CN105950544A - Domestication method of full suspension culture type Marc-145 cell line - Google Patents
Domestication method of full suspension culture type Marc-145 cell line Download PDFInfo
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Abstract
The invention provides a domestication method of a full suspension culture type Marc-145 cell line. The domestication method comprises the following steps: (1) cultivating adherent-culture Marc-145 cells; (2) domesticating a low-serum adherent-culture Marc-145 cell line; (3) domesticating low-serum full suspension culture type Marc-145 cells; and (4) domesticating serum-free full suspension culture type Marc-145 cells. With the application of the cell domestication method disclosed by the invention, the Marc-145 cells of suspension culture can be obtained; the cells are adaptive to low-serum and serum-free full suspension culture; the density of the cells, when subjected to serum-free culture in a 7.5L reactor, can reach 8*10<6> /ml, and at least 4.4*10<10> cells can be obtained, equal to the number of cells obtained from 80-90 15L roller bottles or 18-20 40-layer cell plants; and the domestication method disclosed by the invention is significant in effect.
Description
Technical field
The present invention relates to the acclimation method of full suspension culture type Marc-145 cell line.
Background technology
Marc-145 cell is that Kim HS in 1993 etc. clone the epithelial cell obtained from Rhesus Macacus embryonic kidney cells MA-104, this cell can continuous passage be that adherent type grows, it is research at present and production porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome, PRRSV, it is commonly called as " reproductive and respiratory syndrome ") and the optimal cell strain of vaccine, can be additionally used in avian infectious bronchitis virus (Infectious bronchitis virus, and pig parvoviral (Porcine parvovirus virus IBV), the in-vitro multiplication of virus such as PPV).Because Marc-145 is the growth of adherent type, when Virus culture and production of vaccine, this cell all uses adherent monolayers to cultivate, the amount of cell is limited by container (such as: cell factory, roller bottle) or the surface area of medium (such as: microcarrier), sufficiently large Factory Building or expensive microcarrier and substantial amounts of manual operation is needed during large-scale production, and bioreactor microcarrier is cultivated current many technical barriers that still suffers from when amplification culture and do not solved, popularization and application are the most difficult.During present stage industrialization monolayer culture Marc-145 cell, culture medium all adds a certain proportion of Ox blood serum (including hyclone or new-born calf serum) cell ability normal growth, Ox blood serum not only price, and derived therefrom is in natural biology body, the course of processing itself is carried and gathered in cattle source pollution inoculating microbe and the possibility of virulence factor, and use can exist bio-safety risk.It addition, Ox blood serum is a kind of natural mixture, its concrete composition there is presently no to be analyzed clear completely, and downstream purification technique is caused difficulty after using in producing by biological product, if there being residual that the side reaction of inoculator would generally be caused to affect product quality.During biological product produce, cell is cultivated if using the full suspension process of serum-free, cultivation scale is easy for linear amplification, avoid cell attachment and cultivate and use the defective workmanship that Ox blood serum brings, viral yield and product quality can be improved, but domestication serum-free full suspension culture type cell strain is one of technical bottleneck of current most critical.The present invention provides a kind of low serum and serum-free high density full suspension culture type Marc-145 cell and its preparation method and application method, provides cellular matrix and preparation method and cultural method for producing PRRSV vaccine with full suspension culture Marc-145 cell.
Summary of the invention
The invention provides the acclimation method of full suspension culture type Marc-145 cell line, and provide the amplification culture method of the full suspension culture type Marc-145 cell after domestication.
The present invention provides the acclimation method of full suspension culture type Marc-145 cell line, and step is as follows:
(1) cultivation of adhere-wall culture type Marc-145 cell
Select through aseptic, mycoplasma, exogenous virus calibrating as negative, the adhere-wall culture type Marc-145 cell sensitive to PRRSV, pass in 1:4 ratio sub-bottle with the DMEM culture fluid containing 10% new-born calf serum after the fine and close monolayer of cell growth, put 37 DEG C of 5%CO2Incubator cultivate, form fine and close monolayer at 48-72h inner cell, for sharp-edged squamous epithelium type cell and cell grow normal in the case of can be used for taming；
(2) domestication of low serum adhere-wall culture type Marc-145 cell line
A) step (1) described growth normal adhere-wall culture type Marc-145 cell is respectively cultivated three generations with the DMEM culture fluid containing new-born calf serum 8%, 5% respectively.The standard every time passed on is in 1: 4 ratio sub-bottle, at 37 DEG C of 5%CO2Cultivate, form fine and close monolayer at 48-72h inner cell；
B) add 5% new-born calf serum after changing culture fluid into DMEM/F12 to continue to cultivate three generations, pass on standard synchronisation rapid a)；
C) in culture fluid, new-born calf serum reduces to 3%, and by the method in b step, the ratio of passing on is reduced to 1: 3 and is further cultured for three generations；
D) in culture fluid, new-born calf serum reduces to 1%, adds volumn concentration 20% again and has cultivated culture fluid continuation five generations of cultivation of this cell 48-72h for previous generation time, pass on standard synchronisation rapid c) in culture fluid；Cell forms fine and close monolayer in 48h-72h, and domestication has obtained low serum adhere-wall culture type Marc-145 cell line；
(3) domestication of low serum full suspension culture type Marc-145 cell
A) low serum adhere-wall culture type Marc-145 cell step (2) tamed, after DMEM/F12 and serum-free medium 11: 1 ratio mixing by volume, add 3-5% new-born calf serum and make culture fluid, with culture fluid, cell density is adjusted to 3-5 × 105/ ml, 100-130r/min, 37 DEG C of 5%CO2Shaking table is cultivated；
B) often cultivating 48h sampling counting, cell density is not up to 1 × 106During/ml, use fresh medium Eddy diffusion instead after 800-1000r/min is centrifugal and cultivate；Cell density reaches 1 × 106During/ml, 800-1000r/min is centrifugal to be cultivated in 1: 2 ratio sub-bottle；2 × 10 are reached to cell density6During/ml, 800-1000r/min is centrifugal to be cultivated in 1: 3 ratio sub-bottle；Cultivate 48h cell density continuous three generations in 1: 3 ratio and all reach 2 × 106During/ml, domestication obtains low serum full suspension culture type Marc-145 cell line；
(4) domestication of serum-free full suspension culture type Marc-145 cell
A) in serum-free medium 2 (Lanzhou lark Bioisystech Co., Ltd, article No.: BFLM101.05) in add 3% new-born calf serum, the low serum full suspension culture type Marc-145 cell obtained in step (3) is directly diluted to density 5-7 × 105/ ml, puts 100-130r/min, 37 DEG C of 5%CO2Shaking table is cultivated；
B) often cultivating 24h sampling counting, cell density reaches 3 × 106During/ml, cell suspension is directly diluted to 5 × 105/ ml inoculated and cultured, often cultivates generation new-born calf serum lowering of concentration 1%, reduces to 0 to serum during 4 generation, cultivates with serum-free culture completely, all reaches 3 × 10 at continuous three culture 48h cell densities6During/ml, domestication obtains serum-free full suspension culture type Marc-145 cell line.
The present invention also provides for the full suspension culture type Marc-145 cell that application method described in claim 1 obtains.
The present invention also provides for full suspension culture type Marc-145 cell cultural method in serum-free medium, and step is as follows:
A) seed cell is cultivated, and a full suspension culture type Marc-145 cell of recovering adds 5-10% new-born calf serum with 40-60ml serum-free medium 2, puts 110r/min, 37 DEG C of 5%CO2Cultivate；
When cell grows to 3 × 106During/more than ml, add the culture fluid of 1-3% new-born calf serum cell is diluted to density be 5-7 × 10 with serum-free medium 25/ ml, counts 250-350ml, 100r/min, 37 DEG C of 5%CO2Cultivate；
Amplification culture reaches 3 × 10 to cell density6During/more than ml, more directly cell to be diluted to density with serum-free medium 2 be 5-7 × 105/ ml continues amplification culture, and cumulative volume is 400-600ml；Condition of culture is: 100-130r/min, 37 DEG C of 5%CO2；
Cell density reaches 3 × 106During/more than ml, proceed to bioreactor culture；
B) bioreactor criticizes cultivation: be 5-7 × 10 with No. 2 inoculum densities of serum-free medium5The full suspension culture type Marc-145 cell of/ml to bioreactor culture volume, condition of culture is: rotating speed 70-120r/min, temperature 37 DEG C, dissolved oxygen 40-70%, pH7.15-7.25.
The present invention also provides for full suspension culture type Marc-145 cell cultural method in serum-free medium, and step is as follows:
With the serum-free medium 2 full suspension culture type Marc-145 cell of inoculation to bioreactor culture, initial cell density is 5-7 × 105/ ml, initial volume of culture allows the many 10-20% of minimum volume than bioreactor culture, and condition of culture is: rotating speed 50-70r/min, temperature 37 DEG C, dissolved oxygen 25-40%, pH7.15-7.25；
Cell density reaches 2 × 106Adding the serum-free medium equal with initiateing volume of culture 2 during/more than ml, condition of culture changes into: rotating speed 70-90r/min, temperature 37 DEG C, dissolved oxygen 35-45%, pH7.15-7.25；
Cell density reaches 4 × 106Add serum-free medium 2 during/more than ml to change into the highest volume of culture, condition of culture: rotating speed 90-120r/min, temperature 37 DEG C, dissolved oxygen 50-70%, pH7.10-7.20.
The present invention also provides for full suspension culture type Marc-145 cell cultural method in low blood serum medium, and step is as follows:
With serum-free medium 2 add 1-3% new-born calf serum inoculate full suspension culture type Marc-145 cell to bioreactor culture, initial cell density is 5-7 × 105/ ml, initial volume of culture allows the many 10-20% of minimum volume than bioreactor culture, and condition of culture is: rotating speed 50-70r/min, temperature 37 DEG C, dissolved oxygen 25-40%, pH7.15-7.25；
Cell density reaches 2 × 106Adding the serum-free medium equal with initiateing volume of culture 2 during/more than ml, condition of culture changes into: rotating speed 70-90r/min, temperature 37 DEG C, dissolved oxygen 35-45%, pH7.15-7.25；
Cell density reaches 6 × 106Add serum-free medium 2 during/more than ml to change into the highest volume of culture, condition of culture: rotating speed 90-120r/min, temperature 37 DEG C, dissolved oxygen 50-70%, pH7.10-7.20.
The invention have the benefit that cell acclimation method that the present invention provides and obtain can the Marc-145 cell of suspension culture, this cell adapted low serum and the full suspension culture of serum-free, the reactor (the highest volume of culture 5.5L of reality) at 7.5L presses serum-free culture density up to 8 × 106/ ml, at least can get 4.4 × 1010Cell, be equivalent to the cell quantity of 80-90 15L roller bottle or 18-20 40 layer cell factory results, calculate by this, a volume of culture 500L bioreactor is equivalent to current 300 30 bottles position roller bottle machine production capacities.Apply to the cell of suspension culture actual production also can significantly be saved on place and human input, meet current energy-saving and environmental protection and low-carbon (LC) theory, bringing appreciably while economic benefit, also can produce preferably social benefit.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, is used for together with embodiments of the present invention explaining the present invention, is not intended that limitation of the present invention.In the accompanying drawings:
Fig. 1 is adhere-wall culture type Marc-145 cell before domestication；
Fig. 2 is the suspension culture type Marc-145 cell of domestication；
Fig. 3 is that the suspension culture type Marc-145 cell fluorescence antibody act of domestication checks exogenous virus result figure；
Fig. 4 is suspension culture type Marc-145 cell growth curve chart of serum-free culture in bioreactor of domestication；
Fig. 5 is suspension culture type Marc-145 cell growth curve chart of low serum free culture system in bioreactor of domestication.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is commercially available.
Embodiment 1
1. material:
1) cell strain: Marc-145 Rhesus Macacus embryonic kidney cells, is provided by Gansu Province's System in Animal Cell Biotechnology Technical Research Center；
2) DMEM culture fluid, Gibco, article No.: 02-5062EJ；
3) DMEM/F12 culture fluid, Gibco, article No.: 12500-096；
4) serum-free medium 1, Lanzhou lark Bioisystech Co., Ltd, article No.: BFLM401.05；
5) serum-free medium 2, Lanzhou lark Bioisystech Co., Ltd, article No.: BFLM101.05；
6) low blood serum medium, Lanzhou lark Bioisystech Co., Ltd, article No.: BLLM101.05；
7) new-born calf serum, Lanzhou people's marine growth Engineering Co., Ltd, lot number: 20150716；
8) DMSO (dimethyl sulfoxide), Thermo Fisher Scientific Corporation, article No.: 20,688 8)；
9) trypsin, Gibco, article No.: 27250, it is configured to 0.25% trypsin solution (EDTA-Na 0.3g/L).
2. instrument equipment and vessel
1) bioreactor, B.Braun, model: BIOSTATEOR B plus, maximum volume of culture 5L；
2) shaking table, KUHNER, model: ISF1-XC；
3)CO2Incubator, Thermo Fisher Scientific Corporation, model: 3111；
4) centrifuge, Hunan is triumphant to be reached, model: TD5Z, TDL80-2B；
5) difference inverted microscope, OLYMPUS, OLYMPUS CKX41；
6) fluorescence inverted microscope, OLYMPUS AX71, OLYMPUS；
7) Tissue Culture Flask (Corning, T25 article No.: 430639, T75 article No.s: 430641)；
8) pyramidal cells bottle 150ml, Haimen, Jiangsu, specification: 150ml；
9) suspension culture bottle 500ml, Lanzhou Bailing Plastic Co., Ltd., specification: 500ml；
10) pipettor, BRAND GMBH+CO KG, model: accu-jetORpro.
2. method:
1) cultivation of adhere-wall culture type Marc-145 cell
Choose from Gansu Province's System in Animal Cell Biotechnology Technical Research Center cell bank and be feminine gender through calibrating aseptic, mycoplasma, exogenous virus, and one, adhere-wall culture type Marc-145 cell recovery sensitive for PRRSV is cultivated, the cell of recovery is numbered: GsACC2B0000086, culture fluid is the DMEM of 10% new-born calf serum (following serum-concentration is volumn concentration), condition of culture: 37 DEG C of 5%CO2Incubator cultivate.After cell recovery, vigor is 94.6%, and cell is cultivated 48h and defined fine and close monolayer, also becomes fine and close monolayer in 1: 4 ratio sub-bottle Secondary Culture at 48h, and cell edges is clear, and form is flat, in epitheliated type, sees Fig. 1.Cell growth is good for use in domestication.
2) domestication of low serum adhere-wall culture type Marc-145 cell line
A) by 1) the described growth normal adhere-wall culture type Marc-145 cell DMEM culture fluid of 8% new-born calf serum cultivates three generations, passes in 1: 4 ratio sub-bottle, condition of culture: 37 DEG C of 5%CO2Cultivating, per in generation, forms fine and close monolayer at 48-72h；
B) three generations is cultivated by the method in a step after new-born calf serum reduces to 5% in culture fluid；
C) culture fluid changes into after DMEM/F12 adds 5% new-born calf serum and continues to cultivate three generations by the method in b step；
D) in culture fluid, new-born calf serum reduces to 3%, and by the method in step c, the ratio of passing on is reduced to 1: 3 and is further cultured for three generations；
E) in culture fluid, new-born calf serum reduces to 1%, in culture fluid, add volumn concentration 20% again cultivate culture fluid (be proved cell and can secrete some factors being conducive to cell to grow when low serum free culture system) continuation five generations of cultivation of this cell 48-72h, the same d of cultural method for previous generation time；
F) cell defines fine and close monolayer at 48-72h, illustrate to have tamed adaptation low serum adhere-wall culture type Marc-145 cell line, can be at the cell in liquid nitrogen cryopreservation this stage of a part, in case when later stage domestication process occurs unexpected, directly recovery used with the time of saving.By the conventional method of cell cryopreservation, use frozen stock solution formula: 80%DMEM/F12+10% new-born calf serum+10%DMSO, cell density is 3 × 106/ ml, cell viability is 99%, and the loading amount of every cryopreservation tube is 1.5ml.
3) domestication of low serum full suspension culture type Marc-145 cell line and foundation
A) by above-mentioned 2) the low serum adhere-wall culture type Marc-145 cell tamed, with DMEM/F12 and serum-free medium 1 (purchased from Lanzhou lark Bioisystech Co., Ltd, article No.: BFLM401.05) by volume 1: 1 ratio mixing after, add 3-5% new-born calf serum and make culture fluid, with culture fluid, cell density is adjusted to 4.6 × 105/ ml, with pyramidal cells bottle at 110r/min, 37 DEG C of 5%CO2Shaking table is cultivated；
B) often cultivating 48h sampling counting, cell density is not up to 1 × 106Use fresh medium Eddy diffusion instead after/ml 800-1000r/min is centrifugal and cultivate (also referred to as passing on)；Until cell density reaches 1 × 106During/ml, centrifugal in 1: 2 ratio sub-bottle cultivation as stated above；2 × 10 are reached to cell density6During/ml, centrifugal in 1: 3 ratio sub-bottle cultivation as stated above.2 × 10 are all reached when continuous three generations cultivates 48h cell density in 1:3 ratio6During/ml, illustrate to have tamed low serum full suspension culture type Marc-145 cell line, can liquid nitrogen cryopreservation a part this stage cell.The domestication that entirely suspends of low serum needs 10-20 generation.
Cultivation results and pass on operational circumstances and see table.And the 16th generation cell has been carried out frozen, use frozen stock solution formula: 80% serum-free medium 1+10% new-born calf serum+10%DMSO, cell density is 1.6 × 107/ ml, cell viability is 98.1%, and the loading amount of every cryopreservation tube is 1.5ml.
The full suspension culture of the low serum of table 1 Marc-145 cell is tamed process count results and passes on operational circumstances table
4) domestication of serum-free full suspension culture type Marc-145 cell line and foundation
By No. 2 direct dilution (not being centrifuged) above-mentioned steps 3 of the serum-free medium containing 3% new-born calf serum) in the Marc-145 cell of the 16th generation full suspension culture of low serum, with pyramidal cells bottle with 5 × 105/ ml density is inoculated, and puts 110r/min, 37 DEG C of 5%CO2Shaking table is cultivated.
Every 24h sampling counting, cell density reaches 3 × 106During/ml, cell suspension is directly diluted to 5 × 105/ ml inoculated and cultured, often cultivates generation new-born calf serum lowering of concentration 1%, reduces to 0 to serum during 4 generation, cultivates with serum-free culture completely, cultivates continuous three generations when 48h cell density and all reach 3 × 106During/ml, illustrate to have tamed serum-free full suspension culture type Marc-145 cell line, frozen set up master cell bank and working cell storehouse.Cultivation results and culture fluid situation see table 2, and cellular morphology is shown in Fig. 2.
And the 7th generation cell has been carried out the frozen master cell bank that establishes, use frozen stock solution formula: 80% serum-free medium 1+10% new-born calf serum+10%DMSO, cell density is 1.49 × 107/ ml, cell viability is 98.9%, and the loading amount of every cryopreservation tube is 1.5ml.Cultivating for two generations after master cell bank is recovered and establish working cell storehouse, the working cell storehouse same master cell bank of frozen stock solution formula, cell density is 1.63 × 107/ ml, cell viability is 99.3%, and the loading amount of every cryopreservation tube is 1.5ml.
The full suspension culture of table 2 Marc-145 cell non-serum is tamed process count results and passes on operational circumstances table
5) calibrating of full suspension culture type Marc-145 cell
Examine and determine according to " People's Republic of China's veterinary drug allusion quotation " three 2010 version " Cells for production standard ".
A) steriling test: the supernatant that full suspension culture type Marc-145 cell is cultivated is inoculated in sulphur glycollate culture medium, peptone from casein agar culture medium (inclined-plane) and dextrose peptone medium cultivation and is feminine gender；
B) mycoplasma inspection: full suspension culture supernatant of type Marc-145 cell freeze thawing is inoculated in mycoplasma fluid medium and mycoplasma solid medium cultivation and is feminine gender；
C) exogenous virus inspection: full suspension culture type Marc-145 cell freeze thawing supernatant once is inoculated into Vero cell and ST cell is cultivated, and pathological changes does not occurs in cell；Fluorescent antibody technique checks that bovine viral diarrhea virus (BVDV) and pig parvoviral (PPV) are negative, such as Fig. 3；Hemadsorption test is also negative.
D) chromosome examination, has added up the quantity of 50 chromosomes being in division phases, and 2n=60 accounts for 84%.
6) the bioreactor High Density Cultivation of serum-free full suspension culture type Marc-145 cell
A) seed cell cultivates a full suspension culture type Marc-145 cell of recovering from working cell storehouse, adds 5% new-born calf serum with 50ml serum-free medium 2, puts 110r/min, 37 DEG C of 5%CO2Cultivate.
Cultivate 48h cell density and reach 3.75 × 106/ ml, is diluted to 300ml suspension culture bottle amplification culture with No. 2 culture fluid adding 1% new-born calf serum of serum-free medium, and after dilution, cell density is 6.25 × 105/ ml, condition of culture: 100r/min, 37 DEG C of 5%CO2Cultivate.
Cultivate 48h cell density and reach 4.36 × 106/ ml, obtained cell suspension 140ml, add serum-free medium 2 and divide two suspension culture amplification culture, every bottle of 500ml, condition of culture: 110r/min, 37 DEG C of 5%CO to 1000ml2Cultivating, cell density is 6.1 × 105/ml。
Cultivate 48h cell density and reach 3.91 × 106/ ml, a part proceeds to bioreactor culture, and remainder is with 5-7 × 105/ ml density continuous passage is cultivated.
B) bioreactor is criticized to cultivate and is prepared bioreactor according to bioreactor operation code, with serum-free medium 2 by above-mentioned full suspension culture type Marc-145 cell by with density 6 × 105/ ml is seeded to bioreactor, and culture fluid volume is 5L.Reactor parameter: rotating speed 100r/min, temperature 37 DEG C, dissolved oxygen 45%, pH7.22.Every 12 hours counting cell densities.Cell counts is shown in Table 3, and growth curve is shown in Fig. 4.
C) bioreactor feeding culture prepares bioreactor according to bioreactor operation code, with serum-free medium 2, above-mentioned full suspension culture type Marc-145 cell is pressed density with 7 × 105/ ml is seeded to bioreactor culture, initial volume of culture 2L, reactor parameter: rotating speed 60r/min, temperature 37 DEG C, dissolved oxygen 25%, pH7.25.Every 12 counting cell density and vigor, 36h cell density reaches 26.3 × 105/ ml, adds serum-free medium and changes into 4L, reactor parameter: rotating speed 90r/min, temperature 37 DEG C, dissolved oxygen 40%, pH7.2.Cultivation to 72h cell density reaches 54.3 × 105/ ml, adds serum-free medium and changes into 5.5L, condition of culture: rotating speed 120r/min, temperature 37 DEG C, dissolved oxygen 60%, pH7.10, and cultivation to 96h density reaches 94.2 × 105/ml.Cell counts is shown in Table 3, and growth curve is shown in Fig. 4.
To sum up two training method, the inoculum density 6 × 10 when criticizing cultivation5/ ml, 5L inoculate 3 × 10 altogether9Cell, cultivate 96h cell density 8.54 × 106/ ml total amount reaches 4.27 × 1010, about growth 14 times, cell viability is 93.1%.And feeding culture inoculum density 7 × 105/ ml, initial volume of culture 2L inoculates 1.4 × 10 altogether9Cell, cultivate 96h cell density 9.43 × 106/ ml, total amount reaches 5.19 × 1010, about growth 37 times, cell viability is 98.1%.As can be seen here, feeding culture is more suitable for the bioreactor culture of full suspension culture type Marc-145 cell.
Table 3 full suspension culture type Marc-145 bioreactor serum-free culture count results table
Note: in table, the data before "/" are the value before mending culture fluid, and the data after "/" are the value after mending culture fluid.
7) full suspension culture type Marc-145 cell is cultivated at the low serum high-density of bioreactor
Training method is feeding culture, adds 3% new-born calf serum in bioreactor culture according to the method for 6c with low blood serum medium.With 6.4 × 105/ ml density inoculated and cultured, 36h cell density reaches 24.2 × 105/ ml adds serum-free medium and reaches 60.4 × 10 to 4L, cultivation to 72h cell density5/ ml adds serum-free medium to 5.5L, reaches 105.6 × 10 to 96h cell density5/ml.Cell counts is shown in Table 4, and growth curve is shown in Fig. 5.
Table 4 full suspension culture type Marc-145 bioreactor low serum free culture system count results table
Note: in table, the data before "/" are the value before mending culture fluid, and the data after "/" are the value after mending culture fluid.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature is carried out equivalent.All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (5)
1. the acclimation method of full suspension culture type Marc-145 cell line, it is characterised in that: step is as follows:
(1) cultivation of adhere-wall culture type Marc-145 cell
Select through aseptic, mycoplasma, exogenous virus calibrating as negative, the adhere-wall culture type Marc-145 cell sensitive to PRRSV, pass in 1:4 ratio sub-bottle with the DMEM culture fluid containing 10% new-born calf serum after the fine and close monolayer of cell growth, put 37 DEG C of 5% CO2Incubator cultivate, form fine and close monolayer at 48-72h inner cell, for sharp-edged squamous epithelium type cell and cell grow normal in the case of can be used for taming；
(2) domestication of low serum adhere-wall culture type Marc-145 cell line
A) step (1) described growth normal adhere-wall culture type Marc-145 cell is respectively cultivated three generations with the DMEM culture fluid containing new-born calf serum 8%, 5% respectively；The standard every time passed on is in 1:4 ratio sub-bottle, at 37 DEG C of 5%CO2Cultivate, form fine and close monolayer at 48-72h inner cell；
B) add 5% new-born calf serum after changing culture fluid into DMEM/F12 to continue to cultivate three generations, pass on standard synchronisation rapid a)；
C) in culture fluid, new-born calf serum reduces to 3%, and by the method in b step, the ratio of passing on is reduced to 1:3 and is further cultured for three generations；
D) in culture fluid, new-born calf serum reduces to 1%, adds volumn concentration 20% again and has cultivated culture fluid continuation five generations of cultivation of this cell 48-72h for previous generation time, pass on standard synchronisation rapid c) in culture fluid；Cell forms fine and close monolayer in 48h-72h, and domestication has obtained low serum adhere-wall culture type Marc-145 cell line；
(3) domestication of low serum full suspension culture type Marc-145 cell
A) low serum adhere-wall culture type Marc-145 cell step (2) tamed, after mixing by DMEM/F12 and serum-free medium 1 1:1 ratio by volume, add 3-5% new-born calf serum and make culture fluid, with culture fluid, cell density is adjusted to 3-5 × 105/ ml, 100-130r/min, 37 DEG C of 5% CO2Shaking table is cultivated；
B) often cultivating 48h sampling counting, cell density is not up to 1 × 106During/ml, use fresh medium Eddy diffusion instead after 800-1000r/min is centrifugal and cultivate；Cell density reaches 1 × 106During/ml, 800-1000r/min is centrifugal to be cultivated in 1:2 ratio sub-bottle；2 × 10 are reached to cell density6During/ml, 800-1000r/min is centrifugal to be cultivated in 1:3 ratio sub-bottle；Cultivate 48h cell density continuous three generations in 1:3 ratio and all reach 2 × 106During/ml, domestication obtains low serum full suspension culture type Marc-145 cell line；
(4) domestication of serum-free full suspension culture type Marc-145 cell
A) in serum-free medium 2, add 3% new-born calf serum, the low serum full suspension culture type Marc-145 cell obtained in step (3) is directly diluted to density 5-7 × 105/ ml, puts 100-130r/min, 37 DEG C of 5%CO2Shaking table is cultivated；
B) often cultivating 24h sampling counting, cell density reaches 3 × 106During/ml, cell suspension is directly diluted to 5 × 105/ ml inoculated and cultured, often cultivates generation new-born calf serum lowering of concentration 1%, reduces to 0 to serum during 4 generation, cultivates with serum-free culture completely, all reaches 3 × 10 at continuous three culture 48h cell densities6During/ml, domestication obtains serum-free full suspension culture type Marc-145 cell line.
2. the full suspension culture type Marc-145 cell that application method described in claim 1 obtains.
3. the cultural method in serum-free medium of the full suspension culture type Marc-145 cell described in claim 2, it is characterised in that: step is as follows:
A) seed cell is cultivated, and a full suspension culture type Marc-145 cell of recovering adds 5-10% new-born calf serum with 40-60ml serum-free medium 2, puts 110r/min, 37 DEG C of 5% CO2Cultivate；
When cell grows to 3 × 106During/more than ml, add the culture fluid of 1-3% new-born calf serum cell is diluted to density be 5-7 × 10 with serum-free medium 25/ ml, counts 250-350ml, 100r/min, 37 DEG C of 5% CO2Cultivate；
Amplification culture reaches 3 × 10 to cell density6During/more than ml, more directly cell to be diluted to density with serum-free medium 2 be 5-7 × 105/ ml continues amplification culture, and cumulative volume is 400-600ml；Condition of culture is: 100-130r/min, 37 DEG C of 5% CO2；
Cell density reaches 3 × 106During/more than ml, proceed to bioreactor culture；
B) bioreactor criticizes cultivation: be 5-7 × 10 with No. 2 inoculum densities of serum-free medium5The full suspension culture type Marc-145 cell of/ml to bioreactor culture volume, condition of culture is: rotating speed 70-120r/min, temperature 37 DEG C, dissolved oxygen 40-70%, pH7.15-7.25.
4. the cultural method in serum-free medium of the full suspension culture type Marc-145 cell described in claim 2, it is characterised in that: step is as follows:
With the serum-free medium 2 full suspension culture type Marc-145 cell of inoculation to bioreactor culture, initial cell density is 5-7 × 105/ ml, initial volume of culture allows the many 10-20% of minimum volume than bioreactor culture, and condition of culture is: rotating speed 50-70r/min, temperature 37 DEG C, dissolved oxygen 25-40%, pH7.15-7.25；
Cell density reaches 2 × 106Adding the serum-free medium equal with initiateing volume of culture 2 during/more than ml, condition of culture changes into: rotating speed 70-90r/min, temperature 37 DEG C, dissolved oxygen 35-45%, pH7.15-7.25；
Cell density reaches 4 × 106Add serum-free medium 2 during/more than ml to change into the highest volume of culture, condition of culture: rotating speed 90-120r/min, temperature 37 DEG C, dissolved oxygen 50-70%, pH7.10-7.20.
5. the cultural method in low blood serum medium of the full suspension culture type Marc-145 cell described in claim 2, it is characterised in that: step is as follows:
With serum-free medium 2 add 1-3% new-born calf serum inoculate full suspension culture type Marc-145 cell to bioreactor culture, initial cell density is 5-7 × 105/ ml, initial volume of culture allows the many 10-20% of minimum volume than bioreactor culture, and condition of culture is: rotating speed 50-70r/min, temperature 37 DEG C, dissolved oxygen 25-40%, pH7.15-7.25；
Cell density reaches 2 × 106Adding the serum-free medium equal with initiateing volume of culture 2 during/more than ml, condition of culture changes into: rotating speed 70-90r/min, temperature 37 DEG C, dissolved oxygen 35-45%, pH7.15-7.25；
Cell density reaches 6 × 106Add serum-free medium 2 during/more than ml to change into the highest volume of culture, condition of culture: rotating speed 90-120r/min, temperature 37 DEG C, dissolved oxygen 50-70%, pH7.10-7.20.
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