CN111040986B - Suspension culture cell monoclonal screening culture method - Google Patents
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Abstract
The invention discloses a monoclonal screening culture method for suspension culture cells, and belongs to the technical field of cell culture. The invention adds gelatin solution into a cell culture container for pretreatment, assists in promoting adherence culture medium, acclimates suspension cells to recover adherence characteristics, performs monoclonal culture by a limiting dilution method after adaptation to adherence, and performs suspension culture acclimation by using a suspension acclimation culture medium to complete suspension culture cell monoclonal screening culture. The method effectively solves the difficult problem of screening and culturing the suspension cell monoclonal by restoring the adherent culture characteristic of the suspension cell, the cell has suspension affinity, and the suspension culture acclimatization time is greatly shortened. The invention provides an adherence promoting culture medium and a suspension domestication culture medium, which can be respectively used for recovering adherence characteristics of suspension cells and assisting in suspension culture domestication of the cells. The method can be used for monoclonal screening of CHO and PK15 suspension cells, and is an effective manual screening way for selecting high-quality and high-efficiency cell matrixes.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a monoclonal screening culture method for suspension culture cells.
Background
With the development of bioengineering technology, cell matrix becomes important production data in the biopharmaceutical and biological product industries. In the in vitro cell culture process, cells are influenced by growth environment, nutrient components, illumination radiation and the like, so that genetic character variation can occur in the cell growth process. Differences can occur in the sensitivity of cells to viral infection, integrity of the viral particle packaging, efficiency of antibody or protein or virus production between the same cell line of different generations or between different cell lines of the same generation. In order to separate high-quality and high-yield cells from cell populations, people usually perform cell monoclonal screening, select high-quality and high-yield single cells through detection, amplify, establish cell banks, and use the cell banks for production of related products.
Currently, there are manual and instrumental screening methods (high throughput cell line screening instrument Clone Pix) for cell monoclonal screening. The latter high-throughput cell line screening instrument is expensive and difficult to adopt in common enterprises, so manual screening is a common cell clone screening method. The commonly used manual screening methods are mainly the limiting dilution method and the semi-solid medium culture method.
Cell cloning is often performed in laboratories using limiting dilution, but this method uses liquid media to culture the cells, making tracking the cells difficult or inaccurate. In the process of processing the micro-porous plate, the suspended cells are easy to move in the holes, and the same cell imaged at different time points cannot be ensured, so that the accuracy of monoclonal verification is greatly reduced.
The semi-solid culture medium is used for culturing the cloned cells, a faster and simpler cloning method is provided, the growth of the cells is favorably tracked, the positions of the cells in the semi-solid culture medium are relatively fixed, the positions of the cells cannot move in the conventional experiment operation process, and the cells can grow into independent single cell clones. However, not all cell clones are suitable for semi-solid media, and especially during suspension cell cloning, single cells are difficult to grow and survive in semi-solid media. Therefore, it is a general objective of the prior art to provide an appropriate artificial cell clone screening method to be suitable for more cell clone screening culture applications.
Disclosure of Invention
The invention aims to provide a monoclonal screening culture method for suspension cells.
The invention provides a monoclonal screening culture method for suspension culture cells, which comprises the following steps:
(1) recovering the adherent characteristics of the suspension culture cells;
(2) the suspension culture cells adapting to adherent culture grow to a monolayer, and the digested single cells are subjected to single cell clone screening by a limiting dilution method;
(3) and (4) selecting a cell cluster for single cell growth to digest, perform suspension culture and domesticate, and finishing the suspension culture cell monoclonal screening culture.
The method is suitable for suspension culture monoclonal screening of CHO cells and PK15 cells.
Wherein, the method for recovering the adherence property in the step (1) comprises the following steps: adding gelatin solution or polylysine solution into a cell culture vessel for pretreatment, then adding suspension culture cells and adherence promoting culture medium, incubating the cells for adherence growth, digesting monolayer cells, and repeating the steps for continuous subculturing for 2-3 generations.
The pretreatment solution is PBS solution containing 0.05-0.1 mug/ml gelatin or PBS solution containing 0.05-0.1mg/ml polylysine.
Preferably, the gelatin solution is a 0.1 μ g/ml gelatin PBS solution.
The method for pre-treating by adding gelatin solution comprises adding gelatin solution, incubating at 37 deg.C for 40min, incubating at room temperature for 100min, or standing at 2-8 deg.C overnight, discarding gelatin solution, and washing. Preferably, incubation is carried out at 37 ℃ for 40min, after which the gelatin solution is discarded and washed.
In the embodiment of the invention, a plate is pretreated by adopting 0.1 mu g/ml gelatin PBS solution, incubated at 37 ℃ for 40min, washed 1 time by the PBS solution, and dried for later use.
In the process of adapting suspension cells to adherent culture, the adherence promoting culture medium is DMEM or DMEM/F12 culture medium, wherein the adherence promoting culture medium contains 8% FBS, 5-10 mu mol/L LPA, 2-10 mu g/ml fibronectin and 0.1-0.3mg/ml BSA. Preferably 8% FBS, 5. mu. mol/L LPA, 2. mu.g/ml fibronectin and 0.2mg/ml BSA.
The embodiment of the invention adopts DMEM/F12 or DMEM culture medium, and the culture medium contains 2 mug/ml fibronectin, 0.2g/L BSA and 8% FBS, which can promote the adherent growth of suspension cells and make the suspension cells recover the adherent property; the contained 5 mu mol/L LPA can promote the metabolic growth of cells.
In the suspension culture cell monoclonal screening culture method provided by the invention, the limiting dilution method in the step (2) is carried out by adopting a 96-hole cell culture plate, before adding cell suspension, gelatin solution is added into the hole of the cell culture plate for pretreatment for 40min at 37 ℃, and then the cell culture plate is washed by PBS solution; the gelatin solution was 0.1. mu.g/ml gelatin PBS solution.
In the examples of the present invention, 96-well cell culture plates were used.
And (2) after digesting and blowing into single cells, diluting the cell suspension to 5-8cells/ml by using an adherence promoting culture medium, adding 0.1ml of cell suspension into each hole of the cell culture plate, standing, marking single cell holes, and culturing the cells.
In the examples of the present invention, the cells grown to the monolayer were digested into single cells with cell digest, and DMEM/F12 medium containing 8% FBS, 5. mu. mol/L LPA, 2. mu.g/ml fibronectin and 0.2mg/ml BSA was used. Diluting to 5-8cells/ml, adding 0.1ml cell suspension into each well of the cell culture plate, standing, marking single cell wells, culturing at 37 ℃ for 7-10 days, observing the single cell wells every day, and recording the cell growth condition.
In the monoclonal screening culture method for suspension culture cells provided by the invention, in the step (3), the total number of proliferated cells in a single cell cluster is selected to be about 3 multiplied by 107-4×107cells are digested with appropriate amount of 0.25% recombinant pancreatin or 0.5-1mg/ml collagenase IV (digestion solution is discarded immediately after digestion is finished), blown up, added with suspension culture medium containing 5-10. mu. mol/L LPA and 0.2% -0.5% Clonacell-CHO ACF additive, adjusted to a cell concentration of about 2X 106cells/ml, placing on decolorizing shaker at 60-80rpm, and placing in 5% CO2And (5) in an incubator until the cells adapt to suspension culture, and completing the screening culture of suspension culture cell monoclonals.
Preferably, the suspension acclimatization medium is a CHO cell or PK15 cell serum-free medium containing 5. mu. mol/L LPA and 0.2% Clonacell-CHO ACF additive.
On the other hand, the invention also provides a cell adherence promoting culture medium and a suspension domestication culture medium. The adherence promoting culture medium has the function of promoting cell adherence, is a DMEM or DMEM/F12 culture medium, and contains 8% FBS, 5-10 mu mol/L LPA, 2-10 mu g/ml fibronectin and 0.1-0.3mg/ml BSA; the suspension domestication culture medium has the function of assisting cell suspension culture domestication, is a CHO cell or PK15 cell serum-free culture medium, and contains 5-10 mu mol/L LPA and 0.2-0.5% Clonacell-CHO ACF additive.
The invention provides the monoclonal screening culture method of the suspension culture cells or the application of the cell culture medium in preparing biological products.
According to the method, the culture vessel is treated by the gelatin solution, and the anchorage-promoting cell culture medium is added into the cell culture medium, so that the anchorage-promoting growth of suspension cells can be promoted, and the anchorage-promoting characteristic of the suspension cells can be quickly recovered. In a common culture vessel, the suspension cells are statically cultured in a DMEM/F12 culture medium containing 8% FBS, and the adherent characteristics of the cells can be restored after 5-10 generations on average; and in a gelatin solution treatment culture vessel, the suspension cells are statically cultured in a DMEM/F12 culture medium containing 8% FBS, 2 microgram/ml fibronectin and 0.2mg/ml BSA, and the cell adherence characteristics can be recovered through 1-2 generations of cells, so that the cells have good growth forms and vigorous metabolism. Compared with the conventional adherence method, the method can shorten the re-adherence time of the suspension cells by more than 12 days.
After the suspension cells recover the adherent characteristics, the cell monoclone can be realized by a limiting dilution method. In the present invention, a higher concentration of monoclonal cells (approximately 2X 10) was adjusted by adding 5. mu. mol/L LPA and 0.2% Clonacell-CHO ACF additive to serum-free medium6cells/ml) are cultured in a soft culture environment of a decoloring shaker, and cell suspension culture and domestication can be completed after 1-2 generations. In general, CHO and PK15 adherent cells are subjected to suspension culture domestication, and 10-50 generations of subculture domestication are needed to be carried out so as to adapt to full suspension culture; the CHO and PK15 cells adopted by the invention are both of suspension affinity, and the application of the additive for assisting the growth of the cells can adapt to full suspension culture through 1-2 generations, thereby shortening the acclimation time of the cell suspension culture for months. The method can be used for the monoclonal screening of various suspension culture cells, and isAn effective manual screening approach of high-quality and high-efficiency cell matrixes is selected.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The biochemical materials in the examples are all commercially available unless otherwise specified.
The anchorage-promoting culture media described in the following examples are DMEM/F12 or DMEM culture media containing 8% FBS, 5-10. mu. mol/L LPA, 2-10. mu.g/ml fibronectin and 0.1-0.3mg/ml BSA; the suspension domestication culture medium is a serum-free culture medium containing 5-10 mu mol/L LPA and 0.2% -0.5% Clonacell-CHO ACF additive. Specifically, the adherence-promoting medium of examples 1 and 3 was DMEM/F12 medium containing 8% FBS, 5. mu. mol/L LPA, 2. mu.g/ml fibronectin and 0.2mg/ml BSA; the suspension domestication medium was a CHO cell serum free medium containing 5. mu. mol/L LPA and 0.2% Clonacell-CHO ACF additive. The adherence-promoting medium of examples 2 and 4 was DMEM medium containing 8% FBS, 8. mu. mol/L LPA, 3. mu.g/ml fibronectin and 0.1mg/ml BSA; the suspension acclimatization medium was PK15 cell serum-free medium containing 5. mu. mol/L LPA and 0.2% Clonacell-CHO ACF additive.
The method comprises the key steps of pretreating a culture vessel by using an adherence promoting reagent such as gelatin solution or polylysine PBS solution and the like, assisting the application of an adherence promoting culture medium, restoring adherence characteristics of suspension CHO cells and PK15 cells in advance, performing monoclonal screening on the basis, and performing suspension culture domestication by using a suspension domestication culture medium, so that the monoclonal screening culture efficiency of the suspension CHO cells and PK15 cells is improved, and the problem that the direct monoclonal screening of the suspension cells is difficult to succeed is solved.
Example 1 suspension CHO cell adherence promoting culture and suspension culture acclimatization
1. Suspension CHO cell adherence promoting culture
In cell culture dish
Adding 1ml PBS solution containing 0.1 μ g/ml gelatin, uniformly covering the bottom, and standing and incubating at 37 deg.C for 40 min. The gelatin PBS solution in the culture dish is discarded, the gelatin PBS solution is washed by 2ml of PBS solution for 1 time, and the gelatin PBS solution is dried, sealed and placed at 4 ℃ for standby.
After centrifugation at 800rpml for 5min, suspension-cultured CHO cells were resuspended in DMEM/F12 medium (medium used in the conventional method) and adherence-promoting medium (medium used in the method of the present invention) containing 8% FBS, respectively, and diluted to 1X 106cells/ml, transferring 3ml of cell suspension in sequence, adding the cell suspension into a common culture dish and a cell culture dish treated by the method, respectively supplementing corresponding culture media, and culturing at 37 ℃ for 3-7 days until the cells grow to a monolayer.
Slightly discarding the upper layer of dead cells, wetting a cell monolayer by using 1ml of cell digestive juice, standing and incubating at 37 ℃ for about 3min, discarding the digestive juice, respectively adding appropriate culture medium to blow off the cells, and subculturing according to a 1:3 dish division mode; the passage is repeated in such a way according to the adherent growth condition of the cells, so that the suspension CHO cells recover the adherent property and adapt to adherent culture.
The conventional method comprises the following steps: in a common plate, in a DMEM/F12 culture medium containing 8% FBS, suspension CHO cells are slowly adaptive to adherent culture, the cells need to be adaptive to complete adherent culture after about 6 generations, and single cells can form a cell monolayer after 7 days of culture.
The method comprises the following steps: in a pretreated common plate, the adherence-promoting culture medium suspends cells in weight, and the suspension CHO cells are cultured by 2 generations to adapt to adherence culture, so that the cells grow well adherent. Therefore, the suspension CHO cells can be adapted to adherent culture through 1 generation (4d) by the method, and the cells grow well adherent; while the conventional method needs 5-6 generations (more than 15-18 d) to recover the adherent growth, the method can greatly shorten the time for the suspension CHO cells to recover the adherent growth.
Suspension culture acclimatization of CHO cells
Adopts a CHO cell serum-free culture medium and a suspension domestication culture mediumSeparately diluting adherent CHO cells and CHO cells monocloned by the method of the invention to 2X 106cells/ml, and suspension culture acclimatization, the cell growth conditions are shown in Table 1.
TABLE 1 CHO cell suspension culture acclimatization
The CHO cells domesticated by the method have a suspension affinity type, and the results show that the domestication time required by the method for developing the suspension culture of the CHO cells is shortened by 1-2 generations compared with the domestication time required by the conventional method, and the cell density and the activity are higher than those of the domestication time required by the conventional method.
Example 2 culture of suspension PK15 cells for promoting adherence and acclimatization of suspension culture
1. Suspension PK15 cell adherence-promoting culture
In cell culture dish
Adding 1ml PBS solution containing 0.1 μ g/ml gelatin, uniformly covering the bottom, and standing and incubating at 37 deg.C for 40 min. The gelatin PBS solution in the culture dish is discarded, the gelatin PBS solution is washed by 2ml of PBS solution for 1 time, and the gelatin PBS solution is dried, sealed and placed at 4 ℃ for standby.
After centrifugation at 800rpml for 5min, suspension-cultured PK15 cells were resuspended in DMEM medium (medium used in the conventional method) containing 8% FBS and anchorage-promoting medium (medium used in the method of the present invention) and diluted to 1X 106cells/ml, transferring 3ml of cell suspension in sequence, adding the cell suspension into a common culture dish and a cell culture dish treated by the method, respectively supplementing corresponding culture media, and culturing at 37 ℃ for 3-7 days until the cells grow to a monolayer.
Slightly discarding the upper layer of dead cells, wetting a cell monolayer by using 1ml of cell digestive juice, standing and incubating at 37 ℃ for about 3min, discarding the digestive juice, respectively adding appropriate culture medium to blow off the cells, and subculturing according to a 1:3 dish division mode; the passage is repeated according to the adherent growth condition of the cells, so that the suspended PK15 cells recover adherent characteristics and adapt to adherent culture.
The conventional method comprises the following steps: in a common plate, in a DMEM medium containing 8% FBS, suspended PK15 cells are slowly adaptive to adherent culture, the cells need to be adaptive to complete adherent culture through 4 generations, and single cells can form a cell monolayer after 7 days of culture.
The method comprises the following steps: in a pretreated common plate, the cells are suspended by the adherence-promoting culture medium, and the suspended PK15 cells are cultured by 1 generation to adapt to adherence culture, so that the cells grow well by adherence.
Therefore, the suspension PK15 cells can be adapted to adherent culture through 1 generation (3d) by the method, and the cells grow well adherent; and the conventional method needs more than 4 generations (more than 12 d) to recover the adherent growth, and the method can greatly shorten the time for recovering the adherent growth of the suspended PK15 cells.
Suspension culture acclimatization of PK15 cells
Using PK15 cell serum-free culture medium and suspension domestication culture medium to respectively dilute adherent PK15 cells and PK15 cells monoclonal by the method to 2 x 106cells/ml, and suspension culture acclimatization, the cell growth conditions as shown in Table 2.
TABLE 2 PK15 cell suspension culture acclimatization
The PK15 cells domesticated by the method have a suspension parent type, and the results show that the domestication time for carrying out the suspension culture of the PK15 cells by the method is shortened by more than 20 generations compared with the domestication time of the conventional method, and the cell density and the activity are higher than those of the conventional method.
EXAMPLE 3 CHO cell monoclonal screening culture
0.1ml of PBS solution containing 0.1. mu.g/ml gelatin was added to the 96-well cell culture plate to cover the bottom, and the plate was incubated at 37 ℃ for 40 min. The gelatin PBS solution in the 96-well cell culture plate was discarded, and after washing 1 time with 0.3 ml/well PBS solution, the liquid was spun off at 4 ℃ for use.
Selecting suspension CHO cells and CHO cells recovering the adherence characteristic by the method, respectively diluting the suspension CHO cells and the CHO cells recovering the adherence characteristic by DMEM/F12 culture medium (culture medium used by the conventional method) containing 8% FBS and adherence promoting culture medium (culture medium used by the method of the invention) to 5-8cells/ml, sequentially adding the diluted suspension CHO cells and the CHO cells into a common 96-well plate and a pretreated 96-well plate, inoculating 0.1ml of the suspension CHO cells and the pretreated culture medium into each well, statically culturing the suspension CHO cells at 37 ℃ for 10 days, performing CHO cell monoclonal culture, randomly selecting 20 single cells, and observing the survival state day by day. The number of cells cultured and surviving is shown in Table 3.
TABLE 3 CHO cell monoclonal culture and number of surviving cells
As can be seen from Table 3, the CHO cells were cultured for 10 days by the method of the present invention to obtain 7 strains (7/20 survived) and proliferated cells, and the monoclonal efficiency was higher than that of the conventional method (2/20 survived) monoclonal. Therefore, the method can greatly improve the success rate of the monoclonal screening culture of the CHO cells.
Example 4 monoclonal screening culture of PK15 cells
0.1ml of PBS solution containing 0.1. mu.g/ml gelatin was added to the 96-well cell culture plate to cover the bottom, and the plate was incubated at 37 ℃ for 40 min. The gelatin PBS solution in the 96-well cell culture plate was discarded, and after washing 1 time with 0.3 ml/well PBS solution, the liquid was spun off at 4 ℃ for use.
Suspension PK15 cells and PK15 cells for recovering the adherent property by the method are selected, DMEM medium (culture medium used by the conventional method) containing 8% FBS and adherence promoting medium (culture medium used by the method of the invention) are respectively diluted to 5-8cells/ml, the diluted solution is sequentially added into a common 96-well plate and a pretreated 96-well plate, 0.1ml of the diluted solution is inoculated into each well, standing culture is carried out for 10 days at 37 ℃, PK15 cell monoclonal culture is carried out, 20-well single cells are randomly selected, and the survival state is observed day by day. The number of cells cultured and surviving is shown in Table 4.
TABLE 4 monoclonal culture of PK15 cells and number of surviving cells
As can be seen from Table 3, the PK15 cells were cultured by 10d monoclonal culture by the method of the present invention to obtain 17 strains (17/20 survival) of surviving and proliferating cells, and the monoclonal efficiency was much higher than that of the conventional method (6/20 survival) of the monoclonal. Therefore, the success rate of monoclonal screening culture of PK15 cells can be greatly improved by adopting the method.
In conclusion, the method quickly recovers the adherence characteristics of the suspension CHO cells and the PK15 cells by using the pretreatment culture vessel of adherence promoting reagents such as gelatin and the like and the adherence promoting culture, performs monoclonal screening on the basis, and finally performs suspension culture domestication, thereby completing the monoclonal screening culture of the suspension CHO cells and the PK15 cells. Compared with the conventional method, the method disclosed by the invention can greatly improve the monoclonal screening culture success rate of the suspension CHO cells and the PK15 cells, greatly shorten the suspension culture domestication time of the CHO cells and the PK15 cells, and provide a new screening culture approach for the suspension culture cell monoclonal screening.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (5)
1. A monoclonal screening culture method of suspension culture cells is characterized by comprising the following steps:
(1) recovering the adherent characteristics of the suspension culture cells: adding a PBS solution of gelatin or a PBS solution of polylysine into a cell culture container for pretreatment, then adding suspension culture cells and an adherence promoting culture medium, incubating the cells for adherence growth, digesting monolayer cells, and repeating the step for continuous passage culture for 2-3 generations; the adherence promoting culture medium is DMEM or DMEM/F12 culture medium, wherein the DMEM or DMEM/F12 culture medium contains 8% FBS, 5-10 mu mol/L LPA, 2-10 mu g/ml fibronectin and 0.1-0.3mg/ml BSA;
(2) the suspension culture cells adapting to adherent culture grow to a monolayer, and the digested single cells are subjected to single cell clone screening by a limiting dilution method;
(3) selecting a cell cluster for single cell growth to carry out digestion and suspension culture domestication, wherein the method comprises the following steps: the total number of proliferating cells in a single cell cluster is 3X 107-4×107In cells, 0.25% recombinant pancreatin or 0.5-1mg/ml collagenase IV is digested and blown off, then suspension culture medium containing 5-10 μmol/L LPA and 0.2% -0.5% Clonacell-CHO ACF additive is added, cell concentration is adjusted to 2 × 106cells/ml, placing on decolorizing shaker at 60-80rpm, and placing in 5% CO2In the incubator, suspension culture cell monoclonal screening culture is completed until the cells adapt to suspension culture;
the suspension culture cells are CHO cells or PK15 cells.
2. The method for screening and culturing suspension cultured cells according to claim 1, wherein the pretreatment solution is a PBS solution containing 0.05 to 0.1 μ g/ml of gelatin or a PBS solution containing 0.05 to 0.1mg/ml of polylysine.
3. The method for screening and culturing suspension culture cells according to claim 1, wherein the pretreatment is carried out by adding gelatin solution and incubating at 37 ℃ for 40min, or incubating at room temperature for 100min, or standing at 2-8 ℃ overnight, then discarding the gelatin solution, and washing with PBS solution.
4. The method for screening and culturing suspension cultured cells according to any one of claims 1 to 3, wherein the limiting dilution method in step (2) is to dilute the cell suspension obtained by digesting the monolayer cells into single cells to 5 to 8cells/ml, add the cell suspension to the cell culture plate in an amount of 0.1ml per well, mark the single cell well after standing, continuously culture the cells, and observe the single cells daily until the single cells grow into clusters and are digested and proliferated.
5. Use of the method of monoclonal screening for culture of suspension culture cells according to any of claims 1-4 for the preparation of a biological product.
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| CN102719480A (en) * | 2012-07-08 | 2012-10-10 | 大连医科大学 | Pre-processing method of cell culture plate for suspension cell liposome transfection |
| CN103898044A (en) * | 2014-03-20 | 2014-07-02 | 河南农业大学 | Method for preparing high-sensitivity PK15 cell line L8 of porcine circovirus type 2 |
| CN108570445A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | PK15 cells tame suspension process and second order virus production technique |
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| CN102719480A (en) * | 2012-07-08 | 2012-10-10 | 大连医科大学 | Pre-processing method of cell culture plate for suspension cell liposome transfection |
| CN103898044A (en) * | 2014-03-20 | 2014-07-02 | 河南农业大学 | Method for preparing high-sensitivity PK15 cell line L8 of porcine circovirus type 2 |
| CN108570445A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | PK15 cells tame suspension process and second order virus production technique |
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| PK15 细胞的无血清全悬浮驯化研究;刘天伦等;《中国兽药杂志》;20190930;第53卷(第9期);12-18 * |
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