CN109439542A - A method of preparing cell - Google Patents

Abstract

The present invention provides a kind of methods for preparing cell, equipment of the method using cell is prepared, the equipment is transformed on the basis of full-automatic blood component separator, for cell culture and preparation, grating is substituted using weighing sensor, and increase inlet duct and liquid sensor, relationship is used in conjunction by each device in optimization integral device in the present invention, simplifying operation of equipment process, each each step of device cooperates, reduce production cost, incorporate process flow, so that cell preparation process more intelligent automation, the operation is more convenient, reduce cell contamination, improve the success rate of cell preparation, it has broad application prospects and huge market value.

Description

A method of preparing cell

Technical field

The invention belongs to biomedicine technical fields, are related to a kind of method for preparing cell.

Background technique

Cell therapy is a kind of from the tissue of people or animal, blood etc., and separation and Extraction a portion cell directly returns Be defeated by people perhaps animal or by the part cell of extraction by screening, gene modification, induction differentiation, culture expand etc. means, A kind of therapeutic modality to people or animal is fed back again.Cellular immunotherapy method is in recent years fast-developing for a variety of diseases Cell therapeutic approach, by feed back in-vitro screening, modification, induction, amplification tissue or blood in lymphocyte reach and control More the purpose of disease；Common application includes preparation Dendritic Cells (DC), T cell, NK cell therapy cancer；Prepare Treg cell It treats autoimmune disease and treats a variety of diseases such as infertile, allergy, virus infection.

Cell prepares the preparation process for referring to cell used in cell therapy procedures；With lethal T use for cancer treatment For cell, peripheral blood acquisition → peripheral blood mononuclear cells (PBMC) separation → specific population lymphocyte can be generally divided into Preparation → cell feedback etc. is multiple after separation → cell-stimulating → cell incubation → virus transfection → cell amplification → cell concentration Step；According to different technical principles and clinical application demand, above process step can carry out appropriately combined and adjustment, such as carefully The process of killing cell (CIK) treatment of intracellular cytokine induction can be summarized are as follows: peripheral blood acquisition → PBMC cell separation → cell Preparation → cell is fed back after activation → cell incubation → cell amplification → cell washing；Chimeric antigen receptor T cell (CAR-T) is exempted from Epidemic disease therapy is the relatively advanced and complicated representative of current cell therapy technology means, and the preparation of CAR-T cell needs to complete aforementioned Most or all processes of cell preparation；Since CAR-T technology emerges soon and complex process, lack on the market for it specially The automation equipment of door exploitation optimization, thus the cell preparation process of the clinical research of most CAR-T treatment at present still with Based on the craft preparation method in similar laboratory.With the development of cell therapy industry, the consistency of cell preparation obtains more next How more concerns overcomes the cross contamination of microbial contamination and sample room caused by open manual preparation manipulation Problem, and the difference between batch problem due to caused by the proficiency of different operation technical staff are to push cell therapy technology face The important foundation of cell drug preparation standard can finally be reached to clinic；Therefore cell preparation is individually completed using automation equipment Some step in the process and reach the smallest, stable difference between batch, or even under the premise of single step is stablized and realized, Cell is completed using automation equipment and prepares multiple steps, will complete cell therapy technology to stride forward to cell drug preparation standard Important means.

CN1331610 discloses the system that a kind of separation biological fluids are ingredient, including one group of reception biological fluids to be separated With the container of separated ingredient, optional another container for receiving additive solution and a hollow centrifugal treating room, it has There is axial biological fluids import/export, process chamber has movable piston, to introduce quantitative biological fluids and by outlet extrusion process The ingredients of biological fluid crossed, Optical devices monitor piston position, to control the extrusion of sucking liquid and ingredient, a pressure control valve device The connection process chamber and container of selectivity, or their connection is cut off passes in a standalone mode with dependent by system column are old Mode operation is passed, in particular for listerine is added in separated candidate stem cell；In the independent mode, liquid is inhaled into Process chamber, is centrifuged and is separated into ingredient, and ingredient is squeezed out as far as possible with density gradient product；Under transfer mode, process chamber Liquid is sucked and squeezed out in the stationary case, and valve actuator utilizes the movement of piston, liquid is passed through from a container Process chamber is transmitted to another container, and without being centrifuged or separating, and what the device control for being used to monitor piston position was transmitted does not divide The chaotropic scale of construction；It for mixing under controlled temperatures includes life in reservoir bag flexible that CN105263611A, which is disclosed a kind of, The mixing arrangement of object sample, the mixing arrangement include bracket, for supporting the reservoir bag comprising biological sample to be mixed, are used for So that the sample in reservoir bag on bracket is displaced the component with compound sample；And for during mixing by the mark The temperature control equipment of this holding at controlled, the component for shifting sample includes that at least one may expand/can be received Bag, that is, air bag of contracting, the bag of at least one expandable/collapsible contacts the surface of a part of reservoir bag upon expansion, with gradually Ground squeezes reservoir bag, and the sample for being included is displaced in another part of reservoir bag；CN101146559 discloses one kind It is used in particular for repairing the system for cell subsets extraction, collection, processing and transplanting of organ, the cell in regenerative medicine Subgroup includes adult stem and blood platelet, and the system comprises a set of groups be made of disposable sterilized fluid transport element Part, it is pre-connection or including sterile connector or suitable for being interconnected therebetween with sterile manner that the fluid, which transports element,.It should Component generally includes the complexes of three disposable sterilized elements: it is complete to collect complexes, processing complexes and transplanting Device, three complexes include the blister pack on the supporting member of such as pallet etc, this is packaged with a compartment, is used for Receive the complexes of the component respectively interconnected.The component includes withdrawing device, for example including for piercing through bone or blood The needle of pipe is used to extract marrow or other cell subsets sources out from patient；CN107635668A discloses a kind of for flowing biology Body handles and is separated into the device of component, including hollow centrifugal treating room, is equipped with inlet/outlet head and preferably fills Equipped with the piston that can be axially moved.The inlet/outlet head has two sseparated inlet/outlets, such as axial entrance And side outlet, the process chamber (1) are equipped with internal diversion part, enable devices to operate in continuous processing mode, wherein Biofluid to be processed is continuously introduced by the axial entrance, and at the same time, processed component is via the side outlet Continuous removal.The continuous processing flowing can drive by external peristaltic pump and/or by the axial displacement of piston in the chamber. But the above-mentioned prior art is only capable of the step of completing the part steps of cell preparation or being only used for cell separation, has a single function, Construction is complicated, needs to be advanced optimized.

In conclusion provide it is a kind of can completion prepare multiple steps of immunocyte, it is dirty to reduce more equipment operation brings Dye reduces the frequency of manpower operation, and the method for improving the success rate of cell preparation has broad application prospects and huge city Field value.

Summary of the invention

In view of the deficiencies of the prior art and actual demand, the present invention provide a kind of method for preparing cell, the method Equipment using cell is prepared, the equipment are transformed on the basis of full-automatic blood component separator, are trained for cell It supports and prepares, substitute grating using weighing sensor, and increase inlet duct and liquid sensor, method optimization of the invention Each device is used in conjunction relationship in integral device, and simplifying operation of equipment process, each each step of device cooperates, improve cell system Standby success rate has broad application prospects and huge market value.

To achieve this purpose, the present invention adopts the following technical scheme:

In a first aspect, the present invention provides a kind of method for preparing cell, the method uses a kind of equipment for preparing cell, The equipment includes sampling device, collection device, liquid supply device, centrifugation culture apparatus, liquid sensor device, gas exchanges dress It sets, weigh assembly device and magnet control device；

The liquid sensor device includes the first liquid sensor and second liquid sensor, wherein the first liquid passes Sensor is connected with sampling device, and second liquid sensor is connected with centrifugation culture apparatus；

The weigh assembly device includes weighing sensor, is respectively arranged at the sampling device, collection device, centrifugation training It supports on device and liquid supply device；

Wherein, sampling device, centrifugation culture apparatus, gas exchange device, magnet control device, liquid supply device and collection dress It sets and is sequentially connected；

Described method includes following steps:

(1) it is centrifuged in centrifugation culture apparatus using gradient centrifugation, is cultivated by liquid sensor device, centrifugation Device and weigh assembly device, which cooperate, adjusts liquid flowing, and the cell in sample is separated to collection device；

(2) antibody magnetic bead is added in the cell collected to step (1), is transferred in centrifugation culture apparatus, passes through magnet control Device processed separates target cell；

(3) activator is added in the target cell separated to step (2), carbon dioxide is supplemented using gas exchange device, Mixing is incubated for；

(4) virus is added in the cell after being incubated for step (3), is placed in centrifugation culture apparatus and is incubated for, filled using fluid infusion Supplement or replacement medium are set, after carrying out virus infection, pipeline liquid is emptied by gas exchange device, is received using collection device Collect cell.

The process of cell therapy is as follows: can be generally divided into peripheral blood acquisition → PBMC cell separation → specific population cell Preparation → cell is fed back after separation → cell-stimulating → cell incubation → virus transfection → cell amplification → cell washing；Its China and foreign countries All blood acquisitions and cell feed back the completion operated by manpower, intermediate " PBMC cell separation → specific population cell separation → thin Preparation → cell amplification after born of the same parents' activation → cell incubation → virus transfection → cell washing " is cell preparation process, master of the present invention Solve " PBMC cell separation -> specific population cell separation -> cell-stimulating -> cell incubation -> removal magnetic bead -> virus turn The closed automatic processing technique of these steps of dye ".

In the present invention, inventor realizes the highly efficient cell for preparing, first on the basis of fully automatic blood seperator On improve, in order to which the multi-step for preparing immunocyte is integrated in single equipment, increase inlet duct and liquid face Colour sensor is substituted grating using weighing sensor, reduces machine cost, and have developed methodology according to the equipment, right The entire flow of cell preparation studies intensively practice repeatedly, capture between multiple units can not associated with problem, optimize each step of each device Connection relationship and using sequence, avoid many more manipulations pollution and reduce cost of labor, smoothly realize be concisely and efficiently cell Preparation process.

In the present invention, grating is substituted in the weighing sensor, is set to sampling device, collection device, centrifugation culture dress It sets on liquid supply device, is detected by the weight induction to consumptive materials such as collecting bag or sample sacks, feedback control system；The air inlet Device adjusts the gas exchanges being centrifuged between culture apparatus and pipeline and flowing, the liquid sensor device are respectively arranged at sample introduction Device and centrifugation culture apparatus, monitor the key component of adjustment equipment and the key step of method；Each step in the method It is mutually matched with each device of equipment, collaboration linkage is final to realize the efficiently succinct purpose for preparing cell.

Preferably, the sampling device includes blood bag connector 1, clot filter 31 and the first pipe clamp 8.

Preferably, the blood bag connector 1, clot filter 31 and the first pipe clamp 8 are sequentially connected.

Preferably, the sampling device is connected by the first liquid sensor 4 with the first valve 15.

Preferably, the centrifugation culture apparatus includes lymph separating liquid bag 5, the first liquid filter 21, the second pipe clamp 11, One valve 15, the first injection port 22, the second injection port 23, temperature control module 48, dynamic sealing module 27, centrifugal barrel 29, piston 28 With first gas filter 30.

Preferably, first liquid filter 21, lymph separating liquid bag 5,11 first valve 15 of the second pipe clamp, second liquid Sensor 45, the first injection port 22, the second injection port 23, dynamic sealing module 27, centrifugal barrel 29 and first gas filter 30 according to Secondary connection.

Preferably, piston 28 is contained inside the centrifugal barrel 29.

Preferably, temperature control module 48 is contained outside the centrifugal barrel 29.

Preferably, the equipment further includes centrifugal driving device and pneumatic device.

Preferably, the centrifugal driving device includes rotating electric machine 49 and transmission device 50.

Preferably, the centrifugal driving device is connected with centrifugation culture apparatus.

Preferably, the pneumatic device includes the first gas pressure detector 43 and Pneumatic controller 51.

Preferably, the pneumatic device is connected with centrifugation culture apparatus.

Preferably, the gas exchange device includes the second gas pressure detector 42, the 5th valve 46 and gas path joint 61.

Preferably, the 5th valve 46, the second gas pressure detector 42 and gas path joint 61 are sequentially connected.

Preferably, the liquid supply device includes physiological saline connector 2-3, culture medium bag 4, third pipe clamp 9, third valve 16, the 4th valve 17, the 4th pipe clamp 10 and second liquid filter 20.

Preferably, the physiological saline connector 2-3 is sequentially connected by third pipe clamp 9 with the 4th valve 17.

Preferably, the second liquid filter 20, culture medium bag 4, the 4th pipe clamp 10 are sequentially connected with the 4th valve 17, institute State third valve 16 and the connection of the 4th valve 17.

Preferably, the collection device includes the 6th valve 18, the 5th pipe clamp 12, the 6th pipe clamp 13, the 7th pipe clamp 14, the One collecting bag 6, the second collecting bag 7, third injection port 24, the 4th injection port 25 and sampling bag 26.

Preferably, the sampling bag 26, the 5th pipe clamp 12, the first collecting bag 6, the 6th pipe clamp 13 and the 6th valve 18 be successively Connection.

Preferably, second collecting bag 7 is connected by the 7th pipe clamp 14 with the 6th valve 18.

Preferably, first collecting bag 6 is also connected with third injection port 24 and the 4th injection port 25.

Preferably, the magnet control device includes controllable magnet 47.

Preferably, the controllable magnet 47 includes alnico magnets or non-permanent magnet.

Preferably, the valve include in solenoid valve, pinch valve or rotary valve any one or at least two combination.

Preferably, the equipment includes shell structure.

Preferably, it is passed in the shell structure embedded at least one valve rotating mechanism, centrifugation barrel grab 62, the first liquid Sensor 44, second liquid sensor 45, gas path joint 61 and controllable magnet 47.

Preferably, the valve rotating mechanism of the shell structure includes valve rotating mechanism 52-54.

Preferably, 2-10 weighing sensor is distributed in the shell structure, such as can be 2,3,4,5 A, 6,7,8,9 or 10 weighing sensors.

Preferably, the casing device further includes man-machine interface 56.

Preferably, the method further includes initialization step before step (1).

The step of initialization includes preparing sample, connecting consumptive material and carry out self-test.

Preferably, the consumptive material includes blood sample bag 35, lymph separating liquid bag 5, the first collecting bag 6, the second collecting bag 7, normal saline bag 33, normal saline bag 34, culture medium bag 4 and centrifugal barrel 29.

Wherein, the detailed operation of the initialization are as follows:

Prepare sample: pipe clamps all in pipeline being clamped, closing pipe line, culture medium and lymph separating liquid are passed through into filter respectively Device injects in the specified liquid storing bag of consumptive material, by blood sample and physiological saline, carries out phase respectively by the connecting component of consumptive material Even.

Connection consumptive material: in sequence, blood sample, physiological saline, lymph separating liquid, collecting bag being hung on machine, will Triple valve is connected according to order with the triple valve rotating mechanism of machine, and centrifugal barrel is connected and is fixed with equipment, by gas filter It is connected with equipment, pipeline is caught in the first liquid sensor, second liquid sensor.

Self-test: the pipe clamp (except the pipe clamp of sample collection bag) in pipeline is unclamped, the first liquid sensor, the second liquid are passed through Body sensor detects signal piping, passes through Pneumatic controller, the first baroceptor and the second baroceptor pair Pipeline sealing is detected.

Wherein, the operation of step (1) are as follows:

By the method separating peripheral blood mononuclear cells (PBMC) of lymph separating liquid (Ficoll) gradient centrifugation, step is such as Under: Ficoll is injected centrifugal barrel, sample (peripheral blood or singly adopt component blood) is slowly injected into centrifugal barrel by centrifugal barrel rotation, Under centrifugal action, red blood cell penetrates lymph separating liquid, and PBMC cell reaches separating effect on lymph separating liquid, can pass through The mode of air is added in bucket, increases centrifugation distance, to reduce centrifugation time, then is above pushed away by piston, cooperates the second liquid Body sensor separates heterogeneity, by part blood plasma, PBMC cell, part lymph separating liquid, is collected, is transferred to Part lymph separating liquid, red blood cell are transferred to sample sack by the first collecting bag；Isolated PBMC cell is subjected to physiology salt The subsequent operation of cell preparation is directly collected or carried out to water eccentric cleaning.

Wherein, the detailed operation of step (1) are as follows:

By controlling piston, blood sample is slowly extracted to the first liquid sensor, stops extracting, lymph is separated Liquid is pumped into centrifugal barrel, and starts to be centrifuged, and blood sample is pumped into centrifugal barrel, is waited for a period of time (such as 15 minutes), and liquid will be It is divided into plasma layer, PBMC cellular layer, lymph separating liquid, four layers of red blood cell in centrifugal barrel from the inside to the outside, by piston by centrifugal barrel Interior liquid is slowly released, and under the action of the centrifugal, liquid will be released successively from the inside to the outside, by second liquid sensor to process Liquid is tested and analyzed, and cooperates swivel tee valve and pipeline by different liquids component collection into different collecting bags, by blood Clear first half pushes original blood sample bag to, by serum latter half together with PBMC cellular layer, the first half of lymph separating liquid Part pushes the first collecting bag to, pushes lymph separating liquid latter half and red blood cell to waste fluid bag or blood sample bag.

Preferably, the method further includes the steps that flushing pipeline after step (1) before step (2)；

Preferably, the step of flushing pipeline are as follows: the physiological saline in liquid supply device is pumped into centrifugal barrel 29, then is arranged Into lymph separating liquid bag 5.

Preferably, the number of the flushing pipeline is 1-6 times, such as be can be 1 time, 2 times, 3 times, 4 times, 5 times or 6 times.

Wherein, the operation of the detergent line specifically:

Centrifugal barrel pipeline is connected with physiological saline pipeline, physiological saline is pumped by centrifugal barrel by piston, then by physiology Salt water is expelled to lymph separating liquid bag/waste fluid bag, can be repeated several times to achieve the purpose that thoroughly to clean.

Wherein, the operation of step (2) are as follows:

Antibody magnetic bead is added in the first collecting bag, antibody magnetic bead is allowed to mix well with cell liquid, is recycled to centrifugal barrel, it will Liquid is cleaned in bucket, after by liquid displacement be culture medium, temperature control be incubated for a period of time (antibody magnetic bead by with need to screen Target cell combines, and so as to use magnet to adsorb cell, remaining unwanted cells is discharged), magnet is activated, is allowed thin Cytosol passes through magnet (can be magnet control, be also possible to permanent magnet and pass through the mobile control for being activated and being eliminated), target Cell will be adsorbed by magnet, other unwanted untargeted cells liquid are discharged into waste fluid bag, then to the part of magnet absorption It is rinsed and is discharged into waste fluid bag, finally eliminates magnet, with culture medium by the target cell collection after screening.

Wherein, the detailed operation of step (2) are as follows:

Manpower operation is added magnetic bead antibody and is sufficiently mixed in the first collecting bag, and the cell liquid in the first collecting bag is taken out It being back in centrifugal barrel, adds physiological saline and be diluted, liquid in bucket is slowly pushed out to waste fluid bag by starting centrifugation, there are A small amount of liquid repeats above-mentioned washing step for several times (such as 2 times) in bucket, reconnects culture medium and is pumped into bucket, stops centrifugation, quiet It is only incubated for a period of time (such as 15 minutes), during which centrifugal barrel is rotated left and right (hybrid guided mode several times by (such as 5 minutes) at regular intervals Formula)；

Controllable magnet is activated, the cell liquid in centrifugal barrel is expelled to the first collecting bag, and draw back in bucket again, it will be in bucket Liquid connects waste fluid bag, and all liq is discharged into waste fluid bag, connects centrifugal barrel and culture medium bag, will be in culture medium suction bucket and again It is secondary to be expelled to waste fluid bag, cancel activation magnet, connect centrifugal barrel and culture medium bag, by culture medium together with magnetic bead sorting in pipeline after Cell be pumped into bucket, then the cell liquid in bucket is discharged into the first collecting bag.

In concrete operations, according to experiment or clinical requirement, magnetic bead is if desired removed, then the method also includes removing magnetic bead Operation otherwise need not then carry out the operation of magnetic bead removal.

Preferably, the operation of the removal magnetic bead are as follows: activation is added in cell, step (3) after magnetic bead is added to step (2) The intracellular addition magnetic bead of cell or step (4) virus infection after agent incubation goes to dezymotize, and mixes and incubates in centrifugation culture apparatus After educating, magnetic bead is removed by magnet control device.

Wherein, by taking the cell removal magnetic bead after activator is incubated for is added in step (3) as an example, the step of removal magnetic bead, has Body are as follows:

It is incubated for culture to complete, excessive gas is discharged, magnetic bead is added into centrifugal barrel and goes to dezymotize, and mixing with cells, and incubate A period of time (such as 15-60 minutes) is educated, magnet is activated, centrifugal barrel is connected with the second collecting bag, cell is pushed into the second collection Bag, in the process, magnetic bead will be attracted by magnet, and cell will directly pass through, to reach the target that magnetic bead is separated with cell, then will Magnet is eliminated, and magnetic bead is rinsed to waste fluid bag.

In concrete operations, according to experiment or clinical requirement, magnetic bead is if desired removed, then is removed the operation of magnetic bead, it is no Then, then without removing magnetic bead.

Wherein, the operation of step (3) is as follows:

The density and volume for calculating cell in sampling bag are added suitable activator according to calculated result, are mixed, will Centrifugal barrel left rotation and right rotation is mixed, and is opened gas circuit, is passed through the mixed gas of the prescribed concentration mixed, while controlling centrifugal barrel Temperature, at a suitable temperature carry out cell incubation culture；Incubation process can carry out appropriate mix to cell and (rotate left and right Centrifugal barrel) and ventilation operation, it is incubated for specified time (such as 48 hours).

The detailed operation of step (3) are as follows:

The first collecting bag is mixed and opened the pipe clamp between sampling bag and the first collecting bag manually, cell liquid importing is taken Sample bag, sampling bag is separated, and according to the cell concentration in sampling bag, configures corresponding cell activator, and inject first and collect Bag, manually mix, the cell of the first collecting bag is drawn back in centrifugal barrel, gas piping is connected, by culture with gas suck from Heart bucket, and centrifugal barrel left rotation and right rotation is mixed；To maintain to cultivate gas concentration, can be converted by gas piping will be in bucket Gas discharge, then by the gas sucking bucket of debita spissitudo, cell liquid in bucket is carried out to the culture (such as 48 hours) of certain time.

Wherein, the operation of step (4) is as follows:

The cell liquid of second collecting bag is transferred to centrifugal barrel, virus liquid is added in centrifugal barrel, passes through in centrifugal barrel Temperature control, centrifugation mode allow the viral specified time (such as 120 minutes) of cell infection, finally virus liquid is replaced as by centrifugation thin Born of the same parents' culture medium is packed into the second collecting bag, then is pumped into some gases by air inlet after mixing cell, by the raffinate in pipeline Body is all drained into collecting bag, and cell is directly harvested.

Wherein, the detailed operation of step (4) are as follows:

Cell liquid is pumped into centrifugal barrel from the second collecting bag, virus liquid is added, starting centrifugation carries out virus infection and holds Continue a period of time (such as 120 minutes), in course of infection, mixing with cells is centrifuged by centrifugal barrel again reaches best infection effect several times Rate；Liquid in bucket is slowly pushed out to waste fluid bag later, there are a small amount of liquid in bucket, reconnects culture medium bag and is pumped into bucket Cell washing is carried out, repeats above-mentioned washing step for several times (such as 2 times), stops centrifugation, finally by the cell push-in second after transfection Collecting bag is collected.

The cell that step (4) collection obtains can further culture amplification obtains immunocyte outside equipment.

As optimal technical scheme, a method of cell is prepared, using the equipment, is specifically comprised the following steps:

(1) prepare sample, connect consumptive material and carry out self-test, the consumptive material includes blood sample bag 35, lymph separating liquid bag 5, the first collecting bag 6, the second collecting bag 7, normal saline bag 33-34, culture medium bag 4 and centrifugal barrel 29；

It is centrifuged in centrifugation culture apparatus using gradient centrifugation, passes through liquid sensor device, centrifugation culture dress It sets to cooperate with weigh assembly device and adjusts liquid flowing, the cell in sample is separated to collection device；

Physiological saline in liquid supply device is pumped into centrifugal barrel 29, then is drained into lymph separating liquid bag 5, repeatedly；

(2) antibody magnetic bead is added in the cell collected to step (1), is transferred in centrifugation culture apparatus, passes through magnet control Device processed separates target cell；

(3) activator is added in the target cell separated to step (2), carbon dioxide is supplemented using gas exchange device, And mix incubation；

(4) virus is added in the cell after being incubated for step (3), is placed in centrifugation culture apparatus and is incubated for, filled using fluid infusion Supplement or replacement medium are set, after carrying out virus infection, pipeline liquid is emptied by gas exchange device, is received using collection device Collect cell；

(5) wherein, during preparing cell, activation is added in cell, step (3) after magnetic bead is added to step (2) The intracellular addition magnetic bead of cell or step (4) virus infection after agent incubation goes to dezymotize, and mixes and incubates in centrifugation culture apparatus After educating, magnetic bead is removed by magnet control device.

Specifically, the present invention includes following system and device:

1, consumptive material system

The system includes one group of fluid storage bag and a centrifuge container, there is piston in centrifuge container, piston can more than Lower movement, may be inhaled or push-in liquid to realize variable centrifugation volume cooperates the switching in pipeline fluid path direction, can will Product is collected into corresponding fluid storage bag after centrifugation, which also includes 4 swivel tee valves, can be by valve side To switching realize the flow direction of consumptive material system pipeline liquid.

2, cell preparation system (machine)

1. the system contains a set of consumptive material system；

2. the system includes a set of driving consumptive material centrifuge container rotation and the centrifugal driving device that piston moves up and down, it is somebody's turn to do Device includes motor (driving centrifuge container rotation), air pump, solenoid valve, dynamic seal structure (blow to centrifuge container or Pumping, to realize piston up-down), (pressure of detection centrifuge container, detects the limit position of piston to the second baroceptor It sets)；

3. the system includes a set of temperature control module, which can guarantee centrifuge container at a certain temperature (such as 37 Degree) it works, which has the function of heating, heat preservation, temperature detection, temperature-compensating；

4. the system includes 2 liquid color sensor devices, which can detect to flow through the liquid of the device Color cooperates swivel tee valve, it can be achieved that the liquid of different colors to be assigned to different collecting bags；

5. the system includes the driving device of 4 swivel tee valves, for driving swivel tee valve or the device It can be substituted with other solenoid valves, the change in the flowable direction of consumptive material pipeline may be implemented in the switching of valve；

6. the system includes the gas that can mention air inlet body, signal piping consumptive material system sealing to pipeline consumptive material system Body switch, wherein have pressure sensor (can signal piping pressure), pinch valve (can also other solenoid valves, rotary valve carrys out generation For), to control the on-off that gas enters consumptive material pipeline；

7. the system includes one group of weigh assembly device, each accommodation bag of consumptive material, which can be fixed on one, has weighing function On the device of energy, weighing device can accurately measure the weight of each collecting bag of energy, guarantee the body for entering, releasing centrifuge container Long-pending precision；

8. the system includes a magnet control device, the magnet of the device has moveable function, when needs are to consumption When the magnetic bead of material system is adsorbed, the magnet of the device can be moved adjacent to the position of consumptive material pipeline, conversely, far away from Pipeline, the controllable magnet are also possible to electromagnet, and energization is upper magnetic, have absorption magnetic bead function, and power-off there just is not magnetism, cannot inhale Attached magnetic bead.

3, control principle system

1. air pump inflates centrifuge container, reading of the controller by the pressure to the second baroceptor, detection centrifugation Container whether gas leakage, man-machine interface export next step；

2. air pump is evacuated centrifuge container, controller detects consumptive material by the reading of the pressure to the first baroceptor Pipeline whether gas leakage, man-machine interface export next step；

3. controller reads the weight of consumptive material accommodation bag, on demand, controller controls swivel tee and switches pipeline direction, gas Pump work drives centrifuge container piston, certain liquid of constant weight is pumped into toward centrifuge container.Or from centrifuge container toward some The liquid of accommodation bag release constant weight；

4. controller reads the temperature of temperature control module in real time, the on-off of heating module is controlled；

5. controller controls the movement (or power on/off) for realizing controllable magnet by experiment flow, to adsorb the magnetic of pipeline Pearl.

Compared with prior art, the invention has the following beneficial effects:

Method provided by the invention uses a kind of equipment for preparing cell, compared with the equipment of single technique, by multiple works Skill is realized in an equipment, it is possible to reduce the number of manpower operation, while reducing because carrying out cell transfer in distinct device Caused by pollution, human error the problems such as, method provided by the invention optimizes operating process, reduces and wants to personnel's operation It asks, reduces production cost, incorporate process flow, so that cell preparation process more intelligent automation, the operation is more convenient, The success rate for improving cell preparation, has broad application prospects and huge market value.

Detailed description of the invention

Fig. 1 wastes material system schematic for of the invention；

Fig. 2 is cell preparation system and system connection schematic diagram of the invention；

Fig. 3 is system input and output design and control principle schematic diagram of the invention；

Fig. 4 is the left side view of shell structure of the invention；

Fig. 5 is the right side view of shell structure of the invention；

Fig. 6 (A) is the CD3 positive ratiometric result figure before sorting of the invention；

Fig. 6 (B) is the CD3 positive ratiometric result figure after sorting of the invention；

Fig. 7 (A) is the negative control group result figure that the outer cell culture of equipment of the invention detects slow-virus infection；

Fig. 7 (B) is the result figure for the experimental group culture 2 days that the outer cell culture of equipment of the invention detects slow-virus infection；

Fig. 7 (C) is the result figure for the experimental group culture 8 days that the outer cell culture of equipment of the invention detects slow-virus infection.

Specific embodiment

Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.

The assembling of 1 equipment of embodiment

The present embodiment provides a kind of equipment, the equipment includes sampling device, collection device, liquid supply device, centrifugation culture Device, liquid sensor device, gas exchange device, weigh assembly device and magnet control device；

The liquid sensor device includes the first liquid sensor and second liquid sensor, wherein the first liquid passes Sensor is connected with sampling device, and second liquid sensor is connected with centrifugation culture apparatus；The weigh assembly device includes weighing Sensor is respectively arranged on the sampling device, collection device, centrifugation culture apparatus and liquid supply device；Wherein, sample introduction fills It sets, be centrifuged culture apparatus, gas exchange device, magnet control device, liquid supply device and collection device and be sequentially connected；The sample introduction Device includes blood bag connector 1, clot filter 31 and the first pipe clamp 8；The blood bag connector 1, clot filter 31 and the first pipe Folder 8 is sequentially connected；The sampling device is connected by the first liquid sensor 44 with the first valve 15；The centrifugation culture apparatus Including lymph separating liquid bag 5, the first liquid filter 21, the second pipe clamp 11, the first valve 15, the injection of the first injection port 22, second Mouth 23, temperature control module 48, dynamic sealing module 27, centrifugal barrel 29, piston 28 and first gas filter 30；The first liquid filter Device 21, lymph separating liquid bag 5,11 first valve 15 of the second pipe clamp, second liquid sensor 45, the first injection port 22, the second note Loophole 23, dynamic sealing module 27, centrifugal barrel 29 and first gas filter 30 are sequentially connected；Contain work inside the centrifugal barrel 29 Plug 28；Contain temperature control module 48 outside the centrifugal barrel 29；The equipment further includes centrifugal driving device and pneumatic device；It is described Centrifugal driving device includes rotating electric machine 49 and transmission device 50；The pneumatic device includes the first gas pressure detector 43 and air pressure Control device 51；The centrifugal driving device is connected with centrifugation culture apparatus, and the pneumatic device is connected with centrifugation culture apparatus； The gas exchange device includes the second gas pressure detector 42, the 5th valve 46 and gas path joint 61；5th valve 46, Two gas pressure detectors 42 and gas path joint 61 are sequentially connected；The liquid supply device includes physiological saline connector 2-3, culture medium bag 4, third pipe clamp 9, third valve 16, the 4th valve 17, the 4th pipe clamp 10 and second liquid filter 20；The physiological saline connection Device 2-3 is sequentially connected by third pipe clamp 9 with the 4th valve 17；The second liquid filter 20, culture medium bag 4, the 4th pipe clamp 10 are sequentially connected with the 4th valve 17, the third valve 16 and the connection of the 4th valve 17；The collection device includes the 6th valve The 18, the 5th pipe clamp 12 of door, the 6th pipe clamp 13, the 7th pipe clamp 14, the first collecting bag 6, the second collecting bag 7, third injection port 24, the Four injection ports 25 and sampling bag 26；The sampling bag 26, the 5th pipe clamp 12, the first collecting bag 6, the 6th pipe clamp 13 and the 6th valve 18 are sequentially connected；Second collecting bag 7 is connected by the 7th pipe clamp 14 with the 6th valve 18；First collecting bag 6 is also It is connected with third injection port 24 and the 4th injection port 25；The magnet control device includes controllable magnet 47；The controllable magnet 47 be alnico magnets；The valve is swivel tee valve；The equipment further includes shell structure；The upside of the shell structure Face connects embedded with valve rotating mechanism 52-54, centrifugation barrel grab 62, the first liquid sensor 44, second liquid sensor 45, gas circuit First 61 and controllable magnet 47；There is a weighing sensor in the left side of the shell structure close to the position distribution of upper side, right Close to the position distribution of upper side, there are two weighing sensors for side；It is perpendicular in the middle part of the trailing flank of the shell structure to have upright bar, institute Stating upright bar top braces has an oblate cylinder, and cylindrical sides are uniformly distributed that there are four weighing sensors；The casing device also wraps Man-machine interface 56 is included, the man-machine interface is located at the leading flank of shell structure.

Above equipment is connected according to Fig. 1-connection relationship shown in fig. 5 with sequence of positions, is assembled into and prepares setting for cell It is standby.

Embodiment 2 prepares cell

Cell is prepared using following steps:

(1) prepare 100mL blood sample, connect consumptive material and carry out self-test, the consumptive material includes blood sample bag 35, lymph Separating liquid bag 5, the first collecting bag 6, the second collecting bag 7, normal saline bag 33-34, culture medium bag 4 and centrifugal barrel 29；

It is centrifuged in centrifugation culture apparatus using gradient centrifugation, passes through liquid sensor device, centrifugation culture dress It sets to cooperate with weigh assembly device and adjusts liquid flowing, the cell in sample is separated to collection device；

Physiological saline in liquid supply device is pumped into centrifugal barrel 29, then is drained into lymph separating liquid bag 5, repeatedly；

(2) antibody magnetic bead is added in the cell collected to step (1), is transferred in centrifugation culture apparatus, passes through magnet control Device processed separates target cell；

(3) activator is added in the target cell separated to step (2), carbon dioxide is supplemented using gas exchange device, And mix incubation；

The intracellular addition magnetic bead obtained to step (3) goes to dezymotize, and after mixing is incubated in centrifugation culture apparatus, passes through magnetic Iron control device removes magnetic bead；

(4) virus is added in the cell after being incubated for step (3), is placed in centrifugation culture apparatus and is incubated for, filled using fluid infusion Supplement or replacement medium are set, after carrying out virus infection, pipeline liquid is emptied by gas exchange device, is received using collection device Collect cell.

Detailed operating procedures are as follows:

Prepare sample:

Pipe clamps all in pipeline are clamped, closing pipe line；Culture medium and lymph separating liquid are injected by filter respectively and consumed In the specified liquid storing bag of material, blood sample and physiological saline are carried out connected respectively by the connecting component of consumptive material.

Connect consumptive material:

In sequence, 100mL blood sample, physiological saline, lymph separating liquid, the first collecting bag, the second collecting bag are hung On machine, triple valve is connected according to order with the triple valve rotating mechanism of machine, centrifugal barrel is connected and is fixed with equipment, Gas filter is connected with equipment, pipeline is caught in the first liquid sensor and second liquid sensor.

Self-test:

Pipe clamp (except the pipe clamp of sample collection bag) in pipeline is unclamped, it is right by detector of liquid 1, detector of liquid 2 Signal piping is detected, by Pneumatic controller, the first baroceptor, the second baroceptor to pipeline sealing into Row detection.

PBMC separation:

By controlling piston, blood sample is slowly extracted to the first liquid receptor, stop extracting, lymph is separated Liquid is pumped into centrifugal barrel, and starts to be centrifuged, and blood sample is pumped into centrifugal barrel, waits 15min, liquid will in centrifugal barrel by it is interior extremely It is divided into plasma layer, PBMC cellular layer, lymph separating liquid, four layers of red blood cell outside, is slowly released by piston by liquid in bucket is centrifuged, Under the action of the centrifugal, liquid will be released successively from the inside to the outside, carry out detection point by liquid of the second liquid receptor to process Analysis cooperates swivel tee valve and pipeline by different liquids component collection into different collecting bags.Serum first half is pushed to Original blood sample bag pushes serum latter half to first receipts together with the first half of PBMC cellular layer, lymph separating liquid Collect bag, pushes lymph separating liquid latter half and red blood cell to waste fluid bag or blood sample bag.

Flushing pipeline and centrifugal barrel:

Centrifugal barrel pipeline is connected with physiological saline pipeline, physiological saline is pumped by centrifugal barrel by piston, then by physiology Salt water is expelled to lymph separating liquid bag/waste fluid bag, can be repeated several times to achieve the purpose that thoroughly to clean.

Magnetic bead sorting:

Manpower operation is added CD3+CD28 sorting stimulation magnetic bead and is sufficiently mixed in the first collecting bag, by the first collecting bag In cell liquid draw back into centrifugal barrel, add physiological saline and be diluted, starting centrifugation, slowly liquid in bucket is pushed out to Waste fluid bag, there are a small amount of liquid in bucket, repeats above-mentioned washing step 2 times for several times, reconnects culture medium and is pumped into bucket, stops During which centrifugation, static incubation 15min rotate left and right centrifugal barrel several times (mixed mode) every 5min；

Controllable magnet is activated, the cell liquid in centrifugal barrel is expelled to the first collecting bag, and draw back in bucket again, it will be in bucket Liquid connects waste fluid bag, and all liq is discharged into waste fluid bag；Centrifugal barrel and culture medium bag are connected, it will be in culture medium suction bucket and again It is secondary to be expelled to waste fluid bag；Cancel activation magnet, connect centrifugal barrel and culture medium bag, by culture medium together with magnetic separation in pipeline after Cell is pumped into bucket, then the cell liquid in bucket is discharged into the first collecting bag；

The first collecting bag is mixed and opened the pipe clamp between sampling bag and the first collecting bag manually, cell liquid importing is taken Sample bag, sampling bag is separated, and according to the cell concentration in sampling bag, configures corresponding cell activator, and inject first and collect Bag mixes manually；The cell of first collecting bag is drawn back in centrifugal barrel, gas piping is connected, by culture with gas suck from Heart bucket, and centrifugal barrel left rotation and right rotation is mixed；To maintain to cultivate gas concentration, can be converted by gas piping will be in bucket Gas discharge, then by the gas sucking bucket of debita spissitudo, cell liquid in bucket is carried out to the culture of 48h；

Magnetic bead is added into centrifugal barrel to go to dezymotize, is incubated for 20min；Magnet is activated, centrifugal barrel is connected with the second collecting bag, Cell is pushed into the second collecting bag；In the process, magnetic bead will be attracted by magnet, and cell will directly pass through, thus reach magnetic bead with The target of cell separation；

Magnet is cancelled and is activated, normal saline bag and centrifugal barrel are connected to, the magnetic bead in pipeline is sucked by physiological saline Centrifugal barrel, then centrifugal barrel is connected with waste fluid bag, magnetic bead is expelled to waste fluid bag.

Virus transfection:

Cell liquid is pumped into centrifugal barrel from the second collecting bag, virus liquid is added, starting centrifugation carries out virus infection and holds Continuous 2h, in course of infection, mixing with cells is centrifuged by centrifugal barrel again reaches best efficiency of infection several times；Slowly liquid in bucket is pushed away Out to waste fluid bag, there are a small amount of liquid in bucket, reconnects culture medium bag and is pumped into progress cell washing in bucket, repeats above-mentioned wash Step 2 time is washed, centrifugation is stopped, virus liquid is finally replaced as cell culture medium by centrifugation, is packed into second after cell is mixed Collecting bag, then some gases are pumped by air inlet, the remaining liq in pipeline is all drained into collecting bag, cell is direct Harvest.

Experiment detection

The separation of 100mL blood sample, activation and transfection are as follows:

1, the separation of PBMC:

A. it is greater than the every 100mL whole blood blood sample of 1E8 PBMC cell, highest sample is more than every 100 milliliters of 2.5E8 PBMC cell Whole blood.

Purity: greater than 90% of b.PBMC.

2, CD3 cell sorting

A.CD3+ cell sorting yield: greater than 75%

B. CD3+ cell purity: greater than 95% after sorting

Front and back CD3 positive ratio is sorted using flow cytomery, as a result as shown in Fig. 6 (A)-(B)；

By Fig. 6 (A)-Fig. 6 (B) it is found that positive ratio reaches 96.49% after sorting；

C. Cell viability: greater than 90%

D. every 100 milliliters of acquisition CD3+ positive cells are greater than 2E7 cell

3, cell activation:

A. Cell viability: greater than 85%

4, virus infection

Cell viability is greater than 85% after a.GFP slow-virus infection.

5, cell culture

A. the cell after virus infection is moved to outside equipment and is cultivated, cultivate metainfective CD3+T cell 48 small hour Afterwards, cell density is risen to by being greater than 2E5 cells/ml greater than 1E6 cells/ml.

B. GFP slow-virus infection T cell is used, as a result sees Fig. 7 (A)-Fig. 7 (C), it is positive that GFP in T cell is measured after 48 hours Rate is 59.09%, continues to cultivate cell to 192 hours (8 days), GFP positive rate is 51.55% in T cell.

In conclusion the present invention provides a kind of method for preparing cell, it is described to set using a kind of equipment for preparing cell It is standby to be transformed on the basis of full-automatic blood component separator, it is used for cell culture and preparation, is replaced using weighing sensor For grating, and inlet duct and liquid sensor are increased, the company of each device in method optimization integral device provided by the invention With relationship, simplifying operation of equipment process, each each step of device cooperates, and reduces production cost, incorporates process flow, makes The process more intelligent automation of cell preparation is obtained, the operation is more convenient, reduces pollution, the success rate of cell preparation is improved, It has broad application prospects and huge market value.

The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. a kind of method for preparing cell, which is characterized in that the method using the equipment for preparing cell, the equipment include into Sampling device, collection device, liquid supply device, centrifugation culture apparatus, liquid sensor device, gas exchange device, weigh assembly dress It sets and magnet control device；

The liquid sensor device includes the first liquid sensor and second liquid sensor, wherein the first liquid sensor It is connected with sampling device, second liquid sensor is connected with centrifugation culture apparatus；

The weigh assembly device includes weighing sensor, is respectively arranged at the sampling device, collection device, centrifugation culture dress Set on liquid supply device；

Wherein, sampling device, centrifugation culture apparatus, gas exchange device, magnet control device, liquid supply device and collection device according to Secondary connection；

Described method includes following steps:

(1) it is centrifuged in centrifugation culture apparatus using gradient centrifugation, passes through liquid sensor device, centrifugation culture apparatus It cooperates with weigh assembly device and adjusts liquid flowing, the cell in sample is separated to collection device；

(2) antibody magnetic bead is added in the cell collected to step (1), is transferred in centrifugation culture apparatus, is controlled and filled by magnet Set separation target cell；

(3) activator is added in the target cell separated to step (2), carbon dioxide, mixing is supplemented using gas exchange device It is incubated for；

(4) virus is added in the cell after being incubated for step (3), is placed in centrifugation culture apparatus and is incubated for, mended using liquid supply device It fills or replacement medium, after carrying out virus infection, pipeline liquid is emptied by gas exchange device, is collected using collection device thin Born of the same parents.

2. the method according to claim 1, wherein the sampling device includes blood bag connector (1), clot filter Device (31) and the first pipe clamp (8)；

Preferably, the blood bag connector (1), clot filter (31) and the first pipe clamp (8) are sequentially connected；

Preferably, the sampling device is connected by the first liquid sensor (44) with the first valve (15)；

Preferably, the centrifugation culture apparatus include lymph separating liquid bag (5), the first liquid filter (21), the second pipe clamp (11), First valve (15), the first injection port (22), the second injection port (23), temperature control module (48), dynamic sealing module (27), centrifugation Bucket (29), piston (28) and first gas filter (30)；

Preferably, first liquid filter (21), lymph separating liquid bag (5), (11) first valve (15) of the second pipe clamp, second Liquid sensor (45), the first injection port (22), the second injection port (23), dynamic sealing module (27), centrifugal barrel (29) and One gas filter (30) is sequentially connected；

Preferably, contain piston (28) inside the centrifugal barrel (29)；

Preferably, contain temperature control module (48) outside the centrifugal barrel (29)；

Preferably, the equipment further includes centrifugal driving device and pneumatic device；

Preferably, the centrifugal driving device includes rotating electric machine (49) and transmission device (50)；

Preferably, the centrifugal driving device is connected with centrifugation culture apparatus；

Preferably, the pneumatic device includes the first gas pressure detector (43) and Pneumatic controller (51)；

Preferably, the pneumatic device is connected with centrifugation culture apparatus；

Preferably, the gas exchange device includes the second gas pressure detector (42), the 5th valve (46) and gas path joint (61)；

Preferably, the 5th valve (46), the second gas pressure detector (42) and gas path joint (61) are sequentially connected；

Preferably, the liquid supply device includes physiological saline connector, culture medium bag (4), third pipe clamp (9), third valve (16), the 4th valve (17), the 4th pipe clamp (10) and second liquid filter (20)；

Preferably, the physiological saline connector is sequentially connected by third pipe clamp (9) with the 4th valve (17)；

Preferably, the second liquid filter (20), culture medium bag (4), the 4th pipe clamp (10) successively connect with the 4th valve (17) It connects, the third valve (16) and the connection of the 4th valve (17)；

Preferably, the collection device includes the 6th valve (18), the 5th pipe clamp (12), the 6th pipe clamp (13), the 7th pipe clamp (14), the first collecting bag (6), the second collecting bag (7), third injection port (24), the 4th injection port (25) and sampling bag (26)；

Preferably, the sampling bag (26), the 5th pipe clamp (12), the first collecting bag (6), the 6th pipe clamp (13) and the 6th valve (18) it is sequentially connected；

Preferably, second collecting bag (7) is connected by the 7th pipe clamp (14) with the 6th valve (18)；

Preferably, first collecting bag (6) is also connected with third injection port (24) and the 4th injection port (25).

3. according to the method described in claim 2, it is characterized in that, the magnet control device includes controllable magnet (47)；

Preferably, the controllable magnet (47) includes alnico magnets or non-permanent magnet.

4. according to the method in claim 2 or 3, which is characterized in that the valve includes solenoid valve, pinch valve or rotary valve In any one or at least two combination.

5. method according to any one of claim 1-3, which is characterized in that the equipment further includes shell structure；

Preferably, embedded at least one valve rotating mechanism, centrifugation barrel grab (62), the first liquid sensing in the shell structure Device (44), second liquid sensor (45), gas path joint (61) and controllable magnet (47)；

Preferably, 2-10 weighing sensor is distributed in the shell structure；

Preferably, the casing device further includes man-machine interface (56).

6. method according to any one of claims 1-5, which is characterized in that the method is also wrapped before step (1) Include initialization step；

The step of initialization includes preparing sample, connecting consumptive material and carry out self-test.

7. according to the method described in claim 6, it is characterized in that, the consumptive material includes blood sample bag (35), lymph separation Liquid bag (5), the first collecting bag (6), the second collecting bag (7), normal saline bag, culture medium bag (4) and centrifugal barrel (29).

8. the method according to the description of claim 7 is characterized in that the method after step (1), is gone back before step (2) Include the steps that flushing pipeline；

Preferably, the step of flushing pipeline are as follows: the physiological saline in liquid supply device is pumped into centrifugal barrel (29), then is drained into In lymph separating liquid bag (5)；

Preferably, the number of the flushing pipeline is 1-6 times.

9. method according to claim 1 to 8, which is characterized in that the method also includes removing the behaviour of magnetic bead Make；

Preferably, the operation of the removal magnetic bead are as follows: cell, step (3) after magnetic bead is added to step (2) are added activator and incubate The intracellular addition magnetic bead of cell or step (4) virus infection after educating goes to dezymotize, after mixing is incubated in centrifugation culture apparatus, Magnetic bead is removed by magnet control device.

10. method according to claim 1 to 9, which is characterized in that specifically comprise the following steps:

(1) prepare sample, connect consumptive material and carry out self-test, the consumptive material includes blood sample bag (35), lymph separating liquid bag (5), the first collecting bag (6), the second collecting bag (7), normal saline bag, culture medium bag (4) and centrifugal barrel (29)；

Using gradient centrifugation centrifugation culture apparatus in be centrifuged, by liquid sensor device, centrifugation culture apparatus and Weigh assembly device, which cooperates, adjusts liquid flowing, and the cell in sample is separated to collection device；

Physiological saline in liquid supply device is pumped into centrifugal barrel (29), then is drained into lymph separating liquid bag (5), repeatedly；

(2) antibody magnetic bead is added in the cell collected to step (1), is transferred in centrifugation culture apparatus, is controlled and filled by magnet Set separation target cell；

(3) activator is added in the target cell separated to step (2), carbon dioxide is supplemented using gas exchange device, and mix It closes and is incubated for；

(4) virus is added in the cell after being incubated for step (3), is placed in centrifugation culture apparatus and is incubated for, mended using liquid supply device It fills or replacement medium, after carrying out virus infection, pipeline liquid is emptied by gas exchange device, is collected using collection device thin Born of the same parents；

(5) wherein, during preparing cell, cell, step (3) after magnetic bead is added to step (2) are added activator and incubate The intracellular addition magnetic bead of cell or step (4) virus infection after educating goes to dezymotize, after mixing is incubated in centrifugation culture apparatus, Magnetic bead is removed by magnet control device.