CN109929796A - Serum free suspension cultivation type mdck cell strain and its application - Google Patents
Serum free suspension cultivation type mdck cell strain and its application Download PDFInfo
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Abstract
The invention discloses a kind of serum free suspension cultivation type mdck cell strain and its applications, the serum free suspension cultivation type mdck cell strain is preserved in China typical culture collection center, classification naming: canine kidney cells MDCK-XF03, deposit number are as follows: CCTCC NO:C2018191；The serum free suspension cultivation type mdck cell strain can be applied to the production of influenza vaccines and avian influenza vaccine.The present invention passes through adaptation culture and the culture domestication that suspends, serum free suspension cultivation type MDCK-XF03 cell strain is obtained, canine kidney cells MDCK-XF03 cell is serum free medium suspension culture vigor is high, conglomeration is few and homogeneity is preferable, it adapts to carry out suspension culture in serum free medium completely, the correlative study and production for carrying out influenza and avian influenza vaccine for China provide serum free suspension cultivation type cell strain.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of serum free suspension cultivation type mdck cell strain and its
Using.
Background technique
Influenza, that is, influenza (Influenza) is a kind of acute respiratory infectiousness disease as caused by influenza virus
Disease, bird flu (Bird Flu or Avian Influenza) are the abbreviations of Bird Flu, it is by influenza A virus
A kind of acute infectious disease caused by hypotype (also referred to as avian influenza virus) can also infect the mankind, the death rate after people infected with bird flu
33% or more.Either influenza or bird flu do not find ideal therapeutic agent so far, and vaccine inoculation is still to work as
The present prevents epidemic disease from occurring and popular most effective means.
Influenza vaccines and the traditional production technology of avian influenza vaccine are cultivation in the chick embryo, this using chicken embryo as production matrix
Mode used for many years, technique is naturally very mature, but the production method is there are still many defects, as the production cycle is long, behaviour
Make cumbersome, heavy workload, easy to pollute, such as bird flu large-scale outbreak, very big problem is also will be present in the supply of chicken embryo.In view of
This, WHO proposes the production matrix for using mammal passage cell substitution chicken embryo as influenza vaccines in nineteen ninety-five, relative to
Chicken embryo, zooblast after recovering can with fast breeding and enter scale production of vaccine, and production process it is easy to operate and
It is easily controllable, it can guarantee vaccine quality and safety, will not be limited by natural factors such as chicken embryos using passage cell, such as epidemic situation
Meeting fast reaction can provide a large amount of vaccines supplies market in the short time.
Mdck cell (Madin-Daby Canine Kidney Cells, MDCK) by Madin and Darby in 1958 from
Cocker Spaniel female bent frame dog renal tissue separation in the U.S., which is cultivated, establishes, since its virus infection is high-efficient, proliferation is fast, and
It is not easy to make a variation, is one of the cell line of generally acknowledged most suitable influenza virus culture.It is thin using MDCK to have approval in the world at present
The influenza vaccines of born of the same parents' production, and China is as the production of influenza vaccines and avian influenza vaccine and using big country, there are 13 streams in the whole nation
Influenza vaccine manufacturing enterprise, annual value of production are more than 10,000,000,000 yuan, all chicken embryo technique production；Only have 2 uses in avian influenza vaccine
Mdck cell production is Shandong Xin get animal vaccine Co., Ltd (veterinary drug faces word 153122307) and Jilin Guan Jie biology respectively
Technology Co., Ltd.'s (animal doctor's new word 070372306), but the approval and sign hair number of the avian influenza vaccine of mdck cell matrix production is very
Few, the national total approval and sign hair number of avian influenza vaccine is 1683 batches within 2018, wherein mdck cell matrix is 68 batches, only the total lot number of Zhan
4%, there are also very big room for promotion.
Vaccine is with the biological products of virus preparation, and the method for manually obtaining a large amount of viruses mainly has animal inoculation pvaccination, chicken embryo
Culture and three kinds of enrichment procedures of cell culture, but cell culture is that most economical, quick and safe virus generally acknowledged at present increases
Grow method.In recent years, the fast development of bioreactor mass cell technology, compared with traditional rolling bottle and cell factory culture
Clear superiority is shown, the first choice of production of vaccine technique is had become with bioreactor culture cell and virus of proliferation.Biology
Bioreactor culture cell has the modes such as microcarrier culture, chip carrier culture, low serum suspension culture and serum free suspension culture,
Wherein, microcarrier culture and chip carrier culture category adhere-wall culture mode, need in culture medium plus animal blood serum, and in technique
There is technical difficulty when amplification, domestic single tank volume of culture maximum at present is less than 500L.Low serum suspension culture and serum-free
Its culture scale amplification of the mode that suspension is cultivated is easy, at present country's BHK21 cell and the single tank volume of mdck cell suspension culture
Up to 6000L, but also can be with Linear Amplifer.Either microcarrier culture, chip carrier culture or low serum, which suspend, trains
It supports, because the serum composition in culture medium containing animal origin has side reaction and other security risks to vaccine recipient, so, nothing
Serum suspension culture is the technique that production of vaccine is most favored.
The original cell of mdck cell is adhere-wall culture type cell, and needing to add serum could normal growth.Influenza vaccines/
The difficult point of avian influenza vaccine serum free suspension culture process is that the original type cell line of adhere-wall culture containing serum is domesticated for no blood
Clear suspension cultivation type cell strain.
Summary of the invention
For the shortcomings of the prior art pointed out in above-mentioned background technique, the present invention provides a kind of serum free suspensions
Cultivation type mdck cell strain, the cell strain can be applied in the production of influenza vaccines and avian influenza vaccine.
The first purpose of the invention is to provide a kind of serum free suspension cultivation type mdck cell strain, the patch that ATCC is introduced
Wall cultivation type mdck cell system (CCL-34, P55) has obtained serum free suspension cultivation type mdck cell strain, the nothing by domestication
Serum suspension cultivation type mdck cell strain is preserved in China typical culture collection center, deposit number are as follows: CCTCC NO:
C2018191, classification naming are as follows: canine kidney cells MDCK-XF03, preservation date are as follows: on September 20th, 2018, preservation address are as follows: lake
No. 299 Wuhan Universitys of Bayi Road, the wuchang, wuhan Bei Sheng area.
A second object of the present invention is to provide above-mentioned deposit numbers are as follows: the serum free suspension of CCTCC NO:C2018191
Application of the cultivation type mdck cell strain in influenza vaccines and avian influenza vaccine production.
Preferably, the serum free suspension cultivation type mdck cell strain is flowed as cellular matrix for influenza vaccines and fowl
Influenza vaccine production.
Preferably, the serum free suspension cultivation type mdck cell strain is as cellular matrix to influenza and avian influenza virus
When culture, serum free suspension cultivation type MDCK-XF03 cell is with 1.0-2.0 × 106/ mL density inoculated and cultured.
Compared with the prior art the shortcomings that and deficiency, the invention has the following advantages: the present invention is outstanding by serum-free
Floating culture acclimation method, having obtained the strain of serum free suspension cultivation type mdck cell, (classification naming: canine kidney cells MDCK-XF03 is protected
Hide number CCTCC NO:C2018191), which can be proliferated influenza and avian influenza virus, carry out influenza vaccines for China
Correlative study and production with avian influenza vaccine provide basis.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
Example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is mdck cell form；Wherein, A. adhere-wall culture type (before domestication, 10X), B. suspension cultivation type (after domestication,
20X)；
Fig. 2 is the growth curve chart of suspension cultivation type MDCK-XF03 cell strain different densities culture；
Fig. 3 is the HA column diagram that suspension cultivation type MDCK-XF03 cell strain is proliferated influenza and avian influenza virus.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
Embodiment
1. material
1.1 mdck cell strains (CCL-34) are introduced from ATCC, and cell generation is P55 when introduction, in the deposit number of ATCC:
60245139, freezing the time in ATCC: 2011-10-20 is adhere-wall culture type cell.By " pharmacopeia " 2010 editions after introduction
It is required that establishing master cell bank (number are as follows: MDCK-M-60) at culture to 60 generation, quantity 1560 are frozen；In culture to 63 generations
When establish working cardial cell library (number are as follows: MDCK-W-63) freeze quantity 1680.MDCK-M-60 and MDCK-W-63 difference
It is examined and determine in December, 2014 and inspection in January, 2015 National Institute for Food and Drugs Control, achieves cell in December, 2017
Qualification test report, report number are respectively SH201702634 and SH201702584.
1.2DEME (high-glucous) culture medium, Gibco, article No.: 02-5062EJ；SFM-MDCK free serum culture
Base, Lanzhou lark, article No. are as follows: CFM403；Top grade fetal calf serum (FBS), Lanzhou Min Hai.
1.3 trypsase, Gibco, article No.: 27250, when use, is configured to 0.25% (m/m) and uses liquid；TrypLE pancreas egg
White enzymes extraction object (1X), Gibco, article No.: 12563-029.
1.4 influenza viruses [A/California/7/2009 (NYMC X-179A), A/Texas/50/2012 (NYMC X-
223A), B/Phuket/3073/2013 (hereinafter referred to as: B/P) and B/Brisbane/60/2008 (NYMC BX-35)] by growing
Spring, Co., Ltd, institute of biological products bought from United Kingdom National biological products assay institute (NIBSC)；Recombinant fowl influenza disease
Malicious (H5 hypotype Re-5, H5 hypotype Re-6, H5 hypotype Re-10) is by Jilin Guan Jie Bioisystech Co., Ltd from Harbin animal doctor
Research institute's purchase, the above virus is production of vaccine strain.
1.5CO2Incubator (U.S. ThermoFisher, 3111), CO2Shaking table (knowing Chu, ZCZY-CS8 in Shanghai), cytometer
Number instrument (U.S. Countstar, IC1000).
2. method
2.1 cells, which suspend, tames
1.2.1 low serum adapts to culture
1) with the DMEM culture medium recovery adhere-wall culture type mdck cell (MDCK-W- for containing 10%FBS (V/V, similarly hereinafter)
63), after cell covers with single layer, cell dispersion is digested with 0.25% trypsase (m/m), training is passed on the sub-bottle ratio of 1:6
It supports.(P64 generation)
2) FBS concentration reduces by 1% in every two generation of the secondary culture culture medium of adhere-wall culture type mdck cell, until final culture medium
In serum-concentration be 1% (P84 generation).Keep cell growth speed slack-off because reducing FBS, sub-bottle ratio is also accordingly reduced, specific to grasp
Make as shown in table 1:
The low serum of 1 adhere-wall culture type mdck cell of table adapts to secondary culture information table
| Cell generation | Serum-concentration (%) | Sub-bottle ratio |
| P64、P65 | 10 | 1:6 |
| P66、P67 | 9 | 1:6 |
| P68、P69 | 8 | 1:6 |
| P70、P71 | 7 | 1:5 |
| P72、P73 | 6 | 1:5 |
| P74、P75 | 5 | 1:5 |
| P76、P77 | 4 | 1:4 |
| P78、P79 | 3 | 1:4 |
| P80、P81 | 2 | 1:4 |
| P82、P83 | 1 | 1:4 |
1.2.2 serum free suspension is tamed
1) after dropping to two generation of 1% adaptation culture from serum-concentration, digestive juice is changed to TrypLE trypsase by trypsase
Substitute, and the amount (containing 1%FBS) of DMEM is gradually reduced, it is stepped up the amount of serum free medium, sub-bottle ratio is 1:4, tool
Body is shown in Table 2:
2 serum-free of table tames media components ratio table
| Cell generation | 1%FBS DMEM | Serum free medium CFM403 |
| P84、P85 | 8 | 2 |
| P86、P87 | 6 | 4 |
| P88、P89 | 5 | 5 |
| P90、P91 | 4 | 6 |
| P92、P93 | 3 | 7 |
| P94、P98 | 2 | 8 |
2) expand cell culture amount since when passing in P96 generation, until total number of cells are up to 2.0 × 10 when P988More than a, disappear
Adjusting density with serum free medium entirely after change centrifugation is 3.0 × 106/ mL, take 50mL cell suspension 150mL conical flask (under
Together) in 37 DEG C, 5%CO2It suspends and cultivates with 110rpm shaking table, the serum free medium of centrifugation replacement daily is primary.(P99)
3) viable cell density of mdck cell is cultivated up to 6.0 × 10 wait suspend62.0 × 10 are pressed after being centrifuged when/mL or more6/mL
Density sub-bottle culture, with this continuous passage, every sub-bottle culture is once denoted as a secondary culture.(P100-114)
4) suspension cell that growth is fast, conglomeration is few and homogeneity is good is frozen and establishes the thin of suspension cultivation type mdck cell strain
Born of the same parents library (P114).The formula of frozen stock solution are as follows:+95% serum-free medium CFM403 (v/v) of 5% 2 Party A's base sulfoxide.Because of MDCK
Cell is originally derived from dog kidney, and the number for taming serum free medium used is CFM403, therefore by the suspension cultivation type of domestication
Mdck cell strain name are as follows: canine kidney cells MDCK-XF03, the serum free suspension cultivation type mdck cell strain are preserved in Chinese Typical Representative
Culture collection, deposit number are as follows: CCTCC NO:C2018191, preservation date are as follows: on September 20th, 2018, preservation
Location are as follows: No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road.
The biological characteristics inspection of 2.2 suspension cultivation type mdck cell strains
2.2.1 recovery culture
1) conventional method recovery canine kidney cells MDCK-XF03, and check cell viability.
2) with 2.0 × 106/ mL cell density is inoculated with 37 DEG C of postposition, 5%CO2It suspends and cultivates with 110rpm shaking table.
2.2.2 the growth characteristics of different vaccination density culture
After suspension cultivation type MDCK-XF03 cell recovery culture three generations, with 1.0 × 106/mL、1.5×106/ mL and 2.0 ×
106Three density of/mL are inoculated with 37 DEG C of postposition, 5%CO2It suspends and cultivates with 110rpm shaking table, every group parallel three bottles, every 12h takes
Sample 1ml counts fine and closely woven degree, is that abscissa draws cell growth curve using average cell density as ordinate, sample time, with song
The highest point of line is the maximum proliferation density of the density culture.
2.2.3 adaptability influenza and avian influenza virus culture being proliferated
Cultivation type MDCK-XF03 cell to be suspended grows to density up to 8.0-10.0 × 106When/mL, free serum culture is used
It is 4.0-5.0 × 10 that base, which is diluted to density,6It is inoculated with influenza and avian influenza virus for 0.01 by virus infection plural (MOI) after/mL,
Trypsase is added by 2.5 μ g/mL, every kind of virus is inoculated with three bottles in parallel.Wherein, influenza virus A/California/7/2009
(NYMC X-179A), influenza virus A/Texas/50/2012 (NYMC X-223A) and recombinant fowl influenza virus (H5 hypotype Re-
5), recombinant fowl influenza virus (H5 hypotype Re-6), recombinant fowl influenza virus (H5 hypotype Re-10) are inoculated with postposition 33.5 on cell
DEG C, 5%CO2It is cultivated with 110rpm；Influenza virus B/P and B/Brisbane/60/2008 (NYMC BX-35) connect on cell
Kind 34.2 DEG C of postposition, 5%CO2It is cultivated with 110rpm.After virus inoculation for 24 hours, every bottle of 36h, 48h and 60h each sampling
1ml mixing, with fresh chicken red blood cell by hemagglutinative titer (HA) viral in hemagglutination test (HA test) test sample, with judgement
Proliferative conditions of the virus in cell, and draw HA column diagram.
3 results
The domestication of 3.1 adhere-wall culture type mdck cell serum free suspension cultures
Adhere-wall culture type (MDCK-W-63) gradually reduces fetal calf serum concentration in culture medium, to P83 for when adapted to
1%FBS culture, growth speed are slowed down, and when in 1:4 ratio sub-bottle culture, 48h can still grow into cell monolayer, cellular morphology
It is similar when being cultivated to 10%FBS, but when mutual extrusion or overlapping phenomenon are cultivated not as good as 10%FBS when cell grows up to single layer, is obvious,
The tolerance degree of trypsase is also reduced, therefore uses the relatively low TrypLE trypsase substitute of digestion power instead,
Eliminate dependence of the cell to serum when digestion end needs to neutralize.
After adhere-wall culture type mdck cell adapts to 1%FBS culture, by being stepped up the ratio of serum-free base, cell energy
Free serum culture is adapted to, in serum free medium suspension culture vigor height, conglomeration is few and homogeneity is preferable, the base after 4 times are changed liquid
This has adapted to cultivate under floating condition, and vitro growth rates become faster, and cultivates for 24 hours after changing liquid the 7th time, cell density up to 6.6 ×
106/mL。
2.0 × 10 after the passage for the first time of the mdck cell (P99 generation) of suspension culture6/ mL inoculated and cultured the speed of growth is gradually
Accelerate, as shown in table 3.With the presence of the cell that visible volume is bigger than normal in cell, P107 for when take part to freeze 10 to be protected
Kind, remaining cell continues secondary culture to 114 generations, and maxicell ratio is reduced at this time, freezes 176, and freezing density is 6.0
×107/ mL, cell viability 93.76%, number name are as follows: canine kidney cells MDCK-XF03.
3.2 suspension cultivation type MDCK-XF03 cell strain biological characteristics
3.2.1 recovery vigor and cellular morphology
The suspension cultivation type MDCK-XF03 cell recovery vigor frozen is up to 93.28%%, and cellular morphology is in circle after recovery,
Cell edges are neat, and size is more uniform, with the presence of a small amount of visible volume cell bigger than normal, as shown in Figure 1B.
3 suspension cultivation type MDCK of table tames early growth density meter
3.2.2 growth characteristics
After suspension cultivation type MDCK-XF03 cell recovery culture three generations, with 1.0 × 106/mL、1.5×106/ mL and 2.0 ×
106Three density inoculated and cultureds of/mL, cell growth is fast, and growth curve is at nearly " S " type, as shown in Fig. 2, maximum proliferation is close
Degree is respectively 14.22 × 106/mL、14.36×106/ mL and 14.4 × 106/ mL, maximum proliferation density occur time be respectively
84h, 84h and 72h illustrate the cell with 1.0-2.0 × 106The growth of/mL inoculated and cultured is fast, maximum proliferation density is high.Cell exists
What different time sampling counted the results are shown in Table 4.
4 suspension cultivation type MDCK-XF03 cell different densities inoculated and cultured result table of table
3.2.3 adaptability influenza and avian influenza virus culture being proliferated
Influenza virus A/California/7/2009 (NYMC X- of suspension cultivation type MDCK-XF03 cell inoculation A type
179A), influenza virus A/Texas/50/2012 (NYMC X-223A), recombinant fowl influenza virus (H5 hypotype Re-5), recombination fowl
Influenza virus (H5 hypotype Re-6) and recombinant fowl influenza virus (H5 hypotype Re-10) can be grown well, cultivate 24 hours HA
(log2) reach 3-8, when 60h reaches 6-10.It is inoculated with influenza virus B/P and B/Brisbane/60/2008 (NYMC BX-35)
Growth faster, 24 hours HA (log2) reach 10-12 when reaching 8,60h.Illustrate the cell to influenza and avian influenza virus
It adapts to, and cultivation effect is good, the HA rule characteristic inconsistent with the speed of growth of virus itself and HA height matches.HA takes
Sample measurement result is shown in Table 5, and virus multiplication rule is shown in Fig. 3.
The HA measurement result of table 5 suspension cultivation type MDCK-XF03 cell culture influenza and avian influenza virus
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. a kind of serum free suspension cultivation type mdck cell strain, which is characterized in that the serum free suspension cultivation type mdck cell
Strain is preserved in China typical culture collection center, deposit number are as follows: CCTCC NO:C2018191, classification naming are as follows: dog kidney is thin
Born of the same parents MDCK-XF03, preservation date are as follows: on September 20th, 2018, preservation address are as follows: Wuhan City, Hubei Province Wuchang District Bayi Road 299
Wuhan University.
2. a kind of serum free suspension cultivation type mdck cell strain as described in claim 1 is raw in influenza vaccines and avian influenza vaccine
Application in production.
3. serum free suspension cultivation type mdck cell strain as claimed in claim 2 is in influenza vaccines and avian influenza vaccine production
Application, which is characterized in that the serum free suspension cultivation type mdck cell strain as cellular matrix be used for influenza vaccines and fowl
Influenza vaccines production.
4. serum free suspension cultivation type mdck cell strain as claimed in claim 3 is in influenza vaccines and avian influenza vaccine production
Application, which is characterized in that the serum free suspension cultivation type mdck cell strain is as cellular matrix to influenza and avian flu
When poison culture, serum free suspension cultivation type MDCK-XF03 cell is with 1.0-2.0 × 106/ mL density inoculated and cultured.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111117944A (en) * | 2019-11-22 | 2020-05-08 | 中农威特生物科技股份有限公司 | Anti-apoptosis MDCK host cell strain for large-scale suspension culture and establishment method thereof |
| CN111440762A (en) * | 2020-04-14 | 2020-07-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Full-suspension MDCK cell and method for culturing swine influenza virus by using same |
| CN112210530A (en) * | 2020-10-15 | 2021-01-12 | 山东信得动物疫苗有限公司 | Cell full-suspension domestication method, DF-1 cell full-suspension domestication method and application |
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2019
- 2019-03-27 CN CN201910237651.5A patent/CN109929796A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111117944A (en) * | 2019-11-22 | 2020-05-08 | 中农威特生物科技股份有限公司 | Anti-apoptosis MDCK host cell strain for large-scale suspension culture and establishment method thereof |
| CN111440762A (en) * | 2020-04-14 | 2020-07-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Full-suspension MDCK cell and method for culturing swine influenza virus by using same |
| CN111440762B (en) * | 2020-04-14 | 2020-12-04 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Full-suspension MDCK cell and method for culturing swine influenza virus by using same |
| CN112210530A (en) * | 2020-10-15 | 2021-01-12 | 山东信得动物疫苗有限公司 | Cell full-suspension domestication method, DF-1 cell full-suspension domestication method and application |
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