CN110551631B - Fixed bed bioreactor system for mass production of mesenchymal stem cells - Google Patents
Fixed bed bioreactor system for mass production of mesenchymal stem cells Download PDFInfo
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 28
- 239000001963 growth medium Substances 0.000 claims abstract description 101
- 230000007246 mechanism Effects 0.000 claims abstract description 90
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 239000002699 waste material Substances 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims description 62
- 238000003756 stirring Methods 0.000 claims description 59
- 238000004659 sterilization and disinfection Methods 0.000 claims description 42
- 210000004027 cell Anatomy 0.000 claims description 34
- 238000007789 sealing Methods 0.000 claims description 16
- 238000009833 condensation Methods 0.000 claims description 10
- 230000005494 condensation Effects 0.000 claims description 10
- 239000000969 carrier Substances 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 2
- 230000003116 impacting effect Effects 0.000 claims description 2
- 238000010992 reflux Methods 0.000 description 21
- 210000000130 stem cell Anatomy 0.000 description 16
- 230000009286 beneficial effect Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 7
- 238000010008 shearing Methods 0.000 description 5
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000001079 digestive effect Effects 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
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- 229910001220 stainless steel Inorganic materials 0.000 description 3
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
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- 238000010828 elution Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
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- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- -1 ammonia ions Chemical class 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
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- C12M27/02—Stirrer or mobile mixing elements
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- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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Abstract
The invention discloses a fixed bed bioreactor system for mass production of mesenchymal stem cells, which comprises a bioreactor (1), an external circulation mechanism (2) and a collecting mechanism (3), wherein the bioreactor (1) is used for amplifying the mesenchymal stem cells; the external circulation mechanism (2) is used for receiving the culture medium discharged from the bottom of the bioreactor (1) and conveying the culture medium into the culture medium mixing tank (11) at the upper part of the bioreactor (1) so as to realize the circulation of the culture medium, and the external circulation mechanism (2) is provided with a flowmeter (22) so as to monitor the flow rate of the culture medium and regulate and control the material exchange of the culture medium according to the flow rate; the collecting mechanism (3) can collect the mesenchymal stem cells cultured by the bioreactor (1) and the waste liquid generated during the culture of the mesenchymal stem cells. According to the invention, the flow rate of the culture medium passing through the fixed bed can be accurately controlled through the external circulation of the culture medium, so that the large-scale culture of the mesenchymal stem cells is realized.
Description
Technical Field
The invention belongs to the technical field of mesenchymal stem cell culture, in particular relates to the technical field of mass production of mesenchymal stem cells, and specifically relates to a fixed bed bioreactor system for mass production of mesenchymal stem cells.
Background
The traditional fixed bed reactor generally adopts in-tank circulation, in the in-tank circulation process, the stirring paddle rotates to drive the culture medium to circulate in the tank, but the flow rate of the culture medium passing through the carrier in the circulation process can not be measured, so that the nutrition transmission and the shearing force of the stem cells are difficult to effectively control, the culture quality of the stem cells is difficult to improve, and the mass production is difficult to carry out. At present, a fixed bed reactor has two stirring modes, namely upper stirring and lower stirring, but in any mode, only one stirring paddle is arranged, and the bottom of the tank body is provided with a spherical sealing head.
Disclosure of Invention
The object of the present invention is to solve the problems of the prior art and to provide a fixed bed bioreactor system for mass production of mesenchymal stem cells.
The invention aims at solving the problems through the following technical scheme:
a fixed bed bioreactor system for mass production of mesenchymal stem cells, characterized by: the system comprises a fixed bed type bioreactor, an external circulation mechanism and a collecting mechanism, wherein the bioreactor is used for amplifying mesenchymal stem cells; the external circulation mechanism is used for receiving the culture medium discharged from the bottom of the bioreactor and conveying the culture medium to a culture medium mixing tank at the upper part of the bioreactor so as to realize circulation of the culture medium, and is provided with a flowmeter so as to monitor the flow rate of the culture medium and regulate and control the material exchange of the culture medium according to the flow rate; the collecting mechanism can collect the mesenchymal stem cells cultured by the bioreactor and the waste liquid generated during the culture of the mesenchymal stem cells respectively.
The lower part of the bioreactor is provided with a carrier, the area above the carrier forms a culture medium mixing tank, the bioreactor is respectively provided with a carrier stirrer and a culture medium stirrer, the carrier stirring paddle of the carrier stirrer is positioned in the carrier, and the culture medium stirring paddle of the culture medium stirrer is positioned in the culture medium mixing tank.
The bottom of the bioreactor is a butterfly-shaped end socket.
The carrier is filled in the bioreactor through a porous supporting plate, a butterfly-shaped sealing head is arranged below the porous supporting plate, and a tank bottom valve is arranged at the bottom outlet of the butterfly-shaped sealing head.
The carrier stirring paddles adopt stirring paddles with adjustable blade angles so as to adapt to different carriers, and the blades of the carrier stirring paddles are fixed on the stirring shaft through bolts; for specific cell culture, the carrier stirring paddle can also adopt a stirring paddle with fixed blades according to requirements.
The culture medium stirring paddle adopts a radial stirring paddle, the outlet part of the return pipe in the external circulation mechanism is horizontally arranged, and the outlet of the return pipe corresponds to the blade of the culture medium stirring paddle on the culture medium stirrer.
A microbubble generator is arranged in the bioreactor and is positioned below the culture medium stirring paddle; and a fixed baffle plate is arranged right below the culture medium stirring paddle and used for preventing bubbles generated by the microbubble generator from directly impacting the stirring area of the culture medium stirring paddle and blocking downward axial flow generated by the culture medium stirring paddle.
The inlet of the external circulation pipeline of the external circulation mechanism is connected with the outlet of the tank bottom valve at the outlet of the bottom of the bioreactor, the outlet of the external circulation pipeline is connected with the inlet of the return pipe on the tank cover of the bioreactor, and the external circulation pipeline is sequentially provided with a centrifugal pump, a flowmeter and a heat exchanger along the flow direction of the culture medium.
The system also comprises a sterilization mechanism, wherein three-way valve groups are respectively arranged at the inlet and the outlet of an external circulation pipeline connected with the bioreactor by the external circulation mechanism, the three-way valve groups can close any one passage and keep the communication of the other two passages, two ends of a horizontal main pipe of the three-way valve group at the inlet of the external circulation pipeline are respectively connected with the inlet of the external circulation pipeline and the bottom outlet of the bioreactor, and the branch pipe outlet of the three-way valve group is connected with a steam trap in the sterilization mechanism by corresponding pipelines; the two ends of the horizontal main pipe of the three-way valve group at the outlet are respectively connected with the outlet of the external circulation pipeline and the inlet of the reflux pipe on the tank cover of the bioreactor, and the inlet of the branch pipe of the three-way valve group is used for introducing steam.
The discharge pipeline of the sterilizing mechanism is provided with a temperature detecting element which is arranged on the pipeline in front of the steam trap so as to ensure that the sterilizing temperature of the part to be sterilized reaches over 121 ℃.
A condensation discharge port is arranged at the lowest position of a pump head of a centrifugal pump in the external circulation mechanism, a pipeline with a centrifugal pump discharge valve is arranged at the condensation discharge port, and the pipeline is connected with a steam trap; and a cold end thermometer of the pump serving as a temperature detection element is arranged on a pipeline at the outlet side of the pump outlet valve.
The collecting mechanism comprises a cell collecting tank and a waste liquid collecting tank, wherein the cell collecting tank is connected with an outlet of a tank bottom valve at the outlet of the bottom of the bioreactor through a cell collecting pipe with a cell pipe clamp/valve, and the waste liquid collecting tank is connected with an outlet of a tank bottom valve at the outlet of the bottom of the bioreactor through a waste liquid collecting pipe with a waste liquid pipe clamp/valve.
Further, one technical scheme of the sterilization of the external circulation mechanism is as follows: the three-way valve at the inlet of the external circulation pipeline is split into a discharge three-way valve belonging to a sterilization mechanism and a discharge valve belonging to a bioreactor part, at the moment, the outlet of the tank bottom valve is connected with one end of the discharge valve, the other end of the discharge valve is connected with one end of a horizontal main pipe of the discharge three-way valve, the other end of the horizontal main pipe of the discharge three-way valve is connected with the inlet of the external circulation pipeline, and the outlet of a branch pipe of the discharge three-way valve is connected with a steam trap through a corresponding pipeline; meanwhile, the three-way valve at the outlet of the external circulation pipeline is required to be split into a sterilization three-way valve belonging to a sterilization mechanism and a reflux valve arranged at the inlet side of the reflux pipe, at the moment, one end of a horizontal main pipe of the sterilization three-way valve is connected with the reflux valve, the other end of the horizontal main pipe of the sterilization three-way valve is connected with the outlet of the external circulation pipeline, and a branch pipe inlet of the sterilization three-way valve is used for introducing steam; when the sterilizing device is used, pure steam is introduced into the sterilizing three-way valve and sequentially flows through the sterilizing three-way valve, the external circulation mechanism and the discharge three-way valve and then is discharged to the steam trap, and the external circulation mechanism is sterilized.
Further, in order to reduce the cost and facilitate the operation of the system, the corresponding pipelines of the collecting mechanism and the sterilizing mechanism are integrated; the outlet of the branch pipe of the discharge three-way valve is required to be respectively connected with steam traps in the collecting mechanism and the sterilizing mechanism through corresponding pipelines, at the moment, the outlet of the branch pipe of the discharge three-way valve is connected with the inlet end of a collecting pipe, the outlet end of the collecting pipe is connected with one end of a vertical main pipe of the collecting three-way valve, the other end of the vertical main pipe of the collecting three-way valve is connected with the inlet end of a cell collecting pipe, a branch pipe of the collecting three-way valve is connected with a steam trap with a condensing discharge valve at the end part, a sterilizing cold end thermometer serving as a temperature detecting element is arranged on the steam trap at the rear side of the condensing discharge valve, and the other end of the steam trap is connected with a steam trap with the tail end.
Compared with the prior art, the invention has the following advantages:
according to the invention, the external circulation mechanism capable of precisely controlling the flow rate of the culture medium passing through the fixed bed is arranged on the bioreactor, the external circulation mechanism measures the real-time flow rate of the culture medium through the flowmeter, the centrifugal pump controls the reflux amount of the culture medium in real time according to the measured value of the flowmeter so as to control the flow rate and the shearing force of the culture medium, so that the mesenchymal stem cells can be cultured on a large scale, and meanwhile, the culture quality of the stem cells can be improved and the production cost can be reduced.
The fixed bed bioreactor adopts the structural design of double stirring paddles, and can effectively help cells to elute from a carrier through the arrangement of a carrier stirrer; by limiting the position of the culture medium stirring paddle of the culture medium stirrer, the culture medium can be uniformly stirred, and the rotating speed of the culture medium stirring paddle is matched with the culture medium reflux speed in the reflux pipe, so that the nutrition transfer of stem cells is effectively controlled, and the production efficiency is improved.
The butterfly-shaped sealing head adopted by the invention has small volume, saves a large amount of digestive juice in the process of eluting stem cells, is beneficial to improving the product quality of the stem cells and is extremely beneficial to the subsequent stem cell expansion.
The sterilization mechanism can perform online sterilization under the condition of not disassembling the external circulation mechanism by arranging the valves and the corresponding pipelines, so that the production efficiency of the bioreactor is improved; the external circulation mechanism capable of sterilizing on line can save cost and is beneficial to the sterilization operation of the large bioreactor; if the bioreactor is a stainless steel tank, the external circulation mechanism and the bioreactor can be sterilized together on line, so that large-scale culture is realized.
Drawings
FIG. 1 is a schematic diagram of the structure of a fixed bed bioreactor system of the present invention;
fig. 2 is a block diagram of the structure of fig. 1 with the addition of a control section, auxiliary piping, support section, and monitoring section.
Wherein: 1-a bioreactor; 11-a culture medium mixing tank; 12-a carrier; 13-a carrier stirrer; 131-carrier stirring paddles; 14-a media stirrer; 141-a culture medium stirring paddle; 15-a microbubble generator; 16-a fixed baffle; 17-butterfly end socket; 18-a porous support plate; 2-an external circulation mechanism; 21-a centrifugal pump; 22-a flow meter; 23-a return pipe; 24-a heat exchanger; 3-a collection mechanism; 31-cell collection tube; 32-a cell collection tank; 33-a waste liquid collecting pipe; 34-a waste liquid collection tank; 35-collecting pipe; 4-a sterilization mechanism; 41-steam trap; 42-a hydrophobic pipe; 43-a hydrophobic branch; f1, a tank bottom valve; f2—a return valve; f3—a drain valve; f4, a sterilizing three-way valve; f5—a discharge three-way valve; f6—a condensate drain valve; f7—centrifugal pump discharge valve; f8—a collection three-way valve; n1-sterilizing cold end thermometer; n2-pump cold end thermometer; a-cell clamp/valve; b-waste line clamp/valve.
Detailed Description
The invention is further described below with reference to the drawings and examples.
As shown in fig. 1: a fixed bed bioreactor system for mass production of mesenchymal stem cells, the system comprising a fixed bed bioreactor 1, an external circulation mechanism 2, a collection mechanism 3, wherein the bioreactor 1 is used for amplifying the mesenchymal stem cells; the external circulation mechanism 2 is used for receiving the culture medium discharged from the bottom of the bioreactor 1 and conveying the culture medium to the culture medium mixing tank 11 at the upper part of the bioreactor 1 so as to realize circulation of the culture medium, the external circulation mechanism 2 is provided with a flowmeter 22 for monitoring the flow rate of the culture medium and regulating the rotation speed of the centrifugal pump 21 according to the flow rate, so that the culture medium substance exchange and the shear force regulation in the bioreactor 1 are realized, the mesenchymal stem cells can be cultured on a large scale, the culture quality of the stem cells can be improved, and the production cost can be reduced; the collecting means 3 can collect the mesenchymal stem cells cultured in the bioreactor 1 and the waste liquid generated during the culture of the mesenchymal stem cells.
As shown in fig. 1, a carrier 12 is arranged at the lower part of a bioreactor 1, and a region above the carrier 12 forms a culture medium mixing tank 11, wherein in the invention, the carrier 12 is a PET non-woven fabric paper carrier, a hollow or solid spherical carrier or an unequal polygonal carrier, and a material is a polymer material or inorganic materials such as glass and ceramic; the carrier 12 is packed in the bioreactor 1 through a porous support plate 18. In addition, because the existing fixed bed bioreactor is provided with a spherical sealing head, the volume of the sealing head is large enough to store a large amount of culture medium, and the culture quality of stem cells is not beneficial to improvement, therefore, in the invention, the sealing head below the porous supporting plate 18 is a butterfly-shaped sealing head 17, and the volume of the butterfly-shaped sealing head 17 is only 5% of the volume of the spherical sealing head, so that the digestion liquid can be greatly saved when the butterfly-shaped sealing head 17 is used for harvesting and digesting cells, the elution quality of products is beneficial to improvement, and the cell expansion in the next step is beneficial to; and a tank bottom valve F1 is arranged at the bottom of the butterfly-shaped seal head 17.
As shown in fig. 1, the bioreactor 1 is provided with a carrier stirrer 13 and a medium stirrer 14, wherein a carrier stirrer 131 of the carrier stirrer 13 is positioned in the carrier 12, and a medium stirrer 141 of the medium stirrer 14 is positioned in the medium mixing tank 11. Wherein the carrier stirring paddle 131 is located at the center of the bioreactor 1, preferably, the carrier stirring paddle 131 adopts a stirring paddle with adjustable blade angle to adapt to different carriers, and the blades of the carrier stirring paddle 131 are fixed on the stirring shaft through bolts; activating the carrier paddle 131 during cell digestion can effectively assist in eluting cells from the carrier 12. The culture medium stirring paddle 141 adopts a radial stirring paddle, the outlet part of the reflux pipe 23 on the tank cover of the bioreactor 1 is horizontally arranged, and the outlet of the reflux pipe 23 corresponds to the blade of the culture medium stirring paddle 141 on the culture medium stirrer 14, so that the culture medium refluxed by the reflux pipe 23 can be uniformly mixed with the culture medium in the culture medium mixing tank 11.
In addition, it should be specifically noted that, in the mass production of mesenchymal stem cells, the medium stirrer 14 is in operation for a long period of time throughout the entire production process; whereas the carrier stirrer 13 is activated only at the beginning and end of production to adjust the internal cell distribution of the carrier 12 and to assist in the elution of cells from the carrier 12.
As shown in fig. 1: a microbubble generator 15 is arranged in the bioreactor 1, and the microbubble generator 15 is positioned below the culture medium stirring paddle 141; and be equipped with fixed baffle 16 under culture medium stirring rake 141, fixed baffle 16 is used for preventing the bubble that microbubble generator 15 produced from directly striking the stirring region of culture medium stirring rake 141 to the downward axial flow that the culture medium stirring rake 141 produced, maintain the homogeneity of stirring, the effectual microenvironment of guaranteeing the culture cell is unanimous. It is further defined that the horizontal central axis of the radial paddles is lower than the outlet lower edge of the return pipe 23 and the upper edge of the radial paddles is higher than the outlet lower edge of the return pipe 23; the radial stirring paddle is matched with the microbubble generator 15, so that gas-liquid exchange can be enhanced, the consistency of pH value, DO, temperature, glucose, glutamine, lactic acid and ammonia ions of a culture medium flowing through the bioreactor 1 can be effectively controlled, and the consistency of microenvironment of cultured cells can be effectively ensured.
As shown in fig. 1, the inlet of the external circulation pipeline of the external circulation mechanism 2 is connected with the outlet of a tank bottom valve F1 at the outlet of the bottom of the bioreactor 1, the outlet of the external circulation pipeline is connected with the inlet of a return pipe 23 on the tank cover of the bioreactor 1, and a centrifugal pump 21, a flowmeter 22 and a heat exchanger 24 are sequentially arranged on the external circulation pipeline along the flow direction of the culture medium. Because the reflux is carried out by the external circulation mechanism 2, the temperature of the refluxed culture medium is slightly lower than the temperature of the culture medium in the bioreactor 1, a heat exchanger 24 is arranged on the external circulation pipeline, a temperature control water inlet is arranged at the lower part of the heat exchanger 24, and a temperature control water outlet is arranged at the upper part of the heat exchanger, so that the temperature of the culture medium after the reflux by the external circulation mechanism 2 is consistent with the temperature of the culture medium in the bioreactor 1, and the consistency of the microenvironment of stem cell culture is ensured. Further, in order to achieve stability of medium reflux and sensitivity of adjustment, the centrifugal pump 21 is a magnetic suspension centrifugal pump and the flowmeter 22 is a mass flowmeter, and the flow rate of the medium passing through the fixed bed can be precisely controlled by using the magnetic suspension centrifugal pump and the mass flowmeter.
As shown in fig. 1, the collection means 3 comprises a cell collection tube 31 with a cell clamp a, a waste liquid collection tube 33 with a waste liquid clamp B, a cell collection tank 32 and a waste liquid collection tank 34, wherein the cell collection tank 32 is provided with the cell collection tube 31 with the cell clamp/valve a, the waste liquid collection tank 34 is provided with the waste liquid collection tube 33 with the waste liquid clamp/valve B, and the cell collection tube 31 and the waste liquid collection tube 33 are respectively connected with an outlet pipe of a tank bottom valve F1 at the outlet of the bottom of the bioreactor 1 through a pipe with a tee joint. In one embodiment of the present invention, as shown in FIG. 1, since the sterilization mechanism 4 is added, a part of the piping of the sterilization mechanism 4 coincides with a part of the piping of the collection mechanism 3, the cell collection tank 32 is connected to one end of the vertical main pipe of the collection three-way valve F8 through the cell collection pipe 31 with the cell pipe clamp A, the other end of the vertical main pipe of the collection three-way valve F8 is connected to the outlet end of the collection pipe 35, the inlet end of the collection pipe 35 is connected to the branch pipe outlet of the discharge three-way valve F5, one end of the horizontal main pipe of the discharge three-way valve F5 is connected to the outlet piping of the tank bottom valve F1 at the bottom outlet of the bioreactor 1, and at this time, the inlet of the waste collection pipe 33 is disposed at the upper stage of the cell collection pipe 31 between the inlet end of the cell collection pipe 31 and the cell pipe clamp/valve A.
In one embodiment of the present invention, in order to improve the production efficiency, the online sterilization of the external circulation mechanism 2 can be realized, as shown in fig. 1, the system further comprises a sterilization mechanism 4, the sterilization mechanism 4 is respectively provided with three-way valve groups at the inlet and the outlet of the external circulation pipeline of the external circulation mechanism 2 connected with the bioreactor 1, two ends of a horizontal main pipe of the three-way valve group at the inlet of the external circulation pipeline are respectively connected with the inlet of the external circulation pipeline and the bottom outlet of the bioreactor 1, and the branch pipe outlet of the three-way valve group is connected with a steam trap 41 in the sterilization mechanism 4 through corresponding pipelines; two ends of a horizontal main pipe of the three-way valve set at the outlet are respectively connected with the outlet of an external circulation pipeline and the inlet of a return pipe 23 on the tank cover of the bioreactor 1, and the inlet of a branch pipe of the three-way valve set is used for introducing steam; meanwhile, a condensation discharge port is provided at the lowest position of the pump head of the centrifugal pump 21 in the external circulation mechanism 2, a pipeline with a centrifugal pump discharge valve F7 is provided at the condensation discharge port and connected with the steam trap 41, and a pump cold end thermometer N2 as a temperature detecting element is provided on the pipeline at the outlet side of the pump discharge valve F7. A temperature detecting element is installed on each discharge pipe of the sterilizing unit 4 and is provided on the pipe in front of the steam trap 41 to ensure that the sterilizing temperature of the portion to be sterilized reaches 121 c or higher.
Further, to reduce costs and facilitate operation of the system, the respective pipes of the collection means 3 and the evacuation portion of the sterilization means 4 are integrated. One embodiment provided by the invention is: the outlet of the branch pipe of the discharge three-way valve F5 is respectively connected with the steam trap 41 in the collecting mechanism 3 and the sterilizing mechanism 4 through corresponding pipelines, at the moment, the outlet of the branch pipe of the discharge three-way valve F5 is connected with the inlet end of the collecting pipe 35, the outlet end of the collecting pipe 35 is connected with one end of the vertical main pipe of the collecting three-way valve F8, the other end of the vertical main pipe of the collecting three-way valve F8 is connected with the inlet end of the cell collecting pipe 31, the branch pipe of the collecting three-way valve F8 is connected with the drain branch pipe 43 with the condensing discharge valve F6 at the end part, the drain branch pipe 43 at the rear side of the condensing discharge valve F6 is provided with a sterilizing cold end thermometer N1 serving as a temperature detecting element, and the other end of the drain branch pipe 43 is connected with the drain pipe 42 with the steam trap 41 at the tail end. Further, the combination of the valves in the sterilization mechanism 4 may be various, and as illustrated in fig. 1 of the present invention, one of the combination of the valves in the sterilization mechanism 4, the reflux valve F2 and the sterilization three-way valve F4 may be two independent combinations of valves; the valve can also be a combined valve formed by integrating a reflux valve F2 and a sterilization three-way valve F4 shown in FIG. 2. Likewise, there are various combinations of the discharge three-way valve F5 and the discharge valve F3, the collection three-way valve F8 and the condensation discharge valve F6, all for the purpose of achieving the on-line sterilization of the external circulation line. If the tank body of the bioreactor 1 is a stainless steel tank body, the external circulation mechanism 2 can be sterilized on line together with the tank body of the bioreactor 1.
As shown in FIG. 2, the fixed bed bioreactor system shown in FIG. 1 is arranged on a mounting frame with casters and is provided with a control cabinet, an auxiliary pipeline and a monitoring system positioned at the left upper side of an external circulation mechanism 2, the barrel body of the bioreactor 1 is made of transparent materials (glass or polymer materials), a butterfly-shaped sealing head 17 is adopted, a tank bottom valve F1 is arranged at the bottom of the butterfly-shaped sealing head 17, a porous supporting plate 18 is arranged at the upper parts of the barrel body and the butterfly-shaped sealing head 17 of the bioreactor 1, two stirrers, pH sensors, DO (DO), glucose and temperature sensors are arranged on the upper cover of the bioreactor 1, and four gas inlet pipes, exhaust pipes, perfusion liquid inlet pipes, perfusion liquid outlet pipes, sampling pipes, digestive juice, stopping liquid, PBS (phosphate buffer solution), culture medium and carrier feeding ports are arranged. The control cabinet consists of a touch screen industrial personal computer, a PLC controller, software and the like, can control the flow of external circulation, the pH value, DO, temperature, glucose, lactic acid and the like in the bioreactor 1, and can perform perfusion. The whole production process comprises the steps of adherent expansion of stem cells in a reactor, liquid exchange, cleaning, digestion, termination and cell collection, and can realize full-closed, full-automatic, intelligent and digital production under the control of a computer.
The external circulation mechanism 2 of the invention can perform online sterilization, while the bioreactor 1 and the collecting mechanism 3 need offline sterilization, taking the glass tank type bioreactor 1 as an example, the sterilization flow is as follows:
(1) disconnecting the bioreactor 1 from the external circulation pipeline, placing the bioreactor 1, a reflux valve F2 and a discharge valve F3 which are connected with the external circulation pipeline into an autoclave for sterilization, wherein the reflux valve F2 and the discharge valve F3 are in a closed state, and the sterilization is completed and connected with the external circulation pipeline;
(2) the collecting mechanism 3 and the collecting three-way valve F8 are put into an autoclave for sterilization in advance, the collecting three-way valve F8 is connected with the condensation discharge valve F6 and the collecting pipe 35 after sterilization, and the collecting three-way valve F8 is in a closed state at the moment;
(3) after the sterilized bioreactor 1 and the collecting mechanism 3 are respectively connected with the external circulation mechanism 2, the reflux valve F2, the discharge valve F3 and the collecting three-way valve F8 are in a closed state; opening a discharge three-way valve F5, a condensation discharge valve F6 and a pump discharge valve F7, finally opening a sterilization three-way valve F4, allowing pure steam to enter an external circulation mechanism 2 from the sterilization three-way valve F4 for on-line Sterilization (SIP), starting timing and automatically or manually sterilizing for 30-45 min when the detection temperature of a sterilizing cold end thermometer N1 and a pump cold end thermometer N2 reaches 121 ℃, then closing the sterilization three-way valve F4, the discharge three-way valve F5, the condensation discharge valve F6 and the pump discharge valve F7, and finishing sterilization of the whole external circulation mechanism 2.
It should be noted that, in the protection scope of the present invention, the external circulation mechanism 2 and the collection mechanism 3 using disposable products are also used, the disposable pump head, the disposable hose, the disposable cell collection bag and the disposable waste liquid collection bag in the external circulation mechanism 2 are connected with the bioreactor 1 in a sterile manner, and then the flowmeter 22 and the heat exchanger 24 are installed, so that the function of culturing mesenchymal stem cells in a large scale can be realized.
As shown in fig. 1, when the fixed bed bioreactor system of the invention is used, a tank bottom valve F1 and a centrifugal pump 21 are opened to work, a culture medium stirrer 14 is operated, a flow meter 22 monitors the flow rate of the culture medium in real time and transmits the flow rate of the culture medium to a control mechanism in real time, and the control mechanism controls the operation of the centrifugal pump 21 and the rotation speed of a culture medium stirring paddle 141 of the culture medium stirrer 14 and other components according to the received flow rate to realize the adjustment of the culture medium substance exchange and the control of the shearing force in the bioreactor 1; the heat exchanger 24 keeps the temperature of the culture medium after the backflow through the external circulation mechanism 2 consistent with the temperature of the culture medium in the bioreactor 1, and ensures that the microenvironment of the cultured cells is consistent.
According to the invention, the external circulation mechanism 2 capable of precisely controlling the flow rate of the culture medium passing through the fixed bed is arranged on the bioreactor 1, the external circulation mechanism 2 measures the real-time flow rate of the culture medium through the flowmeter 22, the centrifugal pump 21 controls the reflux amount of the culture medium in real time according to the measured value of the flowmeter 22 so as to control the flow rate and the shearing force of the culture medium, so that the mesenchymal stem cells can be cultured on a large scale, and meanwhile, the culture quality of the stem cells can be improved and the production cost can be reduced; the bioreactor 1 adopts the structural design of double stirring paddles, and the arrangement of the carrier stirrer 13 can effectively help cells to elute from the carrier; by limiting the position of the culture medium stirring paddle 141 of the culture medium stirrer 14, not only can the culture medium be uniformly stirred, but also the rotating speed of the culture medium stirring paddle 141 can be matched with the culture medium reflux speed in the reflux pipe 23, so that the nutrition transmission and the shearing force of stem cells are effectively controlled, and the production efficiency is improved; the adopted butterfly-shaped sealing head 17 has small volume, saves a large amount of digestive juice in the process of eluting stem cells, is beneficial to improving the product quality of the stem cells and is extremely beneficial to the subsequent stem cell expansion; the sterilizing mechanism 4 can perform online sterilization under the condition of not disassembling the external circulation mechanism 2 through the arrangement of a plurality of valves and corresponding pipelines, so that the production efficiency of the bioreactor 1 is improved; the external circulation mechanism 2 capable of sterilizing on line can save cost and is beneficial to the sterilization operation of the large bioreactor 1; if the bioreactor 1 is a stainless steel tank body, the external circulation mechanism 2 and the bioreactor 1 can be sterilized together on line, so that large-scale culture is realized.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by the above embodiments, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
Claims (6)
1. A fixed bed bioreactor system for mass production of mesenchymal stem cells, characterized by: the system comprises a fixed bed type bioreactor (1), an external circulation mechanism (2) and a collecting mechanism (3), wherein the bioreactor (1) is used for amplifying mesenchymal stem cells; the external circulation mechanism (2) is used for receiving the culture medium discharged from the bottom of the bioreactor (1) and conveying the culture medium into the culture medium mixing tank (11) at the upper part of the bioreactor (1) so as to realize the circulation of the culture medium, and the external circulation mechanism (2) is provided with a flowmeter (22) so as to monitor the flow rate of the culture medium and regulate and control the material exchange of the culture medium according to the flow rate; the collecting mechanism (3) can respectively collect the mesenchymal stem cells cultured by the bioreactor (1) and the waste liquid generated during the culture of the mesenchymal stem cells; the lower part of the bioreactor (1) is provided with a carrier (12), the area above the carrier (12) forms a culture medium mixing tank (11), the bioreactor (1) is respectively provided with a carrier stirrer (13) and a culture medium stirrer (14), a carrier stirring paddle (131) of the carrier stirrer (13) is positioned in the carrier (12), and a culture medium stirring paddle (141) of the culture medium stirrer (14) is positioned in the culture medium mixing tank (11); a microbubble generator (15) is arranged in the bioreactor (1), and the microbubble generator (15) is positioned below the culture medium stirring paddle (141); a fixed baffle plate (16) is arranged right below the culture medium stirring paddle (141), and the fixed baffle plate (16) is used for preventing bubbles generated by the microbubble generator (15) from directly impacting a stirring area of the culture medium stirring paddle (141) and blocking downward axial flow generated by the culture medium stirring paddle (141); an inlet of an external circulation pipeline of the external circulation mechanism (2) is connected with an outlet of a tank bottom valve (F1) at the outlet of the bottom of the bioreactor (1), the outlet of the external circulation pipeline is connected with an inlet of a return pipe (23) on a tank cover of the bioreactor (1), and a centrifugal pump (21), a flowmeter (22) and a heat exchanger (24) are sequentially arranged on the external circulation pipeline along the flowing direction of a culture medium; the system also comprises a sterilization mechanism (4), wherein the sterilization mechanism (4) is respectively provided with a three-way valve group at the inlet and the outlet of an external circulation pipeline connected with the bioreactor (1) by an external circulation mechanism (2), two ends of a horizontal main pipe of the three-way valve group at the inlet of the external circulation pipeline are respectively connected with the inlet of the external circulation pipeline and the bottom outlet of the bioreactor (1), and the branch pipe outlet of the three-way valve group is connected with a steam trap (41) in the sterilization mechanism (4) by corresponding pipelines; two ends of a horizontal main pipe of the three-way valve group at the outlet are respectively connected with the outlet of an external circulation pipeline and the inlet of a return pipe (23) on a tank cover of the bioreactor (1), and the inlet of a branch pipe of the three-way valve group is used for introducing steam; the collecting mechanism (3) comprises a cell collecting tank (32) and a waste liquid collecting tank (34), wherein the cell collecting tank (32) is provided with a cell collecting pipe (31) with a cell pipe clamp/valve (A), and the waste liquid collecting tank (34) is provided with a waste liquid collecting pipe (33) with a waste liquid pipe clamp/valve (B).
2. The fixed bed bioreactor system for mass production of mesenchymal stem cells of claim 1, wherein: the bottom of the bioreactor (1) is provided with a butterfly-shaped sealing head (17).
3. The fixed bed bioreactor system for mass production of mesenchymal stem cells of claim 1, wherein: the carrier stirring paddle (131) adopts a stirring paddle with adjustable paddle angles to adapt to different carriers, and the paddles of the carrier stirring paddle (131) are fixed on the stirring shaft through bolts.
4. The fixed bed bioreactor system for mass production of mesenchymal stem cells of claim 1, wherein: the culture medium stirring paddle (141) adopts a radial stirring paddle, the outlet part of the return pipe (23) in the external circulation mechanism (2) is horizontally arranged, and the outlet of the return pipe (23) corresponds to the blade of the culture medium stirring paddle (141) on the culture medium stirrer (14).
5. The fixed bed bioreactor system for mass production of mesenchymal stem cells of claim 1, wherein: the discharge pipeline of the sterilizing mechanism (4) is provided with a temperature detecting element which is arranged on the pipeline in front of the steam trap (41) so as to ensure that the sterilizing temperature of the part to be sterilized reaches more than 121 ℃.
6. The fixed bed bioreactor system for mass production of mesenchymal stem cells of claim 1, wherein: the lowest part of the pump head of the centrifugal pump (21) in the external circulation mechanism (2) is provided with a condensation discharge port, a pipeline with a centrifugal pump discharge valve (F7) is arranged at the condensation discharge port, and the pipeline is connected with the steam trap (41).
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