CN112430541A - Method for culturing cells through multichannel perfusion - Google Patents
Method for culturing cells through multichannel perfusion Download PDFInfo
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- CN112430541A CN112430541A CN202011283099.2A CN202011283099A CN112430541A CN 112430541 A CN112430541 A CN 112430541A CN 202011283099 A CN202011283099 A CN 202011283099A CN 112430541 A CN112430541 A CN 112430541A
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- 230000010412 perfusion Effects 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000012258 culturing Methods 0.000 title claims abstract description 8
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 67
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 46
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 46
- 239000001301 oxygen Substances 0.000 claims abstract description 46
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 239000007789 gas Substances 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 45
- 229920001296 polysiloxane Polymers 0.000 claims description 38
- 239000006228 supernatant Substances 0.000 claims description 31
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 21
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 20
- 239000001569 carbon dioxide Substances 0.000 claims description 18
- 238000005070 sampling Methods 0.000 claims description 17
- 239000002699 waste material Substances 0.000 claims description 15
- 238000005086 pumping Methods 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 11
- 230000008859 change Effects 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 229960002897 heparin Drugs 0.000 claims description 4
- 229920000669 heparin Polymers 0.000 claims description 4
- 230000000149 penetrating effect Effects 0.000 claims description 4
- 230000037452 priming Effects 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000010008 shearing Methods 0.000 abstract description 10
- 238000004113 cell culture Methods 0.000 abstract description 4
- 239000011521 glass Substances 0.000 abstract description 4
- 230000005587 bubbling Effects 0.000 abstract description 3
- 230000002900 effect on cell Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to the technical field of cell culture, in particular to a method for culturing cells by multi-channel perfusion, which realizes that multi-channel peristaltic pump liquid enables liquid in a tank to flow mildly and uniformly, increases the circulation efficiency, generates small shearing force and has no damage to cells; the multi-channel perfusion can be used for a large-volume reactor, so that the culture volume of cells is increased; the dissolved oxygen sensor and the PH sensor patch are fixed inside the reactor glass tank, the sensor probe is fixed outside the reactor glass tank, PH and dissolved oxygen are measured in a non-contact manner, the dissolved oxygen and the PH value in the culture tank can be reflected in real time, and meanwhile, the pollution risk is avoided; the PID closed loop accurately controls the CO2 and 02 flows, can realize quick and accurate adjustment, accurate and active control of dissolved oxygen and accurate and active control of PH value; the oxygenator module is added, so that liquid and gas exchange can be better carried out, the dissolved oxygen efficiency is improved, and the stress and shearing force damage effect on cells caused by the traditional bubbling type dissolved oxygen mode is avoided.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for culturing cells by multi-channel perfusion.
Background
In the cell culture production process, the requirements of the growth of some cells on the environment are strict, the growth of the cells can be influenced by shearing force, temperature change, pH value and dissolved oxygen supply, and the shearing force generated by a reactor with paddles on the market is larger, so that the growth of some cells is not facilitated; when the culture volume in the tank body of the reactor is small, the single-channel perfusion culture cell can obtain a better result; however, if the volume in the culture tank is large, the single-channel perfusion speed is slow, cells can be settled if the perfusion speed is not increased, and the activity of the cells is reduced or even the cells die due to insufficient oxygen supply of the cells; if the rotating speed of the peristaltic pump is simply increased to improve the perfusion speed of the cells, the cells can be driven by the high rotating speed of the peristaltic pump to bear great shearing force, the activity of the cells can be reduced, and the death of the cells is accelerated.
In conclusion, the invention solves the existing problems by designing a method for culturing cells by multi-channel perfusion
Disclosure of Invention
The invention aims to provide a method for multi-channel perfusion culture of cells, which aims to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for culturing cells by multi-channel perfusion comprises the following specific steps:
s1, medium filling: f1, F2, F3, F3, F4, F5, F6, F7 and F8 which are culture bags filled with culture medium, WS1, WS2, WS3, WS4, WS5, WS6, WS7 and WS8 weighing sensors, wherein the culture medium is pumped into a heating module H1 through a peristaltic pump P1, then enters a centrifugal module C tank and is pumped into the culture tank through a peristaltic pump P3 to complete the filling of the culture medium;
s2, cell priming: the injection of the cell sap is finished by penetrating the injector into the filling port of the culture tank;
s3, main loop circulation: the four peristaltic pump heads are driven by two motors, namely two single motors and two pump heads, so that liquid in the culture tank is perfused; the main circulation adopts four-channel perfusion culture, wherein one channel passes through an oxygenator, namely a membrane gas exchange unit; oxygen and carbon dioxide pass through the pneumatic duplex piece, pass through the mass flow controller, pass through the air filter, enter the oxygenator G, dissolve into the culture solution and enter the culture tank, provide sufficient dissolved oxygen and carbon dioxide for cells; a PH and dissolved oxygen sensor patch is arranged inside the culture tank, a probe of the PH and dissolved oxygen sensor is arranged outside the culture tank, and the PH and dissolved oxygen inside the culture tank are detected without directly contacting a culture solution; when the PH is higher, the PID controls the mass flow controller to inject quantitative carbon dioxide into the culture tank to form carbonic acid to adjust the PH; when the pH value is low, the pH value adjusting liquid in the F7 bag is put into the culture tank through a peristaltic pump P5 to adjust the pH value;
s4, sampling module: pumping cell sap out of the culture tank through a peristaltic pump P4 to finish sampling, soaking a needle head in an alcohol cleaning container before sampling, pumping the sampled waste liquor into a waste liquor bag F8 by using a vacuum pump VP after sampling, arranging a weighing sensor WS8 on the upper part of F8, and when the weight reaches a set value, alarming to remind a worker to change the waste liquor bag;
s5, supernatant collection: pumping the culture solution into a centrifugal module C through a peristaltic pump P3, driving a centrifugal tank to move at a high speed by a motor, separating cells from supernatant through centrifugal movement, and pumping the supernatant into a supernatant collection bag F2 through the peristaltic pump;
s6, cell collection: the culture solution is pumped into the centrifugal module C through a peristaltic pump P3, a motor drives a centrifugal tank to move at a high speed, cells are separated from the supernatant through centrifugal movement, and the supernatant is pumped into a supernatant collection bag F2 through the peristaltic pump.
Preferably, in S1, F1, F2, F3, F3, F4, F5, F6, F7 and F8 are respectively hung below the weighing sensors of WS1, WS2, WS3, WS4, WS5, WS6, WS7 and WS8 in a one-to-one correspondence to culture bags filled with culture media, and in S2, a cell filling port is arranged above the culture tank and a heparin cap is arranged at the cell filling port.
Preferably, the culture medium passes through the silicone tube and connects in heating module H1 in S1, heating module H1 passes through the silicone tube and connects the inlet end at peristaltic pump P1, the play liquid end of peristaltic pump P1 passes through the silicone tube and connects on centrifugal module C jar, centrifugal module C jar passes through the silicone tube and connects on the inlet end of peristaltic pump P3, the play liquid end of peristaltic pump P3 passes through the silicone tube and connects on the culture tank.
Preferably, the culture tank in S3 is connected to the oxygenator through a silicone tube, the oxygen and the carbon dioxide are pneumatically connected to the mass flow controller through a pneumatic two-way connector through a silicone tube, the mass flow controller is connected to the air filter through a pipeline, the air filter is connected to the oxygenator G through a pipeline, and the oxygenator G is connected to the culture tank through a silicone tube.
Preferably, the peristaltic pump P4 in S4 is connected to the culture tank through a silicone tube, and the vacuum pump VP is connected to the waste liquid bag F8 through a silicone tube.
Preferably, the peristaltic pump P3 in S5 is connected to the culture solution through a silicone tube, and the peristaltic pump is connected to the collection bag F2 through a silicone tube.
Preferably, the cell sap collection bag F2 is connected to the S5 through a peristaltic pump P2 and a silicone tube.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the multichannel peristaltic pump liquid is designed, so that the liquid in the tank flows mildly and uniformly, the circulation efficiency is increased, the generated shearing force is small, and the cell is not damaged; the multi-channel perfusion can be used for a large-volume reactor, so that the culture volume of cells is increased; the dissolved oxygen sensor and the PH sensor patch are fixed inside the reactor glass tank, the sensor probe is fixed outside the reactor glass tank, PH and dissolved oxygen are measured in a non-contact manner, the dissolved oxygen and the PH value in the culture tank can be reflected in real time, and meanwhile, the pollution risk is avoided; the PID closed loop accurately controls the CO2 and 02 flows, can realize quick and accurate adjustment, accurate and active control of dissolved oxygen and accurate and active control of PH value; the oxygenator module is added, so that liquid and gas exchange can be better carried out, the oxygen dissolving efficiency is improved, and the damage of stress and shearing force generated by the traditional bubbling type oxygen dissolving mode on cells is avoided, so that the problem that the shearing force generated by a reactor with paddles on the market is large and is not beneficial to the growth of some cells is solved; when the culture volume in the tank body of the reactor is small, the single-channel perfusion culture cell can obtain a better result; however, if the volume in the culture tank is large, the single-channel perfusion speed is slow, cells can be settled if the perfusion speed is not increased, and the activity of the cells is reduced or even the cells die due to insufficient oxygen supply of the cells; if the rotating speed of the peristaltic pump is simply increased to improve the perfusion speed of the cells, the cells can be driven by the high rotating speed of the peristaltic pump to bear great shearing force, the activity of the cells can be reduced, and the death of the cells is accelerated.
Drawings
FIG. 1 is a flow chart of multi-channel perfusion culture of cells according to the present invention;
FIG. 2 is a main loop cycle diagram of the present invention;
FIG. 3 is an isometric view of the main circuit cycle of the present invention;
FIG. 4 is a core collection module of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
Referring to fig. 1-4, the present invention provides a technical solution:
a method for culturing cells by multi-channel perfusion comprises the following specific steps:
s1, medium filling: f1, F2, F3, F3, F4, F5, F6, F7 and F8 which are culture bags filled with culture medium, WS1, WS2, WS3, WS4, WS5, WS6, WS7 and WS8 weighing sensors, wherein the culture medium is pumped into a heating module H1 through a peristaltic pump P1, then enters a centrifugal module C tank and is pumped into the culture tank through a peristaltic pump P3 to complete the filling of the culture medium;
s2, cell priming: the injection of the cell sap is finished by penetrating the injector into the filling port of the culture tank;
s3, main loop circulation: the four peristaltic pump heads are driven by two motors, namely two single motors and two pump heads, so that liquid in the culture tank is perfused; the main circulation adopts four-channel perfusion culture, wherein one channel passes through an oxygenator, namely a membrane gas exchange unit; oxygen and carbon dioxide pass through the pneumatic duplex piece, pass through the mass flow controller, pass through the air filter, enter the oxygenator G, dissolve into the culture solution and enter the culture tank, provide sufficient dissolved oxygen and carbon dioxide for cells; a PH and dissolved oxygen sensor patch is arranged inside the culture tank, a probe of the PH and dissolved oxygen sensor is arranged outside the culture tank, and the PH and dissolved oxygen inside the culture tank are detected without directly contacting a culture solution; when the PH is higher, the PID controls the mass flow controller to inject quantitative carbon dioxide into the culture tank to form carbonic acid to adjust the PH; when the pH value is low, the pH value adjusting liquid in the F7 bag is put into the culture tank through a peristaltic pump P5 to adjust the pH value;
s4, sampling module: pumping cell sap out of the culture tank through a peristaltic pump P4 to finish sampling, soaking a needle head in an alcohol cleaning container before sampling, pumping the sampled waste liquor into a waste liquor bag F8 by using a vacuum pump VP after sampling, arranging a weighing sensor WS8 on the upper part of F8, and when the weight reaches a set value, alarming to remind a worker to change the waste liquor bag;
s5, supernatant collection: pumping the culture solution into a centrifugal module C through a peristaltic pump P3, driving a centrifugal tank to move at a high speed by a motor, separating cells from supernatant through centrifugal movement, and pumping the supernatant into a supernatant collection bag F2 through the peristaltic pump;
s6, cell collection: the culture solution is pumped into the centrifugal module C through a peristaltic pump P3, a motor drives a centrifugal tank to move at a high speed, cells are separated from the supernatant through centrifugal movement, and the supernatant is pumped into a supernatant collection bag F2 through the peristaltic pump.
Further, in S1, F1, F2, F3, F3, F4, F5, F6, F7, and F8 are respectively hung below WS1, WS2, WS3, WS4, WS5, WS6, WS7, and WS8 weighing sensors in one-to-one correspondence to culture bags filled with culture media, and in S2, a cell filling port is arranged above the culture tank and a heparin cap is arranged at the cell filling port.
Further, in S1 the culture medium passes through the silicone tube and connects at heating module H1, heating module H1 passes through the silicone tube and connects the feed liquor end at peristaltic pump P1, the play liquid end of peristaltic pump P1 passes through the silicone tube and connects on centrifugal module C jar, centrifugal module C jar passes through the silicone tube and connects on the feed liquor end of peristaltic pump P3, the play liquid end of peristaltic pump P3 passes through the silicone tube and connects on the culture tank.
Further, cultivate the jar in S3 and pass through the silicone tube and connect on the oxygenator, oxygen and carbon dioxide pass through pneumatic duplicate and pass through the silicone tube and connect at mass flow controller, mass flow controller passes through the pipe connection on air cleaner, air cleaner passes through the pipe connection on oxygenator G, oxygenator G passes through the silicone tube and connects on cultivating the jar.
Further, in the S4, a peristaltic pump P4 is connected to the culture tank through a silicone tube, and the vacuum pump VP is connected to the waste liquid bag F8 through a silicone tube.
Further, in the S5, a peristaltic pump P3 is connected to the culture solution through a silicone tube, and the peristaltic pump is connected to the collection bag F2 through the silicone tube.
The specific implementation case is as follows:
step 1, filling a culture medium: as shown in the cell culture flow chart of FIG. 1, F3, F4, F5 and F6 are culture bags filled with culture medium, and load cells WS3, WS4, WS5 and WS6 are arranged above the culture bags; pumping the culture medium into a heating module H1 through a peristaltic pump P1, then entering a centrifugal module C tank, and then pumping into a culture tank through a peristaltic pump P3 to complete the filling of the culture medium;
Step 3, as shown in fig. 2 and as shown in fig. 3, the main loop circulates: the main circulation module is driven by four peristaltic pump heads (namely two single-motor double-pump head drives) driven by two motors, so that liquid in the culture tank is perfused; the main circulation adopts four-channel perfusion culture, wherein one channel passes through an oxygenator (membrane type gas exchange unit); oxygen and carbon dioxide pass through the pneumatic duplex piece, pass through the mass flow controller, pass through the air filter, enter the oxygenator G, dissolve into the culture solution and enter the culture tank, provide sufficient dissolved oxygen and carbon dioxide for cells; the pneumatic duplex piece can stabilize pressure and filter water and impurities in gas, the flow mass sensor can provide quantitative gas, the air filter can filter bacteria in the gas to prevent polluting cells, the oxygenator can enable the gas in the reactor to be exchanged better, a PH and dissolved oxygen sensor patch is arranged inside the culture tank, a PH and dissolved oxygen sensor probe is arranged outside the culture tank, and the PH and dissolved oxygen sensor probe does not directly contact with culture solution to detect the PH and dissolved oxygen inside the culture tank, so that the pollution risk is avoided; when the PH is higher, the PID controls the mass flow controller to inject quantitative carbon dioxide into the culture tank to form carbonic acid to adjust the PH; when the pH is low, the pH is adjusted by feeding the pH adjusting liquid in the F7 bag into the culture tank by a peristaltic pump P5.
Step 4, as shown in fig. 1, fig. 2 and fig. 3, the sampling module: cell sap is pumped out from the culture tank through a peristaltic pump P4 to finish sampling, before sampling, a needle is always immersed in the alcohol cleaning container, waste liquid after sampling is pumped into a waste liquid bag F8 by a vacuum pump VP after sampling, a weighing sensor WS8 is arranged on the upper part of the F8, and when the weight reaches a set value, an alarm is given to remind a worker to change the waste liquid bag.
Step 5, as shown in fig. 1, fig. 2 and fig. 3, supernatant collection: the culture solution is pumped into the centrifugal module C through a peristaltic pump P3, a motor drives a centrifugal tank to move at a high speed, cells are separated from the supernatant through centrifugal movement, and the supernatant is pumped into a supernatant collection bag F2 through the peristaltic pump.
Step 6, cell collection, as in fig. 1, fig. 2, fig. 3 and fig. 4: the culture solution is pumped into the centrifugal module C through a peristaltic pump P3, a motor drives a centrifugal tank to move at a high speed, cells are separated from supernatant through centrifugal movement, the supernatant is pumped into a supernatant collecting bag F2 through the peristaltic pump, and the remaining cell sap is pumped into a cell sap collecting bag F2 through the peristaltic pump P2.
The process achieves the following advantages by design:
(1) the single motor double pump heads, the two motors drive the four pump heads, and the four channels enable the tank body to realize vortex stirring. The number of pump heads and pipelines can be adjusted according to actual requirements;
(2) the required rotational speed of single channel is higher under the large volume, and the shearing force of production is bigger, and the single channel is replaced to the multichannel, and the pump head rotational speed is required lower, can be better faster carry out the circulation of liquid. Cell death due to high shear is eliminated;
(3) the culture medium, the recovered supernatant and the recovered cell sap are placed in a 4-degree refrigerator, so that the culture medium, the recovered supernatant and the recovered cell sap are convenient to store for a long time, and a weighing sensor is arranged above the bag, so that the weight of the liquid can be accurately weighed; when the quantity reaches or is less than the set quantity, the alarm function is provided, and the worker is reminded to operate.
(4) Be equipped with the heating module, the culture medium is gone into in the culture tank through the heating module pump, can realize the intensification fast, reaches the ambient temperature of cell growth, saves the process of slowly heating through the incubator.
(5) Compared with a membrane package, the centrifugal module can quickly collect supernatant, so that time and cost are greatly saved.
(6) The oxygen and the carbon dioxide pass through the pneumatic duplex piece, the mass flow controller, the air filter and the oxygenator to provide the required oxygen and carbon dioxide for the reactor; pneumatic two allies oneself with piece can steady voltage and the moisture and the impurity in the filtering gas, and flow mass sensor can provide quantitative gas, and air cleaner can filter the bacterium in the gas and prevent the pollution cell, and the oxygenator can make better carrying out gas exchange in the reactor. Stress and shear force damage of the traditional bubbling method is avoided;
(7) the PH sensor and the dissolved oxygen sensor are in non-contact with the liquid in the reaction tank, the dissolved oxygen and the PH value of the liquid in the reaction tank are measured, when the dissolved oxygen value is lower than a set value, the PID control flow and quality controller injects a certain amount of gas into the oxygenator, the gas is dissolved in the liquid in the reaction tank, and the dissolved oxygen is increased; when the PH value in the reaction tank is on the high side, flow quality controller lets in quantitative carbon dioxide in the oxygenator, dissolve in the liquid in the reaction tank through the oxygenator, form carbonic acid and reduce the PH value, when the PH value is higher than the setting value, PID closed loop control peristaltic pump goes into the reaction tank with sodium bicarbonate liquid through hydrophilic filter pump in, promote the PH value in the reaction tank, PID closed loop accurate control CO2 and 02 flow, can realize quick accurate regulation, dissolve oxygen volume controllable range +/-1%, the controllable volume of PH value +/-0.2.
The whole device is integrated into a whole, has simple structure, is not in direct contact with the outside, and avoids pollution risks.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for culturing cells by multi-channel perfusion comprises the following specific steps:
s1, medium filling: f1, F2, F3, F3, F4, F5, F6, F7 and F8 which are culture bags filled with culture medium, WS1, WS2, WS3, WS4, WS5, WS6, WS7 and WS8 weighing sensors, wherein the culture medium is pumped into a heating module H1 through a peristaltic pump P1, then enters a centrifugal module C tank and is pumped into the culture tank through a peristaltic pump P3 to complete the filling of the culture medium;
s2, cell priming: the injection of the cell sap is finished by penetrating the injector into the filling port of the culture tank;
s3, main loop circulation: the four peristaltic pump heads are driven by two motors, namely two single motors and two pump heads, so that liquid in the culture tank is perfused; the main circulation adopts four-channel perfusion culture, wherein one channel passes through an oxygenator, namely a membrane gas exchange unit; oxygen and carbon dioxide pass through the pneumatic duplex piece, pass through the mass flow controller, pass through the air filter, enter the oxygenator G, dissolve into the culture solution and enter the culture tank, provide sufficient dissolved oxygen and carbon dioxide for cells; a PH and dissolved oxygen sensor patch is arranged inside the culture tank, a probe of the PH and dissolved oxygen sensor is arranged outside the culture tank, and the PH and dissolved oxygen inside the culture tank are detected without directly contacting a culture solution; when the PH is higher, the PID controls the mass flow controller to inject quantitative carbon dioxide into the culture tank to form carbonic acid to adjust the PH; when the pH value is low, the pH value adjusting liquid in the F7 bag is put into the culture tank through a peristaltic pump P5 to adjust the pH value;
s4, sampling module: pumping cell sap out of the culture tank through a peristaltic pump P4 to finish sampling, soaking a needle head in an alcohol cleaning container before sampling, pumping the sampled waste liquor into a waste liquor bag F8 by using a vacuum pump VP after sampling, arranging a weighing sensor WS8 on the upper part of F8, and when the weight reaches a set value, alarming to remind a worker to change the waste liquor bag;
s5, supernatant collection: pumping the culture solution into a centrifugal module C through a peristaltic pump P3, driving a centrifugal tank to move at a high speed by a motor, separating cells from supernatant through centrifugal movement, and pumping the supernatant into a supernatant collection bag F2 through the peristaltic pump;
s6, cell collection: the culture solution is pumped into the centrifugal module C through a peristaltic pump P3, a motor drives a centrifugal tank to move at a high speed, cells are separated from the supernatant through centrifugal movement, and the supernatant is pumped into a supernatant collection bag F2 through the peristaltic pump.
2. The method for multichannel perfusion culture of cells according to claim 1, wherein: f1, F2, F3, F3, F4, F5, F6, F7 and F8 in the S1 are respectively hung below WS1, WS2, WS3, WS4, WS5, WS6, WS7 and WS8 weighing sensors in a one-to-one correspondence manner, and a cell filling port is arranged above a culture tank in the S2 and is provided with a heparin cap.
3. The method for multichannel perfusion culture of cells according to claim 1, wherein: in S1 the culture medium passes through the silicone tube and connects at heating module H1, heating module H1 passes through the silicone tube and connects the feed liquor end at peristaltic pump P1, peristaltic pump P1 'S play liquid end passes through the silicone tube and connects on centrifugal module C jar, centrifugal module C jar passes through the silicone tube and connects on peristaltic pump P3' S feed liquor end, the play liquid end of peristaltic pump P3 passes through the silicone tube and connects on the culture tank.
4. The method for multichannel perfusion culture of cells according to claim 1, wherein: the culture tank passes through the silicone tube and connects on the oxygenator in S3, oxygen and carbon dioxide pass through pneumatic duplicate and pass through the silicone tube and connect at mass flow controller, mass flow controller passes through the pipe connection on air cleaner, air cleaner passes through the pipe connection on oxygenator G, oxygenator G passes through the silicone tube and connects on the culture tank.
5. The method for multichannel perfusion culture of cells according to claim 1, wherein: the peristaltic pump P4 in the S4 is connected to the culture tank through a silicone tube, and the vacuum pump VP is connected to the waste liquid bag F8 through a silicone tube.
6. The method for multichannel perfusion culture of cells according to claim 1, wherein: the peristaltic pump P3 in the S5 is connected to the culture solution through a silicone tube, and the peristaltic pump is connected to the collection bag F2 through a silicone tube.
7. The method for multichannel perfusion culture of cells according to claim 1, wherein: in the S5, the cell sap collection bag F2 is connected through a peristaltic pump P2 and a silicone tube.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265330A (en) * | 2021-06-04 | 2021-08-17 | 河南大学 | Animal cell high-density culture system suitable for efficient production of vaccines and antibodies |
| CN113564125A (en) * | 2021-08-04 | 2021-10-29 | 河南省肿瘤医院 | Method for culturing tumor cells |
| CN113604342A (en) * | 2021-09-05 | 2021-11-05 | 英诺维尔智能科技（苏州）有限公司 | Full-automatic aseptic connection reactor sampling unit |
| CN114317270A (en) * | 2022-03-12 | 2022-04-12 | 广州赛太特生物医学科技有限公司 | Cell culture device for biological gene research |
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996009876A1 (en) * | 1992-09-11 | 1996-04-04 | Xenogenex, Inc. | Artificial liver apparatus and method |
| CN101899393A (en) * | 2010-08-10 | 2010-12-01 | 中国人民解放军军事医学科学院卫生装备研究所 | Dynamic load and recirculating perfusion bioreactor |
| WO2013116449A1 (en) * | 2012-02-02 | 2013-08-08 | Corning Incorporated | Automatic continuous perfusion cell culture microplate consumables |
| CN105050495A (en) * | 2013-01-23 | 2015-11-11 | 艾利丹尼森公司 | Wireless sensor patches and methods of manufacturing |
| CN105087380A (en) * | 2015-07-31 | 2015-11-25 | 南方医科大学珠江医院 | System for massively culturing animal cells |
| CN208672241U (en) * | 2018-09-11 | 2019-03-29 | 三峡大学 | A kind of water floor heating pipeline leak localization device |
| WO2019168913A1 (en) * | 2018-02-28 | 2019-09-06 | Pop Test Oncology Llc | Medical devices and uses thereof |
| RU2019103277A (en) * | 2018-08-01 | 2020-02-03 | Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ ТИО им. ак. В.И. Шумакова" Минздрава России) | Flowing bioreactor for the cultivation of bioengineered structures |
| CN111154647A (en) * | 2020-01-17 | 2020-05-15 | 英诺维尔智能科技（苏州）有限公司 | Closed circulation external exchange biological product reactor |
| CN211317581U (en) * | 2020-03-02 | 2020-08-21 | 成都伊莱特光测科技有限公司 | Temperature calibration device of surface mount type optical fiber temperature sensor |
-
2020
- 2020-11-17 CN CN202011283099.2A patent/CN112430541B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996009876A1 (en) * | 1992-09-11 | 1996-04-04 | Xenogenex, Inc. | Artificial liver apparatus and method |
| CN101899393A (en) * | 2010-08-10 | 2010-12-01 | 中国人民解放军军事医学科学院卫生装备研究所 | Dynamic load and recirculating perfusion bioreactor |
| WO2013116449A1 (en) * | 2012-02-02 | 2013-08-08 | Corning Incorporated | Automatic continuous perfusion cell culture microplate consumables |
| CN105050495A (en) * | 2013-01-23 | 2015-11-11 | 艾利丹尼森公司 | Wireless sensor patches and methods of manufacturing |
| CN105087380A (en) * | 2015-07-31 | 2015-11-25 | 南方医科大学珠江医院 | System for massively culturing animal cells |
| WO2019168913A1 (en) * | 2018-02-28 | 2019-09-06 | Pop Test Oncology Llc | Medical devices and uses thereof |
| RU2019103277A (en) * | 2018-08-01 | 2020-02-03 | Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ ТИО им. ак. В.И. Шумакова" Минздрава России) | Flowing bioreactor for the cultivation of bioengineered structures |
| CN208672241U (en) * | 2018-09-11 | 2019-03-29 | 三峡大学 | A kind of water floor heating pipeline leak localization device |
| CN111154647A (en) * | 2020-01-17 | 2020-05-15 | 英诺维尔智能科技（苏州）有限公司 | Closed circulation external exchange biological product reactor |
| CN211317581U (en) * | 2020-03-02 | 2020-08-21 | 成都伊莱特光测科技有限公司 | Temperature calibration device of surface mount type optical fiber temperature sensor |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265330A (en) * | 2021-06-04 | 2021-08-17 | 河南大学 | Animal cell high-density culture system suitable for efficient production of vaccines and antibodies |
| CN113564125A (en) * | 2021-08-04 | 2021-10-29 | 河南省肿瘤医院 | Method for culturing tumor cells |
| CN113604342A (en) * | 2021-09-05 | 2021-11-05 | 英诺维尔智能科技（苏州）有限公司 | Full-automatic aseptic connection reactor sampling unit |
| CN114317270A (en) * | 2022-03-12 | 2022-04-12 | 广州赛太特生物医学科技有限公司 | Cell culture device for biological gene research |
| CN114317270B (en) * | 2022-03-12 | 2022-06-17 | 广州赛太特生物医学科技有限公司 | Cell culture device for biological gene research |
| CN117143726A (en) * | 2023-04-28 | 2023-12-01 | 哈尔滨工业大学 | Human-simulated large-scale stem cell culture equipment with low loss rate |
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