Cell culture device, cell culture system, and cell culture method
By designing a three-layer structure in the cell culture device and using a semi-permeable membrane layer for material exchange, efficient 3D cell culture is achieved, solving the problems of mechanical damage and contamination risks in the existing technology, and improving cell harvesting rate and amplification Effect.
Patent Information
- Application Number
- CN202411383709.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-01-07
AI Technical Summary
Existing suspended cell culture methods can easily lead to mechanical damage, risk of contamination and low cell harvest, and it is difficult to achieve efficient 3D cell culture.
Design a cell culture device including cell culture layer, semipermeable membrane layer and perfusion layer. It can realize 3D culture through a three-layer structure, use the semipermeable membrane layer to exchange substances and avoid direct contact, realize automatic fluid replenishment and perfusion, and expand the culture system No manual operation is required.
24-hour uninterrupted 3D cell culture was achieved, avoiding the risks of mechanical damage and contamination, improving the cell expansion multiple and dryness maintenance, significantly improving the cell harvest rate, and automation and sealing.
Smart Images
Figure CN119265039A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of cell culture technology, and in particular to a cell culture device, a cell culture system and a cell culture method. Background Art
[0002] Suspension culture refers to a method for culturing non-anchorage-dependent cells by dispersing and suspending isolated single cells and small cell clusters in liquid culture medium. The usual method is to continuously stir, oscillate or rotate the liquid culture medium. Specifically, if the suspension culture depth exceeds 5 mm, the culture medium needs to be stirred. If it exceeds 10 cm, CO needs to be introduced into the deep layer. 2 However, most suspension cultures currently use cell culture plates, cell culture bags or cell culture bottles for 2D cell culture, which requires frequent fluid replacement, rehydration and subculture, which can easily cause mechanical damage, increase the risk of contamination, and result in low cell yields. Summary of the invention
[0003] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a cell culture device, a cell culture system and a cell culture method.
[0004] In a first aspect of the present application, a cell culture device is provided, comprising:
[0005] The cell culture layer comprises a first culture chamber and a second culture chamber with gradually increasing volumes, the first culture chamber is provided with a cell inlet, a first fluid infusion port and a first fluid transfer outlet, and the second culture chamber is provided with a second fluid transfer inlet, a second fluid infusion port and a cell outlet; the cell culture layer further comprises a culture fluid transfer channel, the culture fluid transfer channel connects the first culture chamber and the second culture chamber through the first fluid transfer outlet and the second fluid transfer inlet;
[0006] Semipermeable membrane layer;
[0007] a perfusion layer, the perfusion layer being located on the other side of the semipermeable membrane layer relative to the cell culture layer, the perfusion layer comprising a first perfusion chamber and a second perfusion chamber, the first perfusion chamber being provided with a first perfusion port, and the second perfusion chamber being provided with a second perfusion port;
[0008] A fluid pipeline, the fluid pipeline is connected to the first perfusion chamber through the first perfusion port, and the fluid pipeline is connected to the second perfusion chamber through the second perfusion port;
[0009] The first culture chamber and the first perfusion chamber correspond to each other on both sides of the semipermeable membrane layer and are connected through the semipermeable membrane layer, and the second culture chamber and the second perfusion chamber correspond to each other on both sides of the semipermeable membrane layer and are connected through the semipermeable membrane layer.
[0010] The beneficial effects of the cell culture device provided in the embodiments of the present application are:
[0011] The cell culture device adopts a three-layer structure design, and a perfusion layer is arranged outside the cell culture layer through a semi-permeable membrane layer, so that 3D co-culture can be achieved. The design of the semi-permeable membrane layer can avoid direct contact between the upper cell culture layer and the lower perfusion layer while exchanging substances, so as to facilitate the separation of cells cultured in the upper cell culture layer and substances in the lower perfusion layer. In the upper cell culture layer, culture chambers corresponding to different specifications are designed. Culture can be carried out based on the starting volume of culture medium in the first culture chamber. When the culture reaches a certain level, it is transferred to the second culture chamber and automatically replenished to a culture system with higher specifications and larger volume, so that 24h uninterrupted or intermittent perfusion culture can be carried out to achieve system expansion without manual operation and centrifugal resuspension, avoiding the risk of contamination and mechanical damage.
[0012] In some embodiments of the present application, the cell culture layer further includes a transit culture chamber having a volume between the first culture chamber and the second culture chamber, the transit culture chamber being provided with a transit transfer liquid inlet, a transit fluid replenishment port and a transit transfer liquid outlet, the culture transfer liquid channel connecting the first culture chamber and the transit culture chamber through the first transfer liquid outlet and the transit transfer liquid inlet, the culture transfer liquid channel connecting the transit culture chamber and the second culture chamber through the transfer transfer liquid outlet and the second transfer liquid inlet; the perfusion layer further includes a transit perfusion chamber, the transit perfusion chamber being provided with a transit perfusion port, the fluid pipeline being connected to the transit perfusion chamber through the transit perfusion port; the transit culture chamber and the transit perfusion chamber correspond to each other on both sides of the semipermeable membrane layer and are connected via the semipermeable membrane layer.
[0013] In some embodiments of the present application, there are multiple transfer culture chambers and transfer perfusion chambers in one-to-one correspondence, the volumes of the multiple transfer culture chambers increase successively along the liquid transfer direction, and the culture liquid transfer channel is connected to the adjacent transfer culture chambers in the liquid transfer direction through the previous liquid transfer outlet and the next liquid transfer inlet in the liquid transfer direction.
[0014] In some embodiments of the present application, the volume of the first culture chamber and the second culture chamber is 20mL-5L.
[0015] In some embodiments of the present application, the perfusion chamber is filled with microcarriers.
[0016] In some embodiments of the present application, the second culture chamber includes a pH detection unit and a dissolved oxygen detection unit.
[0017] In a second aspect of the present application, a cell culture system is provided, which comprises any one of the aforementioned cell culture devices.
[0018] In some embodiments of the present application, the cell culture system further comprises a physical mixing device, which is used to disperse the cells in the cell culture device in the culture medium.
[0019] A third aspect of the present application provides a cell culture method, which comprises using any of the aforementioned cell culture devices or cell culture systems to culture cells in a first culture chamber for a set period of time and then transferring the cells to a second culture chamber for expanded culture.
[0020] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Schematic diagram of the overall structure of a cell culture device in one embodiment of the present application.
[0022] Figure 2 FIG. 1 is a schematic diagram of a cell culture layer of a cell culture device in one embodiment of the present application.
[0023] Figure 3 Schematic diagram of a perfusion layer of a cell culture device in one embodiment of the present application.
[0024] Figure 4 It is a schematic diagram of the cell culture device in Example 1 of the present application.
[0025] Figure 5 These are the cell expansion results after culturing for different periods of time using the cell culture device of Example 1 and conventional 2D culture in Example 2 of the present application.
[0026] Figure 6 It is the ratio of CD34 / CD45 double positive cells after different time of culture using the cell culture device of Example 1 and conventional 2D culture in Example 2 of the present application. Among them, A and B are the flow cytometry results of G1 and G2 on the 14th day, C and D are the flow cytometry results of G1 and G2 on the 21st day, and E is the ratio of CD34 / CD45 double positive cells at different culture time.
[0027] Figure numerals: cell culture layer 100, first culture chamber 110, cell inlet 111, first liquid transfer outlet 112, first liquid infusion port 113, second culture chamber 120, cell outlet 121, second liquid transfer inlet 122, second liquid infusion port 123, culture liquid transfer channel 130, semi-permeable membrane layer 200, perfusion layer 300, first perfusion chamber 310, first perfusion outlet 311, first perfusion inlet 312, second perfusion chamber 320, second perfusion inlet 321, second perfusion outlet 322, fluid pipeline 400. DETAILED DESCRIPTION
[0028] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.
[0029] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.
[0030] In the description of this application, "several" means more than one, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.
[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.
[0032] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means 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. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.
[0033] refer to Figure 1, shows a schematic diagram of the structure of a cell culture device in an embodiment of the first aspect of the present application. The cell culture device includes a cell culture layer 100, a semipermeable membrane layer 200 and a perfusion layer 300 arranged in sequence. The cell culture layer 100 includes a first culture chamber 110 and a second culture chamber 120, and the volume (or capacity) between the first culture chamber 110 and the second culture chamber 120 gradually increases, that is, the volume of the first culture chamber 110 is smaller than the volume of the second culture chamber 120. The perfusion layer 300 is located on the other side of the semipermeable membrane layer 200 relative to the cell culture layer 100, and the perfusion layer 300 includes a first perfusion chamber 310 and a second perfusion chamber 320. The first culture chamber 110 and the first perfusion chamber 310 correspond to each other on both sides of the semipermeable membrane layer 200 and are connected to each other via the semipermeable membrane layer 200, and the second culture chamber 120 and the second perfusion chamber 320 correspond to each other on both sides of the semipermeable membrane layer 200 and are connected to each other via the semipermeable membrane layer 200.
[0034] The first culture chamber 110 is provided with a first liquid transfer outlet 112, the second culture chamber 120 is provided with a second liquid transfer inlet 122, and the cell culture layer 100 is further provided with a culture liquid transfer channel 130, and the culture liquid transfer channel 130 connects the first culture chamber 110 and the second culture chamber 120 through the first liquid transfer outlet 112 and the second liquid transfer inlet 122. Specifically, when it is necessary to transfer the culture medium containing cells in the first culture chamber 110 to the second culture chamber 120, the culture liquid transfer channel 130 between the first culture chamber 110 and the second culture chamber 120 is opened, and under the action of external force, the culture medium containing cells in the first culture chamber 110 can be transferred to the second culture chamber 120 with a larger volume for expanded culture.
[0035] refer to Figure 2 The first culture chamber 110 is provided with a cell inlet 111 and a first liquid transfer outlet 112, and the second culture chamber 120 is provided with a second liquid transfer inlet 122 and a cell outlet 121. Under appropriate conditions, cells are introduced into the first culture chamber 110 through the cell inlet 111 for culture, and when the cells are cultured to a certain extent, the cells cultured in the second culture chamber 120 are collected through the cell outlet 121.
[0036] In some embodiments, the first culture chamber 110 is further provided with a first liquid replenishing port 113, through which the required culture medium is replenished into the first culture chamber 110. In some embodiments, the second culture chamber 120 is further provided with a second liquid replenishing port 123, through which the required culture medium is replenished into the second culture chamber 120.
[0037] In some embodiments, the material added to the perfusion area is pre-stored in a perfusion material container. It is understood that the perfusion material container is connected to the annular pipeline, so that when perfusion is required, the perfusion material container is transported to the perfusion chamber required for perfusion through the annular pipeline. In some embodiments, the perfusion material container is a perfusion device tank.
[0038] refer to Figure 3 , the first perfusion chamber 310 is provided with a first perfusion port, including a first perfusion inlet 312 and a first perfusion outlet 311, and the second perfusion chamber 320 is provided with a second perfusion port, including a second perfusion inlet 321 and a second perfusion outlet 322. In some embodiments, the cell culture device further comprises a fluid pipeline 400, the fluid pipeline 400 is connected to the first perfusion chamber 310 through the first perfusion port (i.e., the first perfusion inlet 312 and the first perfusion outlet 311), and the fluid pipeline 400 is connected to the second perfusion chamber 320 through the second perfusion port (i.e., the second perfusion inlet 321 and the second perfusion outlet 322). An opening and closing unit is provided on the first perfusion port (i.e., the first perfusion inlet 312 and the first perfusion outlet 311) and the second perfusion port (i.e., the second perfusion inlet 321 and the second perfusion outlet 322) to control the opening and closing of the first perfusion port and the second perfusion port. Specifically, an opening and closing unit may be provided on the first perfusion inlet 312, the first perfusion outlet 311, the second perfusion inlet 321, and the second perfusion outlet 322, respectively, to control the opening and closing of the first perfusion inlet 312, the first perfusion outlet 311, the second perfusion inlet 321, and the second perfusion outlet 322, respectively, and to adjust the connection and disconnection between the fluid pipeline 400 and the first perfusion chamber 310 and the second perfusion chamber 320. In some embodiments, the opening and closing unit may be a liquid stop clamp. When liquid needs to be added to the first perfusion chamber 310, liquid flows in from the corresponding first perfusion inlet 312 through the fluid pipeline 400, flows out from the first perfusion outlet 311 after filling the first perfusion chamber 310, and then flows back to the perfusion device tank through the fluid pipeline 400. In addition, when it is necessary to transfer the material in the first perfusion chamber 310 to the second perfusion chamber 320, the liquid stop clamp between the first perfusion chamber 310 and the second perfusion chamber 320 is adjusted, and the material flows out from the corresponding first perfusion outlet 311 through the fluid pipeline 400, and then flows to the second perfusion inlet 321 through the fluid pipeline 400 to pour the required material into the second perfusion chamber 320, and after filling the second perfusion chamber 320, it flows out from the second perfusion outlet 322, and then flows back to the perfusion device tank through the fluid pipeline 400. Under the action of external force, the material in the first perfusion chamber 310 can be transferred to the second perfusion chamber 320.
[0039] In some embodiments, the first perfusion outlet 311 on the first perfusion chamber 310 and the first liquid transfer outlet 112 on the first culture chamber 110 are separate outlets that are not connected, and the second perfusion outlet 322 on the second perfusion chamber 320 and the cell outlet 121 on the second culture chamber 120 are separate outlets that are not connected. Therefore, in some embodiments, the culture liquid transfer channels 130 between the various culture chambers (including the first culture chamber 110, the second culture chamber 120, and possible transfer culture chambers) can be separate channels.
[0040] The cell culture device provided in the embodiment of the present application is designed with a three-layer structure, and a perfusion layer is arranged outside the cell culture layer through a semi-permeable membrane layer, so that 3D co-culture can be achieved. The design of the semi-permeable membrane layer can avoid direct contact between the upper cell culture layer and the lower perfusion layer while exchanging substances, thereby facilitating the separation of cells cultured in the upper cell culture layer and substances in the lower perfusion layer. In the upper cell culture layer, culture chambers corresponding to different specifications are designed, and culture can be carried out based on the first culture chamber with an initial volume of culture medium. When cultured to a certain extent, it is transferred to the second culture chamber and automatically replenished to a culture system with higher specifications and larger volume, so that 24h uninterrupted or intermittent perfusion culture can be carried out to achieve system expansion without manual operation and centrifugal resuspension, avoiding the risk of contamination and mechanical damage.
[0041] In some specific embodiments, the materials added to the perfusion zone include, but are not limited to, at least one of culture medium, cells, exosomes, etc. In some embodiments, the cells may be, for example, any cells that can be cultured on a wall, such as at least one of mesenchymal stem cells, fibroblasts, osteoblasts, chondroblasts, cardiomyocytes, smooth muscle cells, hepatocytes, lung cells, kidney cells, breast cells, epithelial cells, glial cells, endocrine cells, melanocytes, tumor cells, etc. In some embodiments, the exosomes may be any exosomes secreted by cells, such as mesenchymal stem cell exosomes, etc. In some embodiments, the material added to the perfusion zone is a separate culture medium, so that the cells in the cell culture layer are perfused and cultured.
[0042] In some embodiments, the cell culture layer can be used to culture optional cells that can be cultured in suspension, including but not limited to at least one of hematopoietic stem cells, lymphocytes, etc.
[0043] In some embodiments, the cell culture layer also includes a transfer culture chamber whose volume is between the first culture chamber and the second culture chamber, the transfer culture chamber is provided with a transfer liquid inlet, a transfer liquid inlet and a transfer liquid outlet, the culture liquid transfer channel is connected to the first culture chamber and the transfer culture chamber through the first transfer liquid outlet and the transfer liquid inlet, and the culture liquid transfer channel is connected to the transfer culture chamber and the second culture chamber through the transfer liquid outlet and the second transfer liquid inlet. The perfusion layer also includes a transfer perfusion chamber, the transfer perfusion chamber is provided with a transfer perfusion port (including a corresponding transfer perfusion liquid inlet and a transfer perfusion liquid outlet), and the transfer culture chamber and the transfer perfusion chamber correspond to each other on both sides of the semipermeable membrane layer and are connected via the semipermeable membrane layer. In the cell culture process, a third specification of cell culture area can also be added, so that the cells are cultured in the first culture chamber, the transfer culture chamber and the second culture chamber with gradually increasing volumes, thereby realizing the expansion of the system. Correspondingly, in this process, the culture liquid transfer channel is connected to the transfer culture chamber through the transfer liquid port, and the fluid pipeline is connected to the transfer perfusion chamber through the transfer perfusion port. Fluid is replenished into the transfer culture chamber through fluid pipelines to maintain culture, and materials are supplemented into the transfer culture chamber to maintain perfusion or further culture in the perfusion chamber.
[0044] In some embodiments, there are multiple transfer culture chambers and transfer perfusion chambers and they correspond one to one. The volumes of the multiple transfer culture chambers increase in sequence along the liquid transfer direction. The culture liquid transfer channel connects the adjacent transfer culture chambers in the liquid transfer direction through the previous liquid transfer outlet and the next liquid transfer inlet in the liquid transfer direction. Among them, the liquid transfer direction refers to the direction of the cell-containing culture medium from the previous culture chamber to the next culture chamber, or the perfused material from the previous perfusion chamber to the next perfusion chamber, specifically refers to the direction of movement of the cell-containing culture medium from the previous culture chamber with a smaller volume to the next culture chamber with a larger volume. Cells are transferred in this liquid transfer direction, and the culture system is gradually expanded in multiple times. In some embodiments, the volume from the first perfusion chamber to the second perfusion chamber also increases. In some specific embodiments, the volume of multiple transfer perfusion chambers also increases in sequence along the liquid transfer direction, that is, the volume of the first perfusion chamber, transfer perfusion chamber 1, transfer perfusion chamber 2, transfer perfusion chamber 3..., and the second perfusion chamber gradually increases.
[0045] In some embodiments, the volume of the first culture chamber and the second culture chamber is 20mL to 5L, for example, 20mL, 50mL, 100mL, 200mL, 500mL, 1L, 2L, 5L. Specifically, the volume of the first culture chamber is 20 to 100mL, and the volume of the second culture chamber is 1 to 5L. In some embodiments, one or more culture chambers are further included between the first culture chamber and the second culture chamber, and the volume of the one or more transfer culture chambers can be, for example, 20mL, 50mL, 100mL, 200mL, 500mL, 1L, 2L, 5L. It can be understood that the volumes of the first culture chamber, the second culture chamber, and the one or more transfer culture chambers therebetween meet the aforementioned gradually increasing requirements.
[0046] In some embodiments, when cells are cultured in the first culture chamber, the transfer culture chamber or the second culture chamber, the liquid transfer channel or fluid pipeline connecting the culture chamber where the cells are located with the adjacent culture chamber is closed, so that the culture chamber is in an independent culture state disconnected from other culture chambers.
[0047] In some embodiments, the second culture chamber includes at least one of a pH detection unit and a dissolved oxygen detection unit. For example, a pH detection unit, a dissolved oxygen detection unit, or a pH detection unit and a dissolved oxygen detection unit are set in the second culture chamber, so as to detect the pH and / or dissolved oxygen of the culture medium in the second culture chamber, so as to further analyze the culture state of the cells therein, thereby realizing comprehensive monitoring and recording of key process parameters, and gas supplementation, pH adjustment, etc. can be performed according to a preset program. For example, liquid can be directly added to the second culture chamber as needed, or materials can be added to the corresponding second perfusion chamber. In this way, the cell growth state is monitored in real time, and a large number of suspended cells can be fully automatically and closed culture. Since the first culture chamber is small in size, a pH detection unit and a dissolved oxygen detection unit may not be set. In some embodiments, when multiple transfer culture chambers are contained, a pH detection unit and a dissolved oxygen detection unit can be set in a slightly larger transfer culture chamber with a volume sufficient to set the detection unit. For example, the transfer culture chamber adjacent to the second culture chamber in the liquid transfer direction also includes at least one of a pH detection unit and a dissolved oxygen detection unit.
[0048] In some embodiments, the first culture chamber, the transfer culture chamber, and the second culture chamber are provided with pores. In some embodiments, the pores are provided with filters.
[0049] In some embodiments, the pore size of the semipermeable membrane is 0.1 to 5 μm, for example, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.8 μm, 1 μm, 2 μm, 3 μm, 4 μm, or 5 μm.
[0050] The second aspect of the present application provides a cell culture system, which includes any of the aforementioned cell culture devices. In some embodiments, the cell culture system also includes a physical mixing device, which is used to disperse the cells in the cell culture device into the culture medium. In some specific embodiments, the physical mixing device includes but is not limited to a WAVE-type swing bed reaction system, which has a wave-type shaking mixing function, based on the WAVE swing principle, so that the suspended culture cells are fully in contact with the feed liquid, thereby increasing the yield per unit volume of culture.
[0051] The third aspect of the present application provides a cell culture method, which includes using any of the aforementioned cell culture devices or cell culture systems, culturing in a first culture chamber for a set time and then transferring to a second culture chamber for expanded culture. In some embodiments, when one or more transfer culture chambers are included between the first culture chamber and the second culture chamber, the cells are cultured independently in the first culture chamber, the transfer culture chamber, and the second culture chamber for the same or different time periods.
[0052] Example 1
[0053] refer to Figure 4 Combined with Figures 1 to 3, this embodiment provides a cell culture device, including a cell culture layer, a semipermeable membrane layer and a perfusion layer. The cell culture layer is provided with a first culture chamber 110, a first transfer culture chamber 140, a second transfer culture chamber 150, a third transfer culture chamber 160, and a second culture chamber 120, a total of five culture areas of different specifications, corresponding to different cell culture stages. Each culture chamber is an independent culture space, and is provided with a vent with a filter. The volumes of the first culture chamber 110, the first transfer culture chamber 140, the second transfer culture chamber 150, the third transfer culture chamber 160, and the second culture chamber 120 are 20mL, 50mL, 200mL, 1000mL, and 5000mL, respectively. The first culture chamber 110 is provided with a cell inlet 111 for the initial addition of cells and other substances. The first culture chamber 110 is also provided with a first liquid inlet 113 for adding culture fluid to the first culture chamber 110. The first culture chamber 110 is also provided with a first liquid transfer outlet 112, which is connected to the corresponding transfer liquid transfer inlet on the first transfer culture chamber 140 through the culture liquid transfer channel 130. After the culture experiment is completed in the first culture chamber 110, the liquid transfer can be performed to transfer the culture liquid containing cells to the first transfer culture chamber 140. Similar liquid transfer and liquid replenishment structures are provided in the first transfer culture chamber 140 to the third transfer culture chamber 160 and the second culture chamber 120. Among them, the second culture chamber 120 is provided with a cell outlet 121, a second liquid transfer inlet 122, and a second liquid replenishment port 123. The second liquid transfer inlet 122 is connected to the liquid transfer outlet on the third transfer culture chamber 160 through the culture liquid transfer channel 130. A pH sensor and a dissolved oxygen sensor are provided in the third transfer culture chamber 160 and the second culture chamber 120.
[0054] The perfusion layer is provided with a first perfusion chamber, transfer perfusion chambers 1 to 3, and a second perfusion chamber, each of which is provided with a perfusion inlet and a perfusion outlet, which are respectively connected to the outermost annular fluid pipeline 400. When the first culture chamber starts to culture, the liquid stop clamps of the first perfusion chamber inlet and outlet are opened, and the liquid stop clamps between the inlet and outlet of other perfusion chambers and the fluid pipeline and the liquid stop clamp on the fluid pipeline 400 between the perfusion inlet and outlet of the first perfusion chamber are closed, and the perfused material will pass through the first perfusion chamber and flow back to the perfusion device tank through the fluid pipeline 400. The same applies to the transfer perfusion chambers 1 to 3 and the second perfusion chamber.
[0055] Example 2
[0056] 1. Prepare the materials
[0057] (1) Prepare sterile instruments, gun tips, centrifuge tubes, etc., and dry them under high pressure in advance. Expose the room and operating table to ultraviolet light in advance.
[0058] 2 Isolation of mononuclear cells
[0059] 2.1 Sample
[0060] After disinfecting the sample package with 75% alcohol, cut the plastic tube with sterilized scissors and transfer it to a 50mL centrifuge tube.
[0061] 2.2 Separation of plasma
[0062] Balance the centrifuge tube containing the umbilical cord blood and centrifuge.
[0063] 2.3 Density gradient centrifugation to separate mononuclear cells
[0064] (1) After aspirating the plasma layer, resuspend the umbilical cord blood sample in DPBS and mix well.
[0065] (2) slowly adding the diluted sample into a sterile centrifuge tube containing human lymphocyte separation medium and centrifuging;
[0066] (3) Extraction of mononuclear cells: After centrifugation, obvious stratification appears in the centrifuge tube. Carefully aspirate the mononuclear cells and add DPBS;
[0067] (4) Washing: centrifugation;
[0068] 3 Magnetic bead separation of CD34 + cell
[0069] 3.1 Preparation of magnetic beads
[0070] (1) Mix the magnetic beads, calculate the required magnetic beads according to the ratio, add PBS solution to resuspend, and mix well
[0071] (2) Wash the magnetic beads: Wash the magnetic beads with PBS.
[0072] 3.2CD34 + Cell attachment magnetic beads
[0073] (1) After centrifugation of the cell suspension, discard the supernatant, add DPBS to the sample, and then add it to the washed magnetic beads;
[0074] (2) Incubation;
[0075] (3) Wash away non-specifically bound cells: Place the flow cytometry tube on a magnetic stand, aspirate the supernatant, and repeat the wash twice;
[0076] (4) Harvesting CD34 at room temperature + Cells: Add elution buffer to the flow cytometry tube, incubate at room temperature, collect the washing buffer and centrifuge.
[0077] 4. Amplification of CD34 + cell
[0078] The cells were resuspended in human hematopoietic stem cell serum-free culture medium and inoculated into the first culture chamber of the cell culture device in Example 1, and the corresponding perfusion fluid was added to the lower layer for expansion culture. At the same time, a 2D group without a 3D culture device was set as a control.
[0079] 5. Cell state detection
[0080] Culture to D14 and D21, and detect the cell status:
[0081] (1) Cell counting: Collect the cells in the device and blow them evenly, pipette 20 μL into a cell counting plate, and count them using a cell counter.
[0082] (2) Flow cytometry: Flow cytometry is used to analyze cell phenotypic markers to identify cell subpopulations in culture.
[0083] The results are as follows Figures 5-6 As shown, in the process of screening the culture system, hematopoietic stem cells and the lower nutrient solution were co-cultured using different culture systems, expanded in vitro for 21 days, and the cell status was detected at 14 days and 21 days. G1 is the 2D culture result without the co-culture device, and G2 is the 3D culture result using the co-culture device of the present invention. The results showed that the sample was co-cultured for 14 days and the expansion multiple could reach up to about 40 times, and the dryness was maintained at about 10.6%. After 21 days, it could reach about 68 times, and the dryness was maintained at about 5.70%. It can be seen that as the number of cell culture days increases, the cell expansion multiple gradually increases; the cell stemness gradually decreases with the increase in the number of culture days. The 3D co-culture system has higher effects on cell expansion and stemness maintenance than the 2D control group, which is specifically reflected in the cell expansion multiple and the proportion of CD34 / CD45 double-positive cells are higher than the 2D control group, proving that the co-culture system has better effects on the expansion and stemness maintenance of hematopoietic stem cells. Therefore, by using this cell culture device to co-culture the two systems, it can be observed that the cell proliferation multiples and stemness in the 3D co-culture system are better than those in 2D culture.
[0084] The cell culture device provided in the embodiment of the present application can achieve significantly higher effect than 2D planar culture by co-culturing nutrients and suspended cells in 3D, which can greatly increase the yield of the desired cells and maintain the stemness of the cells. During use, the lower layer of material does not directly contact the upper layer of cells, and only exchanges materials through a semipermeable membrane, which can solve the problem of different culture media when different cells are co-cultured and the difficulty of separating different cells when co-cultured. At the same time, it can avoid mutual killing of different cells when they come into contact, realize indirect contact between cells, and complete activation or inhibition between different cells.
[0085] In addition, continuous perfusion can be achieved by transferring between cell culture chambers of different specifications, avoiding mechanical damage such as centrifugation while expanding the culture system, and avoiding contamination caused by manual operation. At the same time, it also has functions such as gas replenishment, real-time monitoring, and wave-like shaking mixing. It can perform multiple fluid perfusions and preliminary concentrations according to preset programs, monitor the cell growth status in real time, and achieve fully automatic and closed culture of large-scale suspended cells. It has obvious advantages in reducing costs, easy operation, and the potential for more precise control, and has broad prospects for mass production and industrial production of target cells.
[0086] In addition, this cell culture device has a wider range of clinical applications. For example, two different systems can be used to culture multiple stem cells at the same time. The upper cell culture chamber can culture various suspended cells, such as hematopoietic stem cells, lymphocytes, etc. The lower perfusion chamber can continuously perfuse various required substances or other cells, such as exosomes, cell suspensions, nutrient solutions, etc. In addition, if it is necessary to co-culture adherent cells and suspended cells, it can be achieved through microcarriers. It is also possible to harvest immune killer factors in the culture fluid and activate cells by culturing tumor cells, immune killer cells, etc.
[0087] The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.
Claims
1. A cell culture device, characterized in that include: A cell culture layer, the cell culture layer comprising a first culture chamber and a second culture chamber with gradually increasing volumes, the first culture chamber being provided with a cell inlet, a first fluid infusion port and a first fluid transfer outlet, the second culture chamber being provided with a second fluid transfer inlet, a second fluid infusion port and a cell outlet; the cell culture layer further comprising a culture fluid transfer channel, the culture fluid transfer channel being connected to the first culture chamber and the second culture chamber through the first fluid transfer outlet and the second fluid transfer inlet; Semipermeable membrane layer; a perfusion layer, the perfusion layer being located on the other side of the semipermeable membrane layer relative to the cell culture layer, the perfusion layer comprising a first perfusion chamber and a second perfusion chamber, the first perfusion chamber being provided with a first perfusion port, and the second perfusion chamber being provided with a second perfusion port; a fluid pipeline, wherein the fluid pipeline is connected to the first perfusion chamber through the first perfusion port, and the fluid pipeline is connected to the second perfusion chamber through the second perfusion port; The first culture chamber and the first perfusion chamber correspond to each other on both sides of the semipermeable membrane layer and are communicated via the semipermeable membrane layer, and the second culture chamber and the second perfusion chamber correspond to each other on both sides of the semipermeable membrane layer and are communicated via the semipermeable membrane layer.
2. The cell culture device according to claim 1, characterized in that: The cell culture layer further comprises a transfer culture chamber with a volume between the first culture chamber and the second culture chamber, the transfer culture chamber is provided with a transfer liquid inlet, a transfer liquid inlet and a transfer liquid outlet, the culture transfer liquid channel is connected to the first culture chamber and the transfer culture chamber through the first transfer liquid outlet and the transfer liquid inlet, and the culture transfer liquid channel is connected to the transfer culture chamber and the second culture chamber through the transfer liquid outlet and the second transfer liquid inlet; The perfusion layer further comprises a transfer perfusion chamber, wherein the transfer perfusion chamber is provided with a transfer perfusion port, and the fluid pipeline is connected with the transfer perfusion chamber through the transfer perfusion port; The transit culture chamber and the transit perfusion chamber correspond to each other at two sides of the semipermeable membrane layer and are connected via the semipermeable membrane layer.
3. The cell culture device according to claim 2, characterized in that: The transfer culture chambers and the transfer perfusion chambers are provided in multiple and one-to-one correspondence, and the volumes of the multiple transfer culture chambers increase successively along the liquid transfer direction, and the culture liquid transfer channel is connected to the adjacent transfer culture chambers in the liquid transfer direction through the previous liquid transfer outlet and the next liquid transfer inlet in the liquid transfer direction.
4. The cell culture device according to claim 1, characterized in that: The volumes of the first culture chamber and the second culture chamber are 20 mL to 5 L.
5. The cell culture device according to claim 1, characterized in that: The perfusion chamber is filled with microcarriers.
6. The cell culture device according to claim 1, characterized in that: The pore size of the semipermeable membrane is 0.1-5 μm.
7. The cell culture device according to claim 1, characterized in that: The second culture chamber includes a pH detection unit and a dissolved oxygen detection unit.
8. A cell culture system, characterized in that A cell culture device comprising any one of claims 1 to 7.
9. The cell culture system according to claim 8, characterized in that: The cell culture system further comprises a physical mixing device, which is used to disperse the cells in the cell culture device in a culture medium.
10. A cell culture method, characterized in that Using the cell culture device according to any one of claims 1 to 7, or the cell culture system according to any one of claims 8 to 9, after culturing in the first culture chamber for a set period of time, the cells are transferred to the second culture chamber for expanded culture.