A culture device capable of continuously coating a cell-containing hydrogel with a protective layer

Abstract

The present invention relates to the technical field of biomedical instruments, and discloses a culture device capable of continuously coating a cell-containing hydrogel with a protective layer. The culture device comprises a cell thread printing module, which is used for printing the cell-containing hydrogel into a cell thread; a cell thread receiving module, which is located below the cell thread printing module and is used for receiving the cell thread; a protective layer forming module, which is located between the cell thread printing module and the cell thread receiving module and comprises a liquid spraying mechanism and a coating mechanism; the liquid spraying mechanism comprises a nozzle for spraying a protective liquid to the cell thread, and the coating mechanism comprises a first electrode and a second electrode connected to the nozzle, the cell thread is allowed to pass between the first electrode and the nozzle, and the first electrode and the second electrode cooperate to evenly distribute the protective liquid on the outer surface of the cell thread.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical instruments, and in particular to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer. Background Art

[0002] Exosomes are nano-sized vesicles with a double lipid membrane structure and a diameter of 30-150nm. They contain rich biological substances such as functional proteins, lipids and nucleic acids (RNA, DNA), which can perform important functions such as intercellular communication and regulation. Exosomes have a wide range of sources, mainly isolated and extracted from the body (blood, saliva and other body fluids, and secretions such as milk) and in vitro (cell culture). At present, exosomes used in the fields of beauty and medicine are mainly extracted based on stem cell culture. Stem cell-derived exosomes have extremely low immunogenicity and good biocompatibility, can efficiently penetrate major biological barriers such as the plasma membrane and blood-brain barrier, and have no risks of embolism or tumorigenesis when used. They play an important role in the regulation of immune response, tissue repair, cancer treatment, and as a natural drug carrier.

[0003] High-quality culture of stem cells is an important basis for the production of exosomes. At present, conventional methods for 3D culture of stem cells include 3D printing technology, microcarriers, etc. 3D printing combines cells and hydrogels together, and stem cells grow inside the hydrogels full of micropores to simulate the three-dimensional microenvironment of cells in the body; the microcarrier process is to plant cells on the surface of microcarriers for culture. After long-term practice, it is found that the existing process still has some shortcomings in production: 1. The long-term stability of hydrogels is poor. The main raw materials of hydrogels are natural polymer materials. Natural polymer materials will gradually degrade under long-term immersion, and the original macroscopic pore structure will collapse after degradation. The penetration of nutrients and oxygen in hydrogels is limited by distance, which will lead to changes in the cell microenvironment or even death, directly causing changes in the quality, type and quantity of exosomes secreted by cells; 2. In order to maintain the stability of the culture structure during the production cycle, it is necessary to add a cross-linking agent, which directly increases the material cost and labor cost. In this process, the cell culture medium must first be drained, and then the cross-linking agent solution is added. After a period of cross-linking, the cross-linking agent is drained, and then saline is pumped into the culture device for cleaning, and then the saline is pumped out, and fresh culture medium is added again; 3. The microcarrier culture is not biomimetic enough to simulate the in vivo 3D microenvironment, and the components that make up the microcarriers will also be broken. The broken debris will enter the culture medium and cause certain troubles for the subsequent exosome extraction; 4. Cell status detection is more difficult. Direct observation is difficult across the culture tank. If partial sampling is tested, it will be time-consuming and labor-intensive, and its microscopic environment is uneven, and even sampling is difficult to reflect the overall situation.

[0004] Based on the traditional cell 3D printing process, the hydrogel microstructure that provides mechanical support will gradually collapse after a period of culture, and the penetration of nutrients and oxygen in the hydrogel is limited by distance, which will lead to changes in the cell microenvironment or even death, directly causing changes in the quality, type, and quantity of exosomes secreted by cells. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a culture device that can continuously coat a cell-containing hydrogel with a protective layer, forming a polymer protective film on the outside of the 3D printed cell filaments, which can ensure the long-term stability and reliability of the cell microenvironment of different batches and different positions in the culture device, facilitate monitoring of the cell state, reduce the use of traditional hydrogel crosslinkers, significantly simplify the process, and reduce costs.

[0006] A culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention comprises:

[0007] A cell thread printing module, used for printing cell-containing hydrogels into cell threads;

[0008] A cell thread receiving module, located below the cell thread printing module, is used to receive the cell thread;

[0009] A protective layer forming module is located between the cell filament printing module and the cell filament receiving module, and includes a liquid spraying mechanism and a laminating mechanism;

[0010] The liquid spraying mechanism includes a nozzle for spraying protective liquid to the cell thread, and the covering mechanism includes a first electrode and a second electrode connected to the nozzle. The cell thread is allowed to pass between the first electrode and the nozzle. The first electrode and the second electrode cooperate to evenly distribute the protective liquid on the outer surface of the cell thread.

[0011] According to an embodiment of the present invention, a culture device that can continuously coat a cell-containing hydrogel with a protective layer has at least the following beneficial effects: the present application realizes continuous printing and uniform coating of a cell-containing hydrogel with a protective layer by integrating multiple functional modules such as cell thread printing, receiving and protective layer formation. Specifically, the cell thread printing module is responsible for printing the cell-containing hydrogel into a thread state, and the thread can pass between the first electrode and the nozzle. The liquid spraying mechanism is responsible for spraying the protective liquid to the thread passing through. The electric field formed by the cooperation of the first electrode and the second electrode allows the protective liquid to be evenly distributed on the cell thread, and the protective liquid forms a protective layer on the outer wall of the cell thread. The cell thread coated with the protective layer enters the cell thread receiving module for storage and culture. Among them, the cell thread printing module can continuously extrude the cell thread, and the cell type in the cell thread can be replaced according to actual needs. Since the outer wall of the cell thread is covered with a protective layer, even if multiple cells are cultured simultaneously in the receiving dish, there will be no interference. The cells can maintain a stable state during the culture cycle, ensuring the long-term stability and reliability of the cell microenvironment in different batches and different positions in the receiving dish. At the same time, for the same type of cell culture, researchers can also sample and test a certain section of the cell thread to determine the overall culture situation. Researchers do not need to print and culture different types of cell cultures separately as in traditional culture methods. The culture device of the present application can not only ensure the long-term stability of the state of the cell microenvironment, ensure the stability and uniformity of cell quality everywhere, but also save manpower and material resources and simplify the production and processing technology.

[0012] According to an embodiment of the present invention, a culture device that can continuously coat a cell-containing hydrogel with a protective layer, the liquid spraying mechanism includes a first liquid supply component, a liquid outlet pipe and a nozzle, the liquid outlet pipe is connected to the first liquid supply component, the nozzle is connected to the liquid outlet pipe, and the multiple nozzles are arranged in a direction away from the cell thread printing module, which can increase the spraying range of the cell thread, improve the spraying efficiency, and ensure that the protective liquid can evenly coat the cell thread.

[0013] According to an embodiment of the present invention, a culture device that can continuously coat a cell-containing hydrogel with a protective layer, the coating mechanism includes a rotating component, a negative plate and a second electrode connected to a nozzle, the negative plate is a first electrode, the second electrode is a positive electrode, the liquid spraying mechanism and the negative plate are both installed on the rotating component, and the negative plate is arranged opposite to the nozzle. When spraying liquid, the rotating component drives the nozzle and the two electrodes to rotate at the same time, and the rotating component enables the nozzle to spray protective liquid around the cell thread. The rotating component cooperates with the electric field formed by the first electrode and the second electrode to make the protective liquid more evenly coat the outside of the cell thread, ensuring that the outer wall of the cell thread can be completely coated with the protective liquid, ensuring that the cell thread will not cause interference in the subsequent culture cycle.

[0014] According to an embodiment of the present invention, a culture device capable of continuously coating a cell-containing hydrogel with a protective layer, a first liquid supply component includes a first extruder and a high-pressure adapter, the high-pressure adapter is installed at the liquid outlet end of the nozzle, and the liquid outlet pipe is connected to the first extruder.

[0015] According to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention, the first extruder is wrapped with a first temperature control unit on the outside.

[0016] According to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention, the exterior of the nozzle is wrapped with a second temperature control unit.

[0017] According to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention, the cell thread printing module includes a second extruder, and the exterior of the second extruder is wrapped with a third temperature control unit.

[0018] According to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention, a cell thread receiving module comprises a receiving dish in which a cell bracket is arranged.

[0019] According to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention, a cell bracket is provided with a monitoring sensor, the monitoring sensor is clamped on a cell thread, and the monitoring sensor is connected to a monitor.

[0020] According to a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention, the cell thread receiving module further comprises a three-axis moving mechanism, and the receiving dish is arranged on the three-axis moving mechanism.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0023] Figure 1 This is a structural diagram of a culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to an embodiment of the present invention;

[0024] Figure 2 It is a structural schematic diagram of the working principle of the cell thread printing module and the protective layer forming module of an embodiment of the present invention;

[0025] Figure 3 4 is a structural diagram of a cell support according to an embodiment of the present invention.

[0026] Description of reference numerals:

[0027] Cytofilament 1;

[0028] Protective fluid 2;

[0029] Cell filament printing module 100; second extruder 110; third temperature control unit 111;

[0030] Protective layer forming module 200; liquid spraying mechanism 210; nozzle 211; second temperature control unit 2111; first liquid supply assembly 212; first extruder 2121; high pressure adapter 2122; first temperature control unit 2123;

[0031] Liquid outlet pipe 213;

[0032] Laminating mechanism 220; negative electrode plate 221; rotating assembly 222;

[0033] Cell thread receiving module 300; receiving dish 310; cell bracket 320; bottom plate 321; connecting column 322; three-axis moving mechanism 330;

[0034] Housing 400;

[0035] Control module 500. DETAILED DESCRIPTION

[0036] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0037] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., and orientations or positional relationships indicated are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0038] In the description of the invention, "several" means one or more, "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 the first or the second, it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0039] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0040] Reference Figures 1 to 3 An embodiment of the present invention provides a culture device that can continuously coat a cell-containing hydrogel with a protective layer, including a cell thread printing module 100, used to print a cell-containing hydrogel into a cell thread 1; a cell thread receiving module 300, located below the cell thread printing module 100, used to receive the cell thread 1; a protective layer forming module 200, located between the cell thread printing module 100 and the cell thread receiving module 300, including a liquid spraying mechanism 210 and a laminating mechanism 220; the liquid spraying mechanism 210 includes a nozzle 211 for spraying a protective liquid 2 to the cell thread 1, and the laminating mechanism 220 includes a first electrode and a second electrode connected to the nozzle 211, the cell thread 1 is allowed to pass between the first electrode and the nozzle 211, and the first electrode and the second electrode cooperate to make the protective liquid 2 evenly distributed on the outer surface of the cell thread 1.

[0041] This application realizes the continuous printing and uniform coating of the cell-containing hydrogel with a protective layer by integrating multiple functional modules such as cell thread printing, receiving and protective layer formation. Specifically, the cell thread printing module 100 is responsible for printing the cell-containing hydrogel into a thread state. The cell thread 1 can pass between the first electrode and the nozzle 211, and the liquid spraying mechanism 210 is responsible for spraying the protective liquid 2 to the cell thread 1 passing through. Through the electric field formed by the cooperation of the first electrode and the second electrode, the protective liquid 2 can be evenly distributed on the cell thread 1, and the protective liquid 2 forms a protective layer on the outer wall of the cell thread 1. The cell thread 1 coated with the protective layer enters the cell thread receiving module 300 for storage and cultivation. Among them, the cell thread printing module 100 can continuously extrude the cell thread 1, and the cell type in the cell thread 1 can be replaced according to actual needs. Since the outer wall of the cell thread 1 is coated with a protective layer, even if multiple cells are cultured simultaneously in the receiving dish 310, there will be no interference, so that the cells can maintain a stable state during the culture cycle, ensuring the long-term stability and reliability of the cell microenvironment in different batches and different positions in the receiving dish 310. At the same time, for the same type of cell culture, researchers can also sample and test a certain section of the cell thread 1 to determine the overall culture situation. Researchers do not need to print and culture different types of cell cultures separately as in traditional culture methods. The culture device of the present application can not only ensure the long-term stability of the state of the cell microenvironment, ensure the stability and uniformity of cell quality everywhere, but also save manpower and material resources and simplify the production and processing technology.

[0042] Among them, the components of the protective solution 2 of the present application include chitosan (0.2%-5%), silk protein (0.1%-15%), polylactic acid PLA (0.1%-15%), polyvinyl alcohol (0.1%-20%), polycaprolactone (0.1%-15%) or polyurethane (0.1%-15%) and the like.

[0043] Hydrogels are biomaterials with good biocompatibility, such as collagen (0.1%-10%), gelatin (0.5%-15%), GelMA (0.1%-10%), matrix gel (0.1%-10%), sodium alginate (0.1%-10%) or fibrinogen (0.1%-10%).

[0044] According to some embodiments of the present application, the liquid spraying mechanism 210 includes a first liquid supply component 212, a liquid outlet pipe 213 and a nozzle 211. The liquid outlet pipe 213 is connected to the first liquid supply component 212, and the nozzle 211 is connected to the liquid outlet pipe 213. The multiple nozzles 211 are arranged in a direction away from the cell thread printing module 100, which can increase the spraying range of the cell thread 1, improve the spraying efficiency, and ensure that the protective liquid 2 can evenly cover the cell thread 1.

[0045] According to some embodiments of the present application, the coating mechanism 220 includes a rotating assembly 222, a negative electrode plate 221 and a second electrode connected to the nozzle 211, the negative electrode plate 221 is a first electrode, the second electrode is a positive electrode, the liquid spraying mechanism 210 and the negative electrode plate 221 are both mounted on the rotating assembly 222, and the negative electrode plate 221 is arranged opposite to the nozzle 211. When spraying liquid, the rotating assembly 222 drives the nozzle 211 and the two electrodes to rotate at the same time, and the rotating assembly 222 enables the nozzle 211 to spray the protective liquid 2 around the cell thread 1. The rotating assembly 222 cooperates with the electric field formed by the first electrode and the second electrode to make the protective liquid 2 more evenly cover the outside of the cell thread 1, ensuring that the outer wall of the cell thread 1 can completely cover the protective liquid 2, and ensuring that there will be no interference in the subsequent culture cycle of the cell thread 1.

[0046] Specifically, the second electrode is a connecting line of a high-voltage electrostatic generator disposed outside the laminating mechanism 220. Further, the first liquid supply assembly 212 includes a first extruder 2121 and a high-voltage adapter 2122. The high-voltage adapter 2122 is installed at the liquid outlet of the nozzle 211, and the liquid outlet pipe 213 is connected to the first extruder 2121. The connecting line of the high-voltage electrostatic generator is connected to the high-voltage adapter 2122. The high-voltage electrostatic generator can generate high voltage electricity. The high-voltage adapter 2122 conducts the high voltage electricity to the nozzle 211. The protective liquid 2 output by the first extruder 2121 forms a Taylor cone under the action of high voltage electricity, thereby ejecting nano-shaped filaments, which will be stacked layer by layer on the outer wall of the cell filament 1 to form a protective layer with abundant gaps. Preferably, the high-voltage adapter 2122 is made of a metal with good electrical conductivity. At the same time, the high voltage electricity generated by the high-voltage electrostatic generator is preferably 5-35KV. The coating mechanism 220 of the present application can form a protective layer with a thickness of not less than 0.5 μm on the outer wall of the cell thread 1. Preferably, the thickness of the protective layer is 0.5 μm-100 μm.

[0047] According to some embodiments of the present application, the nozzle 211 is wrapped with a second temperature control unit 2111. The second temperature control unit 2111 is used to increase the temperature to 60-120°C, and the high temperature accelerates the volatilization of the solvent and reduces the residue.

[0048] According to some embodiments of the present application, the first extruder 2121 is wrapped with a first temperature control unit 2123. The first temperature control unit 2123 is used to increase the temperature of the protective liquid 2 in the first extruder 2121 to 50-90°C. A higher temperature can improve the fluidity of the solution and also provide a certain basis for the temperature increase of the nozzle 211, thereby avoiding excessive temperature difference and causing the temperature increase of the nozzle 211 to fail to meet the standard.

[0049] According to some embodiments of the present application, the cell filament printing module 100 includes a second extruder 110, and the outside of the second extruder 110 is wrapped with a third temperature control unit 111. The third temperature control unit 111 is used to ensure that the temperature of the second extruder 110 is constant within the range of 4-30°C, and the cell activity in the second extruder 110 is guaranteed by controlling the temperature.

[0050] According to some embodiments of the present application, the cell thread receiving module 300 includes a receiving dish 310, and a cell bracket 320 is arranged in the receiving dish 310. Further, the cell bracket 320 is provided with a monitoring sensor, the monitoring sensor is clamped on the cell thread 1, and the monitoring sensor is connected to the monitor. Further, the cell thread receiving module 300 also includes a three-axis moving mechanism 330, and the receiving dish 310 is arranged on the three-axis moving mechanism 330. As shown in the figure, after the cell thread 1 coated with the protective layer enters the rear receiving dish 310, the cell thread 1 is coiled and multi-layered on the cell bracket 320 with the cooperation of the three-axis moving mechanism 330. Because the outer wall of the cell thread 1 is coated with a protective layer, the cell thread 1 can be multi-layered and will not cause interference, which effectively improves the space utilization. In addition, the monitoring sensor is used to monitor the culture state of the cells in real time, so that researchers can observe the state of the cells in time. Specifically, the monitoring sensor is an impedance measurement sensor. The monitoring sensor of the present application has a groove that can clamp the cell thread 1, and an electrode is arranged in the groove, and the electrode is connected to the monitor. Among them, the monitor can use an impedance spectrum measurement host to reflect the cell culture state by measuring the impedance value of the target.

[0051] According to some embodiments of the present application, as shown in the figure, the cell bracket 320 includes a base plate 321 and connecting posts 322 connected to the base plate 321 , and a plurality of connecting posts 322 are radially spaced apart in a direction away from the central axis of the base plate 321 .

[0052] According to some embodiments of the present application, a protective cover (not shown) may be further provided between the receiving dish 310 and the coating mechanism 220 to catch the protective liquid 2 that has not completely evaporated and has dripped, thereby preventing the protective liquid 2 from falling into the receiving dish 310 and contaminating the cells.

[0053] According to some embodiments of the present application, the culture device of the present application is further provided with a housing 400, and the cell filament printing module 100, the cell filament receiving module 300, and the protective layer forming module 200 are all provided in the housing 400. The housing 400 is used to isolate the production of the entire culture device from the external environment, thereby ensuring the cleanliness of the environment for printing the cell filament 1. Furthermore, the housing 400 may also be provided with a clean air circulation system (not shown in the figure), and the clean air circulation system (not shown in the figure) is used to control the cleanliness of the entire interior of the housing 400 to reduce the probability of contamination.

[0054] In the description of this specification, 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 invention. 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.

[0055] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A culture device capable of continuously coating a cell-containing hydrogel with a protective layer, characterized in that: include: A cell thread printing module, used for printing cell-containing hydrogels into cell threads; A cell thread receiving module is located below the cell thread printing module and is used to receive the cell thread. The cell thread receiving module includes a receiving dish, in which a cell bracket is arranged. The cell bracket includes a bottom plate, a connecting column connected to the bottom plate, and a monitoring sensor. A plurality of the connecting columns are radially spaced in a direction away from the central axis of the bottom plate. The monitoring sensor is clamped on the cell thread and connected to a monitor. A protective layer forming module, located between the cell filament printing module and the cell filament receiving module, comprises a liquid spraying mechanism and a laminating mechanism; The liquid spraying mechanism includes a first liquid supply component, a liquid outlet pipe and a nozzle for spraying protective liquid to the cell thread, the liquid outlet pipe is connected to the first liquid supply component, the nozzle is connected to the liquid outlet pipe, and the plurality of nozzles are arranged in a direction away from the cell thread printing module. The laminating mechanism includes a rotating component, a negative plate and a second electrode connected to the nozzle, the negative plate is a first electrode, and the second electrode is a positive electrode. The liquid spraying mechanism and the negative plate are both installed on the rotating component, the negative plate is arranged opposite to the nozzle, the cell thread is allowed to pass between the first electrode and the nozzle, and the first electrode and the second electrode cooperate to make the protective liquid evenly distributed on the outer surface of the cell thread.

2. A culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to claim 1, characterized in that: The first liquid supply assembly includes a first extruder and a high-pressure adapter. The high-pressure adapter is installed at the liquid outlet end of the nozzle, and the liquid outlet pipe is connected to the first extruder.

3. A culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to claim 2, characterized in that: The first extruder is wrapped with a first temperature control unit on the outside.

4. The culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to claim 1, characterized in that: The outside of the nozzle is wrapped with a second temperature control unit.

5. The culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to claim 1, characterized in that: The cell filament printing module comprises a second extruder, and the exterior of the second extruder is wrapped with a third temperature control unit.

6. The culture device capable of continuously coating a cell-containing hydrogel with a protective layer according to claim 1, characterized in that: The cell thread receiving module also includes a three-axis moving mechanism, and the receiving dish is arranged on the three-axis moving mechanism.

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