CN119144448A

CN119144448A - Immune cell in-vitro separation culture equipment and method

Info

Publication number: CN119144448A
Application number: CN202411612338.2A
Authority: CN
Inventors: 黄燕; 郑旭东; 王慧琴; 张磊; 钟丽平
Original assignee: Shenzhen Lailisai Biological Technology Co ltd
Current assignee: Shenzhen Lailisai Biological Technology Co ltd
Priority date: 2024-11-13
Filing date: 2024-11-13
Publication date: 2024-12-17
Anticipated expiration: 2044-11-13
Also published as: CN119144448B

Abstract

The invention relates to an immune cell in-vitro separation culture device and a method, and relates to the technical field of cell culture devices, wherein the device comprises an incubator and a temperature controller arranged on the outer side wall of the incubator, side wall glass is arranged on the incubator, an observation port is arranged on the upper side of the incubator, a box top box is covered on the observation port, high-permeability glass is arranged on the upper side of the box top box, and a culture device is arranged in the incubator; the invention has the advantages that the isolated immune cells can be cultured in a controllable environment, in addition, the cell culture conditions in different areas can be rapidly and accurately checked under the condition of not influencing the culture environment, the operation process is simple, and the effect of reducing the observation time length is facilitated.

Description

Immune cell in-vitro separation culture equipment and method

Technical Field

The application relates to the technical field of cell culture equipment, in particular to immune cell in-vitro separation culture equipment and a method.

Background

Immune cells refer to cells involved in or associated with an immune response. Including lymphocytes, dendritic cells, granulocytes, mast cells, and the like. Immune cells can be divided into a variety of classes, and various immune cells play an important role in the human body, are essential components of the immune system in the body, and are responsible for the recognition and elimination of pathogens, tumor cells, and other foreign substances.

In vitro cell culture allows researchers to better understand the biological properties, developmental processes, functions, and interactions of immune cells. By controlling the culture conditions, the effect of different factors (e.g., cytokines, antigens, etc.) on immune cell function can be systematically studied. The functional state of immune cells can be better monitored and evaluated, and quality assurance is provided for clinical application. Researchers can study the characteristics and functions of cells in a more controllable environment, and provide an important experimental basis for immunology, drug development and clinical treatment.

In the prior Chinese patent with publication number CN114181832A, an auxiliary device for external separation and culture of NK cells is disclosed, which comprises an incubator, wherein a plurality of positioning groove plates are arranged in sequence from top to bottom in the incubator, the top ends of the positioning groove plates are slidably connected with a linkage support plate, one side of the linkage support plate is provided with an inspection mechanism, the inspection mechanism comprises a movable thread block arranged on one side of the linkage support plate, and a driving screw is arranged in the movable thread block. Through above-mentioned prior art, adopt inspection mechanism, can need not to open the incubator and can carry out about contrast to each inside NK cell of culture tank of incubator inside and look over the operation, accurate grasp NK cell cultivation situation improves the cultivation accuracy to can not cause external factor to influence the inside cultivation situation of incubator, the cultivation effect is better.

However, the prior art has the following technical defects:

when observing the cell culture condition in the incubator, the prior art starts the rotating motor to drive the rotating rod to rotate ninety degrees, and the sleeved frame plate drives the movable screw block to rotate, so that the two cameras respectively move above and below the transparent culture plate. The first driving motor is started to drive one camera to move backwards along the transparent culture plate, the second driving motor drives the movable belt to drive through the first belt pulley, and the linkage block drives the two sleeved frame plates to move rightwards to check the culture conditions of the other group of three culture tanks. The second driving motor drives the first belt pulley to rotate reversely, so that the sleeved frame plate resets to the leftmost position of the inner wall of the incubator, the rotating motor drives the rotating rod to rotate for resetting, and the moving motor is started to drive the driving screw to rotate forward so as to drive the moving screw block to move upwards to check another layer of transparent culture plate.

When the staff looks over the cell culture condition in each layer transparent culture plate culture tank, all need use the cooperation in proper order through four motors just can realize to the operation is too loaded down with trivial details, leads to observing required live time longer. Secondly, when the device is used for checking the cell culture condition, the shot picture is mainly transmitted to a display through the camera to be observed by a worker, and when the temperature is regulated in the incubator in a cold-hot circulation mode, on one hand, fog is easy to generate at the lens of the cold-hot alternate camera to bring inconvenience to observation work, on the other hand, the camera is internally provided with electronic devices, and the camera is used for a long time in a cold-hot replacement environment, so that the phenomenon that the internal electronic elements are damaged easily causes that the camera cannot be used and needs to be replaced, and the observation cost is further increased.

Based on the above, on the basis of the existing auxiliary device and the using method for conveniently operating NK cell in vitro separation culture, in order to overcome the technical defects, there is still room for improvement.

Disclosure of Invention

In order to culture isolated immune cells in a controllable environment, and quickly and accurately check cell culture conditions in different areas under the condition of not affecting the culture environment, the application provides the immune cell in-vitro isolated culture equipment and method, which have simple operation process and are beneficial to reducing the observation time of staff.

In a first aspect, the present application provides an immune cell in vitro separation culture apparatus, which adopts the following technical scheme:

The immune cell in-vitro separation culture equipment comprises an incubator and a temperature controller arranged on the outer side wall of the incubator, wherein side wall glass is arranged on the incubator, an observation port is formed in the upper side of the incubator, a box top box is covered on the observation port, high-permeability glass is arranged on the upper side of the box top box, and a culture device is arranged in the incubator;

The culture device comprises a bottom supporting plate which is arranged in the incubator in a limiting sliding manner through a T-shaped bottom rod, a T-shaped limiting groove for the sliding installation of the T-shaped bottom rod is formed in the bottom surface in the incubator, a double-layer refrigerator door is arranged at one end, facing the outside of the incubator, of the bottom supporting plate, a plurality of layered supporting plates are symmetrically arranged at one side, facing the incubator, of the double-layer refrigerator door from top to bottom, a cell culture plate is arranged on the layered supporting plates, and a transfer mechanism for driving the displacement of the cell culture plate from outside is arranged on the layered supporting plates and the double-layer refrigerator door.

Preferably, the transfer mechanism comprises an L-shaped sliding rod which is arranged on the upper side of the layered supporting plate in a limiting sliding manner through clamping blocks at two ends, limiting sliding grooves for installing the clamping blocks at two ends are symmetrically formed in the layered supporting plate, the L-shaped sliding rod is used for lifting a cell culture plate, a rectangular clamping groove matched with the L-shaped sliding rod is formed in the bottom of the cell culture plate, and a middle moving component and a pushing unit which are matched with each other are arranged on the layered supporting plate and the double-layer box door.

Preferably, a plurality of culture dishes are embedded on the upper side of the cell culture plate at equal intervals, and a plurality of circular grooves for mounting the culture dishes are formed in the cell culture plate at equal intervals.

Preferably, the middle moving assembly comprises a transmission belt which is rotatably arranged in the middle of the layered support plate through two inner rotating wheels and is positioned below the L-shaped sliding rod, and a middle section groove for installing the inner rotating wheels and the transmission belt is formed in the middle of the layered support plate.

Preferably, the U-shaped fixture block that is connected with the drive belt is installed to L shape slide bar downside, double-deck chamber door outside rotates installs and pierces through double-deck chamber door and layering layer board to with the drive bull stick that one of them inward-turning wheel is connected, all seted up the round bar jack that supplies the drive bull stick to pierce through on layering layer board and the double-deck chamber door.

Preferably, the pushing unit comprises a Z-shaped push rod which is arranged in the middle of the bottom supporting plate and one end of which penetrates through the double-layer box door to extend outwards, a rectangular bayonet for the installation of the Z-shaped push rod is formed in the bottom supporting plate, a middle sliding port which is matched with the Z-shaped push rod is formed in the double-layer box door in a penetrating mode, and a middle box is arranged at the position, located at the middle sliding port, of the outer side of the double-layer box door.

Preferably, the middle box is internally provided with two limiting round rods vertically arranged through the Z-shaped push rod, limiting round holes for the two limiting round rods to penetrate are formed in the Z-shaped push rod in a penetrating mode, the Z-shaped push rod is internally provided with threaded round rods penetrating through the Z-shaped push rod in a rotating mode, the Z-shaped push rod is internally provided with internal threaded holes matched with the threaded round rods in a penetrating mode, the upper end of the middle box is provided with end round buttons connected with the threaded round rods in an inserting mode, and the middle box is provided with connecting holes for the end round buttons to be installed in an inserting mode.

Preferably, the cell culture plate and the Z-shaped push rod are further provided with a positioning assembly, the positioning assembly comprises a lateral metal rod arranged on the L-shaped slide rod, a strip-shaped magnet is embedded and arranged on one side of the cell culture plate, which faces the lateral metal rod, and a lateral groove for mounting the strip-shaped magnet is formed in one side of the cell culture plate.

Preferably, the cell culture plate bottom is inlayed and is installed two downside magnet, the cell culture plate bottom is offered and is supplied the embedded recess of downside magnet installation, Z shape push rod upside inlays and is equipped with two upside magnet that inhale mutually with downside magnet, set up on the Z shape push rod and inlay and establish the circular slot of upside magnet installation.

In a second aspect, the application also discloses an immune cell in vitro separation culture method, which comprises the following steps:

step one, layered seeding, namely sequentially seeding immune cells in cell culture plates placed on different layered supporting plates, and filling nutrient substances;

Secondly, culturing at constant temperature, namely pushing the double-layer box door to enable the layered supporting plate to enter the incubator, closing the inlet of the incubator by the double-layer box door, opening the incubator to operate, and adjusting the internal temperature of the incubator by the temperature controller;

Thirdly, appointing to check, namely when the cell culture condition on a certain layer of cell culture plate needs to be observed, transferring the cell culture plate at the appointed position to an observation port by operating a transfer mechanism, and checking through high-permeability glass;

And fourthly, accurately resetting, namely after the cell culture condition on the cell culture plate transferred to the observation port is observed, operating the transfer mechanism again to drive the cell culture plate to reset.

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

1. Through the setting of a plurality of layering layer boards, can place a plurality of cell culture boards respectively in different regions, all be provided with a plurality of culture dishes on every cell culture board, can sow multiunit immune cell subregion in the culture dish on corresponding cell culture board, realize can carrying out multiunit immune cell in vitro separation culture in the thermostated container.

2. When the cell culture condition on a certain layer of cell culture plate needs to be observed regularly, a worker drives a transmission belt to operate through a driving rotating rod at the corresponding position in the outside, the L-shaped sliding rod can be driven to move towards the middle of the incubator through a U-shaped clamping block, so that the corresponding cell culture plate can be accurately transferred to the upper part of the Z-shaped pushing rod, then the Z-shaped pushing rod can be driven to move upwards through twisting a round button at the end part, the bottom of the cell culture plate is abutted against in the upward movement process, the cell culture plate is pushed to the observation port, and the worker observes the cell culture condition through high-transmittance glass.

When 3.Z shape push rod moves up and contradicts with cell culture plate bottom, two upside magnet can inhale with two downside magnet respectively for cell culture plate can firmly be in on the Z shape push rod, avoids the in-process cell culture plate of reciprocates to take place the skew, can't be accurate card to go into on the L shape slide bar when the follow-up resets.

Drawings

Fig. 1 is a schematic overall view of the present invention.

FIG. 2 is an exploded view of the roof box and oven of the present invention.

FIG. 3 is a schematic view of the culture apparatus according to the present invention (seen from below to above).

FIG. 4 is a schematic diagram of a culture apparatus according to the present invention.

Fig. 5 is a schematic view of a transfer mechanism according to the present invention.

Fig. 6 is an exploded view of a portion of the components of the transfer mechanism of the present invention.

Fig. 7 is a schematic view of the centering assembly of the present invention (from bottom to top).

Fig. 8 is an enlarged view of area a of fig. 7 in accordance with the present invention.

FIG. 9 is a partial sectional exploded view of the mobile assembly of the present invention.

Fig. 10 is a schematic diagram of a pushing unit of the present invention.

Fig. 11 is an exploded view of the push unit of the present invention.

Fig. 12 is a cross-sectional exploded view of the push unit of the present invention.

FIG. 13 is a schematic view of a positioning assembly of the present invention.

Fig. 14 is an exploded view of a portion of the components of the positioning assembly of the present invention.

Fig. 15 is a schematic view (from bottom to top) of the lower side magnet of the present invention.

Fig. 16 is a schematic view of a filling unit of the present invention.

FIG. 17 is a cross-sectional view of a spoiler of the invention.

FIG. 18 is a flow chart of the cultivation method of the present invention.

The reference numerals indicate that 1, a constant temperature box; 11, a temperature controller; 12, side wall glass; 13, observation port, 14, box top box, 15, high-permeability glass, 2, culture device, 21, T-shaped bottom rod, 22, bottom support plate, 101, T-shaped limit groove, 23, double-layer box door, 24, layered support plate, 25, cell culture plate, 3, transfer mechanism, 31, end side clamping block, 32, L-shaped slide rod, 241, limit slide groove, 251, rectangular clamping groove, 4, middle moving component, 5, pushing unit, 26, culture dish, 252, round groove, 41, inner rotating wheel, 42, driving belt, 242, middle groove, 43, U-shaped clamping block, 44, driving rotary rod, 243, round rod jack, 51, Z-shaped push rod, 221, rectangular bayonet, 231, middle sliding port, 52, box, 53, limit round rod, 511, limit round hole, 54, threaded round rod, 512, internal threaded hole, 55, end round button, connecting hole, 6, positioning component, 61, side metal rod, 62, iron-sucking stone, 253, side groove, 63, lower side iron-sucking stone, 254, embedded groove, 64, inner rotating rod, 243, round rod, 511, air flow-blocking pipe, 75, air inlet pipe, air inlet, air outlet, air inlet air.

Detailed Description

The present application is described in further detail below with reference to fig. 1-18.

The embodiment of the application discloses an in-vitro separation culture device and method for immune cells, which can culture the separated immune cells in a controllable environment, can quickly and accurately check the cell culture conditions of different areas under the condition of not affecting the culture environment, has simple operation process and is beneficial to reducing the observation time of staff.

Embodiment one:

Referring to fig. 1 and 2, in a first aspect, the present application provides an immune cell in vitro separation culture apparatus, which comprises an incubator 1 and a temperature controller 11 disposed on an outer side wall of the incubator 1, wherein the incubator 1 is in the prior art, when opened, the temperature inside the incubator 1 can be adjusted by the temperature controller 11, and when the incubator 1 is adjusted to a situation temperature required for immune cell culture, the real-time temperature inside the incubator 1 can be displayed by the temperature controller 11. The incubator 1 is provided with a side wall glass 12, an observation port 13 is formed in the upper side of the incubator 1, a box top box 14 is covered on the observation port 13, the box top box 14 is fixedly arranged on the upper side of the incubator 1, a high-permeability glass 15 is arranged on the upper side of the box top box 14, a culture device 2 is arranged in the incubator 1, and the incubator is used for in-vitro separation culture of immune cells, and can transfer cells at a designated position to the observation port 13 when the culture condition of the immune cells is required to be observed, so that staff can observe the immune cells from the outside through the high-permeability glass 15, and the original culture environment in the incubator 1 is not influenced.

Referring to fig. 3 and 4, specifically, the culture device 2 includes a bottom plate 22 that is slidably disposed in the incubator 1 through a T-shaped bottom rod 21, a T-shaped limiting groove 101 that is slidably mounted on the T-shaped bottom rod 21 is formed in the inner bottom surface of the incubator 1, a double-layer door 23 is disposed at one end of the bottom plate 22 facing the outside of the incubator 1, eight layered support plates 24 are preferably disposed at equal intervals from top to bottom and symmetrically on one side of the double-layer door 23 facing the incubator 1, and the layered support plates 24 are fixedly connected with the double-layer door 23. The layered support plate 24 is provided with a cell culture plate 25, and the layered support plate 24 and the double-layer box door 23 are provided with a transfer mechanism 3 for driving the cell culture plate 25 to move from outside to drive the cell culture plate 25 at different positions to the observation port 13 for the staff to observe the cell culture condition.

In this embodiment, five culture dishes 26 are preferably embedded on the upper side of the cell culture plate 25 at equal intervals, and five circular grooves 252 for installing the culture dishes 26 are formed on the cell culture plate 25 at equal intervals. Five dishes 26 are provided on each cell culture plate 25. In use, isolated immune cells are seeded at the appropriate cell density in each of the culture dishes 26 at different locations and the desired nutrients are added simultaneously to the dishes 26. The double box door 23 is then pushed to close the double box door 23 at the inlet of the incubator 1, the operation of the incubator 1 is started, and the temperature inside the incubator 1 is adjusted to a proper temperature by the temperature controller 11, so that a plurality of groups of immune cells are cultured in the incubator 1.

Referring to fig. 5 and 6, in order to support and place the cell culture plate 25, and when the condition of cell culture needs to be observed, the cell culture plate 25 at the corresponding position is accurately transferred to the observation port 13, so that a worker can observe and record the condition of cell culture through the high-permeability glass 15, meanwhile, the worker can conveniently take the amplifying equipment used for observation to the top of the box top box 14, the transfer mechanism 3 comprises an L-shaped slide bar 32 which is arranged on the upper side of the layered support plate 24 in a limited sliding manner through two end clamping blocks 31, limiting sliding grooves 241 for installing the two end clamping blocks 31 are symmetrically arranged on the layered support plate 24, the L-shaped slide bar 32 is used for lifting and placing the cell culture plate 25, a rectangular clamping groove 251 matched with the L-shaped slide bar 32 is formed in the bottom of the cell culture plate 25, when the cell culture plate 25 is placed on the layered support plate 24, the cell culture plate 25 is firmly clamped on the L-shaped slide bar 32 through the rectangular clamping groove 251 formed in the bottom of the cell culture plate 25, and when the L-shaped slide bar 32 is stressed, the cell culture plate 25 can be driven to slide horizontally and slidingly move on the layered support plate 24. The layered supporting plate 24 and the double-layer box door 23 are provided with a moving component 4 and a pushing unit 5 which are matched with each other.

In the culturing process, when the condition of the cell culture in a certain layer of culture dish 26 needs to be observed, firstly, a worker can drive the cell culture plate 25 on the designated layered support plate 24 from the outside through the moving component 4, so that the cell culture plate translates to the middle part of the incubator 1 to be positioned right below the observation port 13. Then through pushing unit 5 alright upwards push up this cell culture board 25 to viewing aperture 13 department for the staff to look over from the outside, whole operation process is simple and swift, need not open oven 1, can ensure that the inside original culture environment of oven 1 does not receive external factor influence.

Referring to fig. 7 and 8, since it is required to transfer the cell culture plate 25 to the middle of the incubator 1 by an external operation, the cell culture plate 25 is first moved to the position right below the viewing port 13, so that the cell culture plate 25 can be moved up by the pushing unit 5, the moving assembly 4 includes a driving belt 42 rotatably installed at the middle of the layered support plate 24 by two inner rotating wheels 41 and positioned under the L-shaped sliding rod 32, and a middle groove 242 for installing the inner rotating wheels 41 and the driving belt 42 is provided at the middle of the layered support plate 24. It should be noted that, when the driving belt 42 is installed at the middle groove 242 through the two inner rotating wheels 41, the driving belt 42 is in a tight state, and when one of the inner rotating wheels 41 is forced to rotate, the driving belt 42 can be driven to rotate. Since the layered pallets 24 are provided with eight, in turn, each layered pallet 24 is provided with a shift assembly 4 therein.

Referring to fig. 7 to 9, a U-shaped clamping block 43 connected with a driving belt 42 is installed at the lower side of the L-shaped sliding bar 32, it should be noted that the upper side of the U-shaped clamping block 43 is fixedly connected with the lower side of the L-shaped sliding bar 32, and the U-shaped clamping block 43 is fixedly clamped and connected at the position where the driving belt 42 is in a parallel state, so that when the driving belt 42 is driven to rotate, the L-shaped sliding bar 32 can be driven to translate on the layered support plate 24 by the U-shaped clamping block 43, a driving rotating rod 44 penetrating through the double-layer door 23 and the layered support plate 24 and connected with one inner rotating wheel 41 is rotatably installed at the outer side of the double-layer door 23, and round rod insertion holes 243 for penetrating through the driving rotating rod 44 are formed in the layered support plate 24 and the double-layer door 23.

When observing the cell culture condition on a certain cell culture plate 25, a worker can drive the L-shaped slide bar 32 to translate towards the middle part of the incubator 1 through the rotation of the transmission belt 42 by rotating the driving rotating rod 44 at the corresponding position on the outer side of the double-layer oven door 23 until the U-shaped clamping block 43 is abutted against the side wall of the middle section groove 242 and cannot rotate continuously, and the cell culture plate 25 clamped on the L-shaped slide bar 32 is located under the observation port 13.

Referring to fig. 10 to 12, considering that the cell culture plate 25 moving to the middle of the incubator 1 needs to be pushed to the observation port 13, the cell culture condition in the culture dish 26 is convenient for a worker to observe, the pushing unit 5 comprises a Z-shaped push rod 51 arranged at the middle of the bottom supporting plate 22, one end of the Z-shaped push rod penetrates through the double-layer refrigerator door 23 and extends outwards, a rectangular bayonet 221 for installing the Z-shaped push rod 51 is arranged on the bottom supporting plate 22, a middle sliding port 231 matched with the Z-shaped push rod 51 is arranged on the double-layer refrigerator door 23 in a penetrating manner, and a middle box 52 is arranged at the middle sliding port 231 outside the double-layer refrigerator door 23. In the initial state, the Z-shaped push rod 51 is clamped at the rectangular bayonet 221 of the bottom support plate 22, one end of the Z-shaped push rod 51 is positioned at the inner bottom of the middle box 52, and the Z-shaped push rod 51 can slide up and down at the middle sliding port 231 in a limiting manner when driven.

Referring to fig. 11 and 12, two limit round bars 53 are vertically installed in the middle-set box 52 through the Z-shaped push bar 51, limit round holes 511 through which the two limit round bars 53 penetrate are formed in the Z-shaped push bar 51 in a penetrating manner, the limit round bars 53 are fixedly installed in the middle-set box 52 and play a limiting role on the Z-shaped push bar 51, so that the Z-shaped push bar 51 cannot rotate to generate angular offset, a threaded round bar 54 penetrating the Z-shaped push bar 51 in a threaded manner is rotationally arranged in the middle-set box 52, an internal threaded hole 512 matched with the threaded round bar 54 is formed in the threaded manner on the Z-shaped push bar 51 in a threaded manner, an end round button 55 connected with the threaded round bar 54 is inserted into the upper end of the middle-set box 52, and a connecting hole 521 through which the end round button 55 is inserted and installed is formed in the middle-set box 52.

Likewise, the staff can operate from the outside, rotate tip knob 55 and drive screw thread round bar 54 rotation, through with the internal thread hole 512 cooperation of seting up on the Z shape push rod 51 down, can order about Z shape push rod 51 to move up, the in-process that Z shape push rod 51 moved up can be inconsistent with translation to the cell culture board 25 bottom that incubator 1 middle part is in under the viewing aperture 13 to can move up and promote cell culture board 25 to break away from L shape slide bar 32 and upwards move to viewing aperture 13 department, so that the staff passes through high transparent glass 15 and observes the cell culture situation in this cell culture board 25 culture dish 26.

Referring to fig. 13 and 14, in order to avoid displacement of the cell culture plate 25 during movement of the cell culture plate 25 by pushing the Z-shaped push rod 51, a positional deviation is generated, so that when the cell culture plate 25 needs to be driven to reset later, the cell culture plate 25 cannot be aligned and clamped on the L-shaped slide rod 32 to return to an initial position, the cell culture plate 25 and the Z-shaped push rod 51 are further provided with a positioning assembly 6, the positioning assembly 6 comprises a lateral metal rod 61 arranged on the L-shaped slide rod 32, and when the cell culture plate 25 is placed, a rectangular clamping groove 251 of the cell culture plate 25 needs to be clamped between the lateral wall of the L-shaped slide rod 32 and the lateral metal rod 61, and at this time, one side of the cell culture plate 25 is attached to the lateral metal rod 61. The cell culture plate 25 is embedded and installed with a strip-shaped magnet 62 towards one side of the lateral metal rod 61, and a lateral groove 253 for installing the strip-shaped magnet 62 is formed 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 bar 61, suction is provided between the strip-shaped magnet 62 and the side metal bar 61, so that the cell culture plate 25 can be more stably positioned on the L-shaped slide bar 32.

Referring to fig. 13 to 15, two lower side magnet 63 are embedded in the bottom of the cell culture plate 25, an embedded groove 254 for installing the lower side magnet 63 is formed in the bottom of the cell culture plate 25, two upper side magnet 64 attracted with the lower side magnet 63 are embedded in the upper side of the z-shaped push rod 51, and an embedded circular groove 513 for installing the upper side magnet 64 is formed in the z-shaped push rod 51. When the Z-shaped push rod 51 moves upwards to collide with the bottom of the cell culture plate 25, the two upper side magnet stones 64 are respectively attracted with the two lower side magnet stones 63, 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 cannot deviate in the process of moving upwards or downwards. After the observation of the staff is completed, when the Z-shaped push rod 51 is driven to drive the cell culture plate 25 to move downwards, the rectangular clamping groove 251 formed in the bottom of the cell culture plate 25 can be accurately clamped between the side wall of the L-shaped slide rod 32 and the side metal rod 61, and at the moment, the strip-shaped magnet 62 and the side metal rod 61 have suction force, so that the Z-shaped push rod 51 and the cell culture plate 25 can be ensured to be smoothly separated, and the reset effect is realized.

Embodiment two:

Referring to fig. 16, in order to facilitate the subsequent filling of gases such as oxygen and carbon dioxide into the incubator 1, and to satisfy the culture of microorganism tissues or cells having special demands for the gases and the replenishment of subsequent nutrient solutions, the cells consume nutrient substances in the culture medium during the culture, and to facilitate the accurate filling of nutrient solutions into the culture dish 26 having nutrient deficiency by staff when observing the cell culture conditions, a filling unit 7 is further provided outside the incubator 1, the filling unit 7 comprises an L-shaped configuration plate 71 provided on the outer side wall of the incubator 1, an air storage tank 72 and an air storage tank 73 are respectively provided on the L-shaped configuration plate 71, an air pipe 74 is installed in communication between the air storage tank 72 and the top box 14, and an air control valve 75 is provided on the air pipe 74. The gas tank 72 may store oxygen, carbon dioxide, and other gases as necessary. And the liquid storage tank 73 can be filled with nutrient solution required by cells. The amount of gas injected into the oven 1 can be controlled by the gas control valve 75.

In this embodiment, five spray heads 76 are preferably mounted on the side wall of the box top box 14 obliquely to the inner cavity of the incubator 1, and five inclined holes 141 for mounting the spray heads 76 are formed on the side wall of the box top box 14, and it should be noted that the five spray heads 76 are aligned with the five culture dishes 26 on the cell culture plate 25 which is transferred to the observation port 13. The spray head 76 is provided with bending pipes 77 in a communicating manner, the liquid storage tank 73 is provided with a liquid supply pump 78, and the five bending pipes 77 are connected with the liquid supply pump 78 through a shunt pipe 79. Nutrient solution in the liquid storage tank 73 can be conveyed into the split-flow pipes 79 through the liquid supply pump 78, and is dispersed into each bending pipe 77 through the split-flow pipes 79, and finally sprayed into the culture dish 26 through the spray heads 76.

Referring to fig. 16 and 17, considering the diversity of immune cells in the culturing process, five culture dishes 26 on the same cell culture plate 25 are not all consumed and need to be supplemented at the same time, and the culture dishes 26 with more nutrients do not need to be supplemented with nutrient solution, so as to avoid affecting the normal culturing of immune cells, and also prevent the waste of the nutrient solution, a flow blocking piece 8 is arranged on the bending tube 77, the flow blocking piece 8 comprises an inner through tube 81 fixedly arranged in the bending tube 77, an inner round bead 82 is arranged in the inner through tube 81 in a limiting rotation manner, a limiting rotating groove 811 matched with the inner round bead 82 is formed in the inner through tube 81, and it is required to say that the inner through tube 81 is divided into two parts so that the inner round bead 82 is arranged at the limiting rotating groove 811. The inner round bead 82 is provided with a communication hole 821 in a penetrating way, the bending tube 77 is provided with a toggle rod 83 fixedly connected with the inner round bead 82 in a rotating way, and the bending tube 77 and the inner through tube 81 are provided with round rod holes 771 for the toggle rod 83 to penetrate and be installed.

When a worker observes the cell culture condition on the cell culture plate 25 transferred to the observation port 13, the worker can rotate the toggle rod 83 on the corresponding bending tube 77 by ninety degrees when observing that less nutrient substances in a certain culture dish 26 need to be supplemented, and the toggle rod 83 drives the inner round beads 82 to rotate, so that the communication holes 821 on the inner round beads 82 and the inner through tube 81 are in a dredging state, and nutrient solution is enabled to enter the spray head 76 and be sprayed into the culture dish 26 at the corresponding position.

Referring to FIG. 18, in another aspect, the application also discloses an immune cell in vitro isolated culture method, which comprises the following steps:

In a first step, the immune cells are seeded in layers by sequentially seeding the immune cells in cell culture plates 25 placed on different layered trays 24 and adding nutrients, and in particular, the isolated immune cells are seeded in respective culture dishes 26 at different locations at an appropriate cell density and simultaneously adding desired nutrients to the culture dishes 26.

And secondly, culturing at constant temperature, namely pushing the double-layer box door 23 to enable the layered supporting plate 24 to enter the incubator 1, closing the inlet of the incubator 1 by the double-layer box door 23, opening the incubator 1 to operate, and regulating the internal temperature of the incubator 1 through the temperature controller 11 so as to enable a plurality of groups of immune cells to be cultured in the incubator 1.

And thirdly, appointing to check the cell culture condition on a certain layer of cell culture plate 25, wherein the transfer mechanism 3 is operated to transfer the cell culture plate 25 at the appointed position to the observation port 13 for checking through the high-permeability glass 15, the driving rotary rod 44 at the corresponding position is rotated to drive the L-shaped sliding rod 32 to drive the cell culture plate 25 to translate towards the middle part of the incubator 1 during specific operation, then the end round button 55 is rotated to drive the threaded round rod 54 to drive the Z-shaped push rod 51 to move upwards to be in contact with the bottom of the cell culture plate 25, and the two upper side magnet stones 64 and the two lower side magnet stones 63 are attracted to ensure that the cell culture plate 25 is firmly adsorbed on the Z-shaped push rod 51 to push the cell culture plate 25 to the observation port 13 so as to observe the cell culture condition conveniently.

Fourth, accurate reset, namely, after the observation of the cell culture condition transferred to the cell culture plate 25 at the observation port 13 is completed, the transfer mechanism 3 is operated again to drive the cell culture plate 25 to reset. When observing, it is observed that the nutrient substances in a certain culture dish 26 are less to be supplemented, the toggle rod 83 corresponding to the bending tube 77 is rotated for ninety degrees, so that the communication hole 821 on the inner round bead 82 and the inner through tube 81 are in a dredging state, and nutrient solution is sprayed into the corresponding culture dish 26 through the spray head 76. Finally, the end knob 55 and the drive rod 44 are turned back to reset the cell culture plate 25.

The embodiments of the present invention are all preferred embodiments of the present invention, and are not limited in scope by the present invention, so that all equivalent changes according to the structure, shape and principle of the present invention are covered by the scope of the present invention.

Claims

1. The immune cell in-vitro separation culture equipment comprises an incubator (1) and a temperature controller (11) arranged on the outer side wall of the incubator (1), wherein side wall glass (12) is arranged on the incubator (1), an observation port (13) is formed in the upper side of the incubator (1), a box top box (14) is covered on the observation port (13), and high-permeability glass (15) is arranged on the upper side of the box top box (14), and the immune cell in-vitro separation culture equipment is characterized in that a culture device (2) is arranged in the incubator (1);

Culture apparatus (2) include through the spacing bottom plate (22) of locating in thermostated container (1) of T shape sill bar (21) slip, offer T shape spacing groove (101) of confession T shape sill bar (21) slidable mounting on the bottom surface in thermostated container (1), one end outside floor plate (22) orientation thermostated container (1) is provided with double-deck chamber door (23), a plurality of layering layer boards (24) are installed from last equidistance to lower symmetry towards one side of thermostated container (1) to double-deck chamber door (23), be equipped with cell culture board (25) on layering layer board (24), be equipped with on layering layer board (24) and double-deck chamber door (23) and be used for from transfer mechanism (3) of outside drive cell culture board (25) displacement.

2. The device for in vitro isolation and culture of immune cells according to claim 1, wherein the transfer mechanism (3) comprises L-shaped sliding rods (32) which are arranged on the upper side of the layered supporting plate (24) in a limiting sliding manner through two end-side clamping blocks (31), limiting sliding grooves (241) for installing the two end-side clamping blocks (31) are symmetrically formed in the layered supporting plate (24), the L-shaped sliding rods (32) are used for lifting the cell culture plates (25), rectangular clamping grooves (251) matched with the L-shaped sliding rods (32) are formed in the bottoms of the cell culture plates (25), and the layered supporting plate (24) and the double-layer box door (23) are provided with middle moving components (4) and pushing units (5) which are matched with each other.

3. The device for in vitro isolation and culture of immunocytes according to claim 1, wherein 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 formed in the cell culture plate (25).

4. The device for in vitro isolation and culture of immunocytes according to claim 2, wherein the middle moving component (4) comprises a transmission belt (42) rotatably installed in the middle of the layered support plate (24) through two inner rotating wheels (41) and positioned below the L-shaped sliding rod (32), and a middle groove (242) for installing the inner rotating wheels (41) and the transmission belt (42) is formed in the middle of the layered support plate (24).

5. The device for in vitro isolation and culture of immunocytes according to claim 4, wherein a U-shaped clamping block (43) connected with a transmission belt (42) is arranged on the lower side of the L-shaped sliding rod (32), a driving rotating rod (44) penetrating through the double-layer box door (23) and the layered support plate (24) and connected with one inner rotating wheel (41) is rotatably arranged on the outer side of the double-layer box door (23), and round rod insertion holes (243) for penetrating through the driving rotating rod (44) are formed in the layered support plate (24) and the double-layer box door (23).

6. The device for in vitro isolation and culture of immune cells according to claim 2, wherein the pushing unit (5) comprises a Z-shaped push rod (51) arranged in the middle of the bottom supporting plate (22) and one end of the Z-shaped push rod penetrates through the double-layer box door (23) to extend outwards, a rectangular bayonet (221) for installing the Z-shaped push rod (51) is formed in the bottom supporting plate (22), a middle sliding opening (231) matched with the Z-shaped push rod (51) is formed in the double-layer box door (23) in a penetrating mode, and a middle box (52) is arranged on the outer side of the double-layer box door (23) and located at the middle sliding opening (231).

7. The device for the in vitro isolation and culture of immune cells according to claim 6, wherein two limit round rods (53) are vertically installed in the middle-placed box (52) through a Z-shaped push rod (51), limit round holes (511) for the two limit round rods (53) to penetrate are formed in the Z-shaped push rod (51) in a penetrating mode, a threaded round rod (54) penetrating the Z-shaped push rod (51) in a threaded mode is rotationally arranged in the middle-placed box (52), an inner threaded hole (512) matched with the threaded round rod (54) is formed in the Z-shaped push rod (51) in a threaded mode in a penetrating mode, an end round button (55) connected with the threaded round rod (54) is inserted into the upper end of the middle-placed box (52), and a connecting hole (521) for the end round button (55) to be installed in a inserting mode is formed in the middle-placed box (52).

8. The device for in vitro isolation and culture of immunocytes according to claim 6, wherein the cell culture plate (25) and the Z-shaped push rod (51) are further provided with a positioning component (6), the positioning component (6) comprises a lateral metal rod (61) arranged on the L-shaped slide rod (32), a strip-shaped magnet (62) is embedded and arranged on one side, facing the lateral metal rod (61), of the cell culture plate (25), and a lateral groove (253) for installing the strip-shaped magnet (62) is formed on one side of the cell culture plate (25).

9. The device for in vitro isolation and culture of immunocytes according to claim 8, wherein two lower side magnet stones (63) are embedded and installed at the bottom of the cell culture plate (25), an embedded groove (254) for installing the lower side magnet stones (63) is formed at the bottom of the cell culture plate (25), two upper side magnet stones (64) which are attracted with the lower side magnet stones (63) are embedded on the upper side of the Z-shaped push rod (51), and an embedded circular groove (513) for installing the upper side magnet stones (64) is formed on the Z-shaped push rod (51).

10. An immune cell in vitro separation culture method is characterized by using the immune cell in vitro separation culture equipment according to any one of claims 1-9, and comprises the following operation steps:

Step one, layered seeding, namely sequentially seeding immune cells in cell culture plates (25) placed on different layered supporting plates (24), and filling nutrient substances;

secondly, constant temperature cultivation, namely pushing a double-layer box door (23) to enable a layered supporting plate (24) to enter the incubator (1), closing an inlet of the incubator (1) by the double-layer box door (23), opening the incubator (1) to operate, and adjusting the internal temperature of the incubator (1) through a temperature controller (11);

Thirdly, appointing to check, namely when the cell culture condition on a certain layer of cell culture plate (25) needs to be observed, the cell culture plate (25) at an appointed position is transferred to an observation port (13) by operating a transfer mechanism (3) and checked through a high-permeability glass (15);

and fourthly, accurately resetting, namely after the observation of the cell culture condition transferred to the cell culture plate (25) at the observation port (13) is completed, operating the transfer mechanism (3) again to drive the cell culture plate (25) to reset.