CN112779161B

CN112779161B - Immune cell culture method

Info

Publication number: CN112779161B
Application number: CN202110170617.8A
Authority: CN
Inventors: 梅建勋; 欧阳建华; 汪凯; 何伟贤; 付引梅; 谭慕华; 梁键全
Original assignee: Guangdong Mercells Cell Biotechnology Co ltd
Current assignee: Guangdong Mercells Cell Biotechnology Co ltd
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-07-27
Anticipated expiration: 2041-02-08
Also published as: CN112779161A

Abstract

The invention discloses an immune cell culture method, which comprises the following steps: s1: preparation, S2: sample dilution, S3: gradient centrifugation, S4: collecting the white film layer, S5: cleaning, S6: cleaning, S7: inoculation, S8: feeder cells, S9: initial culture, S10: observation record, S11: and (5) freezing and storing. This immune cell culture method has avoided artifical manual operation in the past to the cell culture solution blow beat waste time and energy and blow beat the poor condition of effect, has improved and has beaten efficiency, can realize the centrifugal treatment to the cell culture solution through having set up first centrifugal mechanism and second centrifugal mechanism, the device is once only being blown and beaten the mixing mechanism outside and set up four first centrifugal mechanisms and four second centrifugal mechanisms, the multistation operation can once only carry out the cultivation of a large amount of immune cells, and is efficient, the facilitate promotion.

Description

Immune cell culture method

Technical Field

The invention relates to the technical field of immune cell culture, in particular to an immune cell culture method.

Background

Natural killer cells are important immune cells in the body, are not only relevant to tumor resistance, virus infection resistance and immune regulation, but also participate in hypersensitivity reaction and autoimmune disease occurrence under certain conditions, can recognize target cells and killing media, and have important medical functions, so that the immune cells need to be cultured.

In the existing immune cell culture, culture liquid needs to be manually blown and beaten and mixed, multiple times of centrifugal treatment is needed in the operation process, the manual operation process in the treatment is complex, the culture cost is high, and in the existing immune cell culture, cells cultured at one time are few, and the culture efficiency is low, so that the immune cell culture method is provided.

Disclosure of Invention

The present invention is directed to a method for culturing immune cells to solve the above problems of the background art.

In order to achieve the purpose, the invention provides the following technical scheme: an immune cell culture method, comprising the following steps:

s1: preparing, namely re-warming the culture solution and the lymphocyte separation solution to room temperature;

s2: diluting a sample, namely adding equal volume of normal saline into the lower layer red blood cells after plasma separation for dilution, fully and uniformly blowing and stirring the mixture by a blowing and stirring mixing mechanism, and transferring the mixture into a 50ml centrifuge tube;

s3: gradient centrifugation, namely slowly adding prepared diluted blood into each 50ml centrifuge tube filled with lymphocyte separation liquid, and centrifuging for 20min by a first centrifugal mechanism under the conditions of 20 ℃ and 800 g;

s4: collecting the tunica albuginea layer, absorbing the middle tunica albuginea layer in a 50ml centrifuge tube by a 25ml pipette of a liquid transfer mechanism after centrifugation, adding physiological saline into each tube to 50ml, and centrifuging for 6min by a second centrifuge mechanism at the rotation speed of 1500 rpm;

s5: cleaning, dumping supernatant, moderately knocking the bottom of the centrifuge tube by fingers until cell sediment is loose, adding normal saline for heavy suspension, combining and uniformly mixing cell suspensions in the centrifuge tubes, and ensuring that the total volume is 30 ml;

s6: cleaning, namely centrifuging the cell suspension with the required volume again at the rotating speed of 1500rpm for 6min, dumping the rest supernatant, and properly knocking the bottom of the centrifuge tube by fingers until cell sediment is loose;

s7: inoculating, re-suspending with 15ml complete culture medium, and inoculating into a culture flask;

s8: adding trophoblast, recovering trophoblast, adding physiological saline to 40ml for re-suspension, sampling, measuring cell viability, centrifuging at 1500rpm for 5min, and adding 15ml of complete culture medium into the culture flask in the seventh step;

s9: starting culture, adding 1.5ml of 5% autologous plasma, 30ml of NK complete medium, 1.5ml of autologous plasma and feeder cells, setting the temperature to 37 ℃ and starting culture in a CO2 incubator;

s10: and (4) observing and recording, observing by naked eyes and a microscope, and detecting and recording related data.

S11: freezing and storing;

the bottom of the blowing and beating mixing mechanism in the step S2 is provided with a bottom plate, the blowing and beating mixing mechanism is fixedly installed at the top center of the bottom plate and comprises a processing box, a box cover, a feeding hole, a hollow pipe, a buffer plate and a blowing and beating pipe, the top center of the bottom plate is fixedly provided with a fixing frame, the top of the bottom plate is fixedly installed inside the fixing frame and is provided with the processing box, the top of the processing box is provided with the box cover, one end of the top of the box cover is provided with the feeding hole, the top center of the box cover is fixedly provided with the hollow pipe, the end part of the hollow pipe extends into the processing box, one end of the hollow pipe inside the processing box is fixedly connected with the buffer plate, the buffer plate is integrally in a circular structure, a cavity is arranged in the buffer plate, the hollow pipe is communicated with the interior of the buffer plate, and the bottom of the buffer plate is fixedly connected with the blowing and beating pipe at equal intervals, the blowing pipes are communicated with the interior of the buffer plate, the end parts of the blowing pipes are of a conical structure, and one-way air valves are mounted on the outer sides of the blowing pipes;

and the first centrifugal mechanism in the step S3 and the second centrifugal mechanism in the step four are both fixedly installed at the top of the bottom plate, the first centrifugal mechanism is positioned between the blowing and beating mixing mechanism and the second centrifugal mechanism, the liquid transfer mechanism in the step four is fixedly installed at the top of the bottom plate, and the liquid transfer mechanism is positioned between the first centrifugal mechanism and the second centrifugal mechanism.

Preferably, one end of the hollow tube, which is positioned outside the case cover, is fixedly connected with a fixing box, the fixing box is integrally in a circular plate-shaped structure, the hollow tube is communicated with the inside of the fixing box, the side wall of the fixing box is fixedly connected with a mounting box, three arc-shaped grooves are equidistantly formed in the outer side of the mounting box, the inside of the mounting box is communicated with the outside through the arc-shaped grooves, a supporting spring is fixedly connected with the inside of one side, which is away from the fixing box, of the fixing box, a plugging plate is fixedly connected with the end part of the supporting spring, the plugging plate is matched and slidably connected with the inside of the mounting box, a communicating port is formed in one side, which is positioned on the mounting box, of the fixing box, the fixing box is communicated with the inside of the mounting box through a communicating port, a sliding rod is fixedly connected with one side, which is positioned on the communicating port, the end part of the sliding rod penetrates through the fixing box, the top of the case cover is slidably connected with a vertical plate, and the end part of the sliding rod is fixedly connected with the vertical plate, and a telescopic spring is sleeved between the vertical plate and the fixed box on the outer side of the sliding rod.

Preferably, the top of case lid is seted up the spout of horizontal direction, the bottom of riser is provided with the stopper, and stopper cooperation sliding connection inside the spout, the riser is through the stopper sliding connection who sets up on it at the case lid top, the case lid top is located the side symmetry fixedly connected with fixed plate of riser, and the top between two fixed plates rotates through the pivot and is connected with the eccentric wheel, the eccentric wheel contacts with the riser, fixed plate top fixed mounting has the rotation motor, and the output shaft tip of rotating the motor passes fixed plate and eccentric wheel fixed connection.

Preferably, first centrifugal mechanism and second centrifugal mechanism all include fixing base, revolving stage, fixed slot, rack and centrifuging tube, the bottom plate top is located the outside equidistance fixed mounting of mount has four fixing bases, and the fixing base internal rotation is connected with the revolving stage, four fixed slots have been seted up to the top equidistance of revolving stage, and four fixed slot internal rotations are connected with the rack, the joint has the centrifuging tube in the rack, the top fixed mounting of mount has the water pump, and the feed liquor end of water pump installs the connecting pipe, and the tip of connecting pipe is connected with the bottom of handling the case, the play water end fixed mounting of water pump arranges the material pipe, and arranges the material pipe and be located the centrifuging tube directly over.

Preferably, the bottom of revolving stage is rotated and is connected with outer ring gear, the bottom of rack is all rotated and is connected with drive gear, and four drive gear all with outer ring gear meshing connection, revolving stage bottom fixed mounting has servo motor, and servo motor's output shaft tip fixed mounting has drive gear, drive gear and outer ring gear meshing connection.

Preferably, one side of the blocking plate, which is located on the fixed box, is provided with a sealing gasket, the sealing gasket is in close contact with the fixed box, the surface of the blocking plate, which is located on the outer side of the sliding rod, is symmetrically fixed with limiting rods, and the end parts of the limiting rods slide to penetrate through the fixed box and be fixedly connected with the vertical plate.

Preferably, the bottom of the inner wall of the fixing seat is rotatably connected with a transmission rod through a bearing, a driven gear is fixedly mounted on the outer side of the transmission rod, the top of the transmission rod is fixed to the rotary table, a driving motor is fixedly mounted at the bottom of the inner wall of the fixing seat, a driving gear is fixedly mounted at the end of an output shaft of the driving motor, and the driving gear is meshed with the driven gear.

Preferably, the liquid transferring mechanism comprises an installation frame, a fixing rod, a supporting rod, a lead screw, a sliding block, a liquid transferring motor, an electric push rod and a liquid transferring pipe, the installation frame is fixedly installed between the first centrifugal mechanism and the second centrifugal mechanism at the top of the bottom plate, the fixing rod is fixedly connected to the top end of the installation frame, the supporting rod is fixedly connected to the end portion of the fixing rod, length scale marks are arranged on the surface of the fixing rod, a groove in the horizontal direction is formed in the top portion of the fixing rod, the lead screw is rotatably connected to the groove, the sliding block is in threaded connection with the outer side of the lead screw and is in sliding connection with the inside of the fixing rod in a matched mode, the liquid transferring motor is fixedly installed at the end portion of the fixing rod, the end portion of the sliding block is, the pipette is located directly above the centrifuge tube.

Preferably, the top of rack inner wall is provided with the sealing washer, the top and the fixed slot inner wall sealing connection of rack, and the rack is inside to communicate with the fixed slot is inside.

Preferably, the cylinder groove has been seted up at the top center of revolving stage, and cylinder inslot fixed mounting has the hot plate, cylinder inslot on the revolving stage is provided with the water tank, and the top center of water tank is provided with the handle, the air pump is installed at the top of water tank, the inlet end and the external intercommunication of air pump, and the end of giving vent to anger of air pump installs the breather pipe, and inside the tip of breather pipe stretched into the water tank, the breather pipe was located one section one-tenth heliciform setting inside the water tank.

Compared with the prior art, the invention has the beneficial effects that: the invention makes the eccentric wheel rotate through the work of the rotating motor on the blowing and beating mixing mechanism, the eccentric wheel continuously pushes the vertical plate to slide on the box cover in the rotating process, the vertical plate slides towards the fixed box to push the sliding rod to slide, the sliding rod pushes the plugging plate to slide in the mounting box, the supporting spring and the telescopic spring compress in the sliding process of the plugging plate towards the supporting spring, the communication port is in an open state at the moment, external gas enters the fixed box through the arc-shaped groove and the communication port, when the end part of the eccentric wheel is far away from the vertical plate, the vertical plate recovers under the action of the telescopic spring and the supporting spring, the plugging plate seals the communication port at the moment, the gas blows towards the inside of the processing box through the blowing and beating pipe, the blowing and beating pipe continuously blows towards the inside of the processing box in the work of the rotating motor, and the situations that the time and labor are wasted and the blowing and beating effect is poor in the cell culture solution through manual operation in the past are avoided, the blowing efficiency is improved, the centrifugal treatment of cell culture solution can be realized by arranging the first centrifugal mechanism and the second centrifugal mechanism, the raw materials are put into each centrifuge tube on the rotary table by controlling the intermittent rotation of the rotary table, then the water in the water tank is heated by controlling the working of the heating plate, then the air pump works, the air is heated to a certain degree by hot water in the water tank, hot air enters the fixed groove to heat the centrifuge tube, meanwhile, the control system controls the servo motor to work to rotate the driving gear, the four transmission gears are rotated by the meshing connection of the driving gear and the outer gear ring, and the placing frame is rotated, so that the rotation of the centrifuge tube is realized, the centrifugal treatment of the liquid in the centrifuge tube can be realized, the process operation is continuous, the time is saved, the centrifugal efficiency is improved, the device is provided with the four first centrifugal mechanisms and the four second centrifugal mechanisms outside the blowing and mixing mechanism in one step, the multi-station operation can be used for culturing a large number of immune cells at one time, the efficiency is high, and the popularization is convenient.

Drawings

FIG. 1 is a schematic view of the process flow of the cultivation method of the present invention;

FIG. 2 is one of schematic views showing an overall connection structure of the apparatus used in the cultivation method of the present invention;

FIG. 3 is a second schematic view showing the overall connection structure of the apparatus used in the cultivation method according to the present invention;

FIG. 4 is a schematic view of a connecting structure of the blowing and beating mixing mechanism of the present invention;

FIG. 5 is a schematic view of a connection structure of a turntable and a fixing base according to the present invention;

FIG. 6 is a schematic view of the inner connection structure of the turntable and the fixing base according to the present invention;

FIG. 7 is a schematic view of the internal connection structure of the fixing base of the present invention;

FIG. 8 is a schematic view of a connecting structure of the blowing and beating mixing mechanism of the present invention;

FIG. 9 is a schematic view of the internal connection structure of the whipping mixing mechanism of the present invention;

fig. 10 is a schematic view of a blowpipe structure of the present invention.

In the figure: 1. a base plate; 2. a blowing and beating mixing mechanism; 3. a first centrifugal mechanism; 4. a second centrifugal mechanism; 5. a pipetting mechanism; 6. a treatment tank; 7. a box cover; 8. a feed inlet; 9. a hollow tube; 10. a buffer plate; 11. blowing and beating the pipe; 12. a chute; 13. a fixing box; 14. a vertical plate; 15. a slide bar; 16. a communication port; 17. a plugging plate; 18. mounting a box; 19. an arc-shaped slot; 20. a support spring; 21. a gasket; 22. a tension spring; 23. a limiting rod; 24. an eccentric wheel; 25. rotating the motor; 26. a fixing plate; 27. a fixed mount; 28. a fixed seat; 29. a turntable; 30. fixing grooves; 31. placing a rack; 32. centrifuging the tube; 33. a seal ring; 34. a driven gear; 35. a driving gear; 36. a transmission rod; 37. a drive motor; 38. a transmission gear; 39. an outer ring gear; 40. a servo motor; 41. a drive gear; 42. a water pump; 43. a connecting pipe; 44. a discharge pipe; 45. a mounting frame; 46. fixing the rod; 47. a support bar; 48. a lead screw; 49. a slider; 50. a pipetting motor; 51. an electric push rod; 52. a pipette; 53. length scale lines; 54. a water tank; 55. an air pump; 56. a breather pipe; 57. heating plates; 58. a handle.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in FIG. 1, the present invention provides an immune cell culture method, comprising the following steps:

step S1: preparation work: the culture medium (37 ℃ incubator) and the lymphocyte separation medium were rewarming (room temperature).

Step S1: sample dilution: diluting the lower layer red blood cells after plasma separation with equal volume of normal saline, fully and uniformly blowing and stirring by a blowing and stirring mechanism, and transferring to a 50ml centrifuge tube.

Step S3: gradient centrifugation: prepared diluted blood was slowly added to 50ml centrifuge tubes each containing a lymphocyte separation medium at 20 ℃ and 800g (about 2000rpm) and centrifuged by a first centrifuge mechanism for 20min (accelerate 1, brake 0).

Step S4: collecting a white film layer: after centrifugation, the middle albuginea layer is sucked by a 25ml pipette of a pipetting mechanism into a 50ml centrifuge tube, physiological saline is added into each tube to 50ml, and the mixture is centrifuged for 6min at 1500rpm by a second centrifuging mechanism (acceleration 9 and brake 9).

Step S5: cleaning: and (3) dumping the supernatant, moderately knocking the bottom of the centrifuge tube by fingers until the cell sediment is loose, adding normal saline for resuspension, and combining and uniformly mixing cell suspensions in the centrifuge tubes to obtain a total volume of 30 ml.

Step S6: cleaning: the desired volume of cell suspension was centrifuged again at 1500rpm for 6min (acceleration 9, brake 9), the remaining supernatant was decanted and the fingers flicked the bottom of the tube until the cell pellet was loose.

Step S7: inoculation: resuspended in 15ml complete medium and inoculated into a culture flask.

Step S8: adding a nourishing cell: recovering the trophoblast, adding physiological saline to 40ml for re-suspension, and sampling to measure the cell viability. Centrifuged at 1500rpm for 5min and suspended with 15ml of complete medium in the flask.

Step S9: initial culture: 1.5ml autologous plasma (5% concentration) PBMC (20 x 10^6cells) + NK complete medium (30ml) + autologous plasma (1.5ml) + feeder cells was added, and culture was initiated in a 5% CO2 incubator at 37 ℃.

Step S10: visual and microscopic observations on day 1 and day 2, and visual and microscopic observations on day 3, fluid change (no bottle change) was started: gently pumping 3-4 times, transferring the culture solution into a 50ml centrifuge tube, centrifuging at 1500rpm for 5min, discarding the supernatant, resuspending with 30ml NK complete medium, adding into the original culture flask, and adding 1.5ml autologous plasma. Observing whether the cells are obviously clustered or not by naked eyes and a microscope every day from the 4 th to the 6 th days, and adding culture solution and autologous plasma according to the observation condition. Transferred to a flask for culture on day 6. On day 7, visually and microscopically, add a2 and add broth: recovering A2 (feeder cells), adding culture medium, autologous plasma, transferring to 2T 175 bottles for culture (transition period at day 7, plasma concentration is preferably 2-3%, and subsequent culture concentration is 1%), observing with naked eye and microscope every day from day 8 to day 9, adding culture medium, autologous plasma, and culturing in bags at day 8-9 to obtain total volume of at least 250 ml.

The culture medium and autologous plasma were added daily for visual and microscopic observation from day 10 to day 12. On day 13, no liquid is added when the volume of day 12 reaches 1800ml through naked eyes and microscope observation, no operation is performed on day 13, if the volume of day 12 does not reach 1800ml, liquid can be added properly according to the situation, and no operation is performed on day 14. And (4) uniformly blowing, sampling and detecting the cell concentration and the cell viability. Cell status was visually and microscopically observed after day 14 and harvested: fully mixing liquid in a culture bag, sampling to Pre meter cell concentration and viability and flow detection, transferring culture solution into a 50ml centrifuge tube, centrifuging at 1400rpm for 5min, discarding supernatant, transferring the culture solution into the 50ml centrifuge tube again for centrifugation (1 time at most), discarding supernatant, resuspending cell sediment to 40ml by using normal saline, sampling Post to meter cell concentration and viability, centrifuging cell suspension at 1400rpm for 5min, taking supernatant for aseptic detection and mycoplasma and endotoxin detection, and preparing frozen stock solution: ml (90% FBS + 10% DMSO), cryopreserved NK cells according to the counting results (Pre as standard, Post as reference): 4 ml/tube.

Step S11: freezing and storing: placing the sealed cryopreservation tube into a program cooling box, temporarily storing the cryopreservation tube in a refrigerator at 4 ℃ for no more than 1h, placing the program cooling box into an ultralow temperature refrigerator at-80 ℃ for cooling (4-24h), detecting the cooling process by using a low-temperature recorder, transferring the cells into a liquid nitrogen tank for long-term storage, and printing a program cooling curve;

referring to fig. 2, a bottom plate 1 is arranged at the bottom of the blow-beating mixing mechanism 2 in the second step, the blow-beating mixing mechanism 2 is fixedly installed at the center of the top of the bottom plate 1, the blow-beating mixing mechanism 2 comprises a treatment tank 6, a tank cover 7, a feed inlet 8, a hollow pipe 9, a buffer plate 10 and a blow-beating pipe 11, a fixing frame 27 is fixedly installed at the center of the top of the bottom plate 1, the treatment tank 6 is fixedly installed inside the fixing frame 27 at the top of the bottom plate 1, and the fixing frame 27 plays a role in fixedly supporting the treatment tank 6;

referring to fig. 2, a box cover 7 is installed at the top of the treatment box 6, a feed inlet 8 is formed in one end of the top of the box cover 7, a hollow tube 9 is fixedly installed in the center of the top of the box cover 7, the end of the hollow tube 9 extends into the treatment box 6, a buffer plate 10 is fixedly connected to one end, located inside the treatment box 6, of the hollow tube 9, the buffer plate 10 is integrally of a circular structure, a cavity is formed in the buffer plate 10, the hollow tube 9 is communicated with the inside of the buffer plate 10, blow-off tubes 11 are fixedly connected to the bottom of the buffer plate 10 at equal intervals, the blow-off tubes 11 are communicated with the inside of the buffer plate 10, the end of the blow-off tube 11 is of a tapered structure, and a one-way air valve is installed on the outer side of the blow-off tube 11;

referring to fig. 2, 3 and 4, the first centrifugal mechanism 3 in the third step and the second centrifugal mechanism 4 in the fourth step are both fixedly mounted on the top of the bottom plate 1, the first centrifugal mechanism 3 is located between the blow-beating mixing mechanism 2 and the second centrifugal mechanism 4, the liquid-transferring mechanism 5 in the fourth step is fixedly mounted on the top of the bottom plate 1, the liquid-transferring mechanism 5 is located between the first centrifugal mechanism 3 and the second centrifugal mechanism 4, centrifugal processing can be performed through the first centrifugal mechanism 3 and the second centrifugal mechanism 4, and liquid in the centrifuge tube 32 on the first centrifugal mechanism 3 can be transferred to the interior of the centrifuge tube 32 on the second centrifugal mechanism 4 through the arrangement of the liquid-transferring mechanism 5.

Referring to fig. 4 and 8, a fixing box 13 is fixedly connected to one end of the hollow tube 9 located outside the box cover 7, the fixing box 13 is integrally in a circular plate-shaped structure, the hollow tube 9 is communicated with the inside of the fixing box 13, a mounting box 18 is fixedly connected to the side wall of the fixing box 13, three arc-shaped grooves 19 are equidistantly formed in the outer side of the mounting box 18, the inside of the mounting box 18 is communicated with the outside through the arc-shaped grooves 19, a supporting spring 20 is fixedly connected to the inside of one side of the fixing box 13 away from the fixing box 13, a blocking plate 17 is fixedly connected to the end of the supporting spring 20, and the communication port 16 can be blocked through the arrangement of the blocking plate 17;

referring to fig. 8, the blocking plate 17 is slidably connected inside the mounting box 18 in a matching manner, a communication port 16 is formed in one side of the mounting box 18 of the fixing box 13, the fixing box 13 is communicated with the inside of the mounting box 18 through the communication port 16, a sliding rod 15 is fixedly connected to one side of the blocking plate 17, which is located at the communication port 16, an end of the sliding rod 15 passes through the fixing box 13, an upright plate 14 is slidably connected to the top of the box cover 7, an end of the sliding rod 15 is fixedly connected to the upright plate 14, a telescopic spring 22 is sleeved between the upright plate 14 and the fixing box 13 outside the sliding rod 15, a rotating motor 25 is controlled by a preset electric control system to rotate an eccentric wheel 24, the upright plate 14 is continuously pushed to slide on the box cover 7 during rotation of the eccentric wheel 24, the upright plate 14 pushes the sliding rod 15 to slide towards the fixing box 13, the sliding rod 15 pushes the blocking plate 17 to slide inside the mounting box 18, in the sliding process of the blocking plate 18 to the supporting spring 20, the supporting spring 20 and the expansion spring 22 are compressed, the communication port 16 is in an open state at the moment, outside gas enters the fixed box 13 through the arc-shaped groove 19 and the communication port 16, when the end part of the eccentric wheel 24 is far away from the vertical plate 14, the vertical plate 14 restores to the position under the action of the expansion spring 22 and the supporting spring 20, the blocking plate 17 blocks the communication port 16 at the moment, and the gas is blown to the inside of the treatment box 6 through the blow pipe 11.

Referring to fig. 8, a horizontal sliding groove 12 is formed in the top of the box cover 7, a limiting block is arranged at the bottom of the vertical plate 14 and is slidably connected inside the sliding groove 12 in a matching manner, the vertical plate 14 is slidably connected to the top of the box cover 7 through the limiting block arranged on the vertical plate, fixing plates 26 are symmetrically and fixedly connected to the top of the box cover 7 at the side of the vertical plate 14, an eccentric wheel 24 is rotatably connected to the top end between the two fixing plates 26 through a rotating shaft, the eccentric wheel 24 contacts with the vertical plate 14, a rotating motor 25 is fixedly mounted at the top end of the fixing plate 26, and the end of an output shaft of the rotating motor 25 penetrates through the fixing plates 26 and is fixedly connected with the eccentric wheel 24.

Referring to fig. 3, 5, 6 and 9, each of the first centrifugal mechanism 3 and the second centrifugal mechanism 4 includes a fixing base 28, a rotating table 29, fixing slots 30, a placing frame 31 and centrifuge tubes 32, the top of the base plate 1 is provided with four fixing bases 28 at equal intervals outside the fixing frame 27, the fixing bases 28 are rotatably connected with the rotating table 29, the top of the rotating table 29 is provided with four fixing slots 30 at equal intervals, the placing frame 31 is rotatably connected with the four fixing slots 30, the centrifuge tubes 32 are clamped in the placing frame 31, the top of the fixing frame 27 is fixedly provided with a water pump 42, a liquid inlet end of the water pump 42 is provided with a connecting tube 43, an end of the connecting tube 43 is connected with the bottom end of the discharge box 6, a water outlet end of the water pump 42 is fixedly provided with a water pipe 44, and the discharge pipe 44 is located right above the centrifuge tube 32;

during centrifugation, through the work of control hot plate 57 with the inside water heating of water tank 54, then air pump 55 work, gas carries out the heating of certain degree through the inside hot water of water tank 54, make inside hot gas gets into fixed slot 30, heat centrifuging tube 32, control system control servo motor 40 work simultaneously makes drive gear 41 rotate, it makes four drive gear 38 rotations to be connected with the meshing of outer ring gear 39 through drive gear 41, and then make rack 31 rotate, thereby realize centrifuging tube 32's rotation, can carry out centrifugal treatment to the inside liquid of centrifuging tube 32.

Referring to fig. 6 and 7, the bottom of the turntable 29 is rotatably connected with an outer gear ring 39, the bottom of the placing frame 31 is rotatably connected with transmission gears 38, the four transmission gears 38 are meshed with the outer gear ring 39, a servo motor 40 is fixedly mounted at the bottom of the turntable 29, a driving gear 41 is fixedly mounted at the end of an output shaft of the servo motor 40, the driving gear 41 is meshed with the outer gear ring 39, the driving gear 41 is rotated by the servo motor 40, the outer gear ring 39 is rotated by the meshed connection of the driving gear 41 and the outer gear ring 39, the rotation of the placing frame 31 is realized by the meshed connection of the outer gear ring 39 and the four transmission gears 38, and the rotation of the centrifuge tube 32 is further realized, so that the centrifugal processing of cells in the centrifuge tube 32 is realized.

Referring to fig. 8, a sealing gasket 21 is disposed on one side of the blocking plate 17 located on the fixing box 13, the sealing gasket 21 is in close contact with the fixing box 13, a limiting rod 23 is symmetrically fixed on the surface of the blocking plate 17 located on the outer side of the sliding rod 15, the end of the limiting rod 23 slides through the fixing box 13 and is fixedly connected with the vertical plate 14, better sealing can be performed through the sealing gasket 21, the blocking plate 17 is in close contact with the fixing box 13, the communicating port 16 can be sealed, and gas is prevented from entering the fixing box 13.

Referring to fig. 6, the bottom of the inner wall of the fixed seat 28 is rotatably connected with a transmission rod 36 through a bearing, a driven gear 34 is fixedly installed on the outer side of the transmission rod 36, the top of the transmission rod 36 is fixed with the turntable 29, a driving motor 37 is fixedly installed at the bottom of the inner wall of the fixed seat 28, a driving gear 35 is fixedly installed at the end of an output shaft of the driving motor 37, the driving gear 35 is in meshed connection with the driven gear 34, the driving gear 35 is rotated by controlling the driving motor 37 to work, the transmission rod 36 is rotated by meshing connection of the driving gear 35 and the driven gear 34, the turntable 29 is driven to rotate by the transmission rod 36, so that the turntable 29 is rotated, the driving motor 37 is controlled to work by a control system arranged in the centrifugal tube centrifuge tube.

Referring to fig. 4 and 10, the liquid-transferring mechanism 5 includes an installation frame 45, a fixing rod 46, a supporting rod 47, a screw 48, a slider 49, a liquid-transferring motor 50, an electric push rod 51 and a liquid-transferring pipe 52, the installation frame 45 is fixedly installed on the top of the bottom plate 1 between the first centrifugal mechanism 3 and the second centrifugal mechanism 4, and the installation frame 45 plays a better role in fixing and supporting;

referring to fig. 4, a fixing rod 46 is fixedly connected to the top end of the mounting rack 45, a supporting rod 47 is fixedly connected to the end portion of the fixing rod 46, length scale marks 53 are arranged on the surface of the fixing rod 46, the position of the sliding block 49 moving can be observed conveniently through the length scale marks 53, a groove in the horizontal direction is formed in the top portion of the fixing rod 46, a lead screw 48 is rotatably connected in the groove, a sliding block 49 is connected to the outer side of the lead screw 48 in a threaded manner, the sliding block 49 is connected to the inside of the fixing rod 46 in a sliding manner, a liquid-moving motor 50 is fixedly mounted at the end portion of the fixing rod 46, the end portion of an output shaft of the liquid-moving motor 50 extends into the fixing rod 46 to be fixedly connected with the lead screw 48, the threaded rod 48 is rotated by controlling the liquid-moving motor 50, the sliding block 49 is moved through the threaded connection of the threaded rod 48 and the sliding block 49, and the liquid-moving pipe 52 is driven;

referring to fig. 4, an end portion of the slide block 49 is fixedly provided with an electric push rod 51, an end portion of the electric push rod 51 is fixedly provided with a pipette 52, the pipette 52 is located right above the centrifuge tube 32, and the electric push rod 51 cooperates with the pipette 52 to enable the middle albedo layer in the centrifuge tube 32 of the first centrifuge mechanism 3 to be located in the centrifuge tube 32 of the 50ml second centrifuge mechanism 4.

Referring to fig. 6, a sealing ring 33 is arranged at the top end of the inner wall of the placing frame 31, the top end of the placing frame 31 is connected with the inner wall of the fixing groove 30 in a sealing manner, the placing frame 31 is communicated with the inside of the fixing groove 30, a good sealing effect is achieved through the sealing ring 33, certain skid resistance can be achieved on the other hand, and the centrifugal tube 32 is prevented from shaking.

Referring to fig. 5 and 6, a cylindrical groove is formed in the center of the top of the turntable 29, a heating plate 57 is fixedly installed in the cylindrical groove, a water tank 54 is arranged in the cylindrical groove on the turntable 29, a handle 58 is arranged in the center of the top of the water tank 54, the water tank 54 is conveniently lifted and pulled through the handle 58, the water tank 54 is conveniently installed through the cylindrical groove, and water in the water tank 54 is conveniently heated through the heating plate 57 installed inside the cylindrical groove;

referring to fig. 10, an air pump 55 is installed at the top of the water tank 54, an air inlet end of the air pump 55 is communicated with the outside, an air outlet end of the air pump 55 is provided with an air pipe 56, an end of the air pipe 56 extends into the water tank 54, the air pipe 56 is located at one section inside the water tank 54 and is arranged in a spiral shape, the centrifugal tube 32 inside the fixing groove 30 can be heated by controlling the operation of the air pump 55, external air is placed inside the air pipe 56 by the operation of the air pump 55, the air pipe 56 is heated by hot water inside the water tank 54, hot air enters the fixing groove 30, and the centrifugal tube 32 is heated.

By combining the process method, the working principle of the device adopted by the invention is as follows: when the centrifugal device is used, centrifuge tubes 32 are placed in a placing rack 31 in a fixed groove 30 on a first centrifugal mechanism 3 and a second centrifugal mechanism 4, lower red blood cells after plasma separation are filled in a treatment box 6, an equal volume of physiological saline is added into the treatment box 6 through a feed inlet 8 for dilution, a rotating motor 25 is controlled by a preset electric control system to work so as to enable an eccentric wheel 24 to rotate, the vertical plate 14 is continuously pushed to slide on a box cover 7 in the rotation process of the eccentric wheel 24, the vertical plate 14 pushes a sliding rod 15 to slide towards the fixed box 13, the sliding rod 15 pushes a blocking plate 17 to slide in the mounting box 18, the supporting spring 20 and a telescopic spring 22 are compressed in the sliding process of the blocking plate 18 towards the supporting spring 20, at the moment, a communication port 16 is in an open state, external gas enters the fixed box 13 through an arc-shaped groove 19 and a communication port 16, when the end part of the eccentric wheel 24 is far away from the vertical plate 14, the vertical plate 14 is restored to the position under the action of the expansion spring 22 and the supporting spring 20, at this time, the communication port 16 is blocked by the blocking plate 17, the gas is blown into the treatment tank 6 through the blow-beating pipe 11, the blow-beating pipe 11 is continuously blown into the treatment tank 6 during the operation of the rotating motor 25, so that the diluted liquid in the treatment tank 6 is fully blown and uniformly mixed, then the water pump 42 works to pump the liquid into the centrifuge tube 32 and transfer the liquid into the centrifuge tube 32 on the 50ml first centrifugal mechanism 3, the driving motor 37 is controlled to work during the transfer process to rotate the driving gear 35, the driving rod 36 is rotated through the meshing connection of the driving gear 35 and the driven gear 34, so as to rotate the rotary table 29, the control system controls the rotary table 29 to intermittently rotate, so that the raw material is put into each centrifuge tube 32 on the rotary table 29, then the heating plate 57 is controlled to work to heat the water in the water tank 54, and then the air pump 55 works, the gas is heated to a certain degree through the hot water in the water tank 54, so that the hot gas enters the fixed groove 30 to heat the centrifuge tube 32, meanwhile, the control system controls the servo motor 40 to work to enable the driving gear 41 to rotate, the four transmission gears 38 are connected through the meshing of the driving gear 41 and the outer gear ring 39 to enable the four transmission gears 38 to rotate, and then the placing frame 31 is rotated, so that the centrifuge tube 32 rotates, the liquid in the centrifuge tube 32 can be centrifugally treated, after the centrifugation is finished, the liquid-transferring motor 50 is controlled to work to enable the threaded rod 48 to rotate, the sliding block 49 is moved through the threaded connection of the threaded rod 48 and the sliding block 49, and then the pipette 52 is driven to move, the electric push rod 51 is matched with the pipette 52 to enable the middle white film layer in the centrifuge tube 32 on the first centrifugal mechanism 3 to be placed in the centrifuge tube 32 on the 50ml second centrifugal mechanism 4, physiological saline is added to 50ml in each tube, the centrifuge tube is centrifuged at 1500rpm for 6min (acceleration 9, brake 9).

Table II and Table III show the data of the immune cell culture assay obtained by sampling the sample with the number NK2001-NK 2006:

table one: flow assay of NK cells

TABLE II NK cell survival rates

Sample numbering	Time	NK cell survival rate (%)
			NK2001	2020.10.15	92.26
NK2002	2020.10.16	91.33
			NK2003	2020.10.17	93.19
NK2004	2020.10.22	92.27
			NK2005	2020.10.23	91.31
NK2006	2020.10.24	92.08

TABLE III, NK cell count

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. An immune cell culture method, comprising the steps of:

s9: starting culture, adding 1.5ml of 5% autologous plasma and 30ml of NK complete medium, setting the temperature to 37 ℃ and starting culture in a CO2 incubator;

s10: observing and recording, observing by naked eyes and a microscope, and detecting and recording related data;

s11: freezing and storing;

the bottom of the blowing and beating mixing mechanism (2) in the S2 is provided with a bottom plate (1), the blowing and beating mixing mechanism (2) is fixedly installed at the top center of the bottom plate (1), the blowing and beating mixing mechanism (2) comprises a processing box (6), a box cover (7), a feeding hole (8), a hollow pipe (9), a buffer plate (10) and a blowing and beating pipe (11), the top center of the bottom plate (1) is fixedly provided with a fixing frame (27), the top of the bottom plate (1) is fixedly provided with the processing box (6) inside the fixing frame (27), the top of the processing box (6) is provided with the box cover (7), one end of the top of the box cover (7) is provided with the feeding hole (8), the top center of the box cover (7) is fixedly provided with the hollow pipe (9), the end part of the hollow pipe (9) extends into the processing box (6), one end of the hollow pipe (9) inside the processing box (6) is fixedly connected with the buffer plate (10), the buffer plate (10) is integrally of a circular structure, a cavity is formed in the buffer plate (10), the hollow pipe (9) is communicated with the interior of the buffer plate (10), blowing pipes (11) are fixedly connected to the bottom of the buffer plate (10) at equal intervals, the blowing pipes (11) are communicated with the interior of the buffer plate (10), the end part of each blowing pipe (11) is of a conical structure, and a one-way air valve is mounted on the outer side of each blowing pipe (11);

the first centrifugal mechanism (3) in the S3 and the second centrifugal mechanism (4) in the fourth step are both fixedly installed on the top of the bottom plate (1), the first centrifugal mechanism (3) is located between the blowing and beating mixing mechanism (2) and the second centrifugal mechanism (4), the liquid transfer mechanism (5) in the fourth step is fixedly installed on the top of the bottom plate (1), and the liquid transfer mechanism (5) is located between the first centrifugal mechanism (3) and the second centrifugal mechanism (4);

the fixing box (13) is fixedly connected to one end, located outside the box cover (7), of the hollow pipe (9), the whole fixing box (13) is of a circular plate-shaped structure, the hollow pipe (9) is communicated with the inside of the fixing box (13), the side wall of the fixing box (13) is fixedly connected with the mounting box (18), three arc-shaped grooves (19) are formed in the outer side of the mounting box (18) in an equal distance mode, the inside of the mounting box (18) is communicated with the outside through the arc-shaped grooves (19), a supporting spring (20) is fixedly connected to the inside of one side, away from the fixing box (13), of the fixing box (13), the end portion of the supporting spring (20) is fixedly connected with a blocking plate (17), the blocking plate (17) is matched and slidably connected to the inside of the mounting box (18), a communicating port (16) is formed in one side, located on the mounting box (18), of the fixing box (13), and the fixing box (13) is communicated, a sliding rod (15) is fixedly connected to one side, located at the communicating opening (16), of the blocking plate (17), the end portion of the sliding rod (15) penetrates through the fixing box (13), a vertical plate (14) is slidably connected to the top of the box cover (7), the end portion of the sliding rod (15) is fixedly connected with the vertical plate (14), and a telescopic spring (22) is sleeved between the vertical plate (14) and the fixing box (13) on the outer side of the sliding rod (15);

a horizontal sliding groove (12) is formed in the top of the box cover (7), a limiting block is arranged at the bottom of the vertical plate (14) and is matched and slidably connected inside the sliding groove (12), the vertical plate (14) is slidably connected to the top of the box cover (7) through the limiting block arranged on the vertical plate, fixing plates (26) are symmetrically and fixedly connected to the side edge of the vertical plate (14) at the top of the box cover (7), an eccentric wheel (24) is rotatably connected to the top end between the two fixing plates (26) through a rotating shaft, the eccentric wheel (24) is in contact with the vertical plate (14), a rotating motor (25) is fixedly installed at the top end of each fixing plate (26), and the end part of an output shaft of the rotating motor (25) penetrates through the fixing plates (26) and is fixedly connected with the eccentric wheel (24);

the first centrifugal mechanism (3) and the second centrifugal mechanism (4) respectively comprise a fixing seat (28), a rotary table (29), fixing grooves (30), a placing frame (31) and a centrifugal tube (32), the top of the base plate (1) is located on a fixing frame (27) and is fixedly provided with four fixing seats (28) at equal intervals outside, the fixing seats (28) are rotatably connected with the rotary table (29), the four fixing grooves (30) are formed at equal intervals at the top of the rotary table (29), the placing frame (31) is rotatably connected with the four fixing grooves (30), the centrifugal tube (32) is clamped in the placing frame (31), a water pump (42) is fixedly installed at the top of the fixing frame (27), a connecting pipe (43) is installed at the liquid inlet end of the water pump (42), the end of the connecting pipe (43) is connected with the bottom end of the treatment box (6), and a discharging pipe (44) is fixedly installed at the water outlet end of the water pump (42), the discharge pipe (44) is positioned right above the centrifugal pipe (32);

the bottom of revolving stage (29) is rotated and is connected with outer ring gear (39), the bottom of rack (31) is all rotated and is connected with drive gear (38), and four drive gear (38) all are connected with outer ring gear (39) meshing, revolving stage (29) bottom fixed mounting has servo motor (40), and the output shaft tip fixed mounting of servo motor (40) has drive gear (41), drive gear (41) are connected with outer ring gear (39) meshing.

2. An immune cell culture method according to claim 1, wherein: one side that shutoff board (17) are located fixed box (13) is provided with sealed pad (21), and sealed pad (21) and fixed box (13) in close contact with, shutoff board (17) surface is located slide bar (15) outside symmetry and is fixed with gag lever post (23), and the tip of gag lever post (23) slides and passes fixed box (13) and riser (14) fixed connection.

3. An immune cell culture method according to claim 1, wherein: the bottom of fixing base (28) inner wall is connected with transfer line (36) through the bearing rotation, and transfer line (36) outside fixed mounting has driven gear (34), the bottom fixed mounting of fixing base (28) inner wall has driving motor (37), and the output shaft end fixed mounting of driving motor (37) has driving gear (35), driving gear (35) and driven gear (34) meshing are connected.

4. An immune cell culture method according to claim 1, wherein: the liquid transferring mechanism (5) comprises an installation frame (45), a fixing rod (46), a supporting rod (47), a lead screw (48), a sliding block (49), a liquid transferring motor (50), an electric push rod (51) and a liquid transferring pipe (52), wherein the top of the bottom plate (1) is positioned between the first centrifugal mechanism (3) and the second centrifugal mechanism (4) and is fixedly provided with the installation frame (45), the top end of the installation frame (45) is fixedly connected with the fixing rod (46), the end part of the fixing rod (46) is fixedly connected with the supporting rod (47), the surface of the fixing rod (46) is provided with length scale marks (53), the top of the fixing rod (46) is provided with a groove in the horizontal direction, the groove is rotatably connected with the lead screw (48), the outer side of the lead screw (48) is in threaded connection with the sliding block (49), and the sliding block (49) is matched and slidably connected inside the fixing rod (46), dead lever (46) tip fixed mounting has liquid-transfering motor (50), and the output shaft tip that liquid-transfering motor (50) stretches into inside and lead screw (48) fixed connection of dead lever (46), the tip fixed mounting of slider (49) has electric putter (51), and the tip fixed mounting of electric putter (51) has pipette (52), pipette (52) are located centrifuging tube (32) directly over.

5. An immune cell culture method according to claim 1, wherein: the top of rack (31) inner wall is provided with sealing washer (33), the top and fixed slot (30) inner wall sealing connection of rack (31), and rack (31) inside and fixed slot (30) inside intercommunication.

6. An immune cell culture method according to claim 1, wherein: the cylinder groove has been seted up at the top center of revolving stage (29), and cylinder inslot fixed mounting has hot plate (57), cylinder inslot on revolving stage (29) is provided with water tank (54), and the top center of water tank (54) is provided with handle (58), air pump (55) are installed at the top of water tank (54), the inlet end and the external intercommunication of air pump (55), and the end of giving vent to anger of air pump (55) install breather pipe (56), and inside the tip of breather pipe (56) stretched into water tank (54), one section that breather pipe (56) are located water tank (54) inside becomes the heliciform setting.