CN115927161A - Method for culturing animal cells by using torrent bioreactor - Google Patents
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Abstract
The invention relates to a method for culturing animal cells by using a torrent bioreactor, which comprises the following steps: providing a torrent bioreactor; adding a cell basic culture medium into the torrent bioreactor, and controlling a pre-culture condition to perform pre-culture to obtain a pre-culture system; inoculating animal cells into a pre-culture system, and controlling batch culture conditions to perform batch culture to obtain the animal cells within a target density range; wherein, in the batch culture process, the temperature is monitored and controlled to be 27.0-37.2 ℃, the pH value is 6.20-7.35, the DO is more than 40%, the rotating speed is 35-60 rpm on line in real time through a control system of the torrent bioreactor, and the concentration of glucose is monitored and controlled to be more than 1.5g/L through periodic sampling. The method can realize high-density large-scale culture of the animal cells, and has high cell survival rate.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for culturing animal cells by using a torrent bioreactor.
Background
The high-density large-scale culture of animal cells is beneficial to improving the yield of vaccines and antibody protein medicines, and plays an important role in the field of biological pharmacy. At present, the conventional bioreactors for animal cell culture are mainly stirred reactors and wave reactors. The stirring reactor has the working principle that the liquid is driven to form a vortex by the rotation of the stirring paddle to realize the mixing process, and the gas is introduced into the bottom foamer to carry out bubbling and aeration so as to realize the gas-liquid exchange between the oxygen and the carbon dioxide and the culture medium. However, in the actual use process, the stirring reactor has the problems of high shear force, high energy dissipation, easy foam accumulation and the like caused by the stirring paddle and the bottom ventilation mode, and the phenomenon is particularly remarkable when mammalian cells and insect cells are cultured at high density. The working principle of the wave reactor is to realize the mixing process in the bag by shaking the waves generated by the swinging of the platform. Although the wave reactor can effectively avoid strong shearing force in the oxygen mass transfer process, realize the bubble-free gas-liquid exchange process beneficial to shearing sensitive cells and avoid the use of an antifoaming agent, the wave reactor is mainly used in the seed expansion culture stage, and is difficult to support large-volume culture due to the limitation of the reaction bag manufacturing process, so that the wave reactor is not suitable for large-scale culture of cells.
Meanwhile, in the large-scale culture of animal cells, the density of the animal cells is high, and the demand for oxygen is very large, so that high oxygen mass transfer efficiency is required. However, the mode of improving the oxygen mass transfer efficiency of both the wave reactor and the stirring reactor is to increase the rotating speed or the aeration quantity of deep oxygen. However, both of these methods bring a large amount of foam and higher shearing force, thereby resulting in higher cell killing efficiency, reducing dissipation efficiency of carbon dioxide in cell fluid, making it difficult to maintain normal cell proliferation and resulting in decreased cell survival rate, and further making it difficult to realize high-density large-scale culture of animal cells, especially animal cells sensitive to shearing force.
Accordingly, the conventional method of culturing animal cells in a bioreactor has problems in that it is difficult to achieve high-density large-scale culture of animal cells and the survival rate of the cells is low.
Disclosure of Invention
In view of the above, there is a need to provide a method for culturing animal cells using a torrent bioreactor, which can realize high-density large-scale culture of animal cells and has a high cell survival rate.
A method for culturing animal cells using a torrent bioreactor, comprising the steps of:
providing a torrent bioreactor;
adding a cell basic culture medium into the torrent bioreactor, and controlling a pre-culture condition to perform pre-culture to obtain a pre-culture system;
inoculating animal cells into the pre-culture system, and controlling batch culture conditions to perform batch culture to obtain the animal cells within a target density range;
wherein, in the batch culture process, the temperature is monitored and controlled to be 27.0-37.2 ℃, the pH value is 6.20-7.35, the DO is more than 40%, the rotating speed is 35rpm-60rpm in an online real-time manner through a control system of the torrent bioreactor, and the concentration of glucose is monitored and controlled to be more than 1.5g/L through periodic sampling.
In one embodiment, in the step of inoculating the animal cells in the pre-culture system, the inoculation density of the animal cells is (0.5-1.2). Times.10 6 cells/mL。
In one embodiment, the animal cells are selected from suspension cells selected from CHO, HEK293, or SF9.
In one embodiment, when the suspension cells are selected from CHO or HEK293, monitoring and controlling the glutamine concentration to be more than 2mM by periodic sampling is further included in the batch culture process.
In one embodiment, when said suspension cells are selected from CHO, said temperature is controlled to 36.8-37.2 ℃, said pH is controlled to 6.95-7.20, said DO value is controlled to 50% or more, said rotation speed is controlled to 55-60 rpm, said glucose concentration is controlled to 3-6 g/L, and said glutamine concentration is controlled to 2-4 mM during said batch culture.
In one embodiment, when said suspension cells are selected from HEK293, said temperature is controlled to 36.8 ℃ to 37.2 ℃, said pH is controlled to 6.95 to 7.35, said DO is controlled to 50% or more, said rotational speed is controlled to 35rpm to 60rpm, said glucose concentration is controlled to 1.5g/L to 6.0g/L, and said glutamine concentration is controlled to 2mM to 4mM during said batch culture.
In one embodiment, when the suspension cells are selected from SF9, the temperature is controlled to be 26.8-27.2 ℃, the pH value is controlled to be 6.20-6.40, the DO value is controlled to be more than 40%, the rotation speed is controlled to be 35-60 rpm, and the concentration of glucose is controlled to be 1.5-5.0 g/L during the batch culture process.
In one embodiment, the pH value is controlled by adding alkali liquor or introducing carbon dioxide into the torrent type bioreactor;
and/or the DO value is controlled by adjusting the surface ventilation or the rotating speed of the torrent bioreactor;
and/or the control of the concentration of the glucose is realized by a mode of supplementing the feed liquid.
In one embodiment, the pre-incubation time is 18h to 24h.
In one embodiment, the batch culture process further comprises monitoring and controlling animal cell density, cell viability, lactate content, ammonium root concentration or osmotic pressure by periodic sampling.
According to the invention, the torrent type bioreactor is adopted for inoculation culture of animal cells, on one hand, because the torrent type bioreactor does not contain a stirring and foaming device, the shearing force on the cells is very low and the gas-liquid mass transfer efficiency is high when high-density animal cells are cultured, so that the dissolved oxygen requirement of the high-density animal cells can be met, and the survival rate of the cells is improved; on the other hand, the control system of the torrent bioreactor monitors and controls batch culture conditions in real time on line and monitors and controls the concentration of glucose by sampling periodically, so that a long-term stable and healthy growth environment can be provided for high-density animal cell culture, and the high-density large-scale culture and survival rate of animal cells are improved.
Therefore, the method for culturing the animal cells by using the torrent bioreactor can realize high-density large-scale culture of the animal cells and has high cell survival rate.
Drawings
FIG. 1 is a graph showing the variation of viable cell density and cell viability at different time points during batch culture of CHO cells in example 2;
FIG. 2 is a graph showing the variation of viable cell density and cell viability at different time points during batch culture of HEK-293 cells of example 5 and HEK-293 cells of comparative example 8;
FIG. 3 is a graph showing the variation of viable cell density, cell viability and clumping rate of SF9 cells at different time points during batch culture in example 9;
FIG. 4 is a graph showing the viable cell density, the cell viability and the clumping rate of SF9 cells at different time points during batch culture according to the present invention;
FIG. 5 is a graph showing the growth of SF9 cells of example 9 and comparative example 9 in a batch culture process;
FIG. 6 is a cell morphology chart of SF9 cells in batch culture time of 4.5h, 101h and 214h in example 9 provided by the invention;
FIG. 7 is a cell morphology chart of SF9 cells in comparative example 9 at batch culture time of 4.5h, 101h and 214 h;
FIG. 8 is a graph showing the variation of viable cell density, cell viability and clumping rate of SF9 cells at different time points during batch culture in example 10;
FIG. 9 is a graph showing viable cell density, cell viability and clumping rate of SF9 cells of comparative example 11 at different time points during batch culture;
FIG. 10 is a graph showing the growth of SF9 cells in example 10 and comparative example 12 during batch culture according to the present invention;
FIG. 11 is a cell morphology diagram of SF9 cells in batch culture time of 4.5h, 101h and 214h in example 10 provided by the present invention;
FIG. 12 is a cell morphology chart of SF9 cells in comparative example 12 at batch culture time of 4.5h, 101h and 214 h.
Detailed Description
The method for culturing animal cells using the torrent bioreactor according to the present invention will be further described below.
The method of the invention is mainly used for high-density large-scale culture of animal cells, so that the inoculation density is (0.5-1.2) multiplied by 10 6 The maximum cell density of the cells/mL can reach (0.9-1.2) × 10 after batch culture 6 cells/mL, and the cell survival rate reaches more than 92%, so that the purposes of high-density large-scale culture and high cell survival rate of animal cells are achieved, and particularly the shear force sensitive animal cells are more favorable for meeting the requirements of the biopharmaceuticals such as vaccine production, gene recombinant proteins and antibody protein drugs on cell density and cell activity, and improving the yield of the biopharmaceuticals.
In the method of culturing animal cells using a bioreactor, the present applicant has found, through long-term and intensive studies, that animal cells are sensitive to shear force due to the absence of cell walls, have low survival rate due to weak cells, and have an increased demand for dissolved oxygen during high-density large-scale culture. However, in the conventional bioreactor (e.g., a wave reactor, a stirring reactor), when animal cells are cultured in a large scale at a high density, the dissolved oxygen is increased by increasing the oxygen mass transfer efficiency, and the oxygen mass transfer efficiency is increased by increasing the rotation speed or increasing the aeration rate of deep oxygen. However, both of these methods bring a large amount of foam and higher shearing force, thereby resulting in higher cell killing efficiency, reducing the dissipation efficiency of carbon dioxide in cell fluid, making it difficult to maintain normal cell proliferation and thus decreasing the cell survival rate, and further making it difficult to realize high-density large-scale culture of animal cells, especially animal cells sensitive to shearing force.
The torrent type bioreactor is a non-bubbling type bioreactor, and the principle is that the reactor rotates around a central shaft at a fixed speed, liquid forms a thin film with high gas-liquid mass transfer efficiency on the wall of the reactor along with inertia, and the thin film is combined with a liquid main body after being saturated by oxygen in a top space. The torrent type bioreactor has the advantages of low shearing force on cells, high gas-liquid mass transfer efficiency, accurate parameter control, simple experiment operation, good scalability and capability of ingeniously avoiding the defects of a wave type reactor and a stirring type reactor because the torrent type bioreactor does not contain a stirring and foaming device, and is a better reactor for culturing animal cells (such as mammalian cells and insect cells).
Accordingly, the present invention provides a method for culturing animal cells using a torrent-type bioreactor, which comprises the steps of:
s1, providing a torrent bioreactor;
s2, adding a cell basic culture medium into the torrent bioreactor, and controlling a pre-culture condition to perform pre-culture to obtain a pre-culture system;
s3, inoculating the animal cells into the pre-culture system, and controlling batch culture conditions to perform batch culture to obtain the animal cells within the target density range; wherein, in the batch culture process, the temperature is monitored and controlled to be 27.0-37.2 ℃, the pH value is 6.20-7.35, the DO is more than 40%, the rotating speed is 35-60 rpm on line in real time by a control system of the torrent bioreactor, and the concentration of the glucose is monitored and controlled to be more than 1.5g/L by periodic sampling.
According to the invention, the torrent type bioreactor is adopted for inoculation culture of animal cells, on one hand, because the torrent type bioreactor does not contain a stirring and foaming device, the shearing force on the cells is very low and the gas-liquid mass transfer efficiency is high when high-density animal cells are cultured, so that the dissolved oxygen requirement of the high-density animal cells can be met, and the survival rate of the cells is improved; on the other hand, the control system of the torrent bioreactor monitors and controls batch culture conditions in real time on line and monitors and controls the concentration of glucose by sampling periodically, so that a long-term stable and healthy growth environment can be provided for high-density animal cell culture, and the high-density large-scale culture and survival rate of animal cells are improved.
Therefore, the method for culturing the animal cells by using the torrent bioreactor can realize high-density large-scale culture of the animal cells and has high cell survival rate.
In the present invention, batch culture refers to a culture method in which only a single component solution such as glucose or glutamine is added during culture, and a complex medium solution such as a basal medium or a feed medium is not added.
In one embodiment, the animal cell is selected from a suspension cell selected from CHO (chinese hamster ovary cells), HEK293 (human embryonic kidney cells) or SF9 (spodoptera frugiperda ovary cells). Further preferably, the CHO is selected from CHO-K1.
It should be noted that the type of the cell basic medium in the present invention can be selected according to the type of the inoculated animal cells, so as to meet the growth requirements of different types of cells. The basic culture medium of the cells such as CHO is a CHO-WM1 basic culture medium; the cell basic culture medium of the HEK293 cell is a Celer-S001S basic culture medium; the cell basic culture medium of the SF9 cells is SF-SFM culture medium.
In the field of biopharmaceutical technology, the density requirements for different kinds of animal cells are different, and the maximum cell density of each animal cell can be cultured is different, and the corresponding initial culture cell density (inoculation density of animal cells) is also required, and the difference of initial culture cell density affects the final growth density and cell activity of animal cells. In the present invention, it is preferable that the inoculation density of the animal cells in step S3 is (0.5-1.2). Times.10 6 cells/mL. Further preferably, the CHO has a seeding density of (0.5-1.0). Times.10 6 cells/mL, the seeding density of the HEK293 is (0.8-1.2) multiplied by 10 6 cells/mL, the inoculation density of the SF9 is (0.5-0.8) multiplied by 10 6 cells/mL。
In order to ensure the sterility of a culture bag in the torrent bioreactor and the stability of the initial cell culture environment, in step S2, adding a cell basic culture medium into the culture bag in the torrent bioreactor, setting physical reaction parameters of the torrent bioreactor and controlling the pre-culture conditions for pre-culture for 18-24 h to obtain a pre-culture system.
In one embodiment, in step S2, the pre-culture conditions include temperature, pH, DO (dissolved oxygen) and rotation speed, and during the pre-culture process, the temperature and rotation speed can be automatically controlled by the control system of the torrent bioreactor, and the control of the pH value is realized by adding lye into the torrent bioreactor or introducing carbon dioxide, preferably, a sodium bicarbonate solution with a lye mass fraction of 8% is used for increasing the pH value, and the carbon dioxide is used for reducing the pH value.
In one embodiment, in step S2, the DO value is controlled by adjusting the surface aeration rate or the rotational speed of the torrent bioreactor during the pre-culture. Preferably, the gas with surface aeration is selected from at least one of oxygen or air, so as to increase the dissolved oxygen in the culture process; the oxygen mass transfer efficiency can be improved by properly increasing the rotating speed, so that the dissolved oxygen content is improved, and the demand of cell growth on the oxygen content is met.
In addition, in step S3, before the batch culture, setting physical reaction parameters of the torrent bioreactor so as to control batch culture conditions, thereby ensuring normal growth and metabolism of animal cells.
In one embodiment, in step S3, the batch culture conditions include temperature, pH, DO (dissolved oxygen) and rotation speed, and preferably, the pH is controlled by adding alkaline solution or introducing carbon dioxide into the torrent bioreactor, and further preferably, the mass fraction of the alkaline solution is 8% of sodium bicarbonate solution to increase the pH, and the carbon dioxide is introduced to reduce the pH.
In one embodiment, the DO value is controlled by adjusting the meter aeration or the rotational speed of the torrent bioreactor. Preferably, the gas with surface aeration is selected from at least one of oxygen or air, so as to increase the dissolved oxygen in the culture process; the oxygen mass transfer efficiency can be improved by properly increasing the rotating speed, so that the dissolved oxygen is increased, and the requirement of cell growth on oxygen is met.
In one embodiment, control of the glucose concentration or glutamine concentration can be achieved by feeding the feed solution. Preferably, the feed liquid is a cell basal medium.
In the invention, the volume of the torrent bioreactor and the setting of physical reaction parameters can be correspondingly adjusted according to the culture conditions, the culture density and the culture scale required by the growth of different animal cells, so that the culture conditions can be better controlled, and the density and the cell activity of the cells can be further controlled.
In one embodiment, the animal cells are selected from suspension cells, and when the suspension cells are CHO cells and cultured in a torrent bioreactor having a volume of 5L, the pre-culture stage: setting physical reaction parameters of the torrent bioreactor: temp:36.8 ℃ -37.2 ℃, pH: 7.05. + -. 0.15, DO: more than or equal to 50 percent, motor:48rpm-52rpm, taoton: 198mL/min-202mL/min of air; batch culture stage: setting physical reaction parameters of the torrent bioreactor: temp:36.8 ℃ -37.2 ℃, pH: 7.05. + -. 0.15, DO: not less than 50%, motor:55rpm-60rpm, taoton: 198mL/min-202mL/min of air. By the arrangement, a stable growth environment can be provided for the CHO animal cells, and the growth density and the activity rate of the cells are further improved.
In one embodiment, the animal cells are selected from suspension cells, and when the suspension cells are HEK293 and cultured in a torrent bioreactor with a volume of 5L, the pre-culture stage: setting physical reaction parameters of the torrent bioreactor: temp. 36.8-37.2 deg.C, pH: 7.15. + -. 0.20, DO: not less than 50%, motor:48rpm-52rpm, taotong: 98-102 mL/min of air; a batch culture stage: setting physical reaction parameters of the torrent bioreactor: temp:36.8 ℃ -37.2 ℃, pH: 7.15. + -. 0.20, DO: more than or equal to 50 percent, motor:45rpm-60rpm, taotong: 98mL/min-102mL/min of air. By the arrangement, a stable growth environment can be provided for the HEK293 cell, and the growth density and activity rate of the cell are further improved.
In one embodiment, the animal cells are selected from suspension cells, and when the suspension cells are SF9 and cultured in a torrent bioreactor with a volume of 50L, the pre-culture stage: setting physical reaction parameters of the torrent bioreactor: temp:26.8 ℃ -27.2 ℃, pH: 6.30. + -. 0.1, DO: not less than 40%, motor:35rpm-45rpm, taoton: 0.3L/min-0.5L/min air; batch culture stage: setting physical reaction parameters of the torrent bioreactor: temp: 26.8-27.2 ℃, pH: 6.30. + -. 0.1, DO: not less than 40%, motor:35rpm-45rpm, taoton: 0.3L/min-0.5L/min of air. The arrangement can provide a stable growth environment for SF9 cells, and further improve the growth density and activity rate of the cells.
Considering that different animal cells need different nutrients and need many nutrients, such as the nutrients needed for the growth of CHO and HEK293 include glutamine in addition to glucose, in order to monitor the change of the cell nutrients in the batch culture process in time and adjust the change accordingly, a steady growth environment is provided for the animal cells. Preferably, when the suspension cells are selected from CHO or HEK293, monitoring and controlling the glutamine concentration to be more than 2mM by periodic sampling is further included in the batch culture process.
In batch culture, batch culture conditions are critical to improve growth density, cell viability, and metabolic status of animal cells.
In one embodiment, when said suspension cells are selected from CHO, said temperature is controlled to 36.8-37.2 ℃, said pH is controlled to 6.90-7.20, said DO value is controlled to 50% or more, said rotation speed is controlled to 55-60 rpm, said glucose concentration is controlled to 3-6 g/L, and said glutamine concentration is controlled to 2-4 mM during said batch culture.
In one embodiment, when said suspension cells are selected from the group consisting of HEK293, said temperature is controlled to be 36.8 ℃ to 37.2 ℃, said pH is controlled to be 6.95 to 7.35, said DO is controlled to be 50% or more, said rotational speed is controlled to be 35rpm to 60rpm, said glucose concentration is controlled to be 1.5g/L to 6.0g/L, and said glutamine concentration is controlled to be 2mM to 4mM during said batch culture.
In one embodiment, when the suspension cells are selected from SF9, the temperature is controlled to be 26.8-27.2 ℃, the pH value is controlled to be 6.20-6.40, the DO value is controlled to be 40% or more, the rotation speed is controlled to be 35-60 rpm, and the concentration of glucose is controlled to be 1.5-5.0 g/L during the batch culture process.
By the arrangement, a healthy and stable growth environment can be provided for CHO, HEK293 or SF9 cells, and the growth density and the cell survival rate of the cells can be further improved.
In order to know the growth and metabolism of cells in the batch culture process in time, in one embodiment, the monitoring and control of animal cell density, cell viability, lactate content, ammonium concentration or osmotic pressure are also included in the batch culture process through periodic sampling. Preferably, sampling is performed every 8h to 12h, and the animal cell density, the cell viability, the glucose concentration, the glutamine concentration, the lactate concentration, the ammonium root concentration or the osmolality is monitored and controlled.
In the present invention, the preculture conditions substantially match the physical reaction parameter settings of the torrent type bioreactor in the preculture stage, and the batch culture conditions substantially match the physical reaction parameter settings of the torrent type bioreactor in the batch culture stage.
Hereinafter, the method for culturing animal cells using the torrent bioreactor will be further described with reference to the following specific examples.
It should be noted that the animal cell types, cell basic media and other reagents of the present invention can be purchased from the market, and the cell density and cell viability of the sample can be measured by a Countstar IC100 cell counter, the biochemical value of the solution can be measured by a sielman science M900 biochemical detector, and the osmotic pressure of the solution can be measured by an osmolarity instrument of hangzhou le qian 6003.
Example 1
Adding 2L of CHO-WM1 basal medium into a culture bag of a torrent bioreactor with the volume of 5L for pre-culture for 24h, and controlling the pre-culture condition to obtain a pre-culture system; wherein, the physical reaction parameters of the torrent type bioreactor are set as follows: temp:37 ℃, pH:7.05, DO:50%, motor:50rpm, taoton: 200mL/min of air. Then, CHO-K1 cells were inoculated in the preculture system and fed with 1L of CHO-WM1 basal medium for batch culture, wherein the CHO-K1 cells were inoculated at a density of 0.558X 10 6 cells/mL, the survival rate is 97.58%, and the physical reaction parameters of the torrent bioreactor are set as follows: temp:37 ℃, pH:7.0, DO:50%, motor:55rpm, taoton: 200mL/min of air; in the batch culture process, the control system of the torrent bioreactor monitors and controls the batch culture condition and the change of glucose content in real time on line, the temperature is controlled at 37 ℃, the pH value is controlled at 7.0, the DO is controlled at 50%, the rotating speed is controlled at 55rpm, the concentration of glucose is controlled at 4g/L, the concentration of glutamine is controlled at 4mM, meanwhile, in the batch culture process, samples are taken every 12h, the detection of animal cell density, cell viability, biochemical value and osmotic pressure is carried out, after the batch culture of 72.89h, the CHO-K1 cell density reaches a plateau stage, namely, is basically unchanged, and the batch culture is ended. This exampleThe maximum cell density of the cultured CHO-K1 cells was 0.926X 10 7 cells/mL, and the survival rate reaches 99.20 percent.
Example 2
Adding 2L of CHO-WM1 basal medium into a culture bag of a torrent bioreactor with the volume of 5L for pre-culture for 24h, and controlling the pre-culture condition to obtain a pre-culture system; wherein, the physical reaction parameters of the torrent type bioreactor are set as follows: temp:37 ℃, pH:7.05, DO:50%, motor:50rpm, taoton: 200mL/min of air. Then, CHO-K1 cells were inoculated in the preculture system and fed with 1L of CHO-WM1 basal medium for batch culture, wherein the CHO-K1 cells were inoculated at a density of 0.505X 10 6 cells/mL, survival rate of 93.73%, setting of physical reaction parameters of the torrent bioreactor: temp:37 ℃, pH:7.0, DO:50%, motor:50rpm, taoton: 200mL/min of air; in the batch culture process, the control system of the torrent bioreactor monitors and controls the batch culture condition and the change of the glucose content in real time on line, the temperature is controlled to be 37 ℃, the pH value is controlled to be 6.9, the DO is controlled to be 50%, the rotating speed is controlled to be 50rpm, the concentration of the glucose is controlled to be 6g/L, the concentration of the glutamine is controlled to be 4mM, meanwhile, in the batch culture process, sampling is carried out every 12h, the detection of the animal cell density, the cell viability, the biochemical value and the osmotic pressure is carried out, after the batch culture is carried out for 118.3h, the CHO-K1 cell density reaches a plateau period, namely, the CHO-K1 cell density is basically unchanged, and the batch culture is finished. The CHO-K1 cells cultured in this example had a maximum cell density of 1.05X 10 7 cells/mL, and the survival rate reaches 97.52 percent.
Example 3
Compared with example 2, the only difference is that in example 3, the batch culture conditions and the change of glucose content are monitored and controlled on line in real time by the control system of the torrent bioreactor during the batch culture, the temperature is controlled at 37 ℃, the pH value is controlled at 6.9, the DO is controlled at 60%, the rotation speed is controlled at 55rpm, the glucose concentration is controlled at 6g/L, and the glutamine concentration is controlled at 4mM. The maximum cell density of CHO-K1 cells cultured in this example was 1.03X 10 7 cells/mL, survivalThe rate reaches 94.75 percent.
Example 4
The only difference compared with example 2 is that in example 4, the batch culture conditions and the change of glucose content were monitored and controlled on-line in real time by the control system of the torrent bioreactor during the batch culture at 37 ℃ with the pH value of 7.05 and the DO of 50%, at 45rpm, at 5g/L and at 3mM by the control system of the torrent bioreactor. The CHO-K1 cells cultured in this example had a maximum cell density of 1.08X 10 7 cells/mL, survival rate reaches 92.16%.
Example 5
Adding 2L of Celer-S001S basal medium into a culture bag of a torrent bioreactor with the volume of 5L for pre-culture for 24h, and controlling the pre-culture conditions to obtain a pre-culture system; wherein, the physical reaction parameters of the torrent type bioreactor are set as follows: temp:37 ℃, pH:7.15, DO:50%, motor:50rpm, taoton: 100mL/min air. Then, HEK293 cells were inoculated in the preculture system and fed with 1L of Celer-S001S basal medium for batch culture, wherein the density of HEK293 cells inoculation was 0.787X 10 6 cells/mL, the survival rate is 98.06%, and the physical reaction parameters of the torrent bioreactor are set as follows: temp:37 ℃, pH:7.0, DO:50%, motor:50rpm, taoton: 200mL/min of air; in the batch culture process, the control system of the torrent bioreactor monitors and controls the batch culture condition and the change of the glucose content in real time on line, the temperature is controlled to be 37 ℃, the pH value is controlled to be 7.0, the DO is controlled to be 50%, the rotating speed is controlled to be 50rpm, the concentration of the glucose is controlled to be 3g/L, the concentration of the glutamine is controlled to be 4mM, meanwhile, in the batch culture process, samples are taken every 12h, the detection of the density, the cell viability, the biochemical value and the osmotic pressure of the animal cells is carried out, after the batch culture is carried out for 5 days, the density of the HEK293 cells reaches a plateau stage, namely, is basically unchanged, and the batch culture is finished. The HEK293 cells cultured in this example had a maximum cell density of 0.733X 10 7 cells/mL, the survival rate reaches 97.42%.
Example 6
Adding 15L of Celer-S001S basal medium into a culture bag of a torrent bioreactor with the volume of 50L for pre-culture for 24h, and controlling the pre-culture conditions to obtain a pre-culture system; wherein, the physical reaction parameters of the torrent type bioreactor are set as follows: temp:37 ℃, pH:7.15, DO:50%, motor:35rpm, taoton: 800mL/min air. Then, HEK293 cells were inoculated in the preculture system and fed with 3L of Celer-S001S basal medium for batch culture, wherein the density of HEK293 cells inoculation was 0.802X 10 6 cells/mL, the survival rate is 97.68%, and the physical reaction parameters of the torrent bioreactor are set as follows: temp:37 ℃, pH:7.15, DO:50%, motor:35rpm, taoton: 200mL/min of air; in the batch culture process, the control system of the torrent bioreactor monitors and controls the batch culture condition and the change of the glucose content in real time on line, the temperature is controlled to be 37 ℃, the pH value is controlled to be 7.15, the DO is controlled to be 50%, the rotating speed is controlled to be 35rpm, the concentration of the glucose is controlled to be 3g/L, the concentration of the glutamine is controlled to be 4mM, meanwhile, in the batch culture process, samples are taken every 12 hours, the detection of the density, the cell viability, the biochemical value and the osmotic pressure of the animal cells is carried out, after the batch culture is carried out for 5 days, the density of the HEK293 cells reaches a plateau stage, namely, is basically unchanged, and the batch culture is finished. The HEK293 cells cultured in this example had a maximum cell density of 0.513X 10 7 cells/mL, the survival rate reaches 92.07%.
Example 7
Compared with example 5, the only difference is that in example 7, the batch culture conditions and the change of glucose content are monitored and controlled on-line in real time by the control system of the torrent bioreactor during the batch culture, the temperature is controlled at 37 ℃, the pH value is controlled at 7.20, the DO is controlled at 50%, the rotation speed is controlled at 45rpm, the glucose concentration is controlled at 3g/L, and the glutamine concentration is controlled at 3mM. HEK293 cells cultured in this example had a maximum cell density of 0.714X 10 7 cells/mL, and the survival rate reaches 92.75 percent.
Example 8
Compared with example 5, the only difference is thatIn example 8, during the batch culture, the batch culture conditions and the change of the glucose content were monitored and controlled on-line in real time by the control system of the torrent bioreactor at 37 ℃ and at 7.0 pH and 50% DO, 50rpm, 4g/L glucose and 2mM glutamine, respectively. HEK293 cells cultured in this example had a maximum cell density of 0.729X 10 7 cells/mL, the survival rate reaches 96.56%.
Example 9
Adding 12L of SF-SFM culture medium into a culture bag of a torrent bioreactor with the volume of 50L for pre-culture for 24h, and controlling the pre-culture condition to obtain a pre-culture system; wherein, the physical reaction parameters of the torrent type bioreactor are set as follows: temp:27 ℃, pH:6.20, DO:50%, motor:35rpm, taoton: 0.4L/min air. Then, inoculating the recovered SF9 cells into the pre-culture system, and supplementing 3L of SF-SFM culture medium to ensure that the final culture volume is 15L for batch culture, wherein the inoculation density of the SF9 cells is 0.794X 10 6 cells/mL, the survival rate is 98.49%, and the physical reaction parameters of the torrent bioreactor are set as follows: temp:37 ℃, pH:6.20, DO:50%, motor:35rpm, taoton: 0.4L/min air; in the batch culture process, the control system of the torrent bioreactor monitors and controls the batch culture condition and the change of the glucose content in real time on line, the temperature is controlled to be 27 ℃, the pH value is controlled to be 6.20, the DO is controlled to be 50 percent, the rotating speed is controlled to be 35rpm, and the concentration of the glucose is controlled to be 5g/L; meanwhile, in the batch culture process, samples are taken every 24 hours to detect the density, the cell viability, the biochemical value and the osmotic pressure of the animal cells, and the density of the SF9 cells reaches a plateau stage after 7 days of batch culture, namely is basically unchanged. The batch culture was terminated. SF9 cells cultured in this example had a maximum cell density of 1.25X 10 7 cells/mL, the survival rate reaches 98.95%.
Example 10
The only difference compared to example 9 is that in example 10, during the batch culture process, the on-line real-time monitoring is performed by the control system of the torrent bioreactorAnd controlling batch culture conditions and glucose content change, controlling temperature at 27 deg.C, pH at 6.30, DO at 50%, rotation speed at 36rpm, and glucose concentration at 5g/L. SF9 cells cultured in this example had a maximum cell density of 1.11X 10 7 cells/mL, survival rate reaches 95.97%.
Example 11
Compared with example 9, the difference is only that in example 11, the batch culture conditions and the change of glucose content are monitored and controlled on line in real time by the control system of the torrent bioreactor during the batch culture, the temperature is controlled to be 27.0 ℃, the pH value is controlled to be 6.4, the DO is controlled to be 50%, the rotating speed is controlled to be 35rpm, and the concentration of glucose is controlled to be 3g/L. SF9 cells cultured in this example had a maximum cell density of 1.18X 10 7 cells/mL, and the survival rate reaches 89.13 percent.
Example 12
Compared with example 9, the difference is only that in example 12, the batch culture conditions and the change of the glucose content are monitored and controlled on line in real time by the control system of the torrent bioreactor during the batch culture process, the temperature is controlled to be 27.0 ℃, the pH value is controlled to be 6.30, the DO is controlled to be 50%, the rotating speed is controlled to be 38rpm, and the concentration of the glucose is controlled to be 4g/L. SF9 cells cultured in this example had a maximum cell density of 1.01X 10 7 cells/mL, and the survival rate reaches 92.35 percent.
Comparative example 1
Compared with example 2, the only difference is that in comparative example 1, the batch culture conditions and the change of glucose content were monitored and controlled on-line in real time by the control system of the torrent bioreactor during the batch culture at 37 ℃ in which the pH was controlled to 7.4, the DO was controlled to 30%, the rotation speed was controlled to 60rpm, the glucose concentration was controlled to 4g/L, and the glutamine concentration was controlled to 2mM. The CHO-K1 cell cultured in this comparative example had a maximum cell density of 0.77X 10 7 cells/mL, the survival rate reaches 70.50%.
Comparative example 2
Compared with the embodiment 2, the difference is only thatIn the proportion 2, in the batch culture process, the batch culture conditions and the change of the glucose content are monitored and controlled on line in real time by a control system of the torrent bioreactor, the temperature is controlled to be 37 ℃, the pH value is controlled to be 7.05, the DO is controlled to be 50%, the rotating speed is controlled to be 70rpm, the concentration of the glucose is controlled to be 1.4g/L, and the concentration of the glutamine is controlled to be 5mM. The CHO-K1 cell cultured in this comparative example had a maximum cell density of 0.35X 10 7 cells/mL, and the survival rate reaches 65.65%.
Comparative example 3
Compared with example 2, the difference is only that in comparative example 3, the wave bioreactor is used to replace the torrent bioreactor for CHO-K1 cell culture, the rocking frequency is 12rpm, the rocking angle is 6 degrees, and the rest conditions are the same. The CHO-K1 cell cultured in this comparative example had a maximum cell density of 0.75X 10 7 cells/mL, and the survival rate reaches 90.4 percent.
Comparative example 4
The only difference from example 2 is that in comparative example 4, the culture of CHO-K1 cells was carried out using a stirred bioreactor instead of the torrent bioreactor, with a stirring speed of 75rpm, and aeration of 0.8L/min, and the other conditions were the same. The CHO-K1 cell cultured in this comparative example had a maximum cell density of 0.84X 10 7 cells/mL, the survival rate reaches 80.5%.
Comparative example 5
Compared with example 5, the difference is only that in comparative example 5, the batch culture conditions and the change of glucose content were monitored and controlled on-line in real time by the control system of the torrent bioreactor during the batch culture at a temperature of 36.8 ℃, a pH of 6.8, a DO of 35%, a rotation speed of 55rpm, a glucose concentration of 1.2g/L and a glutamine concentration of 1.5mM. The HEK293 cells cultured in this comparative example had a maximum cell density of 0.46X 10 7 cells/mL, the survival rate reaches 80.5%.
Comparative example 6
The only difference compared to example 5 is that in comparative example 6, HEK293 cells were carried out using a wave bioreactor instead of a flow bioreactorThe rocking frequency was 12rpm, the rocking angle was 6 °, and the conditions were the same. HEK293 cells cultured in this comparative example had a maximum cell density of 0.43X 10 7 cells/mL, the survival rate reaches 88 percent.
Comparative example 7
The only difference from example 5 is that in comparative example 7, the HEK293 cells were cultured using a stirred bioreactor instead of a turbulent bioreactor, the stirring speed was 75rpm, and the air was introduced at 0.8L/min, and the other conditions were the same. HEK293 cells cultured in this comparative example had a maximum cell density of 0.62X 10 7 cells/mL, the survival rate reaches 82.4%.
Comparative example 8
The only difference compared to example 5 is that in comparative example 8, HEK293 cells were cultured by inoculating them in shake flasks at a volume of 30mL, a temperature of 37 ℃ and a CO2 concentration of 5%, and the other conditions were the same. HEK293 cells cultured in this comparative example had a maximum cell density of 0.7X 10 7 cells/mL, and the survival rate reaches 95.02 percent.
Comparative example 9
Compared with example 9, the difference is only that in comparative example 9, the batch culture conditions and the change of glucose content were monitored and controlled on-line in real time by the control system of the torrent bioreactor during the batch culture at a temperature of 27 ℃, a pH of 6.5, a DO of 30%, a rotation speed of 33rpm, and a glucose concentration of 4g/L. The SF9 cells obtained by the comparative example culture had a maximum cell density of 0.75X 10 7 cells/mL, survival rate reaches 88.23%.
Comparative example 10
The difference from example 9 is only that in comparative example 10, the culture of SF9 cells was carried out using a wave bioreactor instead of the flow bioreactor, and the conditions were the same except that the rocking frequency was 12rpm and the rocking angle was 6 °. The SF9 cells cultured in this comparative example had a maximum cell density of 0.82X 10 7 cells/mL, the survival rate reaches 92.5%.
Comparative example 11
In comparison with example 9, in comparative example 11Inoculating the recovered SF9 cells into a shake flask containing 30mL of SF-SFM medium, setting the temperature at 27 ℃, and 0% CO 2 The concentration and the rotation speed are 110rpm, and the rest conditions are the same. The SF9 cells cultured in this comparative example had a maximum cell density of 1.1X 10 7 cells/mL, and the survival rate reaches 94.35 percent.
Comparative example 12
In comparison with example 10, in comparative example 12, the recovered SF9 cells were inoculated into a shake flask containing 30mL of SF-SFM medium at a temperature set at 27 ℃ with 0% CO 2 The concentration and the rotation speed are 110rpm, and the rest conditions are the same. The SF9 cells cultured in this comparative example had a maximum cell density of 1.02X 10 7 cells/mL, survival rate reaches 93.01%.
As can be clearly seen from fig. 1, the cells cultured in this example 2 had good growth status, were maintained at high viability throughout the process, and were in logarithmic growth phase from the start of inoculation to day 4, with doubling time less than 24h; when the cells reached a plateau by day 5, the peak density was 1.05X 10 7 cells/mL, and the batch culture was terminated on day 5. Meanwhile, in the whole culture period in the embodiment 2, the temperature is very stable without obvious fluctuation, meanwhile, the pH curve is kept stable, and the pH is in a descending trend due to the accumulation of lactic acid, but the fluctuation is small and is within an allowable range; meanwhile, the oxygen consumption is gradually increased due to the increase of the cell density, the dissolved oxygen curve slides down to be below 50%, and the dissolved oxygen curve is increased to be above 50% again after the rotating speed of the torrent type bioreactor is gradually increased to 55rpm from 50 rpm. It can be seen that the torrent bioreactor can rapidly increase dissolved oxygen by increasing the rotation speed under the condition of not changing other conditions, namely, the torrent bioreactor can maintain 1.05X 10 without pure oxygen 7 The CHO cell culture of cells/mL or so shows that the gas-liquid mass transfer efficiency of the torrent type reactor is extremely high, the dissolved oxygen requirement of cells with higher density can be maintained by introducing air into the torrent type reactor, and the oxygen ventilation quantity in the culture process can be greatly reduced. Therefore, the torrent bioreactor only carries out dissolved oxygen transfer by surface ventilation of air and oxygen, so that a large amount of foam generated by bubbling ventilation below the liquid level of the traditional stirring type reactor does not exist, and bubbling ventilation does not existCell damage caused by energy dissipation in the case of gas, and the lethality on cells generated when the foam is broken is greatly reduced.
Referring to FIG. 2, it can be seen from FIG. 2 that the HEK293 cells in the example 5 were in good condition, the viability was higher from the beginning of the culture than the shake flasks in the comparative example 8 and all the time in the high viability state, and the density of the shake flasks in the comparative example 8 increased slowly and the viability decreased gradually. The cells are in the logarithmic growth phase from the beginning of inoculation to the 3 rd day, the doubling time is about 48 hours, and the peak cell density is 0.733 multiplied by 10 7 cells/mL, and the batch culture was terminated on day 5.
Meanwhile, in the whole culture cycle of the HEK-293 cell in the example 5, the temperature is very stable without obvious fluctuation, the pH curve is kept stable, and the pH is in a downward trend due to the accumulation of lactic acid as a whole, but the fluctuation is small and is within an allowable range. Meanwhile, in the embodiment 5, the rotating speed of the torrent bioreactor is initially 50rpm, the oxygen consumption is gradually increased due to the increase of the cell density, the dissolved oxygen curve slides down quickly, the rotating speed is increased to 55rpm in the next day, and the normal culture of the HEK293 cell can be maintained under the condition of no pure oxygen by only introducing air for regulation and control at the back under the condition of 55rpm, which indicates that the gas-liquid mass transfer efficiency of the torrent bioreactor is extremely high, the cell dissolved oxygen requirement with higher density can be maintained by introducing air on the surface, and the oxygen aeration quantity in the culture process can be greatly reduced. Therefore, the torrent reactor only carries out dissolved oxygen transfer by surface ventilation of air and oxygen, so that a large amount of foam generated by bubbling ventilation below the liquid level of the traditional stirring reactor does not exist, cell damage caused by energy dissipation in bubbling ventilation does not exist, and the lethality generated on cells when the foam is broken is also greatly reduced.
Referring to FIGS. 3-12, it can be seen that the maximum density of SF9 cells cultured in the torrent bioreactor in examples 9 and 10 of the present invention can reach 1.11X 10 day 7 7 cells/mL or more. Meanwhile, in the whole culture period of SF9 cells, pH is controlled stably, DO is slowly reduced until stable fluctuation, glucose is stably consumed, lactic acid is not accumulated basically, ammonium ions exist in a basic level close to 4mM in a basic culture medium, and ammonium is slowly cultured after inoculationA slow increase. In addition, the torrent bioreactor can rapidly increase dissolved oxygen only by increasing the rotating speed under the condition that other conditions are not changed, and can maintain 1.11 x 10 without pure oxygen 7 cells/mL SF9 cell culture proves that the gas-liquid mass transfer efficiency of the torrent type reactor is extremely high, the dissolved oxygen requirement of cells with higher density can be maintained by introducing air into the torrent type reactor, and the oxygen ventilation quantity in the culture process can be greatly reduced. Because the torrent type reactor only carries out dissolved oxygen transfer by surface ventilation air and oxygen, a large amount of foam generated by bubbling ventilation below the liquid level of the traditional stirring type reactor does not exist, cell damage caused by energy dissipation during bubbling ventilation does not exist, and the lethality generated to cells when the foam is broken is also greatly reduced.
Therefore, the feasibility and the superiority of the method for culturing the CHO, HEK293 and SF9 cells by adopting the torrent bioreactor are shown in the invention.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. A method for culturing animal cells by using a torrent bioreactor, comprising the steps of:
providing a torrent bioreactor;
adding a cell basic culture medium into the torrent bioreactor, and controlling a pre-culture condition to perform pre-culture to obtain a pre-culture system;
inoculating animal cells into the pre-culture system, and controlling batch culture conditions to carry out batch culture to obtain the animal cells within a target density range;
wherein, in the batch culture process, the temperature is monitored and controlled to be 27.0-37.2 ℃, the pH value is 6.20-7.35, the DO is more than 40%, the rotating speed is 35rpm-60rpm in an online real-time manner through a control system of the torrent bioreactor, and the concentration of glucose is monitored and controlled to be more than 1.5g/L through periodic sampling.
2. The method of claim 1, wherein the animal cells are seeded into the pre-culture system at a seeding density of (0.5-1.2). Times.10 6 cells/mL。
3. The method of claim 1, wherein the animal cell is selected from the group consisting of suspension cells selected from the group consisting of CHO, HEK293, and SF9.
4. The method of claim 3, wherein when the suspension cells are selected from CHO or HEK293, the batch culture process further comprises monitoring and controlling the glutamine concentration to be 2mM or more by periodic sampling.
5. The method of culturing animal cells using a torrent bioreactor according to claim 4, wherein when the suspension cells are selected from CHO, the temperature is controlled to 36.8-37.2 ℃, the pH is controlled to 6.95-7.20, the DO value is controlled to 50% or more, the rotation speed is controlled to 55-60 rpm, the concentration of glucose is controlled to 3-6 g/L, and the concentration of glutamine is controlled to 2-4 mM during the batch culture.
6. The method of claim 4, wherein when the suspension cells are selected from HEK293, the temperature is controlled to 36.8-37.2 ℃, the pH is controlled to 6.95-7.35, the DO is controlled to 50% or more, the rotation speed is controlled to 35-60 rpm, the glucose concentration is controlled to 1.5-6 g/L, and the glutamine concentration is controlled to 2mM-4mM during the batch culture.
7. The method for culturing animal cells using a torrent bioreactor according to claim 3, wherein when the suspension cells are selected from SF9, the temperature is controlled to be 26.8-27.2 ℃, the pH is controlled to be 6.20-6.40, the DO is controlled to be 40% or more, the rotation speed is controlled to be 35-60 rpm, and the concentration of glucose is controlled to be 1.5-5.0 g/L during the batch culture.
8. The method for culturing animal cells by using a torrent bioreactor according to any one of claims 1 to 7, wherein the pH is controlled by supplementing an alkali solution or introducing carbon dioxide into the torrent bioreactor;
and/or the control of the DO value is realized by adjusting the surface ventilation or the rotating speed of the torrent bioreactor;
and/or the control of the concentration of the glucose is realized by a mode of supplementing the feed liquid.
9. The method for culturing animal cells using a torrent bioreactor according to any one of claims 1 to 7, wherein the pre-culture time is 18 to 24 hours.
10. The method for culturing animal cells using a torrent bioreactor according to any one of claims 1 to 7, further comprising monitoring and controlling animal cell density, cell viability, lactate content, ammonium root concentration or osmolarity by periodic sampling during the batch culture.
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