CN111088223A - Microcarrier suspension culture method and application of DF-1 cells - Google Patents
Microcarrier suspension culture method and application of DF-1 cells Download PDFInfo
- Publication number
- CN111088223A CN111088223A CN201911379484.4A CN201911379484A CN111088223A CN 111088223 A CN111088223 A CN 111088223A CN 201911379484 A CN201911379484 A CN 201911379484A CN 111088223 A CN111088223 A CN 111088223A
- Authority
- CN
- China
- Prior art keywords
- culture
- cells
- microcarrier
- cell
- stirring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000004114 suspension culture Methods 0.000 title claims abstract description 28
- 238000004113 cell culture Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims description 35
- 241000700605 Viruses Species 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 241000702626 Infectious bursal disease virus Species 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 6
- 241000287828 Gallus gallus Species 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000013019 agitation Methods 0.000 claims 2
- 230000002062 proliferating effect Effects 0.000 claims 1
- 238000011081 inoculation Methods 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 4
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 238000004264 monolayer culture Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 132
- 230000012010 growth Effects 0.000 description 20
- 239000000243 solution Substances 0.000 description 17
- 230000007423 decrease Effects 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 230000010261 cell growth Effects 0.000 description 5
- 238000010899 nucleation Methods 0.000 description 5
- 239000000969 carrier Substances 0.000 description 3
- 230000000120 cytopathologic effect Effects 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 241000271566 Aves Species 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 210000003837 chick embryo Anatomy 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000005727 virus proliferation Effects 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100001221 nontumorigenic Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/10011—Birnaviridae
- C12N2720/10051—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a suspension culture method of a DF-1 cell microcarrier and an application thereof, wherein the suspension culture method of the DF-1 cell is established by optimizing the suspension culture inoculation and culture process of the DF-1 cell microcarrier, the large-scale proliferation of the DF-1 cell can be realized, the method has the advantages of plate culture and suspension culture, the cells are in relatively uniform environment, the culture conditions such as temperature, pH, CO2 and the like are controllable, compared with the traditional monolayer cell culture, the surface area of cell adherence is greatly increased, the amplification control and the high-density cell culture are convenient, the high-density cells which are 20-50 times more than the original cells can be obtained, the labor intensity and the production cost are reduced, and the method has obvious advantages compared with the traditional culture.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a microcarrier suspension culture method and application of DF-1 cells.
Background
The DF-1 cell line is derived from an ELL-0 (sailing) Chick Embryo Fibroblast (CEF) of 10 days old, is a mature continuous cell line, shows a typical fibroblast shape and can form an obvious cytopathic phenomenon. This cell line was first developed by Dr Douglas Foster, Minnesota university, 1998 (Himly et al, 1998). DF-1 cells have strong proliferation capacity, the cell density can reach more than 4 times of that of Chick Embryo Fibroblast (CEF), the cells belong to reverse transcriptase negative and non-tumorigenic cells, and the cells are authorized to become global commercial cell lines by the Food and Drug Administration (FDA). DF-1 cells are currently widely used for recombinant protein expression, avian viral proliferation, recombinant protein expression, oncoviral research, and the production of human and animal vaccines. At present, DF-1 cell line has been applied to a certain extent in experimental detection and virus culture, and a plurality of research results show that DF-1 cell can adapt to the proliferation of various avian viruses and has obvious cytopathic phenomena. The existing method for culturing DF-1 cells mainly adopts a traditional culture mode of a spinner flask or a cell factory, and the culture mode has the following defects: (1) the culture quantity is small, and the area of a plane needs to be increased when the scale is enlarged; (2) the batch culture quality is uncontrollable, and the batch is unstable; (3) the use amount of the culture medium is large, and the production cost is increased; (4) more than 50 units of processing manpower are very laborious, and the manpower cost is high; (5) the number of matched basic equipment is synchronously increased.
Microcarrier culture systems were first proposed in 1967 by inoculating cells into microcarriers and performing stirred suspension culture when most of the cells are attached to the surface of the microcarriers. Through the development of recent decades, microcarrier culture technology has become more and more mature, and is widely applied to vaccine production and other industries, at present, adherent cells are mainly cultured on a large scale through microcarrier culture technology, and the method is also generally accepted by researchers.
Although microcarriers have been used for mass culture of cells, it is difficult to say that a method for mass culture of DF-1 cells using microcarriers has been established. At present, the existing domestic units utilize the paper carriers for culture, but the paper carriers cannot sample at any time to observe the cell state in the cell culture process, the optimal virus receiving time is difficult to control, compared with the paper carriers, the spherical microcarrier culture can sample at any time to observe the cell state, draw a cell growth curve, accurately master the optimal virus receiving and virus receiving time, and harvest the virus liquid with the highest titer.
Therefore, the technical problems to be solved by the invention are as follows: how to culture DF-1 cells in large quantities by microcarriers.
Disclosure of Invention
Therefore, the invention aims to provide a microcarrier suspension culture method of DF-1 cells and application thereof, the invention establishes the suspension culture method of DF-1 cells by optimizing the suspension culture inoculation and culture process of the DF-1 cells, can realize large-scale proliferation of the DF-1 cells, has the advantages of plate culture and suspension culture, ensures that the cells are in relatively uniform environment, has controllable culture conditions such as temperature, pH, CO2 and the like, greatly increases the surface area of cell adherence compared with the traditional monolayer cell culture, is convenient for controlling amplification and high-density cell culture, can obtain 20-50 times more high-density cells than the original cells, simultaneously reduces labor intensity and production cost, and has obvious advantages compared with the traditional culture.
The technical scheme of the invention is as follows:
a microcarrier suspension culture method of DF-1 cells comprises the following steps:
(1) cytodex1 microcarriers were treated with Ca-free media2+And Mg2+Washing with PBS, sterilizing under high pressure, and soaking in DMEM/F12 culture medium containing FBS overnight to obtain sterilized Cytodex1 microcarrier;
(2) adding a sterilized Cytodex1 microcarrier into a rotary bottle, then inoculating the digested DF-1 cells into the rotary bottle, adding a cell culture solution, and culturing in an incubator by adopting a stirring culture mode;
(3) detecting sugar consumption in the culture solution at intervals, and supplementing sugar or replacing the culture solution according to the sugar consumption condition to keep the concentration of glucose in the culture solution at 1.0-1.5 g/L;
(4) culturing until the cell density of DF-1 reaches the highest.
Further, the DF-1 cells are inoculated in the step (2) at a density of 25-100 cells per microcarrier.
Further, the DF-1 cells were seeded at a density of 50 cells per microcarrier in step (2).
Further, the concentration of the Cytodex1 microcarrier in the spinner flask in the step (2) is 1-10 g/L.
Further, in step (2), the concentration of Cytodex1 microcarrier in the spinner flask was 3 g/L.
Further, the conditions for the cultivation in the incubator in the step (2) are 37 ℃ temperature, 5% CO2。
Further, the stirring mode in the step (2) is as follows: stirring is continuously carried out for 6h at 20r/min, then the culture is carried out at 30r/min with stirring, and the culture volume is 100 mL.
Further, the stirring mode in the step (2) is as follows: stirring at 20r/min for 1min, stopping stirring for 10min for 6h, and continuously stirring at 30r/min for culture with culture volume of 33 mL.
Further, the sugar consumption in the culture broth was measured every 24 hours in step (3).
The application of the microcarrier suspension culture method of the DF-1 cell in culturing the infectious bursal disease virus comprises the following steps:
when the cell density of DF-1 cultured by the microcarrier reaches the highest, the inoculation of the chicken infectious bursal disease virus is carried out according to the MOI of 0.01, the virus is propagated, and the virus is harvested after 48 hours of inoculation.
The invention has the following beneficial effects:
the invention establishes the microcarrier suspension culture method of the DF-1 cell by optimizing the optimal condition of the suspension culture of the DF-1 cell microcarrier, and utilizes the technology to culture the DF-1 cell for inoculation of the chicken infectious bursal disease virus, thereby obtaining good culture effect. The spherical microcarrier suspension culture greatly increases the surface area of cell adherence, is convenient for control amplification and high-density cell culture, can obtain 20-50 times more high-density cells than the original cells, simultaneously reduces the labor intensity and the production cost, and has obvious advantages compared with the traditional culture mode.
Drawings
FIG. 1 is a microscopic image of DF-1 cells cultured with Cytodex1 microcarriers for 72h in example 1;
FIG. 2 is a microscopic image of the lesions 48h after the inoculation of DF-1 cells cultured with Cytodex1 microcarriers in example 1;
FIG. 3 is a graph showing the effect of different stirring modes on DF-1 cell adherence efficiency in test example 1;
FIG. 4 is a graph showing the effect of different DF-1 cell seeding densities on DF-1 cell growth density in test example 2;
FIG. 5 is a graph showing the effect of different DF-1 cell seeding densities on the DF-1 cell specific growth rate in test example 2;
FIG. 6 is a graph of the effect of different Cytodex1 microcarrier concentrations on DF-1 cell growth density in test example 3;
FIG. 7 is a graph of the effect of different Cytodex1 microcarrier concentrations on the specific growth rate of DF-1 cells in test example 3.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the following embodiments, but the present invention is not limited thereto.
DF-1 cells were purchased from ATCC in the following examples and test examples, passaged, banked and preserved by the laboratory; infectious bursal disease virus is IBDV (GT strain) and is identified, stored and supplied by the company.
Example 1
A microcarrier suspension culture method of DF-1 cells comprises the following steps:
(1) cytodex1(GE, USA) was used as the microcarrier for the test, with the specific parameters of about 4.3X 10 per gram of dry weight6Microcarriers providing a cell attachment area of about 4400cm2Per g, dry CytodeCa-free for x1 microcarriers2+And Mg2+The PBS is soaked and swelled according to 50-100 mL of PBS per gram of microcarrier, the microcarrier is maintained at room temperature for not less than 3h, gentle shaking is given in the room, after the microcarrier is settled, supernatant is sucked away, a new PBS solution is added, the microcarrier is washed for 2-3 times, then the microcarrier is autoclaved at 121 ℃ for 30min, and then the microcarrier is soaked overnight in a DMEM/F12 culture medium (containing 10% of FBS, L-glutamine is added until the final concentration is 4mmol/L, and HEPES buffer solution is not contained) to obtain a sterilized Cytodex1 microcarrier;
(2) adding sterilized Cytodex1 microcarriers into a 250mL roller bottle to make the concentration of the microcarriers be 3g/L, digesting the trypsin digestion solution of DF-1 cells growing to a good monolayer in a T225 cell bottle, counting the digestion solution by using a cell counting plate, inoculating 50 DF-1 cells into the roller bottle according to the inoculation density of each microcarrier, adding a cell culture solution to 100mL, and controlling the culture conditions of an incubator to be as follows: temperature 37 ℃ and 5% CO2Continuously stirring and culturing for 6h at 20r/min, and then continuously stirring and culturing at 30 r/min;
(3) detecting sugar consumption in the culture solution every 24h, counting cells, and supplementing sugar or replacing the culture solution according to the sugar consumption condition to keep the concentration of glucose in the culture solution at 1.0-1.5 g/L;
(4) microscopic observation of DF-1 cells at 72h of culture was shown in FIG. 1, where DF-1 cell density reached its highest.
At this time, the microscopic observation of DF-1 cytopathic effect after inoculation of chicken infectious bursal disease virus, virus proliferation and inoculation for 48h is carried out according to the MOI of 0.01, as shown in figure 2, at this time, the virus is harvested, and the virus titer reaches 108.5TCID500.1mL or more.
Test example 1
A microcarrier suspension culture method of DF-1 cells specifically comprises the following steps of 1, and is characterized in that 4 different stirring modes are set in the step (2) for culture:
the first method is as follows: continuously stirring and culturing for 6h at the speed of 20r/min, and then continuously stirring and culturing at the speed of 30r/min, wherein the culture volume is 100 mL;
the second method comprises the following steps: stirring for 1min at a speed of 20r/min, stopping stirring for 10min for 6h, and then continuously stirring and culturing at a speed of 30r/min, wherein the culture volume is 100 mL;
the third method comprises the following steps: stirring for 1min at a speed of 20r/min, stopping stirring for 30min at an intermittent stirring mode for 6h, and then continuously stirring and culturing at a speed of 30r/min, wherein the culture volume is 100 mL;
the method is as follows: the stirring mode in the step (2) is as follows: stirring at 20r/min for 1min, stopping stirring for 10min for 6h, and continuously stirring at 30r/min for culture with culture volume of 33 mL.
Sampling and counting the culture solution in four different stirring modes every 1h, calculating the adherence efficiency of the different stirring modes according to the number of free cells in the culture solution, and obtaining a curve graph of the influence of the different stirring modes on the adherence efficiency of the DF-1 cells as shown in figure 3. As can be seen from figure 3, the stirring modes of the first mode and the fourth mode are more favorable for attaching the cells to the microcarrier, and the other two modes have lower efficiency for attaching the cells to the microcarrier at the beginning.
Test example 2
A method for microcarrier suspension culture of DF-1 cells comprises the following specific steps of operation according to example 1, and is characterized in that 4 different cell inoculation densities are set in step (2), namely, each microcarrier is inoculated with 25, 50, 75 and 100 DF-1 cells, culture solutions with four different cell inoculation densities are sampled and counted every 24 hours, and a graph showing the influence of the different DF-1 cell inoculation densities on the DF-1 cell growth density is shown in figure 4; a graph of the effect of different DF-1 cell seeding densities on the specific growth rate of DF-1 cells is shown in FIG. 5.
As can be seen from FIG. 4, when 75 DF-1 cells were seeded on each microcarrier surface, the highest viable cell density (1.04X 10) was achieved in 48h6/mL), cell seeding density of 100 DF-1 cells per microcarrier also showed the highest viable cell density at 48h (1.07X 10)6and/mL), after 72 hours, the microcarriers form a bridging phenomenon through cells, the cells fall off from the surface of the microcarriers to form cell clusters, so that the total number of the cells begins to decrease, and the plateau period of the cells is short. When the cell inoculation density is that each microcarrier is inoculated with 25 DF-1 cells, the cells are overall slow in growth and long in delay period, and the cells are inoculated for 72hTo reach the highest density (0.53 multiplied by 10)6mL), but the cells do not overgrow the microcarrier surface throughout the culture. When 50 DF-1 cells were seeded on each microcarrier, the cells reached the highest viable cell density at 72h (1.09X 10)6mL), the highest density was higher than that of the group of 75, 100 DF-1 cells seeded per microcarrier, and there was a relatively distinct plateau at 72h to 96h, and the cell clumping was also alleviated compared to 75, 100 DF-1 cells seeded per microcarrier.
As can be seen from the attached FIG. 5, the seeding density of the four cells has no obvious lag phase when growing on the microcarriers, and the smaller the number of the cells seeded on the surface of each microcarrier is, the larger the specific growth rate of the 24h cells is; wherein the specific growth rate of 75 and 100 cell inoculation densities on the surface of each microcarrier is relatively close, and the highest viable cell density is reached within 48h because of the relatively high inoculation density, namely the specific growth rate is a positive value before 48h, and after 72h, the microcarriers fall off to cause cell death and density reduction, and the specific growth rate becomes a negative value; the specific growth rate of 25 cells inoculated by each microcarrier is 0.692 at 24h and is the maximum value in all groups, but then the specific growth rate begins to decrease, cells cannot grow to be full of the microcarriers all the time due to cell density dependence, the specific growth rate gradually decreases, the cells begin to fall off due to the influence of factors such as shearing force, culture medium nutrition and the like along with the progress of the culture process, so that the cell density decreases, and the specific growth rate also becomes a negative value; the specific growth rate of 50 cells inoculated with each microcarrier has a relatively stable value within 72h, although the specific growth rate also shows a trend of reduction, but the reduction range is not large in other groups, and is always higher than 0.409d within 72h-1Thus, the cells had a longer steady increase and cell density was higher than the other groups. However, 72h also became negative indicating that the cell density began to decrease.
Test example 3
A method for the microcarrier suspension culture of DF-1 cells comprises the following specific steps of the operation of the embodiment 1, and is characterized in that 5 different microcarrier concentrations, namely 1, 3, 5, 7 and 10g/L, are set in the step (2), and the culture solution with five different microcarrier concentrations is sampled and counted every 24h, so that the obtained curve graph of the influence of different Cytodex1 microcarrier concentrations on the DF-1 cell growth density is shown in the attached figure 6; a graph of the effect of different Cytodex1 microcarrier concentrations on the specific growth rate of DF-1 cells is shown in FIG. 7.
As can be seen from FIG. 6, when the microcarrier is 7g/L and 10g/L, the highest viable cell density of 72h can reach 5.81X 106mL and 5.58X 106and/mL, when the concentration of the microcarrier is 10g/L, the probability of collision among the microcarriers is increased due to overlarge concentration of the microcarriers, the required culture conditions are stricter, and cells can not grow on the surface of the microcarriers all the time, so that the highest cell density is not maximized, but is slightly smaller than that of a microcarrier concentration group of 7 g/L. While the microcarrier concentration of 1g/L can only reach the highest viable cell density of 1.04X 106Microcarrier concentrations of 3g/L and 5g/L, respectively, can be adjusted to a cell density of 3.15X 106mL and 4.45X 106mL, DF-1 cells were cultured at microcarrier concentrations below 7g/L, the highest viable cell density of the cells appeared around 72h and increased with increasing microcarrier concentration, after 72h the cell density began to decrease, the cells aggregated into clumps, or shed from microcarriers. However, when the microcarrier concentration reaches 10g/L, the highest viable cell density of the cells and the cell density obtained by the microcarrier concentration of 7g/L are lower, and the highest viable cell density is not increased any more, so that the microcarrier concentration should be within 7g/L when selecting microcarrier to culture DF-1 cells, so as to avoid waste of microcarriers.
As can be seen from FIG. 7, the lower the microcarrier concentration is, the greater the specific growth rate of the cells for 24h is, after 24h, the specific growth rate of the microcarrier concentration group of 1g/L starts to decrease, and after 72h to 96h, the value changes from positive to negative, which indicates that the number of cells starts to decrease after 72h reaches the maximum; 5. the growth rate of the microcarrier concentration group of 7g/L and 10g/L slightly increases within 24 h-48 h, and the specific growth rate begins to decrease after 72h, which indicates that the cell acceleration is slow; and in the microcarrier concentration group of 3g/L, the specific growth rate is kept relatively stable within 24-72 h, and no obvious decline trend exists, which indicates that the cells can stably proliferate in the time period.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A microcarrier suspension culture method of DF-1 cells is characterized by comprising the following steps:
(1) cytodex1 microcarriers were treated with Ca-free media2+And Mg2+Washing with PBS, sterilizing at high pressure, and soaking in DMEM/F12 culture medium containing FBS to obtain sterilized Cytodex1 microcarrier;
(2) adding a sterilized Cytodex1 microcarrier into a rotary bottle, then inoculating the digested DF-1 cells into the rotary bottle, adding a cell culture solution, and culturing in an incubator by adopting a stirring culture mode;
(3) detecting sugar consumption in the culture solution at intervals, and supplementing sugar or replacing the culture solution according to the sugar consumption condition to keep the concentration of glucose in the culture solution at 1.0-1.5 g/L;
(4) culturing until the cell density of DF-1 reaches the highest.
2. The method for microcarrier suspension culture of DF-1 cells according to claim 1, wherein the DF-1 cells are seeded at a density of 25 to 100 cells per microcarrier in step (2).
3. The method for microcarrier suspension culture of DF-1 cells according to claim 2, wherein the DF-1 cells are seeded at a density of 50 cells per microcarrier in step (2).
4. The microcarrier suspension culture method of DF-1 cell according to claim 1, wherein the concentration of the Cytodex1 microcarrier in the spinner flask in step (2) is 1-10 g/L.
5. The method of claim 4, wherein the microcarrier concentration of Cytodex1 in the spinner flask of step (2) is 3 g/L.
6. The method of claim 1, wherein the culture conditions in the incubator are 37 ℃ and 5% CO2。
7. The method for microcarrier suspension culture of DF-1 cells according to claim 6, wherein the agitation in step (2) is: stirring is continuously carried out for 6h at 20r/min, then the culture is carried out at 30r/min with stirring, and the culture volume is 100 mL.
8. The method for microcarrier suspension culture of DF-1 cells according to claim 6, wherein the agitation in step (2) is: stirring at 20r/min for 1min, stopping stirring for 10min for 6h, and continuously stirring at 30r/min for culture with culture volume of 33 mL.
9. The method of claim 1, wherein the sugar consumption of the culture medium is measured every 24 h.
10. Use of the microcarrier suspension culture method of any one of claims 1 to 9 of DF-1 cells in the culture of infectious bursal disease virus of chicken, comprising the steps of:
when the cell density of DF-1 cultured by the microcarrier reaches the highest, inoculating the chicken infectious bursal disease virus according to the MOI of 0.01, proliferating the virus, inoculating the virus for 48h, and harvesting the virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911379484.4A CN111088223A (en) | 2019-12-27 | 2019-12-27 | Microcarrier suspension culture method and application of DF-1 cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911379484.4A CN111088223A (en) | 2019-12-27 | 2019-12-27 | Microcarrier suspension culture method and application of DF-1 cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111088223A true CN111088223A (en) | 2020-05-01 |
Family
ID=70397431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911379484.4A Pending CN111088223A (en) | 2019-12-27 | 2019-12-27 | Microcarrier suspension culture method and application of DF-1 cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111088223A (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028514A (en) * | 2006-06-30 | 2007-09-05 | 辽宁成大生物股份有限公司 | Method for producing vaccine for man by biological reactor |
| CN102268411A (en) * | 2011-08-15 | 2011-12-07 | 江苏省农业科学院 | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation |
| CN102282253A (en) * | 2009-04-08 | 2011-12-14 | 赛诺菲巴斯德有限公司 | Purification method of rabies virus |
| CN102690791A (en) * | 2011-10-25 | 2012-09-26 | 哈药集团生物疫苗有限公司 | Method for generating porcine pseudorabies virus by culturing ST cell in microcarrier of bioreactor |
| CN103386127A (en) * | 2013-08-09 | 2013-11-13 | 浙江美保龙生物技术有限公司 | Method for preparing vaccine by Newcastle disease virus cultured by using chick embryo continuous cell line and bioreactor |
| CN105368794A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Method of utilizing stirred bioreactor to produce infectious Bursal disease virus |
| CN105838683A (en) * | 2016-05-18 | 2016-08-10 | 山东省滨州畜牧兽医研究院 | Method for proliferation of mink canine distemper virus by applying novel cell microcarrier |
| CN107058243A (en) * | 2017-03-23 | 2017-08-18 | 华南农业大学 | A kind of suspension culture method of MDV |
| CN107326016A (en) * | 2017-06-26 | 2017-11-07 | 中国水产科学研究院珠江水产研究所 | A kind of microcarrier suspension culture CPB cells production mandarin fish infectious spleen and kidney necrosis virus and the method for mandarin fish rhabdovirus |
| CN108277207A (en) * | 2018-03-28 | 2018-07-13 | 广州齐志生物工程设备有限公司 | A kind of method of online amplification production chicken infectivity bursa of Fabricius virus |
| CN109913404A (en) * | 2018-12-04 | 2019-06-21 | 哈药集团生物疫苗有限公司 | The preparation method of infections chicken cloacal bursa virus live vaccine |
| CN110354259A (en) * | 2019-06-25 | 2019-10-22 | 广东渔跃生物技术有限公司 | A kind of preparation method of duck reovirus inactivated vaccine |
| CN111849987A (en) * | 2020-07-29 | 2020-10-30 | 华农（肇庆）生物产业技术研究院有限公司 | Construction method and application of VIPERIN function-deficient cell line |
| CN112063622A (en) * | 2020-09-04 | 2020-12-11 | 华农（肇庆）生物产业技术研究院有限公司 | Construction method and application of I-type IFNAR function-deficient cell line |
-
2019
- 2019-12-27 CN CN201911379484.4A patent/CN111088223A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028514A (en) * | 2006-06-30 | 2007-09-05 | 辽宁成大生物股份有限公司 | Method for producing vaccine for man by biological reactor |
| CN102282253A (en) * | 2009-04-08 | 2011-12-14 | 赛诺菲巴斯德有限公司 | Purification method of rabies virus |
| CN102268411A (en) * | 2011-08-15 | 2011-12-07 | 江苏省农业科学院 | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation |
| CN102690791A (en) * | 2011-10-25 | 2012-09-26 | 哈药集团生物疫苗有限公司 | Method for generating porcine pseudorabies virus by culturing ST cell in microcarrier of bioreactor |
| CN103386127A (en) * | 2013-08-09 | 2013-11-13 | 浙江美保龙生物技术有限公司 | Method for preparing vaccine by Newcastle disease virus cultured by using chick embryo continuous cell line and bioreactor |
| CN105368794A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Method of utilizing stirred bioreactor to produce infectious Bursal disease virus |
| CN105838683A (en) * | 2016-05-18 | 2016-08-10 | 山东省滨州畜牧兽医研究院 | Method for proliferation of mink canine distemper virus by applying novel cell microcarrier |
| CN107058243A (en) * | 2017-03-23 | 2017-08-18 | 华南农业大学 | A kind of suspension culture method of MDV |
| CN107326016A (en) * | 2017-06-26 | 2017-11-07 | 中国水产科学研究院珠江水产研究所 | A kind of microcarrier suspension culture CPB cells production mandarin fish infectious spleen and kidney necrosis virus and the method for mandarin fish rhabdovirus |
| CN108277207A (en) * | 2018-03-28 | 2018-07-13 | 广州齐志生物工程设备有限公司 | A kind of method of online amplification production chicken infectivity bursa of Fabricius virus |
| CN109913404A (en) * | 2018-12-04 | 2019-06-21 | 哈药集团生物疫苗有限公司 | The preparation method of infections chicken cloacal bursa virus live vaccine |
| CN110354259A (en) * | 2019-06-25 | 2019-10-22 | 广东渔跃生物技术有限公司 | A kind of preparation method of duck reovirus inactivated vaccine |
| CN111849987A (en) * | 2020-07-29 | 2020-10-30 | 华农（肇庆）生物产业技术研究院有限公司 | Construction method and application of VIPERIN function-deficient cell line |
| CN112063622A (en) * | 2020-09-04 | 2020-12-11 | 华农（肇庆）生物产业技术研究院有限公司 | Construction method and application of I-type IFNAR function-deficient cell line |
Non-Patent Citations (12)
| Title |
|---|
| SAMIA ROUROU 等: "Development of an In Situ Detachment Protocol of Vero Cells Grown on Cytodex1 Microcarriers Under AnimalComponent-Free Conditions in Stirred Bioreactor", 《APPL BIOCHEM BIOTECHNOL》 * |
| YANK XIANYUAN 等: "Diamagnetic Levitation Affects Cellular Morphology and Functions of Osteoblast Adhered to Micro}arrier", 《航天医学与医学工程》 * |
| 刘晓航 等: "猪繁殖与呼吸综合征病毒微载体培养试验研究", 《兽医实验室》 * |
| 刘玉鹏 等: "马立克氏疫苗病毒在鸡传代细胞上的培养工艺", 《中国兽医学报》 * |
| 刘红 等: "Vero 细胞微载体培养的化学成分明确无血清培养基的设计", 《现代生物医学进展》 * |
| 李洪艳 等: "猪伪狂犬病病毒天活度苗工艺研究", 《营养与疾病》 * |
| 杨霞 等: "鸡IBDV在微载体微型反应器DF-1细胞系上繁殖特性研究", 《中国兽医学报》 * |
| 梅建国 等: "新型CephodexD微载体在猪瘟病毒大规模增殖中的应用研究", 《畜牧与兽医》 * |
| 温良海: "MDV悬浮培养技术工艺及免疫保护研究", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 王佃亮 等: "微载体高密度培养Vero细胞的研究", 《生物工程学报》 * |
| 王永生 等: "鸡传染性法氏囊病毒在DF-1 细胞系上繁殖特性研究", 《河南农业大学学报》 * |
| 陈以衡 等: "微载体浓度与细胞接种密度对ST细胞生长的影响", 《生物技术通报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100389193C (en) | A method for safe continuous closed cell culture, virus production and inactivation | |
| US11629338B2 (en) | Method for acclimating and suspending Vero and second order production process for virus | |
| CN106011083A (en) | Preparation method of avian influenza virus growing in serum-free full-suspended cultured MDCK cells and obtained avian influenza virus | |
| CN108300704B (en) | Method for suspension culture of infectious bronchitis virus by using continuous cell line | |
| CN102100910B (en) | A method of producing a virus vaccine | |
| CN102807964B (en) | Method for scale-up culture of animal cells | |
| CN110872572B (en) | Method for improving in-vitro suspension culture efficiency of human multifunctional stem cells | |
| CN107841482A (en) | Mdck cell serum free suspension culture technique produces H9 subtype influenza vaccines | |
| WO2024153174A1 (en) | Cell domestication method adapting to carrier-free and serum-free suspension culture, stem cell line and use | |
| CN109913404B (en) | Preparation method of chicken infectious bursal disease virus live vaccine | |
| CN107299078A (en) | One plant adapts to complete the suspend S of cat kidney cell line F 81 cultivated and its application | |
| CN104894054B (en) | A kind of suspension adapted strains of monkey embryo renal epithelial cell Marc 145 and its application in culture reproductive and respiratory syndrome virus, the blue ear viral vaccine of production | |
| CN118755665A (en) | A high-density culture method of porcine myoblasts based on bioreactor | |
| CN117070474B (en) | Method for producing virus by three-dimensional porous microcarrier suspension culture cells | |
| CN111088223A (en) | Microcarrier suspension culture method and application of DF-1 cells | |
| CN109609436A (en) | A kind of no CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely | |
| TW202309265A (en) | Mdck suspension cell lines in serum-free, chemically-defined media for vaccine production | |
| CN102743749A (en) | Method for preparing live attenuated rubella vaccine in human diploid cells by using basket-type bioreactor | |
| CN116926020A (en) | High-density culture method for herpesvirus vector cells | |
| CN112795530A (en) | Method for culturing canine distemper virus and canine parvovirus and application | |
| CN115044529A (en) | Method for producing virus vaccine by large-scale culture of human diploid cells | |
| CN109679900B (en) | Preparation method of avian influenza vaccine and product thereof | |
| CN113444686B (en) | A method of cultivating grouper cells using a bioreactor | |
| CN109602900A (en) | The preparation method and products thereof of chook MDV live vaccine | |
| CN111808799B (en) | A kind of multifunctional stem cell culture method and medium used |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200501 |