CN100497582C

CN100497582C - Bioreactor system for nerve stem cell to amplify in large scale and long time

Info

Publication number: CN100497582C
Application number: CNB2005100245813A
Authority: CN
Inventors: 董良; 齐瀚实; 陈彦田; 张宝红; 郭雅静
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2005-03-24
Filing date: 2005-03-24
Publication date: 2009-06-10
Anticipated expiration: 2025-03-24
Also published as: CN1687385A

Abstract

A long-term large-scale expansion bioreactor system for neural stem cells in the technical field of bioengineering, the invention includes: a gas supply unit, a culture solution supply unit, a cell expansion unit and a first peristaltic pump, the connection mode of which is: supply The end of the gas unit is connected to the cell expansion unit through a silicone gas delivery tube, the end of the culture solution supply unit is connected to the cell expansion unit through a silicone delivery tube, and the first peristaltic pump is in the middle of the silicone delivery tube. The invention gets rid of the limitation of the incubator, is beneficial to long-term large-scale expansion and simultaneous detection, and provides the most suitable environment for long-term high-density culture of neural stem cells.

Description

Bioreactor system for nerve stem cell to amplify in large scale and long time

Technical field

The present invention relates to a kind of bioreactor system, specifically is a kind of bioreactor system for nerve stem cell to amplify in large scale and long time.Be used for technical field of bioengineering.

Background technology

Reynold in 1992 and Weiss have isolated neural stem cell for the first time from the central nervous system of adult mice, and successfully make it at amplification in vitro, indicate that the research of neural stem cell has entered a brand-new stage.Because neurocyte is not special antigen presenting cell (APCs), immune response to the allogeneic nerve tissue transplanted does not have its hetero-organization strong like that, so the allogeneic nerve stem cell at amplification in vitro will break away from these restraining factors in transplantation, become the powerful measure of treatment nervous system disease.Existing neural stem cell is attempted being used for the nervus centralis disease, as the treatment of parkinsonism, multiple sclerosis and huntington's chorea.Neural stem cell and noble cells thereof also can be used as the specific aim screening system of relevant medicine, and cell differentiation of nerve cord is various phenotypes, but in the analogue body in the cental system cell with organize between complex interactions, have purposes widely in the study of pharmacy field.In the pharmacological action and toxicity test of various cells, neural stem cell will provide the research means of cell levels for the aspects such as pharmacology, drug effect, medicine generation and toxicity of cental system disease candidate medicine, significantly reduce the quantity of the required animal of drug testing, reduce cost.In addition, because neural stem cell is similar to the cell of body early embryo, which medicine it also might be used for disclosing can be disturbed fetus brain neurodevelopment and cause inborn defect etc.More than all application to be prerequisite with a large amount of acquisitions of neural stem cell all, therefore must have a kind of bioreactor system to increase on a large scale and could satisfy clinical and needs scientific research it.And the cultivation of domestic and international most of neural stem cell is at present still carried out in tissue culture flasks, obtain a large amount of neural stem cell, and only depending on the number that increases tissue culture flasks is very costliness and poor efficiency, and need expend a large amount of labours.Except that above problem, the danger of microbiological contamination and misoperation (calculating as cell counting/survival rate/inoculum size) is arranged all in each transfer from the culturing bottle to the culturing bottle.

Find by prior art documents, people such as Michael S.Kallos are at " Biotechnology andBioengineering " 1999,65 (5): " the Extended Serial Passaging ofMammalian Neural Stem Cells in Suspension Bioreactors " that delivers on the 589-599 (cultivate in the medium-term and long-term continuous passage of suspended biological reactor by mammalian neural stem cells, " Biotechnology and Bioengineering ") in the literary composition, the suspended biological reactor assembly of amplification neural stem cell has been proposed, this system is mainly by rolling bottle, magnetic stirring apparatus and incubator constitute, when the neural stem cell growth reaches finite concentration, be removed, be inoculated into and continue amplification in the other bio-reactor.This device can not be regulated and control O flexibly ₂, CO ₂Deng the supply of gas, can not substratum be provided continuously and remove metabolic by-prods simultaneously for reactor, thereby can't keep the suitableeest growing environment of neural stem cell.And the danger of microbiological contamination in addition in the seeded process of neural stem cell.In addition, all extremely not convenient owing to this whole culturing process is carried out in incubator for observation and sampling, and incubator inherent volume has limited the scale of neural stem cell amplification to a certain extent.Generally speaking, this system too limits in design and is unfavorable for operation and practical application, promptly fails to break away from the restriction of incubator, is unfavorable for enlarging the amplification scale, can't synchronous detection; Lack flexibility in design, optimum environment can't be provided for the long-term cultivation of neural stem cell.Therefore will not only expend scientific research personnel's time and efforts with this bioreactor system amplification neural stem cell, and the more important thing is, such device can't be realized the long-term high-density culture continuously to neural stem cell.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of bioreactor system for nerve stem cell to amplify in large scale and long time is provided.Make its restriction of having broken away from incubator, help long-term big amplification scale and synchronous detection, and provide optimum environment for the long-term high-density culture of neural stem cell.

The present invention is achieved by the following technical solutions, the present invention includes: air supply unit, nutrient solution feed unit, cell amplification unit and first peristaltic pump, its mode of connection is: air supply unit is terminal to link to each other with the cell amplification unit by the silica gel pneumatic tube, the nutrient solution feed unit is terminal to link to each other with the cell amplification unit by the silica gel tubing, and first peristaltic pump is in the middle of the silica gel tubing.Air supply unit is imported gas with the suitableeest ratio and flow velocity to the cell amplification unit, and the nutrient solution feed unit is carried substratum by regulating first peristaltic pump to the cell amplification unit, to satisfy the demand of cell to oxygen and nutritive substance according to the needs of cell growth.

Described air supply unit comprises: the CO gas bomb, air compressor, first gas meter, second gas meter, the one 0.2 μ m nuclepore membrane filter, the 2 0.2 μ m nuclepore membrane filter, the gas humidification device, gas humidification device water-bath layer, second peristaltic pump, first water-bath, the 3 0.2 μ m nuclepore membrane filter and T tube, the CO gas bomb links to each other with the one 0.2 μ m nuclepore membrane filter with first gas meter successively by the copper gas pipe line, air compressor links to each other with the 2 0.2 μ m nuclepore membrane filter with second gas meter successively by the copper gas pipe line, the one 0.2 μ m nuclepore membrane filter links to each other with T tube by the silica gel pneumatic tube respectively with the 2 0.2 μ m nuclepore membrane filter, T tube is connected with the air intake of gas humidification device by the silica gel pneumatic tube, gas humidification device outside is surrounded by gas humidification device water-bath layer, the inlet of gas humidification device water-bath layer links to each other with first water-bath by rubber hose respectively with the outlet of gas humidifying device water-bath layer, second peristaltic pump is connected with the 3 0.2 μ m nuclepore membrane filter on the air outlet of gas humidification device on the inlet and the first water-bath intermediary rubber hose of gas humidification device water-bath layer.

Described the 3 0.2 μ m nuclepore membrane filter is connected with the end of air supply unit, and the 3 0.2 μ m nuclepore membrane filter is connected with the cell amplification unit by the silica gel pneumatic tube.

Described nutrient solution feed unit comprises: the 4 0.2 μ m nuclepore membrane filter, container for storing liquid water-bath layer, the 3rd peristaltic pump, second water-bath and container for storing liquid, the ventage of container for storing liquid connects the 4 0.2 μ m nuclepore membrane filter, the container for storing liquid outside is surrounded by container for storing liquid water-bath layer, the outlet of the inlet of container for storing liquid water-bath layer and container for storing liquid water-bath layer links to each other with second water-bath by rubber hose respectively, and the 3rd peristaltic pump is located on the inlet and the second water-bath intermediary rubber hose of container for storing liquid water-bath layer.

The ventage of described container for storing liquid is connected with the end of nutrient solution feed unit, and the ventage of container for storing liquid is connected with the cell amplification unit by the silica gel tubing, is provided with drain pipe on container for storing liquid.

Described cell amplification unit comprises: gas distributor, magnetic stir bar, rolling bottle, rolling bottle water-bath layer, the 4th peristaltic pump, the 3rd water-bath, magnetic stirring apparatus, the on-line monitoring probe, data receiving processor, the 5th peristaltic pump, device for trapping, waste liquid cylinder and the 6th peristaltic pump, the rolling bottle outside is surrounded by rolling bottle water-bath layer, rolling bottle is located on the magnetic stirring apparatus, the entrance and exit of rolling bottle water-bath layer links to each other with the 3rd water-bath by rubber hose respectively, the 4th peristaltic pump is located on the inlet and the 3rd water-bath intermediary rubber hose of rolling bottle water-bath layer, rolling bottle is provided with magnetic stir bar, rolling bottle links to each other with gas distributor by the silica gel pneumatic tube, insert various on-line monitoring probes on the arm mouth of rolling bottle, these on-line monitoring probes link to each other with data receiving processor by data line, another arm mouth of rolling bottle connects device for trapping by the silica gel tubing, the 5th peristaltic pump is connected with device for trapping, link to each other by the silica gel tubing between waste liquid cylinder and the device for trapping, the 6th peristaltic pump is on the silica gel tubing between waste liquid cylinder and the device for trapping.

On the Da Kou of described rolling bottle and two arms, respectively be provided with the non-toxic plastic lid, these three non-toxic plastics cover equal perforation, on big mouthful plastic cover, be provided with inlet pipe, liquid-inlet pipe, cell culture fluid outlet, liquid-inlet pipe is connected with the unitary nutrient solution initiating terminal of cell amplification, inlet pipe is connected with the unitary gas initiating terminal of cell amplification, and an arm mouth of rolling bottle is provided with the cell reflux inlet.

Described device for trapping comprises: the device for trapping ventage, the 5 0.2 μ m nuclepore membrane filter, waste liquid outlet, device for trapping inlet and cell outlet, there is the device for trapping inlet device for trapping upper end, waste liquid outlet and device for trapping ventage, the bottom is provided with cell outlet, also be connected with the 5 0.2 μ m nuclepore membrane filter on the device for trapping ventage, the device for trapping inlet is gone into nearly cell outlet place, device for trapping bottom by outer depth always, the device for trapping inlet, cell outlet and waste liquid outlet are respectively by silica gel tubing and cell culture fluid liquid outlet, the cell reflux inlet links to each other with the waste liquid cylinder, the device for trapping main body becomes hollow cylindrical, and the bottom is parabolic shape.

Principle of work of the present invention is: the gas that the moisture in the gas humidification appliance ensure rolling bottle can constantly not fed is taken away, gas humidification device water-bath layer makes the water that feeds rolling bottle remain on optimum temperature, makes and can well grow to the very fastidious neural stem cell of envrionment conditions.CO ₂Gas bomb and air compressor provide the most suitable 5%CO for the neural stem cell growth ₂With the atmosphere surrounding of 95% air, set up dual 0.2 separately

μ m nuclepore membrane filter guarantees to feed the gas absolutesterility of rolling bottle.The ventage of container for storing liquid makes the inside and outside gaseous tension of container for storing liquid keep constant, the 4 0.2 μ m nuclepore membrane filter is guaranteed can not bring external source to pollute when interior gas of container for storing liquid and ambient atmos exchange, and the outer water bath with thermostatic control of container for storing liquid makes the nutrient solution that flows into rolling bottle also remain at the optimum temperuture that suitable cell is grown.The device for trapping ventage makes the inside and outside air pressure of device for trapping keep balance, and the 5 0.2 μ m nuclepore membrane filter has guaranteed aseptic in the device for trapping.Cell suspension enters from the device for trapping inlet, and cell flows out from cell outlet after sedimentation, and waste liquid is pumped out from waste liquid outlet.The on-line monitoring probe comprises pH meter, thermometer, dissolved oxygen meter, CO ₂Determinators etc. can be monitored the growth conditions of neural stem cell in real time by these devices, are beneficial to the flow velocity of adjustments of gas and substratum.The outer water bath with thermostatic control layer of rolling bottle makes the temperature in the whole process of cell cultures keep constant.Fresh culture constantly pumps into rolling bottle, and waste liquid then is pumped out in device for trapping, and neural ball sinks, return rolling bottle, this maintains under the suitable culture base condition whole culturing process always, and does not have the loss of cell, helps long-term extensive amplification neural stem cell.

When the present invention works, the CO in the air supply unit ₂Mixed in the proper ratio after filtering with air, through humidifying device and after filtering again, enter in the rolling bottle by gas distributor; Nutrient solution in the nutrient solution feed unit is pumped in the rolling bottle by peristaltic pump; Neural stem cell is cultivated in rolling bottle with the form of neural ball, have peristaltic pump with suitable, the utmost point slowly speed cell suspension is pumped in the device for trapping, because of the neural ball of action of gravity sinks, pass back into to continue in the rolling bottle to cultivate; The nutrient solution that enters device for trapping with neural ball then is pumped out in the waste liquid cylinder; The variation of the on-Line Monitor Device real time reaction cell growth conditions in the cell amplification unit, thus the rotating speed of each peristaltic pump regulated.

The invention has the beneficial effects as follows: the growth of neural stem cell provides an adapt circumstance the most, be fit to its long-term extensive stable amplification, comprise that culture condition such as air feed, feed flow and engineering parameter the required software and hardware unit of routine test such as grope and all be incorporated among the complete system.Single parameter or multiparameter were carried out synchronously research in real time when cell growth also was convenient in the unit of this integration, and the firsthand information of neural stem cell growth is provided, and made testing data more reliable, convincing.Have the humidifying device of water-bath and the restriction that container for storing liquid has thoroughly broken away from cell culture incubator, have greater flexibility, Modulatory character and observation; On-line monitoring system provides the variation of cell growing environment in real time, provides foundation for adjusting irrigation rate at any time, rather than just detects the situation of cell culture environment after some days together in sampling; The perfusion culture system that has device for trapping pumps into fresh medium when constantly pumping waste liquid, make culture environment remain on the constant state, cell is sedimentation under the effect of gravity then, comes back in the rolling bottle to continue to cultivate, and is reduced to the loss of cell minimum.These designs all provide advantageous conditions the most for the long-term extensive amplification of neural stem cell.

Description of drawings

Fig. 1 is a structural representation of the present invention

Fig. 2 is an air supply unit structural representation of the present invention

Fig. 3 is a nutrient solution feed unit structural representation of the present invention

Fig. 4 is a cell amplification modular construction synoptic diagram of the present invention

Fig. 5 is a device for trapping structural representation of the present invention

Embodiment

Shown in Fig. 1-5, the present invention includes: air supply unit 1, nutrient solution feed unit 2, cell amplification unit 3 and first peristaltic pump 4, its mode of connection is: the air supply unit 1 terminal silica gel pneumatic tube that passes through links to each other with cell amplification unit 3, the nutrient solution feed unit 2 terminal silica gel tubings that pass through link to each other with cell amplification unit 3, and first peristaltic pump 4 is in the middle of the silica gel tubing.

Described air supply unit 1 comprises: CO ₂Gas bomb 5, air compressor 6, first gas meter 7, second gas meter the 8, the 1 μ m nuclepore membrane filter the 9, the 2 0.2 μ m nuclepore membrane filter 10, gas humidification device 11, gas humidification device water-bath layer 14, second peristaltic pump 17, first water-bath the 18, the 3 0.2 μ m nuclepore membrane filter 19 and T tube 20, CO ₂Gas bomb 5 links to each other with the one 0.2 μ m nuclepore membrane filter 9 with first gas meter 7 successively by the copper gas pipe line, air compressor 6 links to each other with the 2 0.2 μ m nuclepore membrane filter 10 with second gas meter 8 successively by the copper gas pipe line, the one 0.2 μ m nuclepore membrane filter 9 links to each other with T tube 20 by the silica gel pneumatic tube respectively with the 2 0.2 μ m nuclepore membrane filter 10, T tube 20 is connected with the air intake 12 of gas humidification device 11 by the silica gel pneumatic tube, gas humidification device 11 outsides are surrounded by gas humidification device water-bath layer 14, the outlet 16 of the inlet 15 of gas humidification device water-bath layer 14 and gas humidifying device water-bath layer 14 links to each other with first water-bath 18 by rubber hose respectively, second peristaltic pump 17 is connected with the 3 0.2 μ m nuclepore membrane filter 19 on the air outlet 13 of gas humidification device 11 on the inlet 15 and first water-bath, 18 intermediary rubber hoses of gas humidification device water-bath layer 14.

Described the 3 0.2 μ m nuclepore membrane filter 19 is connected with the end of air supply unit 1, and the 3 0.2 μ m nuclepore membrane filter 19 is connected with cell amplification unit 3 by the silica gel pneumatic tube.

Described nutrient solution feed unit 2 comprises: the 4 0.2 μ m nuclepore membrane filter 22, container for storing liquid water-bath layer 24, the 3rd peristaltic pump 27, second water-bath 28 and container for storing liquid 29, the ventage 21 of container for storing liquid 29 connects the 4 0.2 μ m nuclepore membrane filter 22, container for storing liquid 29 outsides are surrounded by container for storing liquid water-bath layer 24, the outlet 26 of the inlet 25 of container for storing liquid water-bath layer 24 and container for storing liquid water-bath layer 24 links to each other with second water-bath 28 by rubber hose respectively, and the 3rd peristaltic pump 27 is located on the inlet 25 and second water-bath, 28 intermediary rubber hoses of container for storing liquid water-bath layer 24.

The ventage 21 of described container for storing liquid 29 is connected with the end of nutrient solution feed unit 2, and the ventage 21 of container for storing liquid 29 is connected with cell amplification unit 3 by the silica gel tubing, is provided with drain pipe 23 on container for storing liquid 29.

Described cell amplification unit 3 comprises: gas distributor 31, magnetic stir bar 32, rolling bottle 33, rolling bottle water-bath layer 34, the 4th peristaltic pump 37, the 3rd water-bath 38, magnetic stirring apparatus 39, on-line monitoring probe 40, data receiving processor 41, the 5th peristaltic pump 42, device for trapping 44, waste liquid cylinder 46 and the 6th peristaltic pump 48, rolling bottle 33 outsides are surrounded by rolling bottle water-bath layer 34, rolling bottle 33 is located on the magnetic stirring apparatus 39, the inlet 35 of rolling bottle water-bath layer 34 links to each other with the 3rd water-bath 38 by rubber hose respectively with outlet 36, the 4th peristaltic pump 37 is located on the inlet 35 and the 3rd water-bath 38 intermediary rubber hoses of rolling bottle water-bath layer, rolling bottle 33 is provided with magnetic stir bar 32, rolling bottle 33 links to each other with gas distributor 31 by the silica gel pneumatic tube, insert various on-line monitoring probes 40 on the arm mouth of rolling bottle 33, these on-line monitorings probe 40 links to each other with data receiving processor 41 by data line, another arm mouth of rolling bottle 33 connects device for trapping 44 by the silica gel tubing, the 5th peristaltic pump 42 is connected with device for trapping 44, link to each other by the silica gel tubing between waste liquid cylinder 46 and the device for trapping 44, the 6th peristaltic pump 48 is on the silica gel tubing between waste liquid cylinder 46 and the device for trapping 44.

On the Da Kou of described rolling bottle 33 and two arms, respectively be provided with the non-toxic plastic lid, these three non-toxic plastics cover equal perforation, on big mouthful plastic cover, be provided with inlet pipe 47, liquid-inlet pipe 30, cell culture fluid outlet 43, liquid-inlet pipe 30 is connected with the nutrient solution initiating terminal of cell amplification unit 3, inlet pipe 47 is connected with the gas initiating terminal of cell amplification unit 3, and an arm mouth of rolling bottle 33 is provided with cell reflux inlet 45.

Described device for trapping 44 comprises: device for trapping ventage 49, the 5 0.2 μ m nuclepore membrane filter 50, waste liquid outlet 51, device for trapping inlet 52 and cell outlet 53, there is device for trapping inlet 52 device for trapping 44 upper ends, waste liquid outlet 51 and device for trapping ventage 49, the bottom is provided with cell outlet 53, also be connected with the 5 0.2 μ m nuclepore membrane filter 50 on the device for trapping ventage 49, device for trapping inlet 52 is gone into nearly cell outlet 53 places, device for trapping 44 bottoms by outer depth always, device for trapping inlet 52, cell outlet 53 and waste liquid outlet 51 are respectively by silica gel tubing and cell culture fluid liquid outlet 43, cell reflux inlet 45 links to each other with waste liquid cylinder 46, device for trapping 44 main bodys become hollow cylindrical, and the bottom is parabolic shape.

After the present invention fixedly mounts, at first open magnetic stirring apparatus 39, the 4th peristaltic pump 37 and data receiving processor 41 in the cell amplification unit 3, the cell in the rolling bottle 33 is under the suitable temperature and stir speed (S.S.) at the very start.Next opens second peristaltic pump 17 and first water-bath 18 in the air supply unit 1, and opens CO ₂Gas bomb 5 and air compressor 6, gas humidification device 11 and water wherein, and the silica gel pneumatic tube in this patent and silica gel tubing all pass through sterilization before use.Mixed gas enters in the rolling bottle 33 by gas distributor 31 after the 3 0.2 μ m nuclepore membrane filter 19 filters through 11 back humidification and the preheatings of gas humidification device.When opening air supply unit 1, start first peristaltic pump 4 and second peristaltic pump 17, the fresh medium of preheating in the container for storing liquid 29 is entered in the rolling bottle 33 with suitable flow velocity.At last, after cell cultures for some time (as 1-2 days), open the 5th peristaltic pump 42, cell suspension in the rolling bottle 33 is pumped in the device for trapping 44, by the gravity settling effect, neural ball and waste liquid are separated, turn back to again and continue in the rolling bottle 33 to cultivate, waste liquid is then pumped in the waste liquid cylinder 46 by the 6th peristaltic pump 48.

Claims

1. A long-term large-scale expansion bioreactor system for neural stem cells, comprising: an air supply unit (1) and a cell expansion unit (3), characterized in that it also includes: a culture solution supply unit (2) and a first The peristaltic pump (4), the end of the gas supply unit (1) is connected to the cell expansion unit (3) through a silicone gas delivery tube, the end of the culture solution supply unit (2) is connected to the cell expansion unit (3) through a silicone delivery tube, the second A peristaltic pump (4) is in the middle of the silicone infusion tube;

The gas supply unit (1) consists of a _CO2 gas cylinder (5), an air compressor (6), a first gas flow meter (7), a second gas flow meter (8), a first 0.2 μm microporous filter Membrane filter (9), second 0.2 μm microporous membrane filter (10), gas humidifier (11), gas humidifier water bath layer (14), second peristaltic pump (17), first water bath pot (18), the third 0.2 μm microporous membrane filter (19) and T-shaped tee pipe (20), the _CO2 gas cylinder (5) is connected to the first gas flow meter (7) through the copper gas pipeline in sequence ) is connected with the first 0.2 μm microporous membrane filter (9), and the air compressor (6) is connected with the second gas flowmeter (8) and the second 0.2 μm microporous membrane filter ( 10) are connected, the first 0.2 μm microporous membrane filter (9) and the second 0.2 μm microporous membrane filter (10) are connected to each other with the T-shaped three-way pipe (20) through the silica gel air pipe respectively, and the T-shaped three-way The through pipe (20) is connected to the air inlet (12) of the gas humidification device (11) through a silica gel gas delivery pipe, the gas humidification device (11) is wrapped with a gas humidification device water bath layer (14), and the gas humidification device water bath The inlet (15) of the layer (14) and the outlet (16) of the gas humidification device water bath layer (14) are respectively connected with the first water bath (18) by rubber tubes, and the second peristaltic pump (17) is connected to the gas humidification device. On the rubber tube between the inlet (15) of the water bath layer (14) and the first water bath (18), the gas outlet (13) of the gas humidification device (11) is connected with a third 0.2 μm microporous membrane filter (19).

2. neural stem cell long-term large-scale expansion bioreactor system according to claim 1, is characterized in that, the end of described the 3rd 0.2 μm microporous membrane filter (19) and air supply unit (1) Connected with each other, the third 0.2 μm microporous membrane filter (19) is connected with the cell expansion unit (3) through a silica gel air tube.

3. the neural stem cell long-term large-scale expansion bioreactor system according to claim 1, is characterized in that, described culture solution supply unit (2) comprises: the 4th 0.2 μm microporous membrane filter (22) , liquid storage tank water bath layer (24), the third peristaltic pump (27), the second water bath (28) and liquid storage tank (29), the vent (21) of liquid storage tank (29) connects the fourth 0.2 μm The microporous membrane filter (22), the liquid storage tank (29) is externally wrapped with a liquid storage tank water bath layer (24), the inlet (25) of the liquid storage tank water bath layer (24) and the liquid storage tank water bath layer (24) The outlets (26) of the outlets (26) are respectively connected to each other with the second water bath (28) through rubber tubes, and the third peristaltic pump (27) is arranged in the middle of the inlet (25) of the water bath layer (24) of the liquid storage tank and the second water bath (28) on the rubber tube.

4. the neural stem cell long-term large-scale expansion bioreactor system according to claim 3, is characterized in that, the vent (21) of described liquid storage tank (29) and the end of culture solution supply unit (2) The vent (21) of the liquid storage tank (29) is connected with the cell expansion unit (3) through a silicone infusion tube, and a liquid outlet pipe (23) is provided on the liquid storage tank (29).

5. neural stem cell long-term large-scale expansion bioreactor system according to claim 1, is characterized in that, described cell expansion unit (3) comprises: gas distributor (31), magnetic stirring bar (32) , rotating bottle (33), rotating bottle water bath layer (34), the fourth peristaltic pump (37), the third water bath (38), magnetic stirrer (39), on-line monitoring probe (40), data receiving processor ( 41), the fifth peristaltic pump (42), the retaining device (44), the waste liquid tank (46) and the sixth peristaltic pump (48), the rotating bottle (33) is wrapped with a rotating bottle water bath layer (34), and the rotating bottle (33) be located on the magnetic stirrer (39), the inlet (35) and the outlet (36) of the bottle water bath layer (34) link to each other with the 3rd water bath (38) by rubber tube respectively, the 4th peristaltic pump ( 37) On the rubber tube between the entrance (35) of the water bath layer of the spinner bottle and the third water bath (38), the spinner bottle (33) is provided with a magnetic stirring rod (32), and the spinner bottle (33) is transported through silica gel. The trachea is connected to the gas distributor (31), and various on-line monitoring probes (40) are connected to an arm port of the rotating bottle (33), and these on-line monitoring probes (40) are connected to the data receiving processor (41) through data lines, The other arm port of the rotating bottle (33) is connected to the retaining device (44) through a silicone infusion tube, the fifth peristaltic pump (42) is connected to the retaining device (44), and the waste liquid cylinder (46) and the retaining device (44) are connected to each other. Connect to each other by a silicone infusion tube, and the sixth peristaltic pump (48) is on the silicone infusion tube between the waste liquid cylinder (46) and the retaining device (44).

6. The long-term large-scale expansion bioreactor system for neural stem cells according to claim 5 is characterized in that a non-toxic plastic cover is respectively provided on the big mouth and the two arms of the spinner bottle (33), and the three Holes are all arranged on each nontoxic plastic cover, and air inlet pipe (47), liquid inlet pipe (30), cell culture solution outlet (43), liquid inlet pipe (30) and cell expansion unit are arranged on the plastic cover of big mouth. The initial end of the culture solution of (3) is connected, the air inlet pipe (47) is connected with the gas initial end of the cell expansion unit (3), and an arm mouth of the spinner bottle (33) is provided with a cell return inlet (45).

7. The long-term large-scale expansion bioreactor system for neural stem cells according to claim 5, characterized in that, said retaining device (44) comprises: retaining device vent (49), the fifth 0.2 μm microporous filter Membrane filter (50), waste liquid outlet (51), retention device inlet (52) and cell outlet (53), retention device (44) upper end has retention device inlet (52), waste liquid outlet (51) and retention device Air vent (49), the bottom end is provided with cell outlet (53), is also connected with the fifth 0.2 μm microporous membrane filter (50) on the intercepting device vent (49), and retaining device inlet (52) is opened from the outside all the time. Deep into the bottom of the retaining device (44) near the cell outlet (53), the retaining device inlet (52), the cell outlet (53) and the waste liquid outlet (51) pass through the silica gel infusion tube and the cell culture solution outlet (43) respectively. ), the cell return inlet (45) is connected to the waste liquid cylinder (46), and the main body of the retaining device (44) becomes a hollow cylinder with a parabolic bottom.