CN117757722A - Method for domesticating cells into serum-free suspension culture - Google Patents
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Abstract
The application discloses a method for domesticating cells into serum-free suspension culture, which comprises the following steps: partially serum-free domestication step: performing partial serum-reduction adherent culture on the cells, so that the serum in the culture medium is reduced from the initial serum concentration to 8-20% of the initial serum concentration; suspension and complete serum removal domestication steps: collecting cells suspended in the cell culture solution subjected to partial serum removal domestication, and carrying out subculture at a first rotation speed until the activity rate reaches 90% or more than 90%; sequentially carrying out serum-reduction culture on the cells after subculture, and reserving the suspended cells after each serum-reduction culture for next serum-reduction culture until the serum concentration is reduced to 0; strengthening suspension domestication: and collecting suspension cells in the cell culture solution of serum-free culture, and sequentially increasing the rotating speed to culture until the rotating speed reaches a second rotating speed. The cell domestication method can rapidly domesticate and obtain serum-free suspension cultured cells.
Description
Technical Field
The application relates to the technical field of cell culture, in particular to a method for domesticating cells into serum-free suspension culture.
Background
Host cell culture is one of the important methods for producing target proteins and is widely used in medical research, biotechnology and pharmaceutical industry. The target protein is produced by introducing the target gene into a host cell and then culturing the host cell in a suitable medium and under suitable growth conditions. Among them, mammalian cells such as Chinese Hamster Ovary (CHO) cells are one of the most widely used host cells, which have good genomic background characteristics, relatively fast growth rates and high protein expression yields. Mammalian cells are usually subjected to adherent culture by adding a certain amount of fetal bovine serum (fetal bovine serum, abbreviated as FBS) on the basis of a basic culture medium, and the serum provides hormones, growth factors and other nutrients required by growth and proliferation for the cells, and meanwhile, the serum has a plurality of adverse effects on a large-scale culture process of the animal cells due to complex components, large batch difference, easy pollution by mycoplasma, viruses and the like, high cost and adverse product separation and purification. And compared with cells cultured in suspension, the cell proliferation speed of the adherent property is low, the number of cells capable of growing per unit volume is small, and large-scale production is difficult to realize. For some adherent host cells, acclimatization into suspension cells and serum-free culture cells is a common means of developing engineered cell lines.
The prior cell domestication method is usually a two-step method, and the gradual domestication of one condition is finished and then the domestication of the other condition is finished, namely, the domestication is firstly carried out from the serum culture to the serum-free culture and then the domestication is carried out from the adherence culture to the suspension culture, or the domestication is firstly carried out from the adherence culture to the suspension culture and then the domestication is carried out from the serum culture to the serum-free culture.
However, the idea of the two-step domestication is to gradually change the growth condition of the cells, gradually eliminate the cells which cannot adapt to the change of the condition in the process of culturing for many times, and leave the cells which can adapt to the change of the condition, but the cells which can adapt to the change of the condition are few, especially the two stages from low serum concentration to serum-free process and from adherence to adaptation to low-speed vibration are difficult, and the whole domestication time is long. In addition, in the domestication process from the adherent growth to the suspension growth, cells are digested by pancreatin for each passage, and then the liquid is exchanged by centrifugation, so that the operation steps are complicated, and the long-time pollution-free subculture is a challenge.
Disclosure of Invention
The object of the present application is to provide a method for the domestication of cells into serum-free suspension culture.
The application adopts the following technical scheme:
in one aspect, the present application discloses a method of acclimating cells to serum-free suspension culture comprising: partially serum-free domestication step: sequentially reducing serum to adhere to the cells, so that the serum in the culture medium is reduced from the initial serum concentration to 8-20% of the initial serum concentration; suspension and complete serum removal domestication steps: collecting cells suspended in the cell culture solution subjected to partial serum removal domestication, and carrying out subculture at a first rotational speed until the activity rate reaches 90% or more than 90%; sequentially carrying out serum-reduction culture on the cells after subculture, and reserving the suspended cells after each serum-reduction culture for next serum-reduction culture until the serum concentration is reduced to 0; strengthening suspension domestication: and collecting suspension cells in the cell culture solution of serum-free culture, and sequentially increasing the rotating speed to culture until the rotating speed reaches a second rotating speed.
The cell domestication method of the application firstly carries out partial serum-removing domestication, and then combines two stages of complete serum-removing and suspension culture with higher domestication difficulty and longer time consumption into one step, thereby shortening the domestication time. In addition, the suspension domestication method can replace the traditional method of carrying out pancreatin digestion on adherent cells by screening out suspended cells for culture, so that the suspension domestication method can be beneficial to reducing tedious operations and possibly introduced pollution caused by adding pancreatin, centrifuging and liquid exchange and the like.
In one implementation of the present application, the cell is one of a CHO cell, HEK293 cell, vero cell, NS0 cell and BHK-21 cell.
In one implementation of the present application, in the partial antiserum removal domestication step, the magnitude of each decrease in serum concentration is not less than 50%. In the partial serum-lowering phase, the amplitude of each lowering is higher, and the domestication time can be shortened.
In one implementation of the present application, in the partial antisera acclimation step, serum in the medium is sequentially reduced from the initial serum concentration to 80% of the initial serum concentration, 40% of the initial serum concentration, 20% of the initial serum concentration, and 10% of the initial serum concentration.
In one implementation of the present application, the serum concentration is reduced by a magnitude of 10% to 50% each time during the suspension and complete serum removal acclimation step. The difficulty of domestication from serum-free to serum-free culture is generally high, and a gentle serum-down level is set at this stage to perform domestication, which is advantageous in improving the success rate of domestication.
In one implementation of the present application, the subcultured cells are subjected to sequential serum-down culture, and the serum concentration in the culture medium is sequentially reduced to 8% of the initial serum concentration, 6% of the initial serum concentration, 5% of the initial serum concentration, 4% of the initial serum concentration, 3% of the initial serum concentration, 2% of the initial serum concentration, 1% of the initial serum concentration, 0.5% of the initial serum concentration, and 0% of the initial serum concentration.
In one implementation of the present application, in the suspending and complete serum-free acclimating step, each time the serum-reduced culture is performed until the cell viability reaches 90% or more, and then the next serum-reduced culture is performed. In this case, the success rate of domestication can be advantageously ensured.
In one implementation of the present application, the serum-reduced culture is performed at the first rotational speed in the suspension and complete serum-reduced acclimation step.
In one implementation of the present application, the first rotation speed is 20 rotations per minute, and the second rotation speed is 75 to 125 rotations per minute.
In one implementation of the present application, in the step of enhancing suspension domestication, the rotation speed is doubled in sequence.
The beneficial effects of this application lie in:
according to the cell domestication method, partial serum-removing domestication is performed, and then two stages of complete serum-removing and suspension culture, which are relatively difficult and relatively long in time consumption, are combined into one step, so that the domestication time can be shortened. In addition, the suspension domestication method can replace the traditional method of carrying out pancreatin digestion on adherent cells by screening out suspended cells for culture, so that the suspension domestication method can be beneficial to reducing tedious operations and possibly introduced pollution caused by adding pancreatin, centrifuging and liquid exchange and the like.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations, as will be apparent from the description herein and the general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description are for clarity of description of only certain embodiments, and are not meant to be required, unless otherwise indicated, to be followed.
Herein, the description of numerical ranges includes endpoints and any number within the range, for example, "1 to 10" or "1 to 10" may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In protein production, the ability of host cells to express the protein and cell growth are critical. For some adherent host cells, acclimatization to suspension cells is a common means of developing engineered cell lines. Acclimating the adherent cells into suspension cells can enhance the metabolism efficiency of the nutrient substances of the cells, shorten the replication time and increase the protein yield. The existing cell domestication method is usually a two-step method, namely, from serum culture to serum-free culture, and from adherent growth to suspension shake culture, and the gradual domestication of one condition is finished and then the domestication of the other condition is finished. The method comprises the following specific steps: (1) When the culture is carried out from the serum culture to the serum-free culture domestication, each time the adherent cells grow well and need to be replaced, a culture medium with a certain reduction in serum content is added after the supernatant is discarded, the operation is repeated after the cells are adapted to be recovered under new conditions, and the culture medium with the further reduction in serum content is added, so that the operation is repeated until the cells can grow normally in the serum-free culture medium. (2) And when the cell is acclimatized from adherent growth to suspension growth, the cell is digested by pancreatin and then cultured in a lower rotating speed every time when the cell is changed, the cell is repeatedly cultured until the cell is completely adapted, then the rotating speed is properly increased, and the cell is gradually adapted to suspension growth conditions after repeated. The order of the two steps can be exchanged under different schemes.
However, the idea of two-step acclimation is to gradually change the growth conditions of the cells, and to gradually eliminate cells that cannot adapt to the change in conditions during the course of multiple culturing, leaving cells that can adapt to the change in conditions. However, most of the time, the number of cells that can adapt to the change in conditions is small, so that the time for this acclimatization is long. Especially from low serum concentration to serum free and from adherence to the ability to accommodate low speed concussions. In the acclimation process from adherent growth to suspension growth, cells are digested with pancreatin and centrifuged to replace the liquid for each passage. This procedure is numerous and is a challenge for long-term contamination-free subculture.
After one of the conditions is gradually acclimatized, the other condition is acclimatized, namely, the culture is firstly performed from serum culture to serum-free culture, then the culture is performed from adherence culture to suspension culture, or the culture is firstly performed from adherence culture to suspension culture, and then the culture is performed from serum culture to serum-free culture.
In the present application, cells remaining suspended are creatively used to replace traditional pancreatin digestion to suspend the cells, thereby being capable of reducing tedious operations and possibly introduced pollution caused by adding pancreatin, centrifuging liquid exchange and the like. In addition, the application distinguishes the two stages of serum removal (from low serum concentration to 0 serum concentration) and suspension culture (from adherence to free) which are relatively long in time and difficult to domesticate from other domestication stages, and milder conditions are set to reduce the probability of domestication failure. In addition, the application combines the two stages of serum removal (from low serum concentration to 0 serum concentration) and suspension culture (from adherence to free) which take a long time and are difficult to domesticate, so that the domestication time is shortened.
In one embodiment, the present application relates to a method for cell acclimation to serum-free suspension culture (hereinafter sometimes simply referred to as "cell acclimation method") comprising a partial antisera acclimation step, a suspension and complete antisera acclimation step, and an enhanced suspension acclimation step.
In a specific embodiment, the cell may be one of a CHO cell, a HEK293 cell, a Vero cell, a NS0 cell and a BHK-21 cell.
As described above, the cell acclimation methods of the present application may include a partial antisera acclimation step.
In one embodiment, the partial antiserum removal step may comprise: the cells are subjected to sequential serum-lowering adherent culture, so that the serum in the culture medium is reduced from the initial serum concentration to 8-20% of the initial serum concentration. For example, serum in the medium can be reduced from an initial serum concentration to 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the initial serum concentration. It will be appreciated that many cells are cultured with the addition of a specific amount of serum, i.e., in a medium of an initial serum concentration, and that by means of a "partial antisera-acclimation step" the cells can be gradually adapted to a culture environment of lower serum concentration, eventually allowing the cells to be cultured at 8% to 20% of the initial serum concentration, e.g., if the initial serum concentration in the medium is 10% (mass fraction), by means of a "partial antisera-acclimation step" the cells can be cultured at a serum concentration of 10% (i.e., 1% of the serum mass fraction in the medium).
In one embodiment, the serum concentration can be rapidly reduced to a low level and a good cell state by a partial serum-free acclimation step.
In one embodiment, in the partial serum-free acclimation step, the magnitude of each decrease in serum concentration is no less than 50%. For example, the magnitude of each decrease in serum concentration may be 50%, 55%, 60% or 65%. It can be appreciated that the difficulty of domestication from higher serum content to lower serum content is smaller than that of domestication from lower serum content to zero serum content, and thus, in the "partial serum-free domestication step", the domestication can be reduced by setting a larger serum amplitude reduction.
In a specific embodiment, in a partial serum-removal acclimation step, serum in the medium may be sequentially reduced from the initial serum concentration to 80% of the initial serum concentration, 40% of the initial serum concentration, 20% of the initial serum concentration, and 10% of the initial serum concentration.
In one embodiment, after each serum-reduced anchorage culture, the cell growth status is evaluated as good for the next serum-reduced anchorage culture, after a partial serum-reduced acclimation step. It should be noted that the evaluation of the cell growth state can be evaluated by observing the morphology of the cells, detecting the growth rate or the living rate of the cells, and evaluating the health condition of the cells. In the present application, the cell adhesion morphology is normal, the cell growth is vigorous (the cells are amplified more than one time a day) or the cell viability is more than 95%, and the cell growth state can be evaluated as good.
As described above, the cell acclimation method of the present application may include suspension and complete serum-free acclimation steps.
In one embodiment, the suspending and completely desizing acclimating step may include: collecting cells suspended in the cell culture solution subjected to partial serum removal domestication, and carrying out subculture at a first rotation speed until the activity rate reaches 90% or more than 90%; and carrying out sequential serum-reduction culture on the cells after subculture, and keeping the suspended cells after each serum-reduction culture for next serum-reduction culture until the serum concentration is reduced to 0. It can be appreciated that the collection of the suspension cells for culture is equivalent to the screening of the suspension cells, and can replace the traditional pancreatin digestion mode; the serum concentration is changed after the collected suspension cells are raised to a stable state, and the serum is reduced and cultured, and the suspension cells are collected each time and subjected to the next serum reduction culture, so that the two stages of serum removal (from low serum concentration to 0 serum concentration) and suspension culture (from adherence to free) which take a relatively long time and are difficult to domesticate can be combined together, and the domestication time is shortened.
In one embodiment, the first rotational speed may be 20 rpm during the suspension and complete serum removal acclimation step. In this case, suspension domestication can be performed at a lower rotation speed, and the success rate of domestication is improved.
In one embodiment, the serum concentration is reduced by 10% to 50% each time during the suspension and complete serum removal acclimation steps. It can be appreciated that the difficulty of domestication from serum-free culture to serum-free culture is generally high, and the moderate serum reduction amplitude is set for domestication at this stage, which can be beneficial to improving the success rate of domestication.
In one embodiment, the serum concentration in the medium is reduced to 8% of the initial serum concentration, 6% of the initial serum concentration, 5% of the initial serum concentration, 4% of the initial serum concentration, 3% of the initial serum concentration, 2% of the initial serum concentration, 1% of the initial serum concentration, 0.5% of the initial serum concentration, 0% of the initial serum concentration in the suspension and complete serum removal acclimation steps.
In one embodiment, in the suspension and complete serum-free acclimation step, each serum-free culture is performed until the cell viability reaches 90% or more, and then the next serum-free culture is performed.
In one embodiment, the cells are subjected to suspension and complete serum-free acclimation steps, and the cells are capable of higher viability and good growth under serum-free conditions at lower rotational speeds.
As described above, the cell acclimation method of the present application may include an enhanced suspension acclimation step.
In one embodiment, the step of enhancing suspension domestication may comprise: and collecting suspension cells in the cell culture solution of serum-free culture, and sequentially increasing the rotating speed to culture until the rotating speed reaches a second rotating speed.
In one embodiment, the second rotational speed may be 75 to 125 revolutions per minute during the enhanced suspension acclimation step. For example, the second rotational speed may be 75 revolutions per minute, 80 revolutions per minute, 85 revolutions per minute, 90 revolutions per minute, 95 revolutions per minute, 100 revolutions per minute, 105 revolutions per minute, 110 revolutions per minute, 115 revolutions per minute, 120 revolutions per minute, or 125 revolutions per minute.
In one embodiment, the rotational speed may be doubled sequentially during the enhanced suspension acclimation step. In other words, the rotational speed may be sequentially multiplied to enhance suspension acclimation of the cells. It should be noted that other rotation speed adjustment schemes are also possible, and the rotation speed is only increased in sequence to perform the reinforced suspension domestication.
In one embodiment, the cells are subjected to an enhanced suspension acclimation step, and the cells are capable of higher viability and good growth under serum-free conditions at higher rotational speeds.
The present application is described in further detail below by way of specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application.
Unless otherwise indicated, the reagents, starting materials, and apparatus used in this example were all commercially available, and the operating scheme was carried out according to the product specifications or according to the procedure of steps conventional in the art.
Examples
The culture subjects were ATCC-derived adherent CHO-K1 cells, and F-12K medium was used as a basal medium, 10% by volume of FBS was added thereto, and the cells were cultured in a 37℃incubator in which the concentration of carbon dioxide was 5%. When the waiting cell is cultured until 50% -70% of the T75 culture flask is fully paved, the digestion passage or the pressurized culture is started. In this example, the initial serum content required for ATCC-derived CHO-K1 cell culture was 10% (volume fraction).
(1) Firstly, partially removing serum and domesticating, and rapidly domesticating cells to 10% of the original serum for adherence culture. The culture conditions and observed phenomena in sequence are:
1. adding 80% of the original amount of serum for culture, passage and good growth;
2. adding 40% of the original amount of serum for culture, passage and good growth;
3. adding 20% of the original serum for culture, passage and good growth;
4. adding 10% of the original amount of serum for culture, passage and good growth;
the criteria for judging that the cell growth state is good are as follows: the cells grow vigorously (the cell doubling time is less than 1.5 days or 2 days), and the adherent morphology is normal and has no obvious change. This step can rapidly reduce serum concentration to a low level and cell status is good.
(2) The cells were then acclimatized in a pancreatin-free suspension while gently lowering serum to serum-free:
1. the cells were cultured at a low rotational speed (20 rpm), the cells suspended above were retained and transferred to a new flask for culture. After the cells have grown on the wall in the new bottle, they continue to be passaged in the same way (retaining the cells suspended above).
2. The serum concentration was changed after the collected suspension cells had increased in viability to a steady state (viability was 90% or more). The serum concentration was adjusted to 10%,8%,6%,5%,4%,3%,2%,1%,0.5%,0 in this order.
Wherein the process of passaging and mildly reducing serum is carried out in the same stage.
(3) And finally, increasing the rotating speed for suspension culture to obtain healthy serum-free suspension culture cells: in order to further increase the efficiency of nutrient absorption and waste transport between the cells and the environment, the rotational speed of the cell suspension culture is further increased.
And (3) carrying out suspension culture at the multiplication rotating speed, and continuing to multiply the rotating speed until the rotating speed reaches 80 rpm when the growth state of the cells is good (the cell multiplication time is less than 1.5 days or 2 days).
Description of effects: in the cell acclimation experiment using the acclimation method of this example, 2 experiments were successfully acclimated adherent cells to suspension cells in 4 parallel replicates, and the time spent in each stage was as follows:
| partially serum-removed acclimation | Suspension domestication without pancreatin | Suspension culture with increased rotational speed | |
| Experiment 1 | 0.75 month (pass) | 2.5 months (pass) | 0.5 month |
| Experiment 2 | 0.75 month (pass) | Failure of | NA |
| Experiment 3 | 0.75Moon (through) | Failure of | NA |
| Experiment 4 | 0.75 month (pass) | 2 months (pass) | 0.5 month |
Typically, the conventional acclimation process takes 6 to 9 months and the entire acclimation experiment is easily failed because the cells are unable to adapt to pressure. In this example, the acclimation time was 3.75 months and 3.25 months, which were significantly shorter than the conventional acclimation method. In addition, the suspension domestication is performed by screening out the suspended cells for culture instead of using the traditional way of carrying out pancreatin digestion on the adherent cells, so that the method can be beneficial to reducing tedious operations and possibly introduced pollution caused by adding pancreatin, centrifuging and changing liquid and the like.
The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.
Claims (10)
1. A method of acclimating cells to serum-free suspension culture, comprising:
partially serum-free domestication step: sequentially reducing serum to adhere to the cells, so that the serum in the culture medium is reduced from the initial serum concentration to 8-20% of the initial serum concentration;
suspension and complete serum removal domestication steps: collecting cells suspended in the cell culture solution subjected to partial serum removal domestication, and performing subculture at a first rotating speed until the activity rate reaches 90% or more than 90%; sequentially carrying out serum-reduction culture on the cells after subculture, and reserving the suspended cells after each serum-reduction culture for next serum-reduction culture until the serum concentration is reduced to 0;
strengthening suspension domestication: and collecting suspension cells in the cell culture solution of serum-free culture, and sequentially increasing the rotating speed to culture until the rotating speed reaches a second rotating speed.
2. The method of claim 1, wherein the cells are one of CHO cells, HEK293 cells, vero cells, NS0 cells and BHK-21 cells.
3. The method of claim 1, wherein in the partial antiserum removal step, the magnitude of each decrease in serum concentration is not less than 50%.
4. A method of acclimating cells to serum-free suspension culture according to claim 3, characterized in that in said partial desizing step, serum in the culture medium is reduced from said initial serum concentration to 80% of said initial serum concentration, 40% of said initial serum concentration, 20% of said initial serum concentration, 10% of said initial serum concentration in sequence.
5. A method of acclimating cells to serum-free suspension culture according to claim 3, characterized in that the serum concentration is reduced by a magnitude of 10% to 50% each time during the suspension and complete desizing steps.
6. The method according to claim 5, wherein the cells after subculture are subjected to sequential serum-reduction culture to sequentially reduce the serum concentration in the medium to 8% of the initial serum concentration, 6% of the initial serum concentration, 5% of the initial serum concentration, 4% of the initial serum concentration, 3% of the initial serum concentration, 2% of the initial serum concentration, 1% of the initial serum concentration, 0.5% of the initial serum concentration, and 0% of the initial serum concentration.
7. The method according to claim 1, wherein in the step of suspending and completely removing serum, each time the serum-reduced culture is performed until the cell viability reaches 90% or more, the next serum-reduced culture is performed.
8. The method of claim 1, wherein the serum-reduced culture is performed at the first rotational speed during the suspension and complete serum-free acclimation step.
9. The method of claim 1, wherein the first rotational speed is 20 rpm and the second rotational speed is 75 to 125 rpm.
10. The method of claim 9, wherein the step of enhancing suspension culture comprises doubling the rotational speed.
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| CN118995575A (en) * | 2024-10-22 | 2024-11-22 | 深圳市卫光生物制品股份有限公司 | Cell serum-free suspension culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118995575A (en) * | 2024-10-22 | 2024-11-22 | 深圳市卫光生物制品股份有限公司 | Cell serum-free suspension culture method |
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