An in vitro separation and culture device and method for immune cells

Abstract

The present invention relates to an in vitro separation and culture device and method for immune cells, and relates to the technical field of cell culture devices. The device comprises a thermostatic box and a temperature controller arranged on the outer wall of the thermostatic box, the thermostatic box is provided with side wall glass, the upper side of the thermostatic box is provided with an observation port, and the observation port is covered with a box top box, the upper side of the box top box is provided with high-transmittance glass, and a culture device is arranged in the thermostatic box; the culture device comprises a bottom support plate, a double-layer door is arranged at one end of the bottom support plate facing the outside of the thermostatic box, a layered support plate is arranged on one side of the double-layer door, a cell culture plate is arranged on the layered support plate, and a transfer mechanism for driving the displacement of the cell culture plate from the outside is arranged on the layered support plate and the double-layer door; the present invention has the advantages of being able to culture separated immune cells in a controllable environment, and being able to quickly and accurately view the cell culture conditions in different areas without affecting the culture environment, and having a simple operation process, which helps to reduce the observation time.

Description

Technical Field

[0001] The present application relates to the technical field of cell culture equipment, and in particular to an in vitro separation and culture equipment and method for immune cells. Background Art

[0002] Immune cells refer to cells that participate in or are related to immune responses. They include lymphocytes, dendritic cells, granulocytes, mast cells, etc. Immune cells can be divided into many types, and various immune cells play important roles in the human body. They are the basic components of the immune system in the body, responsible for identifying and eliminating pathogens, tumor cells and other foreign substances.

[0003] In vitro cell culture allows researchers to better understand the biological characteristics, developmental processes, functions, and interactions of immune cells. By controlling the culture conditions, the effects of different factors (such as cytokines, antigens, etc.) on immune cell functions can be systematically studied. The functional status of immune cells can be better monitored and evaluated, providing quality assurance for clinical applications. Researchers can study the characteristics and functions of cells in a more controlled environment, providing an important experimental basis for immunology, drug development, and clinical treatment.

[0004] For example, in the existing Chinese patent with publication number CN114181832A, a convenient NK cell in vitro separation and culture auxiliary device and method of use are disclosed, which includes an incubator, wherein a plurality of positioning slots arranged in sequence from top to bottom are installed inside the incubator, and a linkage support plate is slidably connected to the top of the positioning slot, and an inspection mechanism is installed on one side of the linkage support plate; the inspection mechanism includes a movable threaded block arranged on one side of the linkage support plate, and a driving screw is arranged inside the movable threaded block. Through the above-mentioned prior art, the inspection mechanism can be used to check the NK cells in each culture slot inside the incubator without opening the incubator, accurately grasp the NK cell culture status, improve the culture accuracy, and will not cause external factors to affect the culture status inside the incubator, and the culture effect is better.

[0005] However, the above prior art has the following technical defects:

[0006] When observing the cell culture conditions inside the incubator, the above-mentioned prior art first starts the rotating motor to drive the rotating rod to rotate 90 degrees, and the sleeve frame plate drives the moving screw block to rotate, so that the two cameras move to the top and bottom of the transparent culture plate respectively. The first drive motor can drive one of the cameras to move backward along the upper surface of the transparent culture plate when it is started. The second drive motor drives the moving belt transmission through the first pulley, and drives the two sleeve frames to move right under the drive of the linkage block to view the culture conditions of another group of three culture tanks. The second drive motor drives the first pulley to reverse, so that the sleeve frame plate is reset to the leftmost position of the inner wall of the incubator, the rotating motor drives the rotating rod to rotate and reset, and the moving motor is started to drive the driving screw to rotate forward, so as to drive the moving screw block to move up to view another layer of transparent culture plates.

[0007] When the staff checks the cell culture status in each layer of the transparent culture plate culture tank, they need to use four motors in sequence to achieve it, which makes the operation too cumbersome and results in a long time for observation. Secondly, when checking the cell culture status, the above device mainly transmits the captured images to the display through the camera for the staff to observe. When the temperature inside the above incubator is adjusted by hot and cold cycles, on the one hand, due to the alternating hot and cold, fog is easily generated on the camera lens, which brings inconvenience to the observation work; on the other hand, there are electronic devices inside the camera. If the camera is used for a long time in an environment of alternating hot and cold, the internal electronic components are easily damaged, resulting in the camera being unusable and needing to be replaced, further increasing the observation cost.

[0008] Based on this, on the basis of the existing easy-to-operate NK cell in vitro separation and culture auxiliary device and method of use, in order to overcome the above-mentioned technical defects, there is still room for improvement. Summary of the invention

[0009] In order to culture the separated immune cells in a controllable environment and to quickly and accurately check the cell culture conditions in different areas without affecting the culture environment, the operation process is simple and helps to reduce the observation time of the staff. The present application provides an immune cell in vitro separation and culture device and method.

[0010] In the first aspect, the present application provides an in vitro immune cell separation and culture device, which adopts the following technical solution:

[0011] An in vitro separation and culture device for immune cells, comprising a thermostat and a temperature controller arranged on the outer wall of the thermostat, the thermostat being provided with side wall glass, an observation port on the upper side of the thermostat, a box top box covering the observation port, a high-transmittance glass on the upper side of the box top box, and a culture device in the thermostat;

[0012] The culture device includes a bottom support plate that is slidingly limited by a T-shaped bottom rod in a constant temperature box, a T-shaped limiting groove for the sliding installation of the T-shaped bottom rod is opened on the bottom surface of the constant temperature box, a double-layer door is arranged at one end of the bottom support plate facing the outside of the constant temperature box, and a plurality of layered support plates are equidistantly and symmetrically installed from top to bottom on the side of the double-layer door facing the constant temperature box, a cell culture plate is arranged on the layered support plate, and a transfer mechanism for driving the displacement of the cell culture plate from the outside is arranged on the layered support plate and the double-layer door.

[0013] Preferably, the transfer mechanism includes an L-shaped slide bar which is slidingly limited by side blocks at both ends and is arranged on the upper side of the layered pallet. The layered pallet is symmetrically provided with limiting slide grooves for installing the side blocks at both ends. The L-shaped slide bar is used to lift the cell culture plate. A rectangular slot adapted to the L-shaped slide bar is provided at the bottom of the cell culture plate. The layered pallet and the double-layer box door are provided with mutually cooperating centering components and pushing units.

[0014] Preferably, a plurality of culture dishes are equidistantly embedded on the upper side of the cell culture plate, and a plurality of circular grooves for installing the culture dishes are equidistantly provided on the cell culture plate.

[0015] Preferably, the center shifting assembly includes a transmission belt rotatably mounted in the middle of the layered support plate through two inner rotating wheels and located below the L-shaped slide bar, and a middle groove for mounting the inner rotating wheels and the transmission belt is opened in the middle of the layered support plate.

[0016] Preferably, a U-shaped block connected to the transmission belt is installed on the lower side of the L-shaped sliding rod, and a driving rod is rotatably installed on the outer side of the double-layer door, which penetrates the double-layer door and the layered support plate and is connected to one of the inner rotating wheels. The layered support plate and the double-layer door are both provided with round rod sockets for the driving rod to penetrate.

[0017] Preferably, the pushing unit includes a Z-shaped push rod which is arranged in the middle of the bottom support plate and has one end penetrating through the double-layer door to extend outward, the bottom support plate is provided with a rectangular slot for installing the Z-shaped push rod, the double-layer door is provided with a middle slide opening which is adapted to the Z-shaped push rod, and a center box is provided on the outer side of the double-layer door at the middle slide opening.

[0018] Preferably, two limiting round rods are vertically installed through the Z-shaped push rod in the center box, and a limiting round hole is opened on the Z-shaped push rod for the two limiting round rods to penetrate. A threaded round rod with a thread passing through the Z-shaped push rod is rotatably arranged in the center box, and an internal threaded hole matching the threaded round rod is opened on the Z-shaped push rod. An end round button connected to the threaded round rod is inserted at the upper end of the center box, and a connecting hole is opened on the center box for the end round button to be inserted and installed.

[0019] Preferably, the cell culture plate and the Z-shaped push rod are also provided with a positioning component, the positioning component includes a lateral metal rod arranged on the L-shaped sliding rod, a strip magnet is embedded and installed on the side of the cell culture plate facing the lateral metal rod, and a side groove for installing the strip magnet is opened on one side of the cell culture plate.

[0020] Preferably, two lower magnets are embedded in the bottom of the cell culture plate, an embedded groove for installing the lower magnets is provided at the bottom of the cell culture plate, two upper magnets that attract the lower magnets are embedded on the upper side of the Z-shaped push rod, and an embedded circular groove for installing the upper magnets is provided on the Z-shaped push rod.

[0021] In a second aspect, the present application also discloses a method for isolating and culturing immune cells in vitro, the culturing method comprising the following steps:

[0022] The first step is layered seeding: the immune cells are seeded in sequence in the cell culture plates placed on different layered trays and nutrients are added;

[0023] The second step is constant temperature cultivation: push the double-layer door to allow the layered tray to enter the constant temperature box, and at the same time, the double-layer door closes the entrance to the constant temperature box, starts the operation of the constant temperature box, and adjusts the internal temperature of the constant temperature box through the temperature controller;

[0024] Step 3: Designated viewing: When the cell culture status on a certain layer of the cell culture plate needs to be observed, the cell culture plate at the designated position is moved to the observation port by operating the transfer mechanism, and viewed through the high-transmittance glass;

[0025] Step 4: Accurate resetting: After observing the cell culture status on the cell culture plate transferred to the observation port, operate the transfer mechanism again to drive the cell culture plate to re-set.

[0026] In summary, the present application includes at least one of the following beneficial technical effects:

[0027] 1. Through the setting of multiple layered support plates, multiple cell culture plates can be placed in different areas respectively. Each cell culture plate is provided with multiple culture dishes. Multiple groups of immune cells can be sown in the culture dishes on the corresponding cell culture plates, so that multiple groups of immune cells can be separated and cultured in vitro in a constant temperature box.

[0028] 2. When it is necessary to regularly observe the cell culture status on a certain layer of cell culture plate, the staff can drive the transmission belt by rotating the driving rod at the corresponding position externally, and drive the L-shaped sliding rod to move to the middle of the constant temperature box through the U-shaped block, so as to accurately transfer the corresponding cell culture plate to the top of the Z-shaped push rod, and then drive the Z-shaped push rod to move upward by twisting the round button at the end. During the upward movement, it contacts the bottom of the cell culture plate and pushes it to the observation port, and the staff observes the cell culture status through the high-transmittance glass.

[0029] 3. When the Z-shaped push rod moves upward and contacts the bottom of the cell culture plate, the two upper magnets will attract the two lower magnets respectively, so that the cell culture plate can be firmly placed on the Z-shaped push rod, avoiding the cell culture plate from shifting during the up and down movement, and failing to accurately snap into the L-shaped slide bar during the subsequent resetting. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is an overall schematic diagram of the present invention.

[0031] Figure 2 It is an exploded view of the box top box and the thermostatic box of the present invention.

[0032] Figure 3 The culture device of the present invention is schematically shown in FIG. Figure 1 (View from bottom to top).

[0033] Figure 4 The culture device of the present invention is schematically shown in FIG. Figure 2 .

[0034] Figure 5 It is a schematic diagram of the transfer mechanism of the present invention.

[0035] Figure 6 It is an exploded view of some components of the transfer mechanism of the present invention.

[0036] Figure 7 Schematic diagram of the center shifting component of the present invention (viewed from bottom to top).

[0037] Figure 8 The present invention Figure 7 A magnified image of area A.

[0038] Fig. 9 It is a cross-sectional exploded view of some parts of the shifting assembly of the present invention.

[0039] Fig.10 It is a schematic diagram of a push unit of the present invention.

[0040] Fig.11 It is an exploded view of the push unit of the present invention.

[0041] Fig.12 It is a cross-sectional exploded view of the pushing unit of the present invention.

[0042] Fig.13 It is a schematic diagram of the positioning component of the present invention.

[0043] Fig.14 It is an exploded view of some parts of the positioning assembly of the present invention.

[0044] Fig.15 Schematic diagram of the lower magnet of the present invention (viewed from bottom to top).

[0045] Fig.16 It is a schematic diagram of the filling unit of the present invention.

[0046] Fig.17 It is a cross-sectional view of the baffle of the present invention.

[0047] Fig.18 It is a flow chart of the cultivation method of the present invention.

[0048] Description of the reference numerals: 1. Constant temperature box; 11. Temperature controller; 12. Side wall glass; 13. Observation port; 14. Top box; 15. High-transmittance glass; 2. Culture device; 21. T-shaped bottom bar; 22. Bottom support plate; 101. T-shaped limiting groove; 23. Double-layer door; 24. Layered support plate; 25. Cell culture plate; 3. Transfer mechanism; 31. End block; 32. L-shaped slide bar; 241. Limiting slide groove; 251. Rectangular slot; 4. Centering component; 5. Push unit; 26. Culture dish; 252. Circular groove; 41. Inner rotating wheel; 42. Transmission belt; 242. Middle groove; 43. U-shaped block; 44. Driving rotating rod; 243. Round rod socket; 51. Z-shaped push rod; 221. Rectangular bayonet; 231. Middle slide; 52. Center box; 53, limit round rod; 511, limit round hole; 54, threaded round rod; 512, internal threaded hole; 55, end round button; 521, connecting hole; 6, positioning assembly; 61, lateral metal rod; 62, strip magnet; 253, side groove; 63, lower magnet; 254, embedded groove; 64, upper magnet; 513, embedded round groove; 7, filling unit; 71, L-shaped configuration plate; 72, gas tank; 73, liquid storage tank; 74, gas pipe; 75, gas control valve; 76, nozzle; 141, inclined hole; 77, bending pipe; 78, liquid supply pump; 79, shunt pipe; 8, flow blocking piece; 81, internal through pipe; 82, internal round ball; 811, limit rotation groove; 821, connecting hole; 83, toggle rod; 771, round rod hole. DETAILED DESCRIPTION

[0049] The following is combined with Figure 1-Figure 18 This application is described in further detail.

[0050] The embodiments of the present application disclose an in vitro separation and culture device and method for immune cells, which can culture separated immune cells in a controllable environment and can quickly and accurately check the cell culture conditions in different areas without affecting the culture environment. The operation process is simple, which helps to reduce the observation time of staff.

[0051] Embodiment 1:

[0052] Reference Figure 1 and Figure 2 As shown, in the first aspect, the present application provides an immune cell in vitro separation and culture device, comprising a thermostat 1 and a temperature controller 11 arranged on the outer wall of the thermostat 1. The thermostat 1 is a prior art. When it is turned on, the temperature inside the thermostat 1 can be adjusted by the temperature controller 11, and the temperature inside the thermostat 1 is adjusted to the appropriate temperature required for immune cell culture. The real-time temperature inside the thermostat 1 can be displayed by the temperature controller 11. The thermostat 1 is installed with a side wall glass 12, and the upper side of the thermostat 1 has an observation port 13, and the observation port 13 is covered with a box top box 14, which is fixedly installed on the upper side of the thermostat 1; the upper side of the box top box 14 is provided with a high-transmittance glass 15, and the thermostat 1 is provided with a culture device 2; it is used for in vitro separation and culture of immune cells, and when it is necessary to observe the immune cell culture status, the cells at the designated position can be transferred to the observation port 13, so that the staff can observe from the outside through the high-transmittance glass 15, which will not affect the original culture environment inside the thermostat 1.

[0053] Reference Figure 3 and Figure 4 As shown, specifically, the culture device 2 includes a bottom support plate 22 which is limited and slidably arranged in the thermostat 1 by a T-shaped bottom rod 21, and a T-shaped limit groove 101 for the T-shaped bottom rod 21 to be slidably installed is provided on the bottom surface of the thermostat 1. A double-layer door 23 is provided at one end of the bottom support plate 22 facing the outside of the thermostat 1. In this embodiment, preferably, eight layered support plates 24 are equidistantly and symmetrically installed from top to bottom on the side of the double-layer door 23 facing the thermostat 1, and the layered support plates 24 and the double-layer door 23 are fixedly connected. A cell culture plate 25 is provided on the layered support plate 24, and a transfer mechanism 3 for driving the displacement of the cell culture plate 25 from the outside is provided on the layered support plate 24 and the double-layer door 23, which is used to drive the cell culture plates 25 at different positions to move to the observation port 13 for the staff to observe the cell culture status.

[0054] In this embodiment, five culture dishes 26 are preferably embedded at equal intervals on the upper side of the cell culture plate 25, and five circular grooves 252 for installing the culture dishes 26 are opened at equal intervals on the cell culture plate 25. Five culture dishes 26 are arranged on each cell culture plate 25. When in use, the separated immune cells are sown in each culture dish 26 at different positions at an appropriate cell density, and the required nutrients are added to the culture dish 26 at the same time. Then, the double-layer door 23 is pushed to close the double-layer door 23 at the entrance of the thermostat 1, the thermostat 1 is started, and the temperature inside the thermostat 1 is adjusted to a suitable temperature through the temperature controller 11, so that multiple groups of immune cells can be cultured in the thermostat 1.

[0055] Reference Figure 5 and Figure 6 As shown, in order to support and place the cell culture plate 25, and when the cell culture status needs to be observed, the cell culture plate 25 at the corresponding position can be accurately transferred to the observation port 13, so that the staff can observe and record the cell culture status through the high-transmittance glass 15, and it is also convenient for the staff to take the magnifying equipment used for observation to the top of the box top box 14 for use. The transfer mechanism 3 includes an L-shaped slide bar 32 that is limitedly slidably arranged on the upper side of the layered support plate 24 through the side clamping blocks 31 at both ends, and the layered support plate 24 is symmetrically provided with The limiting slide grooves 241 for installing the side blocks 31 at both ends, and the L-shaped slide bar 32 are used to lift and place the cell culture plate 25. The bottom of the cell culture plate 25 is provided with a rectangular slot 251 adapted to the L-shaped slide bar 32. When the cell culture plate 25 is placed on the layered support plate 24, the rectangular slot 251 provided at the bottom of the cell culture plate 25 is used to firmly clamp the cell culture plate 25 on the L-shaped slide bar 32, and when the L-shaped slide bar 32 is subjected to force, the cell culture plate 25 can be driven to slide horizontally on the layered support plate 24. The layered support plate 24 and the double-layer door 23 are provided with a shifting component 4 and a pushing unit 5 that cooperate with each other.

[0056] During the culture process, when it is necessary to observe the cell culture status in a certain layer of culture dishes 26, the staff can first drive the cell culture plate 25 on the designated layer support plate 24 from the outside through the moving component 4, so that it can be translated to the middle of the incubator 1 and directly below the observation port 13. Then, the pushing unit 5 can push the cell culture plate 25 upward to the observation port 13 for the staff to observe from the outside. The whole operation process is simple and fast, and the incubator 1 does not need to be opened, which can ensure that the original culture environment inside the incubator 1 is not affected by external factors.

[0057] Reference Figure 7 and Figure 8As shown, since the cell culture plate 25 needs to be moved to the middle of the incubator 1 through external operation, the cell culture plate 25 is first moved to the position directly below the observation port 13, so that the cell culture plate 25 can be moved upward under the action of the push unit 5. The center shifting component 4 includes a transmission belt 42 that is rotatably installed in the middle of the layered support plate 24 and located below the L-shaped slide bar 32 through two inner rotating wheels 41. The middle of the layered support plate 24 is provided with a middle groove 242 for installing the inner rotating wheels 41 and the transmission belt 42. It should be noted that when the transmission belt 42 is installed in the middle groove 242 through the two inner rotating wheels 41, the transmission belt 42 is in a taut state; and when one of the inner rotating wheels 41 is rotated by force, the transmission belt 42 can be driven to rotate. Since there are eight layered support plates 24, each layered support plate 24 is provided with a center shifting component 4.

[0058] Reference Figures 7 to 9 As shown, a U-shaped block 43 connected to the transmission belt 42 is installed on the lower side of the L-shaped slide bar 32. It should be noted that the upper side of the U-shaped block 43 is fixedly connected to the lower side of the L-shaped slide bar 32, and the U-shaped block 43 is fixedly connected to the transmission belt 42 at a position where the transmission belt 42 is in a parallel state, so that when the transmission belt 42 is driven to rotate, the L-shaped slide bar 32 can be driven to translate on the layered support plate 24 through the U-shaped block 43; a driving rod 44 that penetrates the double-layer door 23 and the layered support plate 24 and is connected to one of the inner rotating wheels 41 is rotatably installed on the outer side of the double-layer box door 23, and a round rod socket 243 for the driving rod 44 to penetrate is provided on the layered support plate 24 and the double-layer box door 23.

[0059] When it is necessary to observe the cell culture status on a certain layer of the cell culture plate 25, the staff can rotate the driving rod 44 at the corresponding position on the outer side of the double-layer box door 23 to drive the L-shaped slide bar 32 to translate toward the middle of the constant temperature box 1 through the rotation of the transmission belt 42, until the U-shaped block 43 hits the side wall of the middle groove 242 and can no longer rotate. At this time, the cell culture plate 25 stuck on this L-shaped slide bar 32 is directly below the observation port 13.

[0060] Reference Figures 10 to 12As shown, considering that the cell culture plate 25 moved to the middle of the incubator 1 needs to be pushed to the observation port 13, so that the staff can observe the cell culture status in the culture dish 26, the pushing unit 5 includes a Z-shaped push rod 51 arranged in the middle of the bottom support plate 22 and one end of which penetrates through the double-layer door 23 and extends outward, the bottom support plate 22 is provided with a rectangular bayonet 221 for the Z-shaped push rod 51 to be installed, the double-layer door 23 is provided with a middle slide 231 adapted to the Z-shaped push rod 51, and a middle box 52 is provided at the middle slide 231 on the outer side of the double-layer door 23. In the initial state, the Z-shaped push rod 51 is stuck at the rectangular bayonet 221 of the bottom support plate 22, and one end of the Z-shaped push rod 51 is at the bottom of the middle box 52, and when the Z-shaped push rod 51 is driven, it can slide up and down at the middle slide 231.

[0061] Reference Fig.11 and Fig.12 As shown, two limiting round rods 53 are vertically installed through the Z-shaped push rod 51 in the center box 52, and a limiting round hole 511 is opened on the Z-shaped push rod 51 for the two limiting round rods 53 to penetrate. The limiting round rod 53 is fixedly installed in the center box 52, and plays a limiting role on the Z-shaped push rod 51, so that the Z-shaped push rod 51 cannot rotate to produce an angular deviation; a threaded round rod 54 is rotatably arranged in the center box 52, and an internal threaded hole 512 is opened on the Z-shaped push rod 51 that is threaded and compatible with the threaded round rod 54; an end round button 55 connected to the threaded round rod 54 is inserted at the upper end of the center box 52, and a connecting hole 521 is opened on the center box 52 for the end round button 55 to be inserted and installed.

[0062] Similarly, the staff can operate from the outside, rotate the end round button 55 to drive the threaded round rod 54 to rotate, and through the cooperation with the internal threaded hole 512 opened on the Z-shaped push rod 51, the Z-shaped push rod 51 can be driven to move upward. During the upward movement of the Z-shaped push rod 51, it will conflict with the bottom of the cell culture plate 25 that is translated to the middle of the constant temperature box 1 and is directly below the observation port 13, so that the cell culture plate 25 can be pushed upward to break away from the L-shaped sliding rod 32 and move upward to the observation port 13, so that the staff can observe the cell culture status of this cell culture plate 25 and culture dish 26 through the high-transmittance glass 15.

[0063] Reference Fig.13 and Fig.14As shown, in order to prevent the cell culture plate 25 from being displaced and causing position deviation during the process of the Z-shaped push rod 51 pushing the cell culture plate 25 to move, so that the cell culture plate 25 cannot be aligned with the card set on the L-shaped slide bar 32 and return to the initial position when the cell culture plate 25 needs to be reset later, a positioning component 6 is also provided on the cell culture plate 25 and the Z-shaped push rod 51. The positioning component 6 includes a side metal rod 61 provided on the L-shaped slide bar 32. When placing the cell culture plate 25, the rectangular card slot 251 of the cell culture plate 25 needs to be stuck between the side wall of the L-shaped slide bar 32 and the side metal rod 61, and at this time, one side of the cell culture plate 25 is in contact with the side metal rod 61. A strip magnet 62 is embedded and installed on the side of the cell culture plate 25 facing the side metal rod 61, and a side groove 253 for installing the strip magnet 62 is provided on one side of the cell culture plate 25. When the cell culture plate 25 is clamped and placed on the side wall of the L-shaped slide bar 32 and the side metal rod 61 , there is suction between the strip magnet 62 and the side metal rod 61 , which can make the cell culture plate 25 more stably placed on the L-shaped slide bar 32 .

[0064] Reference Figures 13 to 15 As shown, two lower magnets 63 are embedded and installed at the bottom of the cell culture plate 25, and an embedded groove 254 for installing the lower magnets 63 is provided at the bottom of the cell culture plate 25. Two upper magnets 64 that attract the lower magnets 63 are embedded at the upper side of the Z-shaped push rod 51, and an embedded circular groove 513 for installing the upper magnets 64 is provided on the Z-shaped push rod 51. When the Z-shaped push rod 51 moves upward and contacts the bottom of the cell culture plate 25, the two upper magnets 64 will be attracted to the two lower magnets 63 respectively, so that the cell culture plate 25 can be firmly adsorbed on the Z-shaped push rod 51, and the position of the cell culture plate 25 will not be offset during the upward or downward movement. After the staff has finished observing, the Z-shaped push rod 51 is driven to move the cell culture plate 25 downward, so that the rectangular slot 251 at the bottom of the cell culture plate 25 can be accurately stuck between the side wall of the L-shaped slide bar 32 and the side metal rod 61. At this time, since the strip magnet 62 and the side metal rod 61 have suction force, it is ensured that the Z-shaped push rod 51 and the cell culture plate 25 can be separated smoothly, thereby achieving the resetting effect.

[0065] Embodiment 2:

[0066] Reference Fig.16As shown, on the basis of the first embodiment, in order to facilitate the subsequent filling of oxygen, carbon dioxide and other gases into the thermostat 1, to meet the cultivation of microbial tissues or cells with special needs for such gases, and the subsequent supplementation of nutrient solution, the cells will consume nutrients in the culture medium during the cultivation process; it is convenient for the staff to accurately fill the nutrient solution into the culture dish 26 with nutrient deficiency when observing the cell culture status, and the thermostat 1 is also provided with a filling unit 7 on the outside, and the filling unit 7 includes an L-shaped configuration plate 71 arranged on the outer wall of the thermostat 1, and the L-shaped configuration plate 71 is respectively equipped with a gas storage tank 72 and a liquid storage tank 73, and the gas storage tank 72 is connected to the top box 14 and installed with a gas pipe 74, and a gas control valve 75 is provided on the gas pipe 74. Gases such as oxygen and carbon dioxide can be stored in the gas tank 72 as needed. The liquid storage tank 73 can be injected with the nutrient solution required by the cells. The amount of gas injected into the thermostat 1 can be controlled by the gas control valve 75.

[0067] In this embodiment, five nozzles 76 are preferably installed on the side wall of the top box 14, tilted toward the inner cavity of the thermostat 1, and five tilted holes 141 are provided on the side wall of the top box 14 for installing the nozzles 76. It should be noted that the five nozzles 76 are respectively aimed at the five culture dishes 26 on the cell culture plate 25 transferred to the observation port 13. A bending pipe 77 is connected and installed on the nozzle 76, and a liquid supply water pump 78 is provided on the liquid storage tank 73. The five bending pipes 77 and the liquid supply water pump 78 are connected through a shunt pipe 79. The nutrient solution in the liquid storage tank 73 can be transported to the shunt pipe 79 through the liquid supply water pump 78, and then dispersed to each bending pipe 77 by the shunt pipe 79, and finally sprayed to the culture dish 26 through the nozzle 76.

[0068] Reference Fig.16 and Fig.17 As shown, taking into account the diversity of immune cells during the culture process, the five culture dishes 26 on the same cell culture plate 25, not all nutrients in each culture dish 26 are exhausted and need to be replenished at the same time. For culture dishes 26 with more nutrients, there is no need to replenish nutrient solution to avoid affecting the normal culture of immune cells and preventing the waste of nutrient solution. A baffle 8 is provided on the bending tube 77, and the baffle 8 includes an inner tube 81 fixedly arranged in the bending tube 77, an inner round ball 82 is installed in the inner tube 81 for limiting rotation, and a limiting rotation groove 811 adapted to the inner round ball 82 is opened in the inner tube 81. It should be noted that the inner tube 81 is divided into two parts to facilitate the installation of the inner round ball 82 in the limiting rotation groove 811. The inner round ball 82 is penetrated by a connecting hole 821 , a toggle rod 83 fixedly connected to the inner round ball 82 is rotatably mounted on the bending tube 77 , and a round rod hole 771 for the toggle rod 83 to penetrate and install is formed on both the bending tube 77 and the inner through tube 81 .

[0069] When the staff observes the cell culture status on the cell culture plate 25 transferred to the observation port 13, if they find that the nutrients in a certain culture dish 26 are insufficient and need to be supplemented, they can rotate the toggle rod 83 on the corresponding bending tube 77 by 90 degrees, and drive the inner round ball 82 to rotate through the toggle rod 83, so that the connecting hole 821 on the inner round ball 82 and the inner through tube 81 are in a clear state, so that the nutrient solution can enter the nozzle 76 and be sprayed into the culture dish 26 at the corresponding position.

[0070] Reference Fig.18 As shown, on the other hand, the present application also discloses an in vitro isolation and culture method for immune cells, and the culture method comprises the following steps:

[0071] The first step is layered seeding: the immune cells are sequentially seeded in the cell culture plates 25 placed on different layered support plates 24, and nutrients are added; specifically, the separated immune cells are seeded in each culture dish 26 at different positions at an appropriate cell density, and the required nutrients are added to the culture dish 26 at the same time.

[0072] The second step is constant temperature culture: push the double-layer door 23 to allow the layered support plate 24 to enter the constant temperature box 1, and at the same time, the double-layer door 23 closes the entrance of the constant temperature box 1, starts the operation of the constant temperature box 1, and adjusts the internal temperature of the constant temperature box 1 through the temperature controller 11; so that multiple groups of immune cells can be cultured in the constant temperature box 1.

[0073] The third step is to designate viewing: when it is necessary to observe the cell culture status on a certain layer of the cell culture plate 25, the cell culture plate 25 at the designated position is transferred to the observation port 13 by operating the transfer mechanism 3, and viewed through the high-transmittance glass 15; during the specific operation, the driving rotating rod 44 at the corresponding position is rotated to drive the L-shaped sliding rod 32 to drive the cell culture plate 25 to move toward the middle of the constant temperature box 1; then the end round button 55 is rotated to drive the threaded round rod 54 to rotate, driving the Z-shaped push rod 51 to move up and contact with the bottom of the cell culture plate 25, and the two upper side magnets 64 and the two lower side magnets 63 are attracted to each other, so that the cell culture plate 25 is firmly adsorbed on the Z-shaped push rod 51, and the cell culture plate 25 is pushed to the observation port 13, so as to observe the cell culture status.

[0074] Step 4: accurate resetting: After observing the cell culture status on the cell culture plate 25 transferred to the observation port 13, operate the transfer mechanism 3 again to drive the cell culture plate 25 to reset. During the observation, if it is found that the nutrient in a certain culture dish 26 is less and needs to be supplemented, the toggle rod 83 on the corresponding bending tube 77 is rotated 90 degrees to make the connecting hole 821 on the inner round ball 82 and the inner through tube 81 unblocked, so that the nutrient solution can be sprayed into the corresponding culture dish 26 through the nozzle 76. Finally, the end round button 55 and the driving rod 44 are rotated back to realize the resetting of the cell culture plate 25.

[0075] The embodiments of this specific implementation method are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An immune cell in vitro separation and culture device, comprising a constant temperature box (1) and a temperature controller (11) arranged on the outer wall of the constant temperature box (1), the constant temperature box (1) is installed with a side wall glass (12), the constant temperature box (1) has an observation port (13) on the upper side, and a box top box (14) is provided on the observation port (13), and the box top box (14) is provided with a high-transmittance glass (15) on the upper side, characterized in that: The incubator (1) is provided with a culture device (2); The culture device (2) comprises a bottom support plate (22) which is limitedly slidably arranged in a thermostatic box (1) by a T-shaped bottom rod (21); a T-shaped limit groove (101) for slidingly installing the T-shaped bottom rod (21) is provided on the inner bottom surface of the thermostatic box (1); a double-layer door (23) is provided at one end of the bottom support plate (22) facing the outside of the thermostatic box (1); a plurality of layered support plates (24) are symmetrically installed from top to bottom at equal distances on the side of the double-layer door (23) facing the thermostatic box (1); a cell culture plate (25) is provided on the layered support plate (24); and a transfer mechanism (3) for driving the cell culture plate (25) to move from the outside is provided on the layered support plate (24) and the double-layer door (23); The transfer mechanism (3) comprises an L-shaped slide bar (32) which is limitedly slidably arranged on the upper side of the layered support plate (24) through two end side clamping blocks (31); the layered support plate (24) is symmetrically provided with limit slide grooves (241) for installing the two end side clamping blocks (31); the L-shaped slide bar (32) is used to lift the cell culture plate (25); a rectangular clamping groove (251) adapted to the L-shaped slide bar (32) is provided at the bottom of the cell culture plate (25); and the layered support plate (24) and the double-layer box door (23) are provided with a shifting assembly (4) and a pushing unit (5) which cooperate with each other; A plurality of culture dishes (26) are equidistantly embedded on the upper side of the cell culture plate (25), and a plurality of circular grooves (252) for installing the culture dishes (26) are equidistantly provided on the cell culture plate (25); The center shifting assembly (4) comprises a transmission belt (42) which is rotatably mounted on the middle of the layered support plate (24) and located below the L-shaped slide bar (32) via two inner rotating wheels (41); a middle groove (242) for mounting the inner rotating wheels (41) and the transmission belt (42) is provided in the middle of the layered support plate (24); The pushing unit (5) comprises a Z-shaped push rod (51) which is arranged in the middle of the bottom support plate (22) and one end of which penetrates through the double-layer box door (23) and protrudes outwards; the bottom support plate (22) is provided with a rectangular bayonet (221) for installing the Z-shaped push rod (51); the double-layer box door (23) is provided with a middle sliding opening (231) adapted to the Z-shaped push rod (51); and a middle box (52) is provided on the outer side of the double-layer box door (23) at the middle sliding opening (231); A U-shaped block (43) connected to a transmission belt (42) is installed at the lower side of the L-shaped slide bar (32); a driving rod (44) penetrating the double-layer door (23) and the layered support plate (24) and connected to one of the inner rotating wheels (41) is rotatably installed on the outer side of the double-layer door (23); and a round rod insertion hole (243) for the driving rod (44) to penetrate is provided on the layered support plate (24) and the double-layer door (23); Two limiting round rods (53) are vertically installed in the middle box (52) through the Z-shaped push rod (51), and a limiting round hole (511) is opened on the Z-shaped push rod (51) for the two limiting round rods (53) to penetrate. A threaded round rod (54) threadedly penetrating the Z-shaped push rod (51) is rotatably arranged in the middle box (52), and an internal threaded hole (512) adapted to the threaded round rod (54) is opened on the Z-shaped push rod (51) through the thread. An end round button (55) connected to the threaded round rod (54) is inserted at the upper end of the middle box (52), and a connecting hole (521) is opened on the middle box (52) for the end round button (55) to be inserted and installed.

2. The immune cell in vitro separation and culture device according to claim 1, characterized in that: The cell culture plate (25) and the Z-shaped push rod (51) are further provided with a positioning assembly (6), the positioning assembly (6) comprising a lateral metal rod (61) arranged on the L-shaped sliding rod (32), a strip magnet (62) is embedded and installed on one side of the cell culture plate (25) facing the lateral metal rod (61), and a side groove (253) for installing the strip magnet (62) is provided on one side of the cell culture plate (25).

3. The immune cell in vitro separation and culture device according to claim 2, characterized in that: Two lower magnets (63) are embedded and installed at the bottom of the cell culture plate (25), and an embedded groove (254) for installing the lower magnets (63) is provided at the bottom of the cell culture plate (25). Two upper magnets (64) that attract the lower magnets (63) are embedded on the upper side of the Z-shaped push rod (51), and an embedded circular groove (513) for installing the upper magnets (64) is provided on the Z-shaped push rod (51).

4. A method for isolating and culturing immune cells in vitro, characterized in that: Using the immune cell in vitro separation and culture device according to any one of claims 1 to 3, the operating steps are as follows: The first step is layered seeding: the staff sequentially seeds the immune cells in the cell culture plates (25) placed on different layered support plates (24), and adds nutrients; Step 2: Constant temperature culture: Push the double-layer door (23) to allow the layered support plate (24) to enter the constant temperature box (1), and at the same time, close the entrance of the constant temperature box (1) with the double-layer door (23), start the constant temperature box (1) to operate, and adjust the internal temperature of the constant temperature box (1) through the temperature controller (11); Step 3, designated viewing: when it is necessary to observe the cell culture status on a certain layer of the cell culture plate (25), the staff operates the transfer mechanism (3) to transfer the cell culture plate (25) at the designated position to the observation port (13) and observes it through the high-transmittance glass (15); The fourth step is accurate resetting: after observing the cell culture status on the cell culture plate (25) transferred to the observation port (13), the transfer mechanism (3) is operated again to drive the cell culture plate (25) to re-set.

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