CN108441424A - A kind of microcarrier biological reactor and its culture porcine circovirus 2 type method - Google Patents
A kind of microcarrier biological reactor and its culture porcine circovirus 2 type method Download PDFInfo
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Abstract
A kind of method that the present invention discloses microcarrier biological reactor and its cultivates porcine circovirus 2 type, the jacket water (J.W.) that tank wall installs heating tube heating interlayer equipped with interlayer and in interlayer carry out liquid in heating tank body；Telescoping tube, can be easy to operate according to the big minor adjustment sample position of culture carrier amount for sampling in tank body；It installs additional and is connect in observation and tank body with transparent observing area, cooperation is corresponding to cross air filtration head and switching valve body, can flow into transparent pipe carrier cell, set and observe cell state under microscope at any time, and not waste remaining return in tank after observing；One carrier filter screen is installed in the bottom sides of retort, coordinates snorkel, when final antigen removes carrier in harvest, avoids carrier plug filter net.The present invention improves microcarrier biological reactor culture apparatus, facilitates tank inner cell to observe in real time, facilitates removal waste fluid, facilitates filtration supports, improves efficiency, improves cell density for 24 hours, the better cell of cultivation conditions.
Description
Technical field
The present invention relates to biotechnology more particularly to a kind of microcarrier biological reactor and its culture pig circular ring virus
The method of 2 types.
Background technology
Traditional microcarrier biological reactor passes through heating to the mode that the mode of heating of pot liquid is electric blanket heating
Electric blanket, electric blanket are attached to outside reaction tank skin, are heated to pot liquid by heat transfer, the electric blanket temperature when just beginning to warm up
It is very high, it is in direct contact tank surface, causes temperature on the wall in tank excessively high, the cell that can be influenced on the carrier in tank is adherent and raw
It is long.
The length of probe tube is fixed in tank, when the culture of tank inner cell to for 24 hours after need to change liquid, the nutrition in drain tank
Then new nutrient solution is added in liquid, when arranging nutrient solution in tank, need first to adjust sampling length of tube before retort assembling sterilizing,
The length of adjusting can only be estimated according to us using carrier amount, and adjust in advance it is inaccurate, cannot thoroughly draining liq or
Carrier is discharged, Component Vectors are lost.
It needs to take out carrier cell when observing the cell in tank on carrier and goes to observe outside tank, the relatively more tired lock of operation, shadow
The virtual condition for ringing observation cell needs all culture solutions to include that carrier discharges outside the tank, then again when harvesting final antigen
The antigen harvested by one of filter screen filtration is with the mixed liquor of carrier, and time-consuming and laborious, carrier cell takes out and goes to observe outside tank, and one
Aspect wastes carrier cell, and another aspect cell will produce some variations after taking out, and the cell state observed has deviation.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of microcarrier biological reactor and its culture pigs
The method of circovurus type 2.
Method improves microcarrier biological reactor culture apparatus, facilitates tank inner cell to observe in real time, facilitate removal waste fluid, side
Just filtration supports improve efficiency, improve adherence rate, improve cell density for 24 hours, the better cell of cultivation conditions.
The technical solution adopted by the present invention is：
A kind of microcarrier biological reactor comprising tank body, tank body is interior to be equipped with blender, biological respinse detection electrode, defoaming dress
It sets, the upper surface of tank body is correspondingly provided with blender installing port, electrode interface and defoaming device interface, blender is in tank body
Liquid is stirred, and the bottom of tank body is equipped with outlet, and outlet extension has discharge pipe, discharge pipe to be equipped with discharge valve, tank
The inner wall of body corresponds to outlet and goes out equipped with filter screen,
Tank body upper surface is additionally provided with intake interface and liquid material interface, and intake interface is used to be passed through gas, liquid to tank body different location
Material interface includes fluid infusion, sample tap, more than two feed inlets, and fluid infusion is used to supplement culture solution, feed inlet in incubation
For adding culture material into tank body,
Adjusting rod is installed, one end of adjusting rod is stretched into tank body, and the other end of adjusting rod is arranged with stop collar, limit in sample tap
Position set limits the other end of adjusting rod in sample tap, and the length of tank body is stretched by adjusting the position change adjusting rod of stop collar
Degree, is arranged with telescoping tube on one end of adjusting rod, and one end of telescoping tube is fixed on one end end of adjusting rod, telescoping tube it is another
End connection probe tube,
Probe tube is equipped with the first air filtration head and first switch valve body, first switch valve body excessively and is located at the first air filtration head correspondence excessively and takes
One end of sample mouth, the other end of probe tube are separately connected one end of the second branch pipe and observation tube, and the second branch pipe other end, which is equipped with, to be received
Collect bottle, receiving flask is equipped with the 4th and crosses air filtration head, and the second branch pipe is equipped with the 4th switching valve body；Observation tube is in horizontally disposed, sight
Examining pipe, there is a transparent observing area, the other end of observation tube to be connected with second by third switching valve body and cross air filtration head；
The side wall of tank body has interlayer space, is filled with jacket water (J.W.) in interlayer space, interlayer space adds jacket water (J.W.) equipped with heating tube
Heat, the lateral surface of the side wall of tank body are equipped at intervals with the through-hole of two connection interlayer spaces, and two through-holes are respectively connected to a circulating pump
Both ends.
Further, intake interface includes respirator interface, deep layer intake interface, surface layer intake interface and strainer air inlet connecting
Mouthful, a snorkel is passed through in tank body by strainer intake interface and opposite filter screen outlet.
Further, blender includes agitating shaft, and multiple agitating paddles are distributed on agitating shaft, and blender installing port is set to tank
At the center of the upper surface of body, agitating shaft installing port is fixed in one end of agitating shaft, and agitating shaft constitutes blender with agitating paddle, stirs
It is in be vertically arranged in tank body to mix device.
Further, the other end of observation tube is also connected with one end of the first branch pipe, and the first branch pipe is equipped with second switch valve
The other end of body, the first branch pipe is equipped with the spare bottle that keeps sample, and the spare bottle that keeps sample crosses air filtration head equipped with third.
Further, probe tube, the first branch pipe and the second branch pipe are hose, first switch valve body, second switch valve body,
Third switching valve body and the 4th switching valve body are gas folder；Alternatively, probe tube, the first branch pipe and the second branch pipe are stereoplasm tube
Road, first switch valve body, second switch valve body, third switching valve body and the 4th switching valve body are air valve.
The invention also discloses a kind of method of the microcarrier biological reactor culture porcine circovirus 2 type described in application,
It is characterized in that：It includes the following steps：
Step 1, biological reactor opens preheating：
PH electrodes, temperature electrode, the dissolved oxygen electrode of biological reactor are connected, chuck is added into the interlayer space of tank body
Water connects heating tube and opens power supply heating, starts circulating pump and recycle the jacket water (J.W.) in interlayer space so that tank body temperature
Degree is uniform；
Step 2, rolling bottle cell dissociation：
The good rolling bottle PK-15 cells of cell state are selected, pancreatin digestive juice vitellophag are used in combination, then postdigestive cell is filled
Divide and shakes up；
Step 3, cell count：It takes appropriate postdigestive cell to be diluted with culture solution, cell after appropriate dilution is taken after dilution
It adds to cell counting board and carries out cell count, calculate the cell density of cell liquid；
Step 4, tank culture on cell：Addition microcarrier is risen by 3-5g/ into biological reactor, by 30-50 cell/microcarrier
Add cell；Adjust the cell culture parameter of biological reactor respective devices：Aerating system is opened by intake interface to biology
Retort carries out ventilation culture；
Step 5, liquid is changed for 24 hours：When in biological reactor cell culture to for 24 hours after, stop stirring, stand 3-5 minute, adjust stretch
Infratubal port sampling height makes flexible infratubal port sampling height at carrier ullage 0.5cm, cell culture fluid is discharged,
Then the cell maintenance medium for equivalent being added by fluid infusion continues to cultivate；
Step 6, cell observation：First switch valve body and third switch are opened after cell culture is to 48h, while being kept stirring
Valve body closes second switch valve body and the 4th switching valve body, and air-breathing in syringe, biological respinse tank are used crossing air filtration head
Carrier and cell stop pumping when flowing through transparent observing area, close first switch valve body, cell observation area placed micro-
Under the microscope, after observation, open first switch valve body K1, inflated in crossing air filtration head using syringe, by transparent observing area and
The carrier of associated conduit part is squeezed into via original routing sample tap in tank body with cell；
Step 7, antigen is harvested for the first time：After cell culture is to 72h, stop stirring, stand 3-5 minutes, adjusting, which takes, to make to stretch
Infratubal port sampling height to places 0.5cm above carrier in tank, collect first time antigen and will harvest antigen liquid set -15 spend under with
Refrigerator-freezer freezen protective, the cell maintenance medium that equivalent is then added by fluid infusion continue to cultivate；
Step 8, after-crop antigen and liquid culture is changed：After biological reactor cell culture is to 96h, stop stirring, stands
3-5 minutes, adjusting made above flexible infratubal port sampling height to carrier in tank at 0.5cm, to harvest training again by sample tap
Supernatant antigen liquid is supported, the second antigen liquid of harvest is set into -15 degree or less refrigerator-freezer freezen protective；
Step 9, third time harvest antigen：After the cell culture in biological reactor is to 96-120h, adjusting revolution is 100-
150 turns 3-5 minutes, then stop stirring, stand 3-5 minute, by aerating system from intake interface to tank body ventilation pressurization, open
The discharge valve opened on discharge pipe harvests filtered antigen, while opening the snorkel air inlet above filter screen, will finally receive
The third time antigen liquid obtained sets -15 degree or less refrigerator-freezer freezen protective.
Further, in step 2 digestion of cell is carried out using 0.25% pancreatin digestive juice.
Further, the specific method of the cell culture parameter of respective devices is adjusted in step 4：It is adjusted by temperature electrode
Temperature is maintained at 36.5-37.5 DEG C in tank, and adjusting dissolved oxygen by dissolved oxygen electrode is maintained at 50%-60%, passes through PH electrode tune
Section PH is maintained at 7.2-7.4, and the revolution for adjusting blender keeps 40-60r/min.
Further, the specific method of cell culture fluid is discharged in step 5：Open first switch valve body and the 4th switch valve
Body closes second switch valve body and third switching valve body, and aerating system is ventilated to tank body by intake interface and pressurizeed, and waste liquid is used
Bottle is used as receiving flask, and since pressure is more than the pressure outside tank body in tank body, the culture solution of carrier ullage 0.5cm or more is being pressed
Probe tube is flowed through by telescoping tube under the action of power and constantly flows into receiving flask, until the liquid level of culture solution is less than flexible infratubal port
Stop when sampling height；It is crossing air filtration head respectively while closing first switch valve body at this time and is using syringe crossing air filtration head
It inflates, the cell culture fluid in transparent observing area and associated conduit part is squeezed into receiving flask.
Further, the specific method of first time antigen and first time antigen is collected：Open first switch valve body and the 4th
Switching valve body, closes second switch valve body and third switching valve body, and aerating system is ventilated to tank body by intake interface and pressurizeed, made
Use antigen bottle as receiving flask, since pressure is more than the pressure outside tank body, the culture of carrier ullage 0.5cm or more in tank body
Liquid flows through probe tube by telescoping tube under the effect of the pressure and constantly flows into receiving flask, until the liquid level of culture solution is less than telescoping tube
Lower port stops when sampling height；It is crossing air filtration head respectively while closing first switch valve body at this time and is being used crossing air filtration head
Syringe is inflated, and the cell culture fluid in transparent observing area and associated conduit part is squeezed into receiving flask.
The present invention uses above technical scheme, and it is two layers that the mode of heating of biological respinse pot liquid, which is designed as retort,
Centre has chuck to install heating tube, and the water of chuck is constantly recycled by peristaltic pump, the liquid in the water heating tank of chuck.Sampling
Pipe pipe is raised to position appropriate in removal waste fluid when culture carrier amount is big using telescopic pipe in tank inside points, when
When culture carrier amount is big, pipe is dropped into position appropriate in removal waste fluid, carrier is unlikely to be discharged in this way.In tank outer portion
Probe tube connect a device, stop when carrier cell flows through device, this device is placed on microscope observe it is cellular
State.Remaining return in tank can not be wasted after observing simultaneously.One carrier filter screen is installed in the bottom sides of retort,
There are one snorkels for dress above filter screen, when in harvest, final antigen removes carrier, the gas above unlatching filter screen,
It is unlikely to carrier plug filter net when filtering, filtering is unobstructed.The invention has the advantages that：1, sample tap is designed to rise in tank
Falling tone section, the height of sample tap can be adjusted according to the height of liquid level of carrier amount in removal waste fluid, is conveniently operated while can arrange
Liquid is more thorough.2, heating pot liquid uses heating tube heating tank internal jacket water, flows jacket water (J.W.), jacket water (J.W.) water by pump circulation
Bath heating pot liquid, such pot liquid temperature is uniform, and will not be locally too high.3, observation cell passes through installation outside tank online
Connect in one pipeline and tank, a transparent glass tube customized is installed additional in tank Outer Tube, it can be by suction by the load in tank
Body cell flows into transparent pipe, sets and observes cell state under microscope at any time, while can be returned remaining in tank after observing
It does not waste.4, a filtering mouth is filled in tank bottom side, filtering mouth is equipped with carrier filter screen, can pass through filtering when harvesting antigen liquid
Net filtration removes carrier, and carrier is easy filter screen to block in filter process, we install an air inlet additional above filter screen
Pipe opens ventilation in filter process, can drain the carrier of filter screen and be unlikely to plug filter net, and much smoother harvest is anti-
It is former.
Being filtered again after previous harvest carrier and antigen needs 2-3 people to operate, on-line filtration when harvesting antigen after applying the present invention
Carrier only needs 1 people to operate, and improves efficiency, saves labour.The present invention improves microcarrier biological reactor culture apparatus, facilitates tank
Inner cell is observed in real time, facilitates removal waste fluid, facilitates filtration supports, improves efficiency, is improved adherence rate, is improved cell for 24 hours
Density, the better cell of cultivation conditions.
Description of the drawings
The present invention is described in further details below in conjunction with the drawings and specific embodiments；
Fig. 1 is a kind of structural schematic diagram of microcarrier biological reactor of the present invention；
Fig. 2 is a kind of schematic cross-sectional view of microcarrier biological reactor of the present invention.
Specific implementation mode
As shown in the figures 1 and 2, the present invention discloses a kind of microcarrier biological reactor comprising tank body 1, interior be equipped with of tank body 1 are stirred
Mix device, biological respinse detection electrode, defoaming device, the upper surface of tank body 1 be correspondingly provided with blender installing port 11, electrode interface and
Defoaming device interface 12, blender are stirred for liquid in tank body 1, and the bottom of tank body 1 is equipped with outlet 13, outlet 13
Extension has discharge pipe 2, discharge pipe 2 to be equipped with discharge valve 21, and the inner wall of tank body 1 corresponds to outlet 13 and goes out equipped with filter screen
22,
1 upper surface of tank body is additionally provided with intake interface and liquid material interface, and intake interface is used to be passed through gas to 1 different location of tank body,
Liquid material interface includes fluid infusion 14, sample tap 15, more than two feed inlets 10, and fluid infusion 14 is used to supplement culture in incubation
Liquid, feed inlet 10 are used for the addition culture material into tank body 1,
Adjusting rod 3 is installed, one end of adjusting rod 3 is stretched into tank body 1, and the other end of adjusting rod 3 is arranged limited in sample tap 15
Position set 31, stop collar 31 limits the other end of adjusting rod 3 in sample tap 15, and by adjusting the position change tune of stop collar 31
Pole 3 stretches into the length of tank body 1, and telescoping tube 4 is arranged on one end of adjusting rod 3, and one end of telescoping tube 4 is fixed on adjusting rod 3
One end end, the other end of telescoping tube 4 connects probe tube 5,
Probe tube 5 is equipped with first and crosses air filtration head F1 and first switch valve body K1, and first switch valve body K1 is located at first and crosses air filtration
One end of head F1 corresponding samples mouth 15, the other end of probe tube 5 are separately connected one end of the second branch pipe 30 and observation tube 6, and second
30 other end of branch pipe is equipped with receiving flask 40, and receiving flask 40 is equipped with the 4th and crosses air filtration head F4, and the second branch pipe 30 is opened equipped with the 4th
Close valve body K4；In being horizontally disposed with, there is observation tube 6 observation tube 6 a transparent observing area 61, the other end of observation tube 6 to pass through third
Switching valve body K3 is connected with second and crosses air filtration head F2；
The side wall of tank body 1 has interlayer space, and jacket water (J.W.) is filled in interlayer space, and interlayer space is equipped with heating tube 60 to jacket water (J.W.)
Heating, the lateral surface of the side wall of tank body 1 are equipped at intervals with the through-hole 22 of two connection interlayer spaces, and two through-holes 22 are respectively connected to one
The both ends of circulating pump 23.
Further, biological respinse detection electrode includes PH electrodes, temperature electrode and dissolved oxygen electrode, and electrode interface includes PH
Electrode interface 17, temperature electrode interface 18 and dissolved oxygen electrode interface 19.
Further, intake interface includes respirator interface 24, deep layer intake interface 25 and surface layer intake interface 26.
Further, intake interface further includes strainer intake interface 27, and a snorkel 50 is logical by strainer intake interface 27
Enter 22 outlet of interior and opposite filter screen of tank body 1.
Further, blender includes agitating shaft 28, and multiple agitating paddles 29, blender installing port are distributed on agitating shaft 28
11 are set at the center of the upper surface of tank body 1, and 28 installing port of agitating shaft, agitating shaft 28 and stirring are fixed in one end of agitating shaft 28
Paddle 29 constitutes blender, and blender is in be vertically arranged in tank body 1.
Further, the other end of receiving flask observation tube 6 is also connected with one end of the first branch pipe 7, and the first branch pipe is equipped with the
The other end of two switching valve body K2, the first branch pipe 7 are equipped with the spare bottle 9 that keeps sample, and the spare bottle 9 that keeps sample crosses air filtration head equipped with third
F3.When the spare bottle 9 that keeps sample being set needing small-lot testing, the spare bottle 9 that keeps sample is used to collect sample.
Further, probe tube 5, the first branch pipe 7 and the second branch pipe 30 are hose, and first switch valve body K1, second open
It is gas folder to close valve body K2, third switching valve body K3 and the 4th switching valve body K4.
Further, probe tube 5, the first branch pipe 7 and the second branch pipe 30 are hard pipeline, first switch valve body K1,
Two switching valve body K2, third switching valve body K3 and the 4th switching valve body K4 are air valve.
Further, the present invention further discloses a kind of application microcarrier biological reactor culture 2 porcine circovirus
The method of type comprising following steps：
Step 1, biological reactor opens preheating：The PH of biological reactor, temperature, dissolved oxygen electrode are connected, retort has been added
The water of chuck connects heating tube 60 and opens power supply heating, and open the circulation pump 23, which allows the water in tank to carry out cycle, keeps temperature equal
It is even.
Step 2, rolling bottle cell dissociation：The good rolling bottle PK-15 cells of cell state are selected, are disappeared using 0.25% pancreatin
Change liquid vitellophag, postdigestive cell fully shakes up.
Step 3, cell count：Appropriate postdigestive cell is taken to be diluted with maintaining liquid, after appropriate dilution is taken after dilution
Cell adds to cell counting board and carries out cell count, calculates the cell density of cell liquid.
Step 4, tank culture on cell：Addition microcarrier is risen by 3-5g/ into biological reactor, by 30-50 cell/micro-
Carrier adds cell；
Cell is used according to needed for cell density calculating after the quantity of microcarrier, every glomus cell number and rolling bottle digestion in retort
Required cell concentration is added by cell bottle pipeline corresponding with feed inlet in biological reactor amount；Adjust biological reactor phase
Close the cell culture parameter of device：Temperature：36.5-37.5 DEG C, DO：50%-60%, PH：7.2-7.4, revolution：40-60r/
Min selects aerating system pattern (Air/O2/CO2), opens aerating system and is ventilated to biological reactor by intake interface
Culture.
Step 5, liquid is changed for 24 hours：When in biological reactor cell culture to for 24 hours after, stop stirring, stand 3-5 minute, adjusting
4 lower port of telescoping tube sampling height makes 4 lower port of telescoping tube sampling height at carrier ullage 0.5cm, discharge cell training
Nutrient solution, the cell maintenance medium that equivalent is then added by fluid infusion 14 continue to cultivate.
The specific method of cell culture fluid is discharged in step 5：Open first switch valve body K1, third switching valve body K3 and the
Four switching valve body K4 close second switch valve body K2, and aerating system is ventilated to tank body by intake interface and pressurizeed, and waste liquid bottle is used
As receiving flask, since pressure is more than the pressure outside tank body in tank body, the culture solution of carrier ullage 0.5cm or more is in pressure
Under the action of probe tube flowed through by telescoping tube constantly flow into receiving flask, until the liquid level of culture solution is less than 4 lower port of telescoping tube
Stop when sampling height；It is crossing air filtration head F1 respectively while closing first switch valve body K1 at this time and is being used crossing air filtration head F2
Syringe is inflated, and the cell culture fluid in transparent observing area 61 and associated conduit part is squeezed into receiving flask.
Step 6, cell observation：After cell culture is to 48h, first switch valve body K1 and the is opened while being kept stirring
Three switching valve body K3 close second switch valve body K2 and the 4th switching valve body K4, the air-breathing in crossing air filtration head F2 and using syringe,
The carrier and cell of biological respinse tank 1 stop pumping when flowing through transparent observing area 61, close first switch valve body K1, will
Microscopically observation is placed in cell observation area, after observation, first switch valve body K1 is opened, using syringe in crossing air filtration head F2
It inflates, the carrier of transparent observing area 61 and associated conduit part and cell is squeezed by the sampled mouth in former road in tank body.
Step 7, antigen is harvested for the first time：After cell culture is to 72h, stop stirring, stand 3-5 minutes, adjusting, which takes, to be made
Above 4 lower port of telescoping tube sampling height to carrier in tank at 0.5cm, collects first time antigen and harvest antigen liquid is set -15
With refrigerator-freezer freezen protective under degree, the cell maintenance medium that equivalent is then added by fluid infusion 14 continues to cultivate.
Collect the specific method of first time antigen：First switch valve body K1 and the 4th switching valve body K4 is opened, closes second
Switching valve body K2 and third switching valve body K3, aerating system by intake interface to tank body ventilate pressurize, using antigen bottle as
Receiving flask, since pressure is more than the pressure outside tank body, the work of the culture solution of carrier ullage 0.5cm or more in pressure in tank body
It probe tube is flowed through by telescoping tube is constantly flowed into receiving flask with lower, until the liquid level of culture solution is sampled less than 4 lower port of telescoping tube
Stop when height；It is crossing air filtration head F1 respectively while closing first switch valve body K1 at this time and is using injection crossing air filtration head F2
Device is inflated, and the cell culture fluid in transparent observing area 61 and associated conduit part is squeezed into receiving flask.
Step 8, after-crop antigen and liquid culture is changed：After biological reactor cell culture is to 96h, stop stirring,
3-5 minutes are stood, adjusting makes above 4 lower port of telescoping tube sampling height to carrier in tank at 0.5cm, again by sample tap 15
Culture supernatant antigen liquid is harvested, the second antigen liquid of harvest is set under -15 degree with refrigerator-freezer freezen protective.
Collect the specific method of second of antigen：First switch valve body K1 and the 4th switching valve body K4 is opened, closes second
Switching valve body K2 and third switching valve body K3, aerating system by intake interface to tank body ventilate pressurize, using antigen bottle as
Receiving flask, since pressure is more than the pressure outside tank body, the work of the culture solution of carrier ullage 0.5cm or more in pressure in tank body
It probe tube is flowed through by telescoping tube is constantly flowed into receiving flask with lower, until the liquid level of culture solution is sampled less than 4 lower port of telescoping tube
Stop when height；It is crossing air filtration head F1 respectively while closing first switch valve body K1 at this time and is using injection crossing air filtration head F2
Device is inflated, and the cell culture fluid in transparent observing area 61 and associated conduit part is squeezed into receiving flask.
Step 9, third time harvest antigen：After the cell culture in biological reactor is to 96-120h, adjusting revolution is
100-150 turns 3-5 minutes, then stops stirring, stands 3-5 minutes, is added from intake interface to tank body ventilation by aerating system
Pressure, open discharge pipe 2 on discharge valve 21 harvest filtered antigen, while open the snorkel 50 above filter screen into
Gas ensures to harvest filtered antigen and carries out the detection antigen valence that keeps sample, smoothly harvests antigen, the third that will finally harvest
Secondary antigen liquid is set under -15 degree with refrigerator-freezer freezen protective.
A kind of method of the application microcarrier biological reactor culture porcine circovirus 2 type of the present invention has the following advantages that：
1, the adherent efficiency of cell is improved, adherent rear cell density is increased.Using neoteric retort culture cell, can improve
The adherent efficiency of cell, adherent rate can keep 95% or more, and the dead cell of liquid is less during culture, be improved after adherent
Cell density, for 24 hours cell density be above 2.00 × 106A/ml.
Table one be the present invention using the adherence rate of the volume of 10 liters of 5 liters of microcarrier biological reactor culture and
Cell density for 24 hours corresponds to the contrast table of 5 batches of experiments.
Table one
2, it is conveniently operated time saving, saving consumptive material.Sample tap can be directly adjusted in removal waste fluid using neoteric retort
Height, it is convenient and efficient, it is not easy to carrier discharge do not waste material, cell state can be observed online, carrier is thin after observation
Born of the same parents are again reflowable to be observed in tank, need not taking out tank, and carrier and cell are not wasted.
The present invention uses the carrier cells of 5 batches of experiments of difference of the cell of 10 liters of 5 liters of microcarrier biological reactor culture
Loss late is almost nil, can be neglected.
3, efficiency is improved, labour is saved.Being filtered again after previous harvest carrier and antigen needs 2-3 people to operate, using this hair
On-line filtration carrier only needs 1 people to operate when harvest antigen after bright, improves efficiency, can save 1 hour time, saves labour.
The present invention uses above technical scheme, and it is two layers that the mode of heating of biological respinse pot liquid, which is designed as retort,
Centre has chuck to install heating tube 60, and the water of chuck is constantly recycled by peristaltic pump, the liquid in the water heating tank of chuck.It takes
Pipe is raised to position appropriate by the pipe of sample when culture carrier amount is big using telescopic pipe in tank inside points in removal waste fluid,
When culture carrier amount is big, pipe is dropped into position appropriate in removal waste fluid, carrier is unlikely to be discharged in this way.In tank outer portion
Probe tube 5 connect a device, stop when carrier cell flows through device, this device be placed on microscope and observes cell
State.Remaining return in tank can not be wasted after observing simultaneously.One carrier filtering is installed in the bottom sides of retort
Net, there are one snorkels 50 for dress above filter screen, when in harvest, final antigen removes carrier, the gas above unlatching filter screen
Body, is unlikely to carrier plug filter net in filtering, and filtering is unobstructed.The invention has the advantages that：1, sample tap 15 is set in tank
Liftable adjusting is counted into, the height of sample tap 15 can be adjusted according to the height of liquid level of carrier amount in removal waste fluid, be conveniently operated
It simultaneously can be more thorough with drain.2, heating pot liquid uses 60 heating tank internal jacket water of heating tube, is flowed and is pressed from both sides by pump circulation
Water is covered, jacket water (J.W.) heating water bath pot liquid, such pot liquid temperature is uniform, and will not be locally too high.3, online observation is thin
Born of the same parents are connected by being installed additional in a pipeline and tank outside tank, are installed the transparent glass tube of a customization additional in tank Outer Tube, can be passed through
Suction flows into the carrier cell in tank in transparent pipe, sets and observes cell state under microscope at any time, while can be incited somebody to action after observing
Remaining return in tank does not waste.4, a filtering mouth is filled in tank bottom side, filtering mouth is equipped with carrier filter screen, in harvest antigen
Carrier can be removed by filter screen filtration when liquid, carrier is easy filter screen to block in filter process, we are on filter screen
Side installs an air inlet pipe additional, and ventilation is opened in filter process, can drain the carrier for filtering mouth and be unlikely to plug filter mouth, very
Smoothly harvest antigen.Being filtered again after previous harvest carrier and antigen needs 2-3 people to operate, when harvesting antigen after applying the present invention
Line filtration supports only need 1 people to operate, and improve efficiency, save labour.The present invention improves microcarrier biological reactor culture apparatus,
Facilitate tank inner cell to observe in real time, facilitate removal waste fluid, facilitate filtration supports, improve efficiency, improve adherence rate, improves for 24 hours
Cell density, the better cell of cultivation conditions.
Claims (10)
1. a kind of microcarrier biological reactor, it is characterised in that：It includes tank body, blender is equipped in tank body, biological respinse is visited
Electrode, defoaming device are surveyed, the upper surface of tank body is correspondingly provided with blender installing port, electrode interface and defoaming device interface, stirring
Device is stirred for liquid in tank body, and the bottom of tank body is equipped with outlet, and outlet extension has discharge pipe, discharge pipe to be equipped with
Discharge valve, the inner wall of tank body, which corresponds to, is equipped with filter screen at outlet,
Tank body upper surface is additionally provided with intake interface and liquid material interface, and intake interface is used to be passed through gas, liquid to tank body different location
Material interface includes fluid infusion, sample tap, more than two feed inlets, and fluid infusion is used to supplement culture solution, feed inlet in incubation
For adding culture material into tank body,
Adjusting rod is installed, one end of adjusting rod is stretched into tank body, and the other end of adjusting rod is arranged with stop collar, limit in sample tap
Position set limits the other end of adjusting rod in sample tap, and the length of tank body is stretched by adjusting the position change adjusting rod of stop collar
Degree, is arranged with telescoping tube on one end of adjusting rod, and one end of telescoping tube is fixed on one end end of adjusting rod, telescoping tube it is another
One end connects probe tube,
Probe tube is equipped with the first air filtration head and first switch valve body, first switch valve body excessively and is located at the first air filtration head correspondence excessively and takes
One end of sample mouth, the other end of probe tube are separately connected one end of the second branch pipe and observation tube, and the second branch pipe other end, which is equipped with, to be received
Collect bottle, receiving flask is equipped with the 4th and crosses air filtration head, and the second branch pipe is equipped with the 4th switching valve body；Observation tube is in horizontally disposed, sight
Examining pipe, there is a transparent observing area, the other end of observation tube to be connected with second by third switching valve body and cross air filtration head；
The side wall of tank body has interlayer space, is filled with jacket water (J.W.) in interlayer space, interlayer space adds jacket water (J.W.) equipped with heating tube
Heat, the lateral surface of the side wall of tank body are equipped at intervals with the through-hole of two connection interlayer spaces, and two through-holes are respectively connected to a circulating pump
Both ends.
2. a kind of microcarrier biological reactor according to claim 1, it is characterised in that：Intake interface includes that respirator connects
Mouth, deep layer intake interface, surface layer intake interface and strainer intake interface, a snorkel are passed through by strainer intake interface in tank body
And opposite filter screen outlet.
3. according to a kind of microcarrier biological reactor described in claim 1, it is characterised in that：Blender includes agitating shaft,
Multiple agitating paddles are distributed on agitating shaft, blender installing port is set at the center of the upper surface of tank body, and one end of agitating shaft is solid
Due to agitating shaft installing port, agitating shaft constitutes blender with agitating paddle, and blender is in be vertically arranged in tank body.
4. according to a kind of microcarrier biological reactor described in claim 1, it is characterised in that：The other end of observation tube also connects
One end of the first branch pipe is connect, the first branch pipe is equipped with second switch valve body, and the other end of the first branch pipe is equipped with the spare bottle that keeps sample, standby
With keeping sample, bottle crosses air filtration head equipped with third.
5. according to a kind of microcarrier biological reactor described in claim 4, it is characterised in that：Probe tube, the first branch pipe and
Two branch pipes are hose, and first switch valve body, second switch valve body, third switching valve body and the 4th switching valve body are gas folder；
Alternatively, probe tube, the first branch pipe and the second branch pipe are hard pipeline, first switch valve body, second switch valve body, third switch
Valve body and the 4th switching valve body are air valve.
6. a kind of method using the microcarrier biological reactor culture porcine circovirus 2 type described in one of claim 1-5,
It is characterized in that：It includes the following steps：
Step 1, biological reactor opens preheating：
PH electrodes, temperature electrode, the dissolved oxygen electrode of biological reactor are connected, chuck is added into the interlayer space of tank body
Water connects heating tube and opens power supply heating, starts circulating pump and recycle the jacket water (J.W.) in interlayer space so that tank body temperature
Degree is uniform；
Step 2, rolling bottle cell dissociation：
The good rolling bottle PK-15 cells of cell state are selected, pancreatin digestive juice vitellophag are used in combination, then postdigestive cell is filled
Divide and shakes up；
Step 3, cell count：It takes appropriate postdigestive cell to be diluted with culture solution, cell after appropriate dilution is taken after dilution
It adds to cell counting board and carries out cell count, calculate the cell density of cell liquid；
Step 4, tank culture on cell：Addition microcarrier is risen by 3-5g/ into biological reactor, by 30-50 cell/microcarrier
Add cell；Adjust the cell culture parameter of biological reactor respective devices：Aerating system is opened by intake interface to biology
Retort carries out ventilation culture；
Step 5, liquid is changed for 24 hours：When in biological reactor cell culture to for 24 hours after, stop stirring, stand 3-5 minute, adjust stretch
Infratubal port sampling height makes flexible infratubal port sampling height at carrier ullage 0.5cm, cell culture fluid is discharged,
Then the cell maintenance medium for equivalent being added by fluid infusion continues to cultivate；
Step 6, cell observation：First switch valve body and third switch are opened after cell culture is to 48h, while being kept stirring
Valve body closes second switch valve body and the 4th switching valve body, and air-breathing in syringe, biological respinse tank are used crossing air filtration head
Carrier and cell stop pumping when flowing through transparent observing area, close first switch valve body, cell observation area placed micro-
Under the microscope, after observation, open first switch valve body K1, inflated in crossing air filtration head using syringe, by transparent observing area and
The carrier of associated conduit part is squeezed into via original routing sample tap in tank body with cell；
Step 7, antigen is harvested for the first time：After cell culture is to 72h, stop stirring, stand 3-5 minutes, adjusting, which takes, to make to stretch
Infratubal port sampling height to places 0.5cm above carrier in tank, collect first time antigen and will harvest antigen liquid set -15 spend under with
Refrigerator-freezer freezen protective, the cell maintenance medium that equivalent is then added by fluid infusion continue to cultivate；
Step 8, after-crop antigen and liquid culture is changed：After biological reactor cell culture is to 96h, stop stirring, stands
3-5 minutes, adjusting made above flexible infratubal port sampling height to carrier in tank at 0.5cm, to harvest training again by sample tap
Supernatant antigen liquid is supported, the second antigen liquid of harvest is set under -15 degree with refrigerator-freezer freezen protective；
Step 9, third time harvest antigen：After the cell culture in biological reactor is to 96-120h, adjusting revolution is 100-
150 turns 3-5 minutes, then stop stirring, stand 3-5 minute, by aerating system from intake interface to tank body ventilation pressurization, open
The discharge valve opened on discharge pipe harvests filtered antigen, while opening the snorkel air inlet above filter screen, will finally receive
The third time antigen liquid obtained is set under -15 degree with refrigerator-freezer freezen protective.
7. a kind of method using microcarrier biological reactor culture porcine circovirus 2 type according to claim 6, special
Sign is：In step 2 digestion of cell is carried out using 0.25% pancreatin digestive juice.
8. a kind of method using microcarrier biological reactor culture porcine circovirus 2 type according to claim 6, special
Sign is：The specific method of the cell culture parameter of respective devices is adjusted in step 4：Temperature in tank is adjusted by temperature electrode to protect
It holds at 36.5-37.5 DEG C, adjusting dissolved oxygen by dissolved oxygen electrode is maintained at 50%-60%, is maintained at by PH electrode regulatings PH
7.2-7.4, the revolution for adjusting blender keep 40-60r/min.
9. a kind of method using microcarrier biological reactor culture porcine circovirus 2 type according to claim 6, special
Sign is：The specific method of cell culture fluid is discharged in step 5：First switch valve body and the 4th switching valve body are opened, closes the
Two switching valve bodies and third switching valve body, aerating system are ventilated to tank body by intake interface and are pressurizeed, using waste liquid bottle as receipts
Collect bottle, since pressure is more than the pressure outside tank body, the effect of the culture solution of carrier ullage 0.5cm or more in pressure in tank body
Probe tube is flowed through by telescoping tube down and constantly flows into receiving flask, until the liquid level of culture solution is less than flexible infratubal port sampling height
When stop；It is crossing air filtration head respectively while closing first switch valve body at this time and is being inflated using syringe crossing air filtration head, it will
The cell culture fluid of transparent observing area and associated conduit part squeezes into receiving flask.
10. a kind of method using microcarrier biological reactor culture porcine circovirus 2 type according to claim 6,
It is characterized in that：Collect the specific method of first time antigen and first time antigen：First switch valve body and the 4th switching valve body are opened,
Second switch valve body and third switching valve body are closed, aerating system is ventilated to tank body by intake interface and pressurizeed, and antigen bottle is used
As receiving flask, since pressure is more than the pressure outside tank body in tank body, the culture solution of carrier ullage 0.5cm or more is in pressure
Under the action of probe tube flowed through by telescoping tube constantly flow into receiving flask, taken until the liquid level of culture solution is less than flexible infratubal port
Stop when sample height；It is crossing air filtration head respectively while closing first switch valve body at this time and is being beaten using syringe crossing air filtration head
The cell culture fluid in transparent observing area and associated conduit part is squeezed into receiving flask by gas.
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| CN116574605A (en) * | 2023-05-23 | 2023-08-11 | 青岛浩大生物科技工程有限责任公司 | Collagen peptide efficient enzymolysis tank |
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