CN108396006A - A kind of BHK-21 cell strains and its cultural method and purposes - Google Patents
A kind of BHK-21 cell strains and its cultural method and purposes Download PDFInfo
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Abstract
The present invention relates to it is a kind of can in a variety of serum free mediums 21 cell strains of BHK of suspension growth cultural method, include the following steps：S1. 21 cell lines of recovery BHK；The sorting and clone cell culture of 21 cell lines of S2.BHK expand；The serum free suspension culture of 21 clonal cell populations of S3.BHK.The present invention also provides can in a variety of serum free mediums suspension growth 21 cell strain strains of BHK and application thereof.Method of the present invention, as attachment carrier, maintains to roll training mode without rolling bottle, culture medium is free of serum, no protein ingredient, the CD grade business serum free mediums being made of inorganic salts and nutrients without microcarrier.In addition, 21 cell strains of serum free suspension culture BHK can directly be frozen by serum free medium addition DMSO, when recovery, also can directly be recovered with serum free medium.New thinking and method are provided for technical grade expanding production viral vaccine.
Description
Technical field
It a kind of can hang the present invention relates to a kind of cultural method of zooblast more particularly in a variety of serum free mediums
The cultural method and purposes of the BHK-21 cell strains for length of growing floating on water.
Background technology
The serum free suspension culture of zooblast is always that genetically engineered drug and viral vaccine large-scale production are pursued
Target, but successful example is few so far.Chinese hamster ovary celI (Chinese hamster ovary cell) is example the most successful, it can be with
Continuously suspended in the container of hundreds of liters with serum free medium culture 2-3 weeks, and the density of cell growth can reach 108A/ml,
Solves the technical barrier of the efficient amplification of cell great expression genetically engineered drug and virus, the cell is in the gene in the whole world
Extensive use in engineering drug (protein medicaments, polypeptide drugs, the therapeutic monoclonal antibody in people source) large-scale production.Chinese hamster ovary celI is without blood
Clear culture medium continuously suspends the success of culture, the serum free suspension cultivating system to establish zooblast open it is new by way of.
BHK-21 cells (Syria's nephrocyte) be include adenovirus, herpes simplex virus, reovirus, vesiculovirus mouth
The ideal host of many viruses including scorching virus, swine fever virus and foot and mouth disease virus, usually cultures of these viruses and largely
Amplification, especially viral vaccine production are all made of BHK-21 cells and are host.Since BHK-21 cells are that property is relied to grow cell, need
A certain amount of animal blood serum cell ability adherent growth is added, conventional suspension culture is hardly possible.Therefore, technical grade is with containing
When there are the medium culture BHK-21 cells of serum to carry out the expanding production of virus, mainly Rotary Machine is seeded in using circular rolling bottle
Upper rolling culture, limited rolling bottle surface area constrains the raising of virus yield, to solve the problems, such as that virus yield is relatively low, research
Persons have developed a kind of micro-carriers cell culture system, allow cell to be attached on microballon and grow again into virus infection, are increased with reaching
Surface area is added to improve the purpose of virus yield, but efficiency is not still high.Furthermore high concentration (10-15%) is added when cell adhere-wall culture
The serum of animal origin can cause difficulty to downstream viral purification.For a long time, researcher borrows the training of Chinese hamster ovary celI serum free suspension
Foster acclimation method, it is intended to which serum-concentration is reduced using successive；The nutriments such as growth factor, hormone, amino acid are added to carry out
Serum-free selects；The different methods such as continuous culture selection tame BHK-21 cells, change cultivation conditions (the suspension training of cell
Support), so that it is used for the industrialized production of vaccine, but still successful example is seen at end.As it can be seen that really one kind of BHK-21 cells is very
It is difficult to the cell tamed, is intended to adherent growth in the culture medium containing serum, without in the serum free medium of increase serum
It does not just grow, or even dead in a short time.Apparently, it to be successfully established the cell suspension cultures system of serum-free, innovation need to be used
Strategy.
There are many kinds of single-cell techniques, is directly separated technology, Beads enrichment technology, laser there are commonly microscope and catches
Microdissection technology, patch clamp technique etc. are obtained, the selected by flow cytometry apoptosis technology of developed recently maturation can quickly and efficiently go respectively
The unicellular separation of kind.The present invention detaches unicellular combination raising layer method using selected by flow cytometry apoptosis technology and screens BHK-21 grams
Grand cell colony is used in combination serum free medium to tame clonal cell population, and obtaining 3 plants can continuously suspend in serum free medium
Culture, and big rule touch the BHK-21 suspended culture cell strains of proliferation；Prove that the cell strain can support foot and mouth disease virus efficient simultaneously
Amplification makes it possible using BHK-21 cells to be that the big rule of host touch amplification virus.
Invention content
The problem of for the industrial production for producing vaccine using zooblast, the present invention provides a kind of BHK-21
The serum free suspension cultural method of cell, this method detach unicellular combination using selected by flow cytometry apoptosis technology and raise layer method
BHK-21 clonal cell populations are screened, serum free medium is used in combination to tame.
For this purpose, the technical solution adopted by the present invention is as follows：
It is a kind of can in a variety of serum free mediums the BHK-21 cell strains of suspension growth cultural method, including following step
Suddenly：
S1. recovery BHK-21 cells
The BHK-21 cells for taking out preservation in liquid nitrogen container are transferred in 37 DEG C of water-bath and thaw (0-2 minutes rapidly
It is interior), it is inoculated into T25 Tissue Culture Flasks after cell dissolution, adds the MEM trainings that 7ml contains 10% (v/v) fetal calf serum thereto
Base is supported, mixing is gently blown and beaten, after cultivating 4-6hr, cell culture medium, the upper fresh MEM containing 10% (v/v) fetal calf serum are abandoned in shifting
Culture medium cultivates 2-3d, for sorting individual cells in 37 DEG C, 5% (v/v) carbon dioxide incubator；
The sorting and clone cell culture of S2.BHK-21 cell lines expand
(1) abdominal cavity cell (mainly macrophage and other immune thin is taken out in sterile working from kunming mice abdominal cavity
Born of the same parents), 1000 turns/min centrifuges 10min, abandons supernatant, with fresh commercially available MEM culture mediums suspension cell, counts, adjustment cell is dense
Degree is 106A/ml, in inoculating cell to 96 porocyte culture plates, the holes 0.1ml/；96 porocyte culture plates are placed in 37 DEG C,
24-36hr is cultivated in 5% (v/v) carbon dioxide incubator, prepares feeder cells, to obtain containing 1.0% (v/v) fetal calf serum
96 hole feeder cells culture plates；
(2) one bottle of BHK-21 cells (it is required that cell growth to 95% degree of merging) prepared by step S1 are taken, are abandoned in culture
Clearly, 1.0ml is added, for 0.25% (W/V) pancreatin to digest cell dispersion, micro- sem observation ensures that all cells are in single point
Dissipate cell state；
(3) the BHK-21 cells of dispersion are placed in flow cytometer and sort individual cells, later by the single thin of sorting
Born of the same parents are directly inoculated into 96 hole feeder cells culture plates made from above-mentioned steps (1), a unicellular/hole；96 hole is raised
It supports confluent monolayer cells culture plate and is placed in 37 DEG C, cultivated 2-3 weeks in 5% (v/v) carbon dioxide incubator；
(4) upgrowth situation of cell in the 96 hole feeder cells culture plate, the good cell of growth selection are hole-specifically observed
In clone's passage to 12 porocyte plates, it is transferred to amplifying cells in T25 culture bottles after cell growth is good, obtains genetic background one
The BHK-21 clonal cell populations of cause, when cell secondary culture reaches 5 × 107When a cell, conventional liquid nitrogen cryopreservation is spare；
The serum free suspension culture of S3.BHK-21 clonal cell populations
(1) recover the BHK-21 clonal cell populations that freeze, backward serum free medium in enter the tire of 2.0% (v/v)
Cow's serum, inoculation 104The BHK-21 clone cells of a/ml, culture are transferred them to after a week containing 1.0% (v/v) fetal calf serum
Serum free medium in tame (12 porocyte culture plates), such operation order is by serum-concentration (v/v) from 0.5%, 0.2%
It is gradually decreased to 0, tames BHK-21 clone cells repeatedly；
(2) after taming repeatedly, the cell in 1/5 hole can gradually suspend in serum free medium BHK-21 clone cells
Growth, then preferably goes out well-grown suspension BHK-21 cells and it is more than generation to pass 5 repeatedly with serum free medium again, will stablize
The suspension cell of passage growth is transferred in T75 bottles, is placed in 37 DEG C, amplifying cells in 5% (v/v) carbon dioxide incubator；
(3) the BHK-21 suspension cells dilution that will stablize passage counts, with 4.5 × 105A/ml, total 30ml are inoculated in
Sterilized CELSIR double side wall suspension blake bottles supplement 5% sterile (v/v) CO2Gas, calcination bottleneck are simultaneously tightened immediately；
Magnetic drive is provided with miniature magnetic blender, the rotating speed of adjustment magnetic stirring apparatus setting rolling bottle is that 30-80 turns/min；It is placed in 37
It is cultivated in DEG C standard incubator；
(4) BHK-21 suspension cells after serum free suspension culture 7-10d, take out amplification in CELSIR double side wall culture bottles
Suspension cell, 800-1000rpm centrifuge 10min, be inoculated into respectively after dilution in 10 T75 culture bottles, every bottle addition 20ml
Blood serum medium supplements 5% sterile (v/v) CO2Gas to 10 T75 Tissue Culture Flasks in 37 DEG C, it is quiet in standard incubator
Only cultivate 7-10d；
(5) by the cell of large amplification (total number of cells about 3-5 × 108/ 10 T75 bottles) it freezes in liquid nitrogen and preserves for a long time.
It is a kind of can in a variety of serum free mediums suspension growth BHK-21 cell strains, pass through above-mentioned serum free suspension
Cultural method obtain, and the BHK-21 cell strains can under conditions of stationary culture suspension growth, without blood in shaking table of ventilating
Clear suspended culture cell concentration is up to 107-8The efficiency of a/ml, amplification aftosa live virus are 106CFU/ml。
A kind of above-mentioned BHK-21 cell strains are for expanding the purposes in foot and mouth disease virus.
Method of the present invention, as attachment carrier, suspension training mode, culture is maintained without rolling bottle without microcarrier
Base is free of serum, no protein ingredient, the CD grade business serum free mediums being made of inorganic salts and nutrients.In addition, the nothing
The serum culture BHK-21 cell strains that suspend can directly be frozen by serum free medium addition DMSO, and when recovery also can be directly with no blood
Clear culture medium recovery.New thinking and method are provided for technical grade expanding production viral vaccine.
The invention has the advantages that：
1. free serum culture based component understands, widely used, easily buy, the BHK-21 suspension cell strains for taming acquisition adapt to
Property is strong：
Serum free suspension domestication culture medium (code name X) used in the present invention derives from the CD of Gibco companies
FortiCHOTMMedium.Tame that successful suspension BHK-21 cell strains are adaptable, and the cell density for the culture that suspends is up to 108/
ml。
2.BHK-21 suspended culture cell strain can support virus amplification
The BHK-21 cell strains tamed of the present invention can the serum free suspension culture under standing or dynamic both of which, and expand
Increase foot and mouth disease virus titre up to 106CFU/ml.The 3D base mesh copy number 4.31 × 10 of virus8copies/μg。
3. freezing, recovery cellular processes are simple, conveniently
Normal BHK-21 attached cells system is required to certain density serum ability successful resuscitation when freezing, and originally
The tamed BHK-21 cell strains of invention, the cell after serum free suspension culture can be added directly with serum-free cell culture medium
10% DMSO freezes and passage growth of recovering.Suspension cell cryopreservation step is enormously simplified, real free serum culture is reached
Serum-free freezes, and is conducive to industrialized production.
Description of the drawings
Figure 1A is the aspect graph (× 100) of the normal adherent growth of BHK-21 cells；
Figure 1B is the aspect graph (× 100) of BHK-21 cell strain serum free suspensions domestication culture；
Fig. 2A is the BHK-21 cells growthform figure (× 100) through freezing；
Fig. 2 B are the aspect graph (× 100) of the BHK-21 cell strain serum free suspensions domestication culture through freezing；
Fig. 3 is the CPE process decision charts (× 400) that BHK-21 suspension cell strains infect that FMDV occurs；
Fig. 4 is the kinetic curve that FMDV is expanded in BHK-21 suspension cell strains.
Specific implementation mode
The following example will be further illustrated other features and advantages of the present invention, but such embodiment it is merely illustrative and
With not limitation of the present invention.
Material or reagent used herein below are commercially available unless otherwise noted.
1 recovery BHK-21 cells of embodiment
The BHK-21 cells (being purchased from China typical culture collection center (CCTCC)) for taking out preservation in liquid nitrogen container, turn
It moves in 37 DEG C of water-bath and thaws (in 0-2 minutes) rapidly.It is inoculated into after cell dissolution in T25 Tissue Culture Flasks, thereto
The MEM culture mediums that 7ml carries 10% (v/v) fetal calf serum are added, mixing is gently blown and beaten, after cultivating 4-6hr, cell culture is abandoned in shifting
Base is changed the fresh MEM culture mediums containing 10% (v/v) fetal calf serum, is trained in 37 DEG C, 5% (v/v) carbon dioxide incubator
2-3d is supported, for sorting individual cells.
The sorting and clone cell culture of embodiment 2BHK-21 cells expand
(1) abdominal cavity cell (mainly macrophage and other immune thin is taken out in sterile working from kunming mice abdominal cavity
Born of the same parents), 1000 turns/min centrifuges 10min, abandons supernatant, with fresh commercially available (Gibco companies) MEM culture medium suspension cells, counts,
It is 10 to adjust cell concentration6A/ml, in inoculating cell to 96 porocyte culture plates, the holes 0.1ml/.96 porocyte plates are placed in
, 24-36hr is cultivated in 5% carbon dioxide incubator, prepares feeder cells, to obtain containing 1.0% (v/v) fetal calf serum by 37 DEG C
96 hole feeder cells culture plates.
(2) BHK-21 cells (it is required that cell growth cultivates 2-3d to 95% degree of merging) prepared by one bottle of step S1 are taken,
Culture supernatant is abandoned, 1.0ml is added, 0.25% (W/V) pancreatin digests cell dispersion, and micro- sem observation ensures that all cells are in
Single cell dispersion state.
(3) the BHK-21 cells of dispersion are set and sorts individual cells in flow cytometer, later by the individual cells of sorting
It is directly inoculated into 96 hole feeder cells culture plates made from above-mentioned steps (1), a unicellular/hole.96 hole is raised
Confluent monolayer cells culture plate is placed in 37 DEG C, is cultivated 2-3 weeks in 5% (v/v) carbon dioxide incubator.Cell growth state is observed every other day.
(4) upgrowth situation of cell in the 96 hole feeder cells culture plate, the good cell of growth selection are hole-specifically observed
(microscopy observation cell density), which is transferred in T25 culture bottles, during clone's passage is pulled to 12 hole cells, after cell growth is good expands
Cell obtains the consistent BHK-21 clone cell reproductive populations of genetic background, when cell secondary culture reach a certain concentration (5 ×
107A cell) when routine liquid nitrogen cryopreservation it is spare.It must pay attention to one-to-one passage, ensure the cell colony source of culture amplification
In the unicellular of the same sorting.
The serum free suspension culture of embodiment 3BHK-21 clonal cell populations is tamed
(1) the BHK-21 clonal cell populations that recovery freezes, for culture.The commercialization serum-free of separate sources is trained
Foster base is divided into B, Q, X, Y, Z groups.Enter the fetal calf serum of 2.0% (v/v), inoculation 10 into serum free medium respectively4A/ml's
BHK-21 clone cells, culture, which is transferred them to after a week in the serum free medium containing 1.0% (v/v) fetal calf serum, tames,
Serum-concentration is gradually decreased to 0 by such operation order from 0.5%, 0.2%, tames the BHK-21 clone cell (mesh of domestication repeatedly
Be cell free serum culture, repeatedly domestication be exactly the serum-concentration for continuously decreasing cell culture medium, until not increase serum it is thin
Until born of the same parents normally can cultivate growth, actually a kind of process constantly screened).
(2) after taming repeatedly, the cell in 1/5 hole can gradually suspend in serum free medium BHK-21 clone cells
Growth, then respectively from B, Q, X, five kinds of serum free mediums of Y, Z filter out well-grown suspension BHK-21 cells, with no blood
It is more than generation to pass 5 repeatedly for clear culture medium, and the suspension cell that will stablize passage growth is transferred in T75 bottles, is placed in 37 DEG C, 5% (v/
V) amplifying cells (static gas wave refrigerator) in carbon dioxide incubator.In static gas wave refrigerator, every other day takes out a part of cell and carry out carefully
Born of the same parents count anyway, as a result (table 1).Investigate the growth efficiency of stationary state low suspension cell.The data of table 1 show that cell is in quiet
Only when the culture of state low suspension, the thin similar density of 3-6 days cells (106/ ml), but incubation time extends cell mortality and increases
(6 days up to 24.2%), it is clear that cell is not best training method in the culture of stationary state low suspension.
The growth efficiency of 1 stationary state low suspension cell of table
| Number of days (days) | Cell number (cells/ml) | Cell mortality (%) |
| 1 | 4.5×105 | N/A |
| 2 | 5.0×105 | 4.2 |
| 3 | 1.0×106 | 7.9 |
| 4 | 3.5×106 | 10.7 |
| 5 | 6.3×106 | 24.2 |
(3) passage BHK-21 suspension cells dilution will be stablized to count, with 4.5 × 105A/ml, total 30ml are inoculated in sterilized
The CELSIR double side wall suspension blake bottles of bacterium supplement 5% sterile (v/v) CO2Gas (adjusts the pH value of culture medium, it is ensured that thin
The pH value that born of the same parents cultivate the culture medium of early stage increases), calcination bottleneck is simultaneously tightened immediately；Magnetic force is provided with miniature magnetic blender to drive
Dynamic, the rotating speed of adjustment magnetic stirring apparatus setting rolling bottle is that 30-80 turns/min, is placed in dynamic cultivation in 37 DEG C of standard incubators.It stirs
It mixes in spinner culture, every other day takes out a part of cell and carry out cell counting anyway, as a result (table 2).It investigates under stirring
The growth efficiency of suspension cell.The data of table 2 show, when cell stirring low suspension, 6-7 days fine and closely woven degree of cell are up to 108/
Ml, but incubation time extends cell mortality and increases (8 days up to 20.0%), and therefore, cell is in stirring low suspension culture
Preferable training method cultivates 6 days best results.
The growth efficiency of 2 stirring low suspension cell of table
| Number of days (days) | Cell number (cells/ml) | Cell mortality (%) |
| 1 | 2.7×105 | N/A |
| 2 | 8.0×105 | 4.2 |
| 3 | 4.0×106 | 3.8 |
| 4 | 3.5×106 | 7.1 |
| 5 | 6.3×107 | 7.8 |
| 6 | 1.8×108 | 10.0 |
| 7 | 2.4×108 | 20.0 |
(4) BHK-21 suspension cells after serum free suspension culture 7-10d, take out amplification in CELSIR double side wall culture bottles
Suspension cell, 800-1000rpm centrifuge 10min, be inoculated into respectively after dilution in 10 T75 culture bottles, every bottle addition 20ml
Blood serum medium (Gibco companies) supplements 5% sterile (v/v) CO2In gas to each T75 Tissue Culture Flasks, in 37 DEG C,
Static gas wave refrigerator 7-10d (explanations in standard incubator：Carbon dioxide incubator leads to 5% (v/v) CO2Gas, standard incubator are not added with
CO2Gas), continue large amplification；
(5) by the cell of large amplification (total number of cells about 3-5 × 108/ 10 T75 bottles) it freezes in liquid nitrogen and preserves for a long time.
Tame the form (Figure 1B) of successfully cell.The aspect graph of the normal adherent growth of BHK-21 cells is shown in Figure 1A, from Figure 1A
In it can be seen that normal BHK-21 cells be typical adherent growth.It is outstanding that BHK-21 cell strain serum-frees are shown in Figure 1B
The aspect graph of floating domestication culture, it can be seen that BHK-21 cell strains are typical suspension growth from Figure 1B.
The preservation and recovery of embodiment 4BHK-21 suspension cells
(1) suspension BHK-21 cells are collected, 10min is centrifuged in 1000rpm, remove supernatant.
DMSO (dimethyl sulfoxide (DMSO)) and commercially available serum free suspension culture medium are premixed, DMSO final concentration of 10% (v/v) is made.
It is placed in 4 DEG C of precoolings.
(3) programmed cooling instrument is set according to following procedure：It is outstanding with the serum free suspension culture medium containing DMSO10% (v/v)
After floating off the heavy collection cell of the heart) it is put into cryopreservation tube, 1.0ml/ manages (2-5 × 106/ ml), it is placed in 4 DEG C of refrigerator 20min；Program is arranged to drop
Temperature temperature per minute reduces by 0.5 DEG C, until after -30 DEG C, then it is set as 1 DEG C of reduction per minute, -100 DEG C are down to, cryopreservation tube is taken out,
It is preserved in liquid nitrogen container.
(4) the BHK-21 cell strains frozen are taken out from liquid nitrogen after a week, melt rapidly in 37 DEG C of water-baths (0~
1.0min).The cell liquid that 1ml is sucked out is mixed with corresponding 9ml serum free suspensions culture medium.800-1000rpm centrifuges 2min
Supernatant is removed, fresh commercially available serum free medium is added, 37 DEG C is placed in, is cultivated in 5% (v/v) carbon dioxide incubator.Fig. 2A
For the BHK-21 cells growthform figure through freezing, Fig. 2 B are the BHK-21 cell strain serum free suspensions domestication culture through freezing
Aspect graph, as can be seen that the BHK-21 suspension cells that freeze can be with successful resuscitation from Fig. 2 B.
The virus amplification of embodiment 5BHK-21 suspension cell strains
(1) judgement of virus infection suspension cell and its induced cytopathic effect (CPE)
- 80 DEG C of foot and mouth disease viruses frozen are taken, 200PFU/ml is diluted to, add 5 μ l dilution virus liquids infection 1 × 107It is a
BHK-21 suspension cells.37 DEG C are placed in, is cultivated in 5% carbon dioxide incubator.The CPE judgements of BHK-21 suspension cells are as schemed
Shown in 3A, Fig. 3 B.As can be seen that living cells surrounding index of refraction is high from Fig. 3 A, in the case where commonly learning microscope, BHK-21 suspends thin
Born of the same parents show one week bright annulus.As can be seen that sick cell is shown, index of refraction is low, and cell is intense darkness without light from Fig. 3 B,
Part BHK-21 suspension cells have deformed, cellular content outflow.Judge suspension cell CPE accordingly.
(2) ability of BHK-21 serum free suspensions culture cell support virus amplification
A) the 5 μ l infection 1 × 10 of 200PFU/ml hoof-and-mouth diseases venom is taken7A BHK-21 serum free suspensions culture cell.Every
12 hours take the supernatant of a virus infected cell.
B) TCID is used50Method measures the virus liquid titre in each period, as a result (table 3 and Fig. 4).
3 TCID50 methods of table measure the virus liquid titre in each period
Table 3 and Fig. 4's the results show that mouth disease virus infection 84-96hr, virus titer reach the peak value of 6Iog, this is
The maximum value of foot and mouth disease virus vaccine industrialized production, but time shortening one third.As it can be seen that BHK-21 serum free suspension cultures
Cell can provide new approach for foot and mouth disease virus vaccine industrialized production.
(3) ability of amplification virus when BHK-21 cell strains suspend
Infect 4 T-75 bottles of BHK-21 suspension cells strain (2 × 10 respectively with 1000 PFU viruses7A/20ml/ bottles),
Each 2 bottles abilities (being shown in Table 1, Fig. 2 and Fig. 4) for investigating amplification virus with dynamic both of which culture with standing.It collects respectively above-mentioned
The cell pyrolysis liquid and culture supernatant of two kinds of cultivation conditions, use TCID50Method detects virus titer in culture supernatant.As a result it shows
Show, dynamically can foot and mouth disease virus be supported to be proliferated with the BHK-21 cells of stationary state low suspension culture, and dynamic suspension culture
When, virus titer reaches6Iog/ml。
Simultaneously foot and mouth disease virus 3D gene copy numbers in cell pyrolysis liquid are detected with fluorescence quantitative PCR method.Comparative measurements is dynamic
State and copy number of the 3D genes in cell under static condition culture situation, find 3D genes in the cell total rna of stationary culture
Copy number be 7.55 × 106Copies/ μ g, in the cell total rna of dynamic cultivation 3D gene copy numbers be 4.31 ×
108Copies/ μ g, 3D gene copy numbers are 100 times more compared with the cell of static gas wave refrigerator in dynamic cultivation cell.
The technique effect of the present invention
1, free serum culture based component understands, widely used, easily buys, and the BHK-21 suspension cell strains for taming acquisition adapt to
Property is strong：
Serum free suspension domestication culture medium (code name X) used in the present invention derives from the CD of Gibco companies
FortiCHOTMMedium.Tame that successful suspension BHK-21 cell strains are adaptable, and the cell density for the culture that suspends is up to 108/
ml。
2, the strain of BHK-21 suspended culture cells can support virus amplification
The BHK-21 serum free suspensions cell strain that the present invention is tamed can be cultivated under standing or dynamic both of which, and expand
Increase virus titer up to 106/ml.The 3D base mesh copy number 4.31 × 10 of virus8copies/μg。
3, it freezes, recovery cellular processes are simple, conveniently
Normal BHK-21 attached cells, be required to when freezing certain density serum could successful resuscitation, and this hair
Bright tamed BHK-21 serum free suspensions cell can directly with serum-free cell culture medium addition 10% DMSO after freeze simultaneously
Recovery passage growth.Suspension cell cryopreservation step is enormously simplified, reaches real free serum culture serum-free and freezes, be conducive to
Industrialized production.
In conclusion the foregoing is merely presently preferred embodiments of the present invention, the patent for not thereby limiting the present invention is protected
Shield range, therefore description of the invention and the made equivalence changes of attached drawing etc. such as, should all be included in protection scope of the present invention.
Claims (9)
1. it is a kind of can in a variety of serum free mediums the BHK-21 cell strains of suspension growth cultural method, which is characterized in that
Include the following steps：
S1. recovery BHK-21 cells；
The sorting and clone cell culture of S2.BHK-21 cell lines expand；
The serum free suspension culture of S3.BHK-21 clonal cell populations.
2. cultural method as described in claim 1, which is characterized in that the recovery BHK-21 cells of step S1 specifically wrap
It includes：
The BHK-21 cells for taking out preservation in liquid nitrogen container are transferred in 37 DEG C of water-bath in thawing in 0-2 minutes, and cell is molten
It is inoculated into after solution in T25 Tissue Culture Flasks, adds the MEM culture mediums that 7ml contains 10% (v/v) fetal calf serum thereto, gently
Mixing is blown and beaten, after cultivating 4-6hr, cell culture medium is abandoned in shifting, changes the fresh MEM culture mediums containing 10% (v/v) fetal calf serum,
2-3d is cultivated in 37 DEG C, 5% (v/v) carbon dioxide incubator, for sorting individual cells.
3. cultural method as described in claim 1, which is characterized in that the sorting of the BHK-21 cells of step S2 and clone are thin
Born of the same parents cultivate amplification and specifically include following steps：
(1) abdominal cavity cell is taken out in sterile working from kunming mice abdominal cavity, and 1000 turns/min centrifuges 10min, abandons supernatant, use is fresh
MEM culture medium suspension cells, count, adjustment cell concentration be 106A/ml, in inoculating cell to 96 porocyte culture plates,
The holes 0.1ml/；96 porocyte culture plates are placed in 37 DEG C, 24-36hr is cultivated in 5% (v/v) carbon dioxide incubator, are prepared
Feeder cells, to obtain the 96 hole feeder cells culture plates containing 1.0% (v/v) fetal calf serum；
(2) one bottle of BHK-21 cells for taking step S1 to prepare, abandon culture supernatant, are added 1.0ml, 0.25% (W/V) pancreatin with
Cell dispersion is digested, micro- sem observation ensures that all cells are in single cell dispersion state；
(3) the BHK-21 cells of dispersion are placed in flow cytometer and sort individual cells, it is later that the individual cells of sorting are straight
It is inoculated into 96 hole feeder cells culture plates made from above-mentioned steps (1), a unicellular/hole；By the 96 hole feeder layer
Tissue culture plate is placed in 37 DEG C, is cultivated 2-3 weeks in 5% (v/v) carbon dioxide incubator；
(4) upgrowth situation of cell in the 96 hole feeder cells culture plate, the good cell clone of growth selection are hole-specifically observed
It passes on into 12 porocyte plates, is transferred to amplifying cells in T25 culture bottles after cell growth is good, it is consistent to obtain genetic background
BHK-21 clonal cell populations, when cell secondary culture reaches 5 × 107When a cell, conventional liquid nitrogen cryopreservation is spare.
4. cultural method as claimed in claim 3, which is characterized in that the abdominal cavity cell in step (1) includes mainly huge
Phagocyte and other immunocytes.
5. cultural method as claimed in claim 3, which is characterized in that the BHK-21 cells in step (2) have
95% degree of merging.
6. cultural method as described in claim 1, which is characterized in that BHK-21 clonal cell populations in step S3 without blood
The clear culture that suspends specifically includes following steps：
(1) recover the BHK-21 clonal cell populations that freeze, backward serum free medium in enter the tire ox blood of 2.0% (v/v)
Clearly, it is inoculated in BHK-21 clone cells to 12 porocyte culture plates, 104A/hole, culture are transferred them to after a week containing 1.0%
(v/v) tamed in the serum free medium of fetal calf serum, such operation order by serum-concentration (v/v) from 0.5%, 0.2% by
It is gradually down to 0, tames BHK-21 clone cells repeatedly；
(2) after taming repeatedly, the cell in 1/5 hole can gradually suspend in serum free medium gives birth to BHK-21 clone cells
It is long, then preferably go out well-grown suspension BHK-21 cells and it is more than generation to pass 5 repeatedly with serum free medium again, biography will be stablized
The generation suspension cell of growth is transferred in T75 bottles, is placed in 37 DEG C, amplifying cells in 5% (v/v) carbon dioxide incubator；
(3) the BHK-21 suspension cell strains dilution that will stablize passage counts, with 4.5 × 105A/ml, total 30ml are inoculated in sterilized
The CELSIR double side wall suspension blake bottles of bacterium supplement the CO of 5% sterile (v/v)2Gas, calcination bottleneck are simultaneously tightened immediately；With
Miniature magnetic blender provides magnetic drive, and the rotating speed of adjustment magnetic stirring apparatus setting rolling bottle is that 30-80 turns/min；It is placed in 37 DEG C
It is cultivated in standard incubator；
(4) strain of BHK-21 suspension cells takes out amplification in CELSIR double side wall culture bottles after serum free suspension culture 7-10d
Suspension cell, 800-1000rpm centrifuge 10min, be inoculated into respectively after dilution in 10 T75 culture bottles, every bottle be added 20ml without
Blood serum medium supplements 5% sterile (v/v) CO2Gas is to per in each and every one T75 Tissue Culture Flasks, in 37 DEG C, standard incubator
Middle static gas wave refrigerator 7-10d；
(5) cell strain of large amplification is frozen in liquid nitrogen and is preserved for a long time.
7. cultural method as claimed in claim 6, which is characterized in that the total number of cells in step (5) are 3-5 × 108/
10 T75 bottles.
8. it is a kind of can in a variety of serum free mediums suspension growth BHK-21 cell strains, which is characterized in that wanted by right
1-7 any one of them serum free suspension cultural methods are asked to obtain, and the BHK-21 cell strains can be in the condition of static gas wave refrigerator
Low suspension is grown, and serum free suspension culture cell concentration is up to 10 in shaking table of ventilating7-8A/ml expands the effect of aftosa live virus
Rate is 106CFU/ml。
9. it is a kind of using BHK-21 cell strains according to any one of claims 8 for expanding the purposes in foot and mouth disease virus.
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| CN115521902A (en) * | 2022-10-14 | 2022-12-27 | 广州斯坦达生物科技有限公司 | Cell line for improving virus production capacity and application thereof |
| CN115521902B (en) * | 2022-10-14 | 2023-04-21 | 广州斯坦达生物科技有限公司 | Cell line for improving virus production capacity and application thereof |
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