Automatic perfusion culture equipment and method for NK cell culture

Abstract

The present invention discloses an automatic perfusion culture device for NK cell culture, which belongs to the technical field of cell culture devices, and includes a liquid storage cylinder, a waste liquid cylinder, a mounting frame, a culture box, a first conveying mechanism, a second conveying mechanism, a driving mechanism, and an anti-slip fixing mechanism. The automatic perfusion culture device for NK cell culture in the present invention can automatically convey the nutrient solution in the liquid storage cylinder to the culture box, and convey the waste nutrient solution in the culture box to the waste liquid cylinder by setting the first conveying mechanism, the second conveying mechanism, and the driving mechanism, thereby realizing the automatic perfusion culture of NK cells. Through the flow of the nutrient solution, the mass transfer between nutrients and metabolic waste products and cells can be achieved, thereby reducing the concentration accumulation of harmful metabolic wastes, which is conducive to the cultivation of NK cells.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture equipment, and in particular relates to automatic perfusion culture equipment and method for NK cell culture. Background Art

[0002] In the field of immune cell therapy, NK (natural killer) cells have attracted much attention due to their powerful tumor killing ability and immunomodulatory function. NK cells can directly recognize and kill a variety of tumor cells without relying on the restrictions of antibodies or major histocompatibility complex (MHC), which gives them unique advantages in cancer immunotherapy. However, the efficient and stable preparation and expansion of NK cells to meet clinical needs has always been an important challenge facing this field.

[0003] Traditional NK cell culture methods often have problems such as complex operation, high cost, low amplification efficiency and easy contamination. For example, the traditional static culture method easily leads to uneven distribution of nutrients in the culture medium, affecting the growth and proliferation of cells; at the same time, the accumulation of waste metabolites may also cause toxicity to cells, further reducing the culture efficiency. Summary of the invention

[0004] The purpose of the present invention is to provide an automatic perfusion culture device and method for NK cell culture, which is used to solve the technical problem that the static culture method in the prior art easily leads to uneven distribution of nutrients in the culture solution, affecting the growth and proliferation of cells; at the same time, the accumulation of waste metabolites may also cause toxicity to the cells, further reducing the culture efficiency.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The automatic perfusion culture equipment for NK cell culture comprises: a liquid storage cylinder for containing nutrient solution; a waste liquid cylinder mounted on the bottom surface of the liquid storage cylinder; a mounting frame mounted on the top surface of the liquid storage cylinder; a culture box mounted on the mounting frame for culturing NK cells; a first conveying mechanism mounted on the mounting frame for conveying the nutrient solution in the liquid storage cylinder to the culture box; a second conveying mechanism mounted on the mounting frame for conveying the nutrient solution in the culture box to the waste liquid cylinder; a driving mechanism mounted on the mounting frame for driving the first conveying mechanism and the second conveying mechanism to operate; and an anti-slip fixing mechanism.

[0007] Preferably, the first conveying mechanism includes: a first piston cylinder, mounted on the mounting frame, with a first piston plate slidably connected inside the first piston cylinder, a first piston rod mounted on the first piston plate, and the first piston rod is slidably connected to the first piston cylinder; a first push plate, fixedly connected to the first piston rod; a first spring, one end of which is connected to the first piston cylinder, and the other end of which is fixedly connected to the first push plate; a first liquid extraction tube, one end of which is fixedly connected to the first piston cylinder, and the other end of which extends into the liquid storage cylinder; a first infusion tube, one end of which is plugged into the first piston cylinder, and the other end of which is plugged into the culture box.

[0008] Preferably, the second conveying mechanism includes: a second piston cylinder, mounted on the mounting frame, with a second piston plate slidably connected inside the second piston cylinder, a second piston rod mounted on the second piston plate, and the second piston rod is slidably connected to the second piston cylinder; a second push plate, fixedly connected to the second piston rod; a second spring, one end of which is connected to the second piston cylinder, and the other end of which is fixedly connected to the second push plate; a second liquid extraction tube, one end of which is plugged into the second piston cylinder, and the other end of which is plugged into the culture box; a second infusion tube, one end of which is fixedly connected to the second piston cylinder, and the other end of which extends to the waste liquid cylinder.

[0009] Preferably, the driving mechanism comprises: a motor mounted on the bottom surface of the culture box; and a cam mounted on a power output shaft of the motor and in contact with the first push plate and the second push plate.

[0010] Preferably, the automatic perfusion culture equipment for NK cell culture also includes a stirring mechanism, which includes: an axis rod, which passes through the top surface of the liquid storage cylinder and is rotatably connected to the liquid storage cylinder; a plurality of stirring rods, which are all located in the liquid storage cylinder and are fixedly connected to the axis rod; a gear, which is installed at the upper end of the axis rod; a rack, which is meshed with the gear, slidably connected to the top surface of the liquid storage cylinder, and fixedly connected to the first push plate.

[0011] Preferably, the anti-slip fixing mechanism includes: a fixing cylinder, which is plugged into and cooperates with the liquid storage cylinder and the waste liquid cylinder; a support ring, which is installed on the inner wall of the fixing cylinder and is used to support the waste liquid cylinder; a through groove, which is opened on the fixing cylinder; a fixing ring, which is fixedly connected to the bottom surface of the fixing cylinder; and a plurality of suction cups, which are equidistantly installed on the bottom surface of the fixing ring.

[0012] Preferably, the anti-slip fixing mechanism further comprises: an ultraviolet lamp installed on the inner bottom surface of the fixing cylinder.

[0013] Preferably, the anti-slip fixing mechanism also includes: two first insert cylinders, both installed on the bottom surface of the fixed cylinder, and plugged with the first infusion tube and the second extraction tube; a rotating plate, hinged to the bottom surface of the fixed cylinder; a second insert cylinder, installed through the rotating plate, and plugged with the first infusion tube and the second extraction tube; and a limiting component.

[0014] Preferably, the limiting assembly includes: a plurality of rotating shafts, all of which are rotatably mounted on the fixed cylinder; and a plurality of baffles, which are respectively mounted on the plurality of rotating shafts.

[0015] The method for using the automatic perfusion culture equipment for NK cell culture comprises the following steps: step 1, checking whether all parts of the automatic perfusion culture equipment for NK cell culture are complete and intact; step 2, placing the anti-skid fixing mechanism upside down on the liquid storage cylinder and the waste liquid cylinder, and starting the ultraviolet lamp to sterilize the culture box, the first conveying mechanism and the second conveying mechanism; step 3, placing the fixing cylinder on the desktop, allowing the suction cup to adsorb on the desktop, and then placing the liquid storage cylinder and the waste liquid cylinder in the anti-skid fixing mechanism respectively; step 4, opening the culture box, adding the NK cell suspension into the culture box, starting the motor, and driving the cam to rotate Step 5: During the culture process, the growth of NK cells in the culture box is regularly observed, and the composition or concentration of the culture solution is adjusted as needed; Step 6: When the NK cells achieve the expected culture effect, the stop button of the driving mechanism is pressed to end the automatic perfusion culture; Step 7: After the culture is completed, the storage cylinder, waste liquid cylinder, culture box and other components are cleaned in time, and disinfected and sterilized for the next use.

[0016] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0017] 1. The automatic perfusion culture equipment for NK cell culture in the present invention can automatically transport the nutrient solution in the liquid storage cylinder to the culture box, and transport the waste nutrient solution in the culture box to the waste liquid cylinder by setting a first conveying mechanism, a second conveying mechanism and a driving mechanism, thereby realizing automatic perfusion culture of NK cells. Through the flow of nutrient solution, the mass transfer between nutrients and metabolic waste products and cells can be achieved, thereby reducing the accumulation of harmful metabolic waste concentrations, which is beneficial to the cultivation of NK cells.

[0018] 2. The stirring mechanism in the present invention is equipped with a shaft, a stirring rod, a gear and a rack. The stirring mechanism can stir the nutrient solution in the liquid storage cylinder to avoid uneven distribution of components after the culture solution has been standing for a long time, thereby ensuring that the cells obtain balanced nutrition during the culture process. The stirring mechanism can change the stirring direction. Compared with the method of not changing the stirring direction, it can more effectively promote the uniform distribution of the components in the culture solution, reduce the uneven distribution of components, and further improve the mixing efficiency.

[0019] 3. The driving mechanism in the present invention is provided with a motor and a cam. The driving mechanism is not only responsible for providing power to the first conveying mechanism and the second conveying mechanism, so that they can respectively convey the nutrient solution to the culture box and the waste nutrient solution to the waste liquid cylinder, but also undertakes the task of driving the stirring mechanism to work. Only by starting the driving mechanism, two functions can be realized at the same time, avoiding the need to use multiple independent driving sources, thereby saving energy and equipment manufacturing costs.

[0020] 4. The automatic perfusion culture equipment for NK cell culture in the present invention is provided with an anti-skid fixing mechanism, which can prevent the waste liquid cylinder and the liquid storage cylinder from sliding on the desktop, thereby ensuring the stability of the equipment; the anti-skid fixing mechanism can be inverted on the liquid storage cylinder and the waste liquid cylinder, so that the culture box, the first conveying mechanism and the second conveying mechanism are in the anti-skid fixing mechanism, and then the culture box, the first conveying mechanism and the second conveying mechanism are sterilized by starting the ultraviolet lamp to reduce the risk of contamination during cell culture; when the device needs to be moved, the first infusion tube, the second infusion tube are connected with the first insert and the second insert to form a temporary handle, thereby facilitating the movement of the device and improving the portability and flexibility of the device. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The three-dimensional automatic perfusion culture device for NK cell culture in the present invention Figure 1 ;

[0023] Figure 2 The three-dimensional automatic perfusion culture device for NK cell culture in the present invention Figure 2 ;

[0024] Figure 3 It is a schematic diagram of the internal structure of the fixed cylinder, the liquid storage cylinder and the waste liquid cylinder in the present invention;

[0025] Figure 4 It is a schematic diagram of the assembly structure of the first conveying mechanism, the second conveying mechanism, and the driving mechanism in the present invention;

[0026] Figure 5 For the present invention Figure 4 Exploded diagram of

[0027] Figure 6 It is a three-dimensional diagram of the fixed cylinder in the present invention;

[0028] Figure 7 It is a schematic diagram of the second state of the automatic perfusion culture device for NK cell culture in the present invention;

[0029] Figure 8 The third state of the automatic perfusion culture device for NK cell culture in the present invention is shown in FIG. Figure 1 ;

[0030] Fig. 9 The third state of the automatic perfusion culture device for NK cell culture in the present invention is shown in FIG. Figure 2 ;

[0031] Reference numerals: 100, liquid storage cylinder; 101, waste liquid cylinder; 102, mounting frame; 103, culture box; 104, box cover; 110, first conveying mechanism; 111, first piston cylinder; 112, first piston rod; 113, first push plate; 114, first spring; 115, first liquid extraction tube; 116, first infusion tube; 120, second conveying mechanism; 121, second piston cylinder; 122, second piston rod; 123, second push plate; 124, second spring; 125, second liquid extraction tube; 126, second liquid infusion tube; 6. Second infusion tube; 130. Driving mechanism; 131. Motor; 132. Cam; 140. Stirring mechanism; 141. Shaft; 142. Stirring rod; 143. Gear; 144. Rack; 200. Anti-skid fixing mechanism; 201. Fixing cylinder; 202. Support ring; 203. Ultraviolet lamp; 204. Through groove; 205. First insert cylinder; 206. Rotating plate; 207. Second insert cylinder; 208. Fixing ring; 209. Suction cup; 210. Limiting assembly; 211. Rotating shaft; 212. Baffle. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1: Figure 1-Figure 4As shown, the automatic perfusion culture device for NK cell culture includes a liquid storage cylinder 100, a waste liquid cylinder 101, a mounting frame 102, a culture box 103, a first conveying mechanism 110, a second conveying mechanism 120, a driving mechanism 130 and an anti-slip fixing mechanism 200.

[0034] The liquid storage cylinder 100 is used to hold the nutrient solution, and a liquid adding tube is installed on the liquid storage cylinder 100; the waste liquid cylinder 101 is installed on the bottom surface of the liquid storage cylinder 100; a liquid discharge tube is installed on the waste liquid cylinder 101, and the liquid discharge tube is located directly below the liquid adding tube. The mounting frame 102 is installed on the top surface of the liquid storage cylinder 100; the culture box 103 is installed on the mounting frame 102, and the culture box 103 is used to culture NK cells; the culture box 103 is provided with a box cover 104.

[0035] The first conveying mechanism 110 is installed on the mounting frame 102, and the first conveying mechanism 110 is used to convey the nutrient solution in the liquid storage cylinder 100 to the culture box 103; the second conveying mechanism 120 is installed on the mounting frame 102, and the second conveying mechanism 120 is used to convey the waste nutrient solution in the culture box 103 to the waste liquid cylinder 101; the driving mechanism 130 is installed on the mounting frame 102, and the driving mechanism 130 is used to drive the first conveying mechanism 110 and the second conveying mechanism 120 to operate; the anti-slip fixing mechanism 200 is used to prevent the waste liquid cylinder 101 from sliding on the desktop.

[0036] Specifically, the culture fluid for culturing NK cells is added into the liquid storage cylinder 100 , and then the liquid storage cylinder 100 and the waste liquid cylinder 101 are placed in the anti-slip fixing mechanism 200 .

[0037] Then, the target culture is placed in the culture box 103, and the driving mechanism 130 is started to start the operation of the first conveying mechanism 110 and the second conveying mechanism 120. When the first conveying mechanism 110 is in operation, the first conveying mechanism 110 will convey the nutrient solution in the liquid storage cylinder 100 to the culture box 103; when the second conveying mechanism 120 is in operation, the second conveying mechanism 120 will convey the waste nutrient solution in the culture box 103 to the waste liquid cylinder 101. Through the flow of the nutrient solution, the mass transfer between nutrients and metabolic waste products and cells can be achieved, thereby reducing the accumulation of harmful metabolic waste concentrations.

[0038] like Figure 4 and Figure 5As shown, the first conveying mechanism 110 includes a first piston cylinder 111, a first push plate 113, a first spring 114, a first liquid extraction tube 115 and a first liquid infusion tube 116. The first piston cylinder 111 is mounted on the mounting frame 102, and a first piston sheet is slidably connected inside the first piston cylinder 111, and a first piston rod 112 is mounted on the first piston sheet, and the first piston rod 112 is slidably connected to the first piston cylinder 111; the first push plate 113 is fixedly connected to the first piston rod 112; one end of the first spring 114 is connected to the first piston cylinder 111, and the other end of the first spring 114 is fixedly connected to the first push plate 113.

[0039] One end of the first liquid extraction tube 115 is fixedly connected to the first piston cylinder 111, and the other end of the first liquid extraction tube 115 extends into the liquid storage cylinder 100; one end of the first liquid infusion tube 116 is plugged into the first piston cylinder 111, and the other end of the first liquid infusion tube 116 is plugged into the culture box 103. A one-way valve is installed in both the first liquid extraction tube 115 and the first liquid infusion tube 116, so that the nutrient solution in the liquid storage cylinder 100 can only enter the first piston cylinder 111 in one direction through the first liquid extraction tube 115, and the nutrient solution in the first piston cylinder 111 can only enter the culture box 103 in one direction through the first delivery tube. The first liquid infusion tube 116 is a hard tube, such as a hard plastic tube or a metal tube.

[0040] Specifically, when the first push plate 113 is moved toward the first piston cylinder 111, the first push plate 113 drives the first piston rod 112 and the first piston sheet to move, thereby allowing the nutrient solution in the first piston cylinder 111 to enter the culture box 103 through the first delivery pipe;

[0041] When the first push plate 113 is released, the resilience of the first spring 114 pushes the push plate to move away from the first piston cylinder 111 , thereby pumping the nutrient solution in the liquid storage cylinder 100 into the first piston cylinder 111 .

[0042] like Figure 4 and Figure 5 As shown, the second conveying mechanism 120 includes a second piston cylinder 121, a second push plate 123, a second spring 124, a second liquid extraction tube 125 and a second liquid infusion tube 126. The second piston cylinder 121 is mounted on the mounting frame 102, and a second piston sheet is slidably connected inside the second piston cylinder 121, and a second piston rod 122 is mounted on the second piston sheet, and the second piston rod 122 is slidably connected to the second piston cylinder 121; the second push plate 123 is fixedly connected to the second piston rod 122; one end of the second spring 124 is connected to the second piston cylinder 121, and the other end of the second spring 124 is fixedly connected to the second push plate 123.

[0043] One end of the second liquid extraction tube 125 is plugged into the second piston cylinder 121, and the other end of the second liquid extraction tube 125 is plugged into the culture box 103; one end of the second liquid infusion tube 126 is fixedly connected to the second piston cylinder 121, and the other end of the second liquid infusion tube 126 extends to the waste liquid cylinder 101. A one-way valve is installed in both the second liquid extraction tube 125 and the second liquid infusion tube 126, so that the waste nutrient solution in the culture box 103 can only enter the second piston cylinder 121 in one direction through the second liquid extraction tube 125, and the waste nutrient solution in the second piston cylinder 121 can only enter the waste liquid cylinder 101 in one direction through the second liquid infusion tube 126. The second liquid infusion tube 126 is a hard tube, such as a hard plastic tube or a metal tube.

[0044] Specifically, when the second push plate 123 is moved toward the second piston cylinder 121 , the second push plate 123 drives the second piston rod 122 and the second piston sheet to move, thereby transporting the waste nutrient solution in the second piston cylinder 121 to the waste liquid cylinder 101 through the second delivery pipe.

[0045] When the second push plate 123 is released, the rebound force of the second spring 124 pushes the second push plate 123 to move, thereby causing the waste nutrient solution in the culture box 103 to be pumped into the second piston cylinder 121 through the second pumping tube 125 .

[0046] like Figure 4 and Figure 5 As shown, the driving mechanism 130 includes a motor 131 and a cam 132. The motor 131 is mounted on the bottom surface of the culture box 103; the cam 132 is mounted on the power output shaft of the motor 131, and the cam 132 contacts the first push plate 113 and the second push plate 123.

[0047] Specifically, when the motor 131 is running, the power output shaft of the motor 131 will drive the cam 132 to rotate, and the rotating cam 132 will intermittently push the first push plate 113 and the second push plate 123 to move.

[0048] like Figure 5 As shown, the automatic perfusion culture apparatus for NK cell culture further includes a stirring mechanism 140 , and the stirring mechanism 140 includes a shaft 141 , a plurality of stirring rods 142 , a gear 143 and a rack 144 .

[0049] The shaft 141 passes through the top surface of the liquid storage cylinder 100 and is rotatably connected to the liquid storage cylinder 100; multiple stirring rods 142 are located in the liquid storage cylinder 100, and the multiple stirring rods 142 are fixedly connected to the shaft 141; the gear 143 is installed at the upper end of the shaft 141; the rack 144 is meshed with the gear 143, the rack 144 is slidably connected to the top surface of the liquid storage cylinder 100, and the rack 144 is fixedly connected to the first push plate 113.

[0050] Specifically, when the first push plate 113 moves, it will drive the rack 144 to move. Since the rack 144 is meshed with the gear 143, when the rack 144 moves, the gear 143 will rotate, thereby driving the shaft 141 to rotate, and then driving the multiple stirring rods 142 to rotate. The rotating stirring rods 142 will stir the nutrient solution in the liquid storage cylinder 100 to avoid uneven distribution of components of the culture solution after standing for a long time. Stirring can help the components to be evenly mixed again, ensuring that the cells obtain balanced nutrition during the culture process.

[0051] When the first push plate 113 moves reciprocatingly horizontally, it will drive the rack 144 to move reciprocatingly horizontally, and then drive the gear 143 and the rotating shaft 211 to rotate intermittently clockwise and counterclockwise, and then drive the stirring rod 142 to rotate clockwise and counterclockwise. By changing the stirring direction, compared with not changing the stirring direction, it can more effectively promote the uniform distribution of the components in the culture solution and reduce the uneven distribution of the components. Changing the stirring direction also helps to break the stable vortex formed, further improving the mixing efficiency.

[0052] like Figure 2 , Figure 3 and Figure 6 As shown, the anti-slip fixing mechanism 200 includes a fixing cylinder 201, a supporting ring 202, a through groove 204, a fixing ring 208 and a plurality of suction cups 209. The fixing cylinder 201 is plugged and matched with the liquid storage cylinder 100 and the waste liquid cylinder 101; the supporting ring 202 is installed on the inner wall of the fixing cylinder 201, and the supporting ring 202 is used to support the waste liquid cylinder 101; the through groove 204 is provided on the fixing cylinder 201; the fixing ring 208 is fixedly connected to the bottom surface of the fixing cylinder 201; and a plurality of suction cups 209 are equidistantly installed on the bottom surface of the fixing ring 208.

[0053] Specifically, by placing the fixed cylinder 201 on the table, the suction cup 209 is adsorbed on the table, and then the waste liquid cylinder 101 and the liquid storage cylinder 100 are placed in the fixed cylinder 201, so that the supporting ring 202 supports the liquid storage cylinder 100 and the waste liquid cylinder 101, the liquid filling pipe of the liquid storage cylinder 100 and the liquid discharge pipe of the waste liquid cylinder 101 will be in the through groove 204.

[0054] like Figure 3 , Figure 6 and Figure 7 As shown, the anti-slip fixing mechanism 200 further includes an ultraviolet lamp 203. The ultraviolet lamp 203 is installed on the inner bottom surface of the fixing cylinder 201.

[0055] Specifically, before using the device, the fixed cylinder 201 is turned upside down on the liquid storage cylinder 100 and the waste liquid cylinder 101, so that the culture box 103, the first conveying mechanism 110 and the second conveying mechanism 120 are located in the fixed cylinder 201, and the ultraviolet lamp 203 is located above the first conveying mechanism 110, the second conveying mechanism 120 and the culture box 103. Then, the ultraviolet lamp 203 is started so that the ultraviolet lamp 203 emits ultraviolet light, and the culture box 103, the first conveying mechanism 110 and the second conveying mechanism 120 are sterilized by the ultraviolet light.

[0056] Working principle: By placing the fixed cylinder 201 upside down on the liquid storage cylinder 100 and the waste liquid cylinder 101, the culture box 103, the first conveying mechanism 110 and the second conveying mechanism 120 are placed in the fixed cylinder 201, and then the ultraviolet lamp 203 is started to emit ultraviolet light, thereby sterilizing the culture box 103, the first conveying mechanism 110 and the second conveying mechanism 120.

[0057] After sterilization is completed, the opening of the fixed cylinder 201 is facing upwards, and then the fixed cylinder 201 is placed on the table so that the suction cup 209 on the fixed ring 208 is adsorbed on the table. Then, the waste liquid cylinder 101 and the liquid storage cylinder 100 are placed in the fixed cylinder 201, and the support ring 202 supports the waste liquid cylinder 101, and then the target culture is placed in the culture box 103.

[0058] Then, by starting the motor 131, the power output shaft of the motor 131 drives the cam 132 to rotate, so that the cam 132 intermittently pushes the first push plate 113 and the second push plate 123 to move, and under the cooperation of the first spring 114 and the second spring 124, the first push plate 113 and the second push plate 123 move back and forth horizontally. When the first push plate 113 moves back and forth, the first piston cylinder 111 will transport the nutrient solution in the liquid storage cylinder 100 to the culture box 103; when the second push plate 123 moves back and forth, the second piston cylinder 121 will transport the waste nutrient solution in the culture box 103 to the waste liquid cylinder 101. Through the flow of the nutrient solution, the mass transfer between nutrients and metabolic waste products and cells can be achieved, thereby reducing the accumulation of harmful metabolic waste concentrations and facilitating the cultivation of NK cells.

[0059] When the first push plate 113 moves, it will drive the rack 144 to move, and then the rack 144 will drive the gear 143 to rotate, and then drive the shaft 141 and the stirring rod 142 to rotate, so as to stir the nutrient solution in the liquid storage cylinder 100, so as to avoid uneven distribution of components of the culture solution after a long period of standing. Stirring can help to re-mix the components evenly, ensuring that the cells obtain balanced nutrition during the culture process.

[0060] When the first push plate 113 moves back and forth, it will drive the rack 144 to move back and forth horizontally, thereby driving the gear 143 and the rotating shaft 211 to rotate intermittently clockwise and counterclockwise, thereby driving the stirring rod 142 to rotate clockwise and counterclockwise. By changing the stirring direction, compared with not changing the stirring direction, it can more effectively promote the uniform distribution of the components in the culture solution and reduce the uneven distribution of the components. Changing the stirring direction also helps to break the stable vortex formed, thereby further improving the mixing efficiency.

[0061] Example 2: Figure 8 and Fig. 9 As shown, when other parts are the same as those of Example 1, the difference between this embodiment and Example 1 is that the anti-slip fixing mechanism 200 further includes two first inserting cylinders 205 , a rotating plate 206 , a second inserting cylinder 207 and a limiting assembly 210 .

[0062] The two first insert tubes 205 are both mounted on the bottom surface of the fixed tube 201, and the two first insert tubes 205 are plugged and matched with the first infusion tube 116 and the second extraction tube 125; the rotating plate 206 is hinged to the bottom surface of the fixed tube 201; the second insert tube 207 is installed through the rotating plate 206, and the second insert tube 207 is plugged and matched with the first infusion tube 116 and the second extraction tube 125. The limiting assembly 210 includes a plurality of rotating shafts 211 and a plurality of baffles 212. The plurality of rotating shafts 211 are all rotatably mounted on the fixed tube 201; and the plurality of baffles 212 are respectively mounted on the plurality of rotating shafts 211.

[0063] Working principle: When the device needs to be moved, the first infusion tube 116 is separated from the first piston cylinder 111 and the culture box 103 , and the second infusion tube 126 is separated from the second piston cylinder 121 and the culture box 103 .

[0064] The fixed cylinder 201 is then turned over so that the opening of the fixed cylinder 201 is at the bottom, and then the fixed cylinder 201 is covered on the liquid storage cylinder 100 and the waste liquid cylinder 101 .

[0065] Then, the four baffles 212 are rotated in sequence so that the baffles 212 are directly below the waste liquid cylinder, so that the waste liquid cylinder cannot be moved out of the fixed cylinder 201 by itself. Then, by rotating the rotating plate 206 and the second insert cylinder 207, the rotating plate 206 is perpendicular to the second insert cylinder 207, and the first infusion tube 116 and the second infusion tube 126 are plugged into the first insert cylinder 205 and the second insert cylinder 207, so that the first infusion tube 116, the second infusion tube 126, the first insert cylinder 205 and the second insert cylinder 207 form a temporary handle, so as to facilitate the lifting of the device and thus facilitate the movement of the device.

[0066] Example 3: Figure 1-Figure 7 As shown, the method for using the automatic perfusion culture device for NK cell culture comprises the following steps:

[0067] Step 1: Check whether all components of the automatic perfusion culture equipment for NK cell culture are complete and intact, including the liquid storage cylinder 100, the waste liquid cylinder, the mounting frame 102, the culture box 103, the first conveying mechanism 110, the second conveying mechanism 120, the driving mechanism 130, the anti-slip fixing mechanism 200 and the stirring mechanism 140;

[0068] Step 2: Place the anti-slip fixing mechanism 200 upside down on the liquid storage cylinder 100 and the waste liquid cylinder, and start the ultraviolet lamp 203 to sterilize the culture box 103, the first conveying mechanism 110 and the second conveying mechanism 120;

[0069] Step 3: Place the fixed cylinder 201 on the desktop, so that the suction cup 209 is adsorbed on the desktop, and then place the liquid storage cylinder 100 and the waste liquid cylinder in the anti-slip fixing mechanism 200 respectively;

[0070] Step 4: Open the cover of the culture box 103, carefully add the NK cell suspension into the culture box 103, start the motor 131, drive the cam 132 to rotate, and the cam 132 will intermittently push the first push plate 113 and the second push plate 123 to move. Under the action of the first push plate 113, the first conveying mechanism 110 conveys the nutrient solution in the liquid storage cylinder 100 to the culture box 103; under the action of the second push plate 123, the second conveying mechanism 120 conveys the waste nutrient solution in the culture box 103 to the waste liquid cylinder, thereby realizing the automatic perfusion culture of NK cells;

[0071] Step 5: During the culture process, regularly observe the growth of NK cells in the culture box 103, and adjust the composition or concentration of the culture solution as needed;

[0072] Step 6: When the NK cells reach the expected culture effect, press the stop button of the driving mechanism 130 to end the automatic perfusion culture;

[0073] Step 7: After the culture is completed, the liquid storage cylinder 100, the waste liquid cylinder 101, the culture box 103 and other components are cleaned in time and disinfected for next use.

[0074] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0075] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An automatic perfusion culture device for NK cell culture, characterized in that: include: A liquid storage cylinder (100) for containing nutrient solution; A waste liquid cylinder (101) mounted on the bottom surface of the liquid storage cylinder (100); A mounting frame (102) mounted on the top surface of the liquid storage cylinder (100); A culture box (103), mounted on the mounting frame (102), and used for culturing NK cells; A first conveying mechanism (110), mounted on the mounting frame (102), and used for conveying the nutrient solution in the liquid storage cylinder (100) to the culture box (103); A second conveying mechanism (120) is mounted on the mounting frame (102) and is used to convey the nutrient solution in the culture box (103) to the waste liquid cylinder (101); A driving mechanism (130) mounted on the mounting frame (102) and used to drive the first conveying mechanism (110) and the second conveying mechanism (120) to operate; Anti-slip fixing mechanism (200); The first conveying mechanism (110) comprises: A first piston cylinder (111) is mounted on the mounting frame (102), a first piston plate being slidably connected therein, a first piston rod (112) being mounted on the first piston plate, and the first piston rod (112) being slidably connected to the first piston cylinder (111); A first push plate (113) fixedly connected to the first piston rod (112); A first spring (114), one end of which is connected to the first piston cylinder (111), and the other end of which is fixedly connected to the first push plate (113); A first liquid extraction tube (115), one end of which is fixedly connected to the first piston cylinder (111), and the other end of which extends into the liquid storage cylinder (100); a first infusion tube (116), one end of which is plugged into the first piston cylinder (111), and the other end of which is plugged into the culture box (103); The second conveying mechanism (120) comprises: A second piston cylinder (121) is mounted on the mounting frame (102), a second piston plate being slidably connected therein, a second piston rod (122) being mounted on the second piston plate, and the second piston rod (122) being slidably connected to the second piston cylinder (121); A second push plate (123) fixedly connected to the second piston rod (122); A second spring (124), one end of which is connected to the second piston cylinder (121), and the other end of which is fixedly connected to the second push plate (123); A second liquid extraction tube (125), one end of which is plugged into the second piston cylinder (121), and the other end of which is plugged into the culture box (103); A second infusion tube (126), one end of which is fixedly connected to the second piston cylinder (121), and the other end of which extends to the waste liquid cylinder (101); The anti-slip fixing mechanism (200) comprises: A fixed cylinder (201) is plugged into and matched with the liquid storage cylinder (100) and the waste liquid cylinder (101); A supporting ring (202) is mounted on the inner wall of the fixed cylinder (201) and is used to support the waste liquid cylinder (101); A through groove (204) is formed on the fixing cylinder (201); A fixing ring (208) fixedly connected to the bottom surface of the fixing cylinder (201); A plurality of suction cups (209) are equidistantly mounted on the bottom surface of the fixing ring (208); The anti-slip fixing mechanism (200) further comprises: Two first insertion tubes (205) are both mounted on the bottom surface of the fixed tube (201) and are plugged into and matched with the first infusion tube (116) and the second extraction tube (125); A rotating plate (206) is hingedly connected to the bottom surface of the fixed cylinder (201); A second insert tube (207) is installed through the rotating plate (206) and is plugged into and matched with the first infusion tube (116) and the second extraction tube (125); Limiting assembly (210); The limiting component (210) comprises: A plurality of rotating shafts (211) are rotatably mounted on the fixed cylinder (201); A plurality of baffles (212) are respectively mounted on the plurality of rotating shafts (211).

2. The automatic perfusion culture device for NK cell culture according to claim 1, characterized in that: The driving mechanism (130) comprises: A motor (131) mounted on the bottom surface of the culture box (103); The cam (132) is mounted on the power output shaft of the motor (131) and is in contact with the first push plate (113) and the second push plate (123).

3. The automatic perfusion culture device for NK cell culture according to claim 2, characterized in that: It also includes a stirring mechanism (140), wherein the stirring mechanism (140) includes: A shaft (141) passes through the top surface of the liquid storage cylinder (100) and is rotatably connected to the liquid storage cylinder (100); A plurality of stirring rods (142), all located in the liquid storage cylinder (100) and all fixedly connected to the shaft rod (141); A gear (143) mounted on the upper end of the shaft (141); The rack (144) is meshingly connected to the gear (143), slidably connected to the top surface of the liquid storage cylinder (100), and fixedly connected to the first push plate (113).

4. The automatic perfusion culture device for NK cell culture according to claim 3, characterized in that: The anti-slip fixing mechanism (200) further comprises: The ultraviolet lamp (203) is mounted on the inner bottom surface of the fixing cylinder (201).

5. The method for using the automatic perfusion culture device for NK cell culture according to claim 4, characterized in that: The following steps are involved: Step 1: Check whether all parts of the automatic perfusion culture equipment for NK cell culture are complete and intact; Step 2: Place the anti-slip fixing mechanism (200) upside down on the liquid storage cylinder (100) and the waste liquid cylinder, and start the ultraviolet lamp (203) to sterilize the culture box (103), the first conveying mechanism (110), and the second conveying mechanism (120); Step 3: Place the fixed cylinder (201) on the table, so that the suction cup (209) is adsorbed on the table, and then place the liquid storage cylinder (100) and the waste liquid cylinder in the anti-slip fixing mechanism (200) respectively; Step 4: open the culture box (103), add the NK cell suspension into the culture box (103), start the motor (131), drive the cam (132) to rotate, and make the first conveying mechanism (110) convey the nutrient solution in the liquid storage cylinder (100) into the culture box (103); make the second conveying mechanism (120) convey the waste nutrient solution in the culture box (103) into the waste liquid cylinder, so as to realize the automatic perfusion culture of NK cells; Step 5: During the culture process, regularly observe the growth of NK cells in the culture box (103), and adjust the composition or concentration of the culture solution as needed; Step 6: When the NK cells reach the expected culture effect, the stop button of the driving mechanism (130) is pressed to end the automatic perfusion culture; Step 7: After the culture is completed, the liquid storage cylinder (100), the waste liquid cylinder (101) and the culture box (103) are cleaned in time and disinfected for next use.

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