CN113881620A - Efficient and low-cost MDCK suspension serum-free domestication process platform - Google Patents
Efficient and low-cost MDCK suspension serum-free domestication process platform Download PDFInfo
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Abstract
The invention discloses a high-efficiency low-cost MDCK suspension serum-free domestication process platform, which is a whole set of technical method from adherence to full suspension domestication of serum-free domesticated serum-free culture medium and MDCK cells; the serum-free culture medium contains amino acid, inorganic salt, vitamin, lipid, hydrolysate and other additives, and is suitable for serum-free adherent and suspension culture of MDCK cells; the suspension process platform adopts a method of adding an anti-caking agent and natural sedimentation to solve the cell caking problem, adopts a low-speed centrifugation method to remove cells and impurities with poor states, and can obtain suspension cells from adherent cells containing serum to serum-free cells by alternately using the two methods for 20-30 times, and the suspension process platform has good cell state and normal multiplication speed.
Description
Technical Field
The invention relates to a method for producing biological products, in particular to a high-efficiency low-cost MDCK suspension serum-free domestication process platform.
Background
Avian Influenza (Bird Flu or Avian Influenza) is an acute infectious disease caused by a subtype of Influenza a virus (also called Avian Influenza virus), and can infect humans, and the mortality rate of humans infected with Avian Influenza is more than 30%. In recent years, the market demand of the global seasonal influenza vaccine is steadily increasing year by year, and the fact that the vaccination rate of a plurality of developing countries is low reflects the huge potential market development prospect of the seasonal influenza vaccine. So far, no ideal therapeutic drug for treating avian influenza is found, and vaccination is still the most effective means for preventing epidemic diseases at present.
The traditional production process of the avian influenza vaccine is a chicken embryo culture method, the mode of taking chicken embryos as production matrix has been used for many years, the process is mature, but the production period is long, the operation is complicated, the workload is large, the pollution is easy, and if the avian influenza is outbreaked on a large scale, the supply of the chicken embryos has great problems. The use of animal cells for the production of influenza vaccines is becoming a mainstream production method compared to traditional chick embryo production methods.
The canine kidney (MDCK) cells are established by Madin and Darby separation culture, are easy to culture, fast to proliferate and high in influenza virus infection efficiency, and are recognized cell lines which are most suitable for avian influenza virus culture at home and abroad. In the last decades, great progress has been made in the expansion of influenza viruses using adherent MDCK cells, however this culture mode still has a number of drawbacks, such as: adherent culture is limited by the surface area of the substrate resulting in reduced virus production; the procedures of liquid changing and the like in the adherent culture process are complex and tedious, the use of serum is easily polluted by microorganisms, viruses, mycoplasma and the like, and the serum-free culture technology is an effective tool for clarifying basic problems of cell generation, proliferation, differentiation and gene expression regulation, but the serum-free adherent culture is only suitable for experimental research, is not suitable for large-scale culture and cannot meet the actual production requirements of virus vaccines and the like. The suspension culture of the MDCK cells in a serum-free or chemically defined culture medium can solve the defects, so that the production process is simple to operate, stable to control, simple in downstream purification operation and easier to realize the large-scale production of the influenza vaccine. Therefore, domesticating the MDCK cells to adapt to serum-free suspension culture and producing the avian influenza virus vaccine by taking the MDCK cells as a carrier becomes the key for preparing the next generation of safe, effective and quality-controllable influenza vaccine.
The domestication of MDCK adherent cells is required to obtain a suspension-culturable MDCK cell line. The commonly used methods at present are a gradual serum reduction method and a one-step domestication method. However, the problems encountered in the acclimatization process are various, such as: the gradual serum-reducing method has the defects of long cell acclimation time, complicated steps, cell agglomeration and the like, and cannot support high-density growth. Many of the disclosed domestication methods have poor universality, have great effect difference on different experimental platforms, and cannot be used as a very stable cell domestication process platform.
Disclosure of Invention
The invention aims to provide an efficient and low-cost MDCK suspension serum-free domestication process platform, according to which adherent MDCK cells can be domesticated into single suspension cells in a serum-free culture medium, and serum-free suspension culture of the cells is realized.
An efficient and low-cost MDCK suspension serum-free domestication process platform comprises the following specific operation steps:
1) recovering one MDCK cell by using an original culture medium, wherein the original culture medium is 5% FBS DMEM, adding 1ml of frozen cell sap into 20ml of DMEM culture medium containing 5% FBS, and culturing in an incubator with the concentration of 5% carbon dioxide and the temperature of 37 ℃;
2) when the confluence degree of the cells reaches about 70%, replacing the original culture medium with a serum-free culture medium;
3) when the confluence degree of the cells in the serum-free culture medium reaches more than 90%, digesting the adherent MDCK cells for 5-8 minutes by using 0.05% of pancreatin at 37 ℃; after the cells become round, gently patting the bottom of the container to dissociate the MDCK cells, and adding a stop solution containing 5% serum PBS (phosphate buffer solution) in a volume which is 3 times larger than that of the digestion solution to stop digestion; 1000rmp, 5min, RT centrifugation, removing the stop solution;
4) after serum removal, the serum was removed as follows 1: 3, rotating the bottle for culture; after 2-3 days of culture, the growth rate of the cells gradually becomes slow due to the existence of serum-free cells, the cells gradually gather and grow, and most of the cells naturally suspend;
5) the cells are continuously passaged for 2-3 times, the multiplication rate of the cells can gradually return to normal, but the adherent state becomes worse, at the moment, the cells can be blown down without being processed by pancreatin, and a small amount of adherent cells can be continuously cultured after being added into a serum-free culture medium;
6) settling the agglomerated cells by using a centrifugal tube natural settling method before replacing the liquid, wherein the settling time is determined according to the size of the cell clusters and is 5-30 minutes, and removing the cell clusters;
7) collecting the cells in all the bottles in a 125ml shake flask with a final volume of 50-60ml and a density of 3E 5-1E 6, and performing suspension acclimation of the cells in a constant temperature carbon dioxide shaker under the culture conditions of 37 ℃ and 110rpm and 8% CO 2;
8)800rmp, 5min, RT centrifugation to remove poor cell and impurity;
9) replacing the culture medium 3-5 days before the cells recover the doubling state, and repeating for many times;
10) when the culture medium is completely single suspension cells and the cell viability is greater than 95%, the cells can be doubled within 24 hours and are considered to be acclimatized;
11) the whole acclimatization process time period is 1-2 months; compared with the traditional culture method for gradually reducing serum, the method greatly shortens the domestication time.
The invention also discloses a serum-free culture medium in the efficient low-cost MDCK suspension serum-free domestication process platform, which comprises amino acid, vitamin, inorganic salt, lipid, hydrolysate and other additives, and the components and the content ranges thereof are as follows:
amino acid group
Vitamin group
Inorganic salts and trace elements
Lipid, hydrolysate and additive group
The invention also discloses application of the MDCK suspension serum-free domestication process platform with high efficiency and low cost in the suspension domestication process of other adherent cells.
Preferably, the MDCK cell is MDCK [ NBL-2 ].
The serum-free culture medium used in the invention is a serum-free culture medium independently developed by Shanghai duoning organisms. The domestication culture platform uses reagents comprising: serum-free medium (shanghai doning), glutamine (sigma), dextran sulfate (sigma), pancreatin (Gbico), fetal bovine serum (shanghai doning), DMEM medium (Gbico); the used equipment comprises a carbon dioxide incubator (WIGGENS), a constant temperature carbon dioxide shaker (Shanghai duoning), a cell counter (CountStar), an optical microscope (Mshot), a sterile operating platform (Sujing Antai) and a centrifuge (BioTek); the cell line used was the MDCK adherent cell line donated by the guangzhou zizhi organism, and the original adherent medium was 5% FBS DMEM.
The settling time in the step 6) is determined according to the size of the cell mass, and is determined by a 50ml centrifugal tube natural settling method, and the settling time of the cell mass with the diameter of more than 5mm is 5 minutes; settling time of cell mass with diameter of 3-5mm is 10 min; settling time of cell mass with diameter of 1-3mm is 15 min; the settling time of the cell mass with the diameter of 0.2mm-1mm is 20 min.
Due to the adoption of the technical scheme, the MDCK suspension serum-free domestication process platform with high efficiency and low cost comprises the following specific operation steps: 1) recovering one MDCK cell by using an original culture medium, wherein the original culture medium is 5% FBS DMEM, adding 1ml of frozen cell sap into 20ml of DMEM culture medium containing 5% FBS, and culturing in an incubator with the concentration of 5% carbon dioxide and the temperature of 37 ℃; 2) when the confluence degree of the cells reaches about 70%, replacing the original culture medium with a serum-free culture medium; 3) when the confluence degree of the cells in the serum-free culture medium reaches more than 90%, digesting the adherent MDCK cells for 5-8 minutes by using 0.05% of pancreatin at 37 ℃; after the cells become round, gently patting the bottom of the container to dissociate the MDCK cells, and adding a stop solution containing 5% serum PBS (phosphate buffer solution) in a volume which is 3 times larger than that of the digestion solution to stop digestion; 1000rmp, 5min, RT centrifugation, removing the stop solution; 4) after serum removal, the serum was removed as follows 1: 3, rotating the bottle for culture; after 2-3 days of culture, the growth rate of the cells gradually becomes slow due to the existence of serum-free cells, the cells gradually gather and grow, and most of the cells naturally suspend; 5) the cells are continuously passaged for 2-3 times, the multiplication rate of the cells can gradually return to normal, but the adherent state becomes worse, at the moment, the cells can be blown down without being processed by pancreatin, and a small amount of adherent cells can be continuously cultured after being added into a serum-free culture medium; 6) settling the agglomerated cells by using a centrifugal tube natural settling method before replacing the liquid, wherein the settling time is determined according to the size of the cell clusters and is 5-30 minutes, and removing the cell clusters; 7) collecting the cells in all the bottles in a 125ml shake flask with a final volume of 50-60ml and a density of 3E 5-1E 6, and performing suspension acclimation of the cells in a constant temperature carbon dioxide shaker under the culture conditions of 37 ℃ and 110rpm and 8% CO 2; 8)800rmp, 5min, RT centrifugation to remove poor cell and impurity; 9) replacing the culture medium 3-5 days before the cells recover the doubling state, and repeating for many times; 10) when the culture medium is completely single suspension cells and the cell viability is greater than 95%, the cells can be doubled within 24 hours and are considered to be acclimatized; 11) the whole acclimatization process time period is 1-2 months; compared with the traditional culture method for gradually reducing serum, the method greatly shortens the domestication time.
The invention has the following beneficial effects:
the domestication method adopts a rapid two-step method, firstly MDCK adherent cells are adapted to a serum-free culture medium, the cells can be rapidly adapted to the serum-free culture medium after 2-3 generations of treatment, and the normal multiplication rate is recovered.
The novel cell line obtained by the platform domestication has good growth state, normal and stable multiplication rate and more than 95 percent of cell survival rate. Compared with other methods, the method has the advantages that the domestication time is greatly shortened, the instability factors existing in the domestication are reduced, and the dependence of the domestication on a laboratory platform is reduced.
It is another object of the present invention to provide a medium for serum-free and suspension acclimation of adherent cells that supports both adherent and suspension passaging of MDCK cells.
And (4) directly resuspending the adapted cells into a shake flask for high-speed suspension adaptation. In the process, a natural sedimentation method is used for removing large cell masses, a low-speed centrifugation method is used for removing cells and impurities in poor states, and a high-concentration anti-caking agent method is used for maintaining single dispersed cells.
Compared with the gradual serum reduction method, the method is faster and more efficient, and a completely serum-free suspension cell line can be obtained within 1-2 months, which is much higher than the gradual serum reduction method. In addition, pancreatin digestion and termination are needed only for the first passage in the whole domestication process, and pancreatin digestion is not needed in the subsequent serum-free culture medium culture process, so that much physical labor and cost consumption are saved.
The domestication platform of the invention has the advantages that the used serum-free culture medium does not contain animal-derived components such as animal serum and the like, the components are simple, the cost is low, and the preparation and the use are convenient.
The domestication platform can be suitable for serum-free suspension domestication process of any adherent cell in a stretching way, and the cells obtained by domestication have the characteristics of single suspension growth, full shape, high and stable specific growth rate, high cell survival rate and the like.
Drawings
FIG. 1 is a microscopic morphology of MDCK cells in an adherent state;
FIG. 2 is a microscopic image of MDCK suspension cells acclimatized in chemically defined medium;
FIG. 3 is a passage chart of MDCK cell lines growing in suspension in serum-free medium used in the method of the present invention.
Detailed Description
In order to make up for the defects, the invention provides an efficient and low-cost MDCK suspension serum-free domestication process platform to solve the problems in the background art.
An efficient and low-cost MDCK suspension serum-free domestication process platform comprises the following steps:
1) recovering one MDCK cell by using an original culture medium, wherein the original culture medium is 5% FBS DMEM, adding 1ml of frozen cell sap into 20ml of DMEM culture medium containing 5% FBS, and culturing in an incubator with the concentration of 5% carbon dioxide and the temperature of 37 ℃;
2) when the confluence degree of the cells reaches about 70%, replacing the original culture medium with a serum-free culture medium;
3) when the confluence degree of the cells in the serum-free culture medium reaches more than 90%, digesting the adherent MDCK cells for 5-8 minutes by using 0.05% of pancreatin at 37 ℃; after the cells become round, gently patting the bottom of the container to dissociate the MDCK cells, and adding a stop solution containing 5% serum PBS (phosphate buffer solution) in a volume which is 3 times larger than that of the digestion solution to stop digestion; 1000rmp, 5min, RT centrifugation, removing the stop solution;
4) after serum removal, the serum was removed as follows 1: 3, rotating the bottle for culture; after 2-3 days of culture, the growth rate of the cells gradually becomes slow due to the existence of serum-free cells, the cells gradually gather and grow, and most of the cells naturally suspend;
5) the cells are continuously passaged for 2-3 times, the multiplication rate of the cells can gradually return to normal, but the adherent state becomes worse, at the moment, the cells can be blown down without being processed by pancreatin, and a small amount of adherent cells can be continuously cultured after being added into a serum-free culture medium;
6) settling the agglomerated cells by using a centrifugal tube natural settling method before replacing the liquid, wherein the settling time is determined according to the size of the cell clusters and is 5-30 minutes, and removing the cell clusters;
7) collecting the cells in all the bottles in a 125ml shake flask with a final volume of 50-60ml and a density of 3E 5-1E 6, and performing suspension acclimation of the cells in a constant temperature carbon dioxide shaker under the culture conditions of 37 ℃ and 110rpm and 8% CO 2;
8)800rmp, 5min, RT centrifugation to remove poor cell and impurity;
9) replacing the culture medium 3-5 days before the cells recover the doubling state, and repeating for many times;
10) when the culture medium is completely single suspension cells and the cell viability is greater than 95%, the cells can be doubled within 24 hours and are considered to be acclimatized;
11) the whole acclimatization process time period is 1-2 months; compared with the traditional culture method for gradually reducing serum, the method greatly shortens the domestication time.
The invention also discloses a serum-free culture medium in the efficient low-cost MDCK suspension serum-free domestication process platform, which comprises amino acid, vitamin, inorganic salt, lipid, hydrolysate and other additives, and the components and the content ranges thereof are as follows:
amino acid group
Vitamin group
Inorganic salts and trace elements
Lipid, hydrolysate and additive group
The invention also discloses application of the MDCK suspension serum-free domestication process platform with high efficiency and low cost in the suspension domestication process of other adherent cells.
The MDCK cell is MDCK [ NBL-2 ].
The serum-free culture medium used in the invention is a serum-free culture medium independently developed by Shanghai duoning organisms. The domestication culture platform uses reagents comprising: serum-free medium (shanghai doning), glutamine (sigma), dextran sulfate (sigma), pancreatin (Gbico), fetal bovine serum (shanghai doning), DMEM medium (Gbico); the used equipment comprises a carbon dioxide incubator (WIGGENS), a constant temperature carbon dioxide shaker (Shanghai duoning), a cell counter (CountStar), an optical microscope (Mshot), a sterile operating platform (Sujing Antai) and a centrifuge (BioTek); the cell line used was the MDCK adherent cell line donated by the guangzhou zizhi organism, and the original adherent medium was 5% FBS DMEM.
The settling time in the step 6) is determined according to the size of the cell mass, and is determined by a 50ml centrifugal tube natural settling method, and the settling time of the cell mass with the diameter of more than 5mm is 5 minutes; settling time of cell mass with diameter of 3-5mm is 10 min; settling time of cell mass with diameter of 1-3mm is 15 min; the settling time of the cell mass with the diameter of 0.2mm-1mm is 20 min.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Example 1:
the culture medium of the following examples of the present invention was prepared as follows: the present example is a serum-free medium, the components of which include 21 amino acids, 18 inorganic salts, 13 vitamins, 1 lipid, 1 plant hydrolysate, 8 additives; the materials used were purchased from sigma without specific reference. The apparatus used for the validation process was a 125ml shake flask (supplied by Dow.Shanghai, Inc.), a carbon dioxide constant temperature shaker model DNSI0101 (supplied by Dow.Shanghai, Inc.), a cytometer model IC1000 (available from CountStar Inc.), an optical microscope model MF52-N (available from Mshot Inc.), and a carbon dioxide incubator model WCI-180 (available from WIGGENS Inc.). The cell line used was the MDCK adherent cell line donated by the guangzhou zizhi organism, and the original adherent medium was 5% FBS DMEM. The culture medium comprises the following specific components:
amino acid group
Vitamin group
| Component (A) | Content mg/L | Component (A) | Content mg/L |
| Alpha-lipoic acid | 1.2 | Nicotinamide | 3.8 |
| Cyanocobalamin | 1.25 | Thiamine hydrochloride | 3.2 |
| Biotin | 3.3 | Riboflavin | 0.38 |
| Folic acid | 4.5 | Para aminobenzoic acid | 1.75 |
| Vitamin C | 22 | Pyridoxine | 4.5 |
| Calcium pantothenate | 3.2 | Choline chloride | 105 |
| Inositol | 88 |
Inorganic salts and trace elements
| Component (A) | Content mg/L | Component (A) | Content mg/L |
| Potassium chloride | 695 | Ferrous sulfate | 0.5 |
| Sodium bicarbonate | 2200 | Sodium silicate | 0.45 |
| Calcium chloride | 25 | Manganese chloride | 0.009 |
| Disodium hydrogen phosphate | 650 | Sodium metavanadate | 0.002 |
| Stannous chloride | 0.0003 | Sodium molybdate | 0.003 |
| Zinc sulfate | 2.6 | Sodium selenite | 0.003 |
| Magnesium sulfate | 72 | Copper sulfate | 0.1 |
| Aluminium chloride | 0.05 | Cobalt chloride | 0.05 |
| Lithium chloride | 0.05 | Germanium dioxide | 0.05 |
Lipid, hydrolysate and additive group
| Component (A) | Content mg/L | Component (A) | Content mg/L |
| Linoleic acid | 0.12 | P188 | 1860 |
| Pyruvic acid sodium salt | 165 | HEPES | 1500 |
| Glucose | 6000 | Soybean hydrolysate | 1200 |
| Ferric ammonium citrate | 45 | Dextran sulfate | 30 |
The liquid medium configuration method for this universal medium is as follows:
step 1, adding ultrapure water filtered by an RO membrane into a container to reach 90% of the total volume;
and 3, after the amino acid is completely dissolved, sequentially adding inorganic salt, water-soluble vitamin, alkali-soluble vitamin and other additives when the solution is cooled to 24-35 ℃, wherein ferrous sulfate heptahydrate, sodium bicarbonate, foaming substances such as P188, hydrolysate and the like are uniformly and finally added.
step 5, carrying out positive pressure filtration sterilization by using a filter membrane of 0.2 mu m after the volume is determined to the final volume;
and 6, sealing and storing at 2-8 ℃ in a dark place after the solution is prepared.
The method for using the culture medium prepared according to the preparation method for cell acclimation culture is as follows:
1. recovering one MDCK cell by using an original culture medium (5% FBS DMEM), and after the cell recovers a multiplication state, replacing the original culture medium with a universal veterinary vaccine culture medium used by a platform when the cell confluency reaches about 70%, wherein the culture medium is a complete serum-free animal source-free culture medium.
2. When the cell confluence in this serum-free medium reached 90% or more, adherent MDCK cells were digested with 0.05% pancreatin at 37 ℃ for 5-8 minutes. After the cells become round, gently patting the bottom of the container to dissociate the MDCK cells, and adding a stop solution containing 5% serum PBS (phosphate buffer solution) in a volume which is 3 times larger than that of the digestion solution to stop digestion; 1000rmp, 5min, RT centrifugation, removal of stop solution, and then the use of equal volume of general serum-free medium heavy suspension cleaning cells, completely removal of serum.
3. After serum removal, the serum was removed as follows 1: the mode 3 is spinner bottle culture, after 2-3 days of culture, the growth rate of the cells gradually becomes slow due to the existence of serum-free, the cells gradually gather and grow, and most of the cells are naturally suspended.
4. When the cells are passaged for 2-3 times, the doubling rate of the cells can gradually return to normal, but the adherent state becomes worse, and the cells can be blown down without pancreatin treatment.
5. All cells in the flasks were collected in a T125ml flask, and the cells were acclimatized in suspension at 37 ℃ at 110rpm in 8% CO2 culture conditions at a final volume of 50-60ml (too small for cell growth). The cells in the initial stage of the suspension domestication can have serious wall-hanging agglomeration, but the cells with excellent single suspension cell state can still be seen through microscopic examination and counting.
6. The aim is to screen out the excellent cells by using a method of settling the agglomerated cells by using a centrifugal tube natural settling method before liquid is changed every time, wherein the settling time is determined according to the size of a cell mass. The cell mass is larger at the beginning, the settling time is 5-10 minutes, and the larger cell mass is removed. 800rmp, 5min, RT centrifugation removed cells and impurities in poor state.
7. The medium was changed 3-5 days before the cells returned to a doubling state. Sedimentation and centrifugation operations were performed before each medium change.
8. And after the multiplication rate of the cells is recovered, the number of good cells is already increased, but the agglomerated unfavorable cells still exist in the culture medium, the method adopted at the moment is to prolong the natural settling time of the centrifugal tube for 10-20min, and only 80% of the suspension culture medium of the supernatant is taken for continuing suspension domestication.
9. When the medium was completely single suspension cells and the cell viability was > 95%, the cells were considered to be 90% acclimatized when they could be doubled within 24 hours. In the acclimation process, cells for suspension acclimation can be continuously supplied by an adherent serum-free culture medium, and the acclimation period is shortened.
10. The whole acclimatization process time period is 1-2 months. Compared with the traditional culture method for gradually reducing serum, the method greatly shortens the domestication time.
The morphology comparison of the MDCK cells obtained in this example and the original adherent MDCK cells in the chemically defined medium is shown in fig. 1, fig. 2 and fig. 3, and the suspension MDCK cell line obtained in this example has single suspension growth, full morphology, stable specific cell growth rate, seeding density of 0.5 × 105 cells/mL, ability of growing to 1.8 × 106 cells/mL after 2 days, specific growth rate of 0.033-0.038 h-1, and cell viability rate higher than 96%.
Finally, the description is as follows: the present invention is not limited to the above embodiments, which are only test embodiments, and are not limited to the present invention, and for those skilled in the art, the application range of the domestication mode platform can still be expanded, and the corresponding platform process can be supplemented and optimized. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. An efficient and low-cost MDCK suspension serum-free domestication process platform is characterized by comprising the following steps:
1) recovering one MDCK cell by using an original culture medium, wherein the original culture medium is 5% FBSDMEM, adding 1ml of frozen cell sap into 20ml of DMEM culture medium containing 5% FBS, and culturing in an incubator at 37 ℃ and with the concentration of 5% carbon dioxide;
2) when the confluence degree of the cells reaches about 70%, replacing the original culture medium with a serum-free culture medium;
3) when the confluence degree of the cells in the serum-free culture medium reaches more than 90%, digesting the adherent MDCK cells for 5-8 minutes by using 0.05% of pancreatin at 37 ℃; after the cells become round, gently patting the bottom of the container to dissociate the MDCK cells, and adding a stop solution containing 5% serum PBS (phosphate buffer solution) in a volume which is 3 times larger than that of the digestion solution to stop digestion; 1000rmp, 5min, RT centrifugation, removing the stop solution;
4) after serum removal, the serum was removed as follows 1: 3, rotating the bottle for culture; after 2-3 days of culture, the growth rate of the cells gradually becomes slow due to the existence of serum-free cells, the cells gradually gather and grow, and most of the cells naturally suspend;
5) the cells are continuously passaged for 2-3 times, the multiplication rate of the cells can gradually return to normal, but the adherent state becomes worse, at the moment, the cells can be blown down without being processed by pancreatin, and a small amount of adherent cells can be continuously cultured after being added into a serum-free culture medium;
6) settling the agglomerated cells by using a centrifugal tube natural settling method before replacing the liquid, wherein the settling time is determined according to the size of the cell clusters and is 5-30 minutes, and removing the cell clusters;
7) collecting the cells in all the bottles in a 125ml shake flask with a final volume of 50-60ml and a density of 3E 5-1E 6, and performing suspension acclimation of the cells in a constant temperature carbon dioxide shaker under the culture conditions of 37 ℃ and 110rpm and 8% CO 2;
8)800rmp, 5min, RT centrifugation to remove poor cell and impurity;
9) replacing the culture medium 3-5 days before the cells recover the doubling state, and repeating for many times;
10) when the culture medium is completely single suspension cells and the cell viability is greater than 95%, the cells can be doubled within 24 hours and are considered to be acclimatized;
11) the whole acclimatization process time period is 1-2 months; compared with the traditional culture method for gradually reducing serum, the method greatly shortens the domestication time.
2. The serum-free medium for the efficient low-cost MDCK suspension serum-free acclimatization process platform according to claim 1, wherein the serum-free medium comprises amino acids, vitamins, inorganic salts, lipids, hydrolysates and other additives, and the content ranges of the components are as follows:
amino acid group
Vitamin group
Inorganic salts and trace elements
Lipid, hydrolysate and additive group
3. The application of the efficient low-cost MDCK suspension serum-free domestication process platform according to claim 1 in other adherent cell suspension domestication processes.
4. Use according to claim 3, characterized in that: the MDCK cell is MDCK [ NBL-2 ].
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| CN115094023A (en) * | 2022-07-04 | 2022-09-23 | 无锡多宁生物科技有限公司 | MDCK cell microcarrier culture and suspension domestication process |
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