable area for cell attachment.
[0016] Preferably, the thickness of the thick film is set within the range of 0.2-5 mm.
[0017] By adopting the above technical solution, the thick film has certain stretching and bending properties, thereby improving the adaptability of cell attachment.
[0018] Preferably, the size of the second diamond-shaped hole is set within the range of 2mm*4mm to 6mm*10mm.
[0019] By adopting the above technical solution, the flow of cells can be satisfied within this size range, and there is a reasonable area for cell attachment.
[0020] Preferably, the thin film and the thick film are arranged in an alternating stacked state.
[0021] By adopting the above technical solution, the requirements of waterfall flow guidance and cell flushing can be met, while also allowing for partial cell retention.
[0022] In summary, this application includes at least one of the following beneficial technical effects:
[0023] 1. Utilizing the principle that after cell expansion reaches a plateau, no digestion process is required; simply increasing the rotation speed allows 10%–90% of the intracellular cells to detach from the membrane sidewall, with a free cell viability greater than 80%. Cell recovery can be achieved through centrifugation and other operations, greatly simplifying the cell expansion and recovery process. Intracellular cells can be continuously expanded and harvested through continuous beneficial detachment and renewal. Theoretically, for cancer cell lines and cell lines capable of unlimited expansion, cells can be harvested indefinitely. After separation, these cells can be used as seed cells for large-scale scaling and continuous production, and cell recovery is convenient.
[0024] 2. The new structure employs an alternating layered membrane structure consisting of a cell growth layer, a thin film, and a thick film. Microscopically, this increases cell shear stress stimulation by improving the rotational speed, promoting exocytosis within the carrier, and increasing the secretion of exosomes. It also addresses the damage to cells caused by contact inhibition and metabolic waste accumulation after the cells reach the plateau phase, maintaining high cell viability and continuous expansion within the cell culture medium. Attached Figure Description
[0025] Figure 1 This is an overall view of the cell culture vector for recyclable cells in this application;
[0026] Figure 2 This is an overall view of the cell culture vector for recyclable cells in this application;
[0027] Figure 3 This is a diagram illustrating the control structure of the cell culture vector for recyclable cells in this application;
[0028] Figure 4 This is a graph showing the cell expansion curve data within the membrane of the cell culture carrier for retrievable cells in this application.
[0029] Figure 5 This is a comparison chart of exosome particle concentration data for the cell culture vector of retrievable cells in this application.
[0030] Figure 6 This figure illustrates the density and viability of free cells in the supernatant of the cell culture vector containing retrievable cells, as presented in this application.
[0031] Figure 7 This is a diagram illustrating the honeycomb-shaped replacement structure of the cell culture carrier for recyclable cells in this application.
[0032] Figure label:
[0033] 1. Cell growth layer; 2. Thin film; 3. Thick film; 4. Rhomboid pore one; 5. Rhomboid pore two. Detailed Implementation
[0034] The following is in conjunction with the appendix Figures 1-7 This application will be described in further detail.
[0035] This application discloses a cell culture vector with recyclable cells.
[0036] Reference Figure 1 - Figure 7 A cell culture carrier for recyclable cells includes a cell growth layer 1 for cell growth, a thin film 2 attached to the sidewall of the cell growth layer 1 on one side, and a thick film 3 attached to the side of the thin film 2 away from the cell growth layer 1 on one side. The cell growth layer 1, thin film 2, and thick film 3 are fixedly connected according to the requirements of the culture steps. Materials such as polybutylene terephthalate, polypropylene terephthalate fiber, or polyethylene terephthalate can be used as the material for the cell growth layer 1. The porosity of the cell growth layer 1 is between 10% and 90%. Both thin film 2 and thick film 3 meet the requirements of a single-layer loose porous form (other honeycomb-like structures can be used for the spacer flow guide layer to eliminate fluid dead zones), thereby satisfying waterfall flow guidance and cell flushing. Multiple rhomboid pores are arranged in a 4-way configuration. Multiple rhomboid pores 5 are formed on the sidewall of the membrane 2 and the sidewall of the thick membrane 3, facilitating cell flow and attachment through rhomboid pores 4 and 5 of different sizes. The thickness of membrane 2 can be set in the range of 0.1-2 mm, the size of rhomboid pore 4 can be set in the range of 1 mm*1 mm to 5 mm*5 mm, and the thickness of thick membrane 3 can be set in the range of 0.2-5 mm, thus forming flow cross sections of different sizes. This avoids excessive cell detachment, which could affect subsequent cell proliferation. The size of rhomboid pore 5 can be set in the range of 2 mm*4 mm to 6 mm*10 mm. Membrane 2 and thick membrane 3 are arranged in an alternating stacked state, which obstructs cell flow at different pore sizes. Membrane 2 and thick membrane 3 can be made of materials that can meet the requirements of dissolved oxygen, nutrient supply, and cell free entry into the culture medium after detachment during cell culture.
[0037] Cell growth layer 1, thin film 2, and thick film 3 were all soaked in 0.2M-0.5M NaOH for more than 4 hours to remove endotoxins, then thoroughly washed with ultrapure water and dried at 70℃ for 72 hours to complete the sterilization process. Cell culture was then performed, followed by the use of Tongteng Xinchuang's independently developed... CultureAd60 and Ad600 fixed-bed bioreactors were used to inoculate HEK293T cells, followed by cyclic culture. Cell density counting, supernatant free cell detection, and supernatant biochemical assays were performed daily until the cell density reached 2E5 cells / cm³. 2Then, the rotation speed of the equipment is increased, thereby increasing the linear velocity. When the cells expand to the plateau phase, the amount and viability of free cells during cell expansion are detected. Combined with biochemical indicators such as glucose consumption and lactate accumulation, the harvest point is determined. Subsequently, 300g of the harvested supernatant is sampled, centrifuged for 10 minutes, and the concentration of exosome particles in the supernatant is detected using a nanoparticle tracking analyzer (NTA), thus completing the harvesting of cells and data acquisition.
[0038] in, CultureAd60, Ad600 fixed-bed bioreactors (Ad60FBR / Ad600FBR), and nanoparticle tracking analyzers (NTA) are all existing technologies, and their structural principles will not be elaborated here.
[0039] The implementation principle of the cell culture carrier with recyclable cells in this application embodiment is as follows:
[0040] Cell growth layer 1, thin film 2, and thick film 3 were soaked in 0.2M-0.5M NaOH for 4 hours to remove endotoxins, rinsed with ultrapure water, and then dried at 70℃ for 72 hours for sterilization. HEK293T cells were cultured in CultureAd60 and Ad600 fixed-bed bioreactors in circulating mode. Cell density, free cells in the supernatant, and biochemical parameters were monitored daily, reaching a cell density of 2E5 cells / cm³. 2 Then, the rotation speed was increased until the cell expansion plateau phase. Based on the amount of free cells, viability, and biochemical indicators such as glucose consumption and lactate accumulation, the culture medium was replaced with fresh medium for multiple harvests. The harvested supernatant was centrifuged at 300g for 10min, and the concentration of exosome particles was detected by NTA. Harvesting and data collection were completed.
[0041] The above are merely optional embodiments of this disclosure and are not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.
Claims
1. A cell culture carrier with recyclable cells, characterized in that: It includes a cell growth layer (1) for cell growth, the outer wall of the cell growth layer (1) is provided with a thin film (2), the outer wall of the thin film (2) is provided with a thick film (3), the side wall of the thin film (2) is provided with a plurality of rhomboid holes (4), and the side wall of the thick film (3) is provided with a plurality of rhomboid holes (5).
2. The cell culture carrier for recyclable cells according to claim 1, characterized in that: The cell growth layer (1) can be selected from polybutylene terephthalate, polypropylene terephthalate fiber or polyethylene terephthalate.
3. The cell culture carrier for recyclable cells according to claim 1, characterized in that: The thickness of the film (2) is set in the range of 0.1-2 mm.
4. The cell culture carrier for recyclable cells according to claim 1, characterized in that: The size of the rhomboid hole (4) is set within the range of 1mm*1mm to 5mm*5mm.
5. The cell culture carrier for recyclable cells according to claim 1, characterized in that: The thickness of the thick film (3) is set in the range of 0.2-5 mm.
6. The cell culture carrier for recyclable cells according to claim 1, characterized in that: The size of the second rhomboid hole (5) is set within the range of 2mm*4mm—6mm*10mm.
7. The cell culture carrier for recyclable cells according to claim 1, characterized in that: The thin film (2) and the thick film (3) are arranged in an alternating stacked state.