CN108504622A - A kind of high-flux cell suspension culture method - Google Patents
A kind of high-flux cell suspension culture method Download PDFInfo
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Abstract
The present invention relates to a kind of high-flux cell suspension culture methods, and using 24 well culture plates as culture materials, cell-seeding-density is 0.2~0.5 × 106Cell/mL, volume of culture are 0.6~1.2mL, and shaking speed is used to carry out shaking table culture for 115~200rpm.The present invention carries out the cell strain high flux screening that shake culture reaches early stage using common 24 orifice plate for the first time, the problem of not only solving bio-pharmaceutical early stage development phase high flux screening, and the usage amount due to significantly reducing culture medium, R＆D costs are significantly reduced under the premise of not influencing screening effect, the R＆D cycle is shortened, the development rate of drug can be accelerated.
Description
Technical field
The present invention relates to a kind of 24 well culture plate high-flux cell suspension culture methods, belong to technical field of bioengineering.
Background technology
It in the upstream conceptual phase of bio-pharmaceutical, is especially screened early stage in cell strain, often needs to carry out a large amount of high-throughput sieves
Work, including cell strain screening, nutrient media components screening and process parameter optimizing etc. are selected, this process is a work taken time and effort
Make, high flux screening can ensure to obtain yield height, quality cell strain from source, mitigate downstream research work, accelerate item
Purpose research speed, Study of Lifting quality.
Currently, the screening of early stage cell strain is general, static gas wave refrigerator, static gas wave refrigerator can not really reflect that the later stage shakes in the orifice plate
Bottle suspend culture virtual condition, institute in this way can not accurate evaluation cell upgrowth situation and expression, and acquisition
Sample size is few, it is difficult to further carry out quality analysis.Although shaking flask/shake pipe cultural method can really reflect that later stage cell is given birth to
Long real conditions, but it is cumbersome, it is of high cost, the hardware facilities such as shaking table are required with high and low because of flux, unsuitable early stage
The high flux screening of cell strain.
As Chinese patent literature CN104404082A (application number 201410662495.4) discloses a kind of efficient external source
The screening technique of protein expressing cells strain.Include the following steps：Destination protein expression vector transfection host cell；Screening drug adds
Pressure is stablized cell pool and is formed；Stablize cell pool cell inoculation in semisolid culturemedium；By cell clone from semisolid culturemedium
In choose into 96 porocyte plates, continue culture 4-5 days, detection cell conditioned medium in destination protein expression quantity；Before expression quantity ranking
50 cells expand to 24 orifice plates and continue culture 5 days；Cell in 24 orifice plates is further expanded into 6 orifice plates and continues culture 5 days；It will
Cell expands in 6 orifice plates cultivates into shaking flask, destination protein expression quantity of the check and evaluation difference cell clone under suspended state；It will
According to shaking flask assessment result, the cell of expression quantity ranking top 10 is subjected to stability passage experiment；Mesh is expressed according to cell strain
Albumen level and continuous expression albumen stability establish candidate cell strain.The technical solution is completed using static gas wave refrigerator
Then high flux screening process uses shaking flask culture to screen a small amount of target strain after screening again.
Currently, high-throughput research equipment includes multi-joint microreactor, flux is still relatively low, and instrument consumptive material etc. is made
High price is expensive.Therefore, a kind of high, at low cost, easy-operating high-flux cell suspension culture method of flux is established, just becomes very must
It wants.
Invention content
The present invention is in view of the deficienciess of the prior art, provide a kind of high-flux cell suspension culture method.Profit of the invention
Use common 24 hole Nostoc commune Vanch plate as material, it is final to establish one kind efficiently by a series of research of condition of culture, optimization
High-flux cell suspension culture method, have been used for the screening of multiple project cell strains and medium optimization at present, obtain good effect
Fruit.
Technical scheme is as follows：
An object of the present invention is to provide a kind of 24 well culture plate high-flux cell suspension culture methods, and technical solution is such as
Under：
A kind of high-flux cell suspension culture method, using 24 well culture plates as culture materials, cell-seeding-density is
0.2~0.5 × 106Cell/mL, volume of culture are 0.6~1.2mL, and shaking speed is used to carry out shaking table training for 115~200rpm
It supports.
According to currently preferred, the volume of culture is 0.8~1.0mL, more preferably 1.0mL.
According to currently preferred, the cell-seeding-density is 0.3 × 106Cell/mL.
According to currently preferred, the shaking speed is 150rpm.
According to the present invention, above-mentioned technical proposal can cultivate under common condition of culture, for example cultivation temperature is set as
36~37 DEG C, relative humidity 75%~95%, CO2Concentration 5% (v/v).
It is 3.4mL according to the every pore volume of 24 currently preferred, described well culture plates.
In cell cultivation process, inoculum density has an impact cell growth, and suitable inoculum density can promote cell
Proliferation, inoculum density is too low or the too high growing multiplication for being all unfavorable for cell.Different culture vessels, such as 96 orifice plates, 48 orifice plates,
Its most suitable inoculum density is different, and inoculum density difference can lead to different results of study.The present invention is using 24 orifice plates as culture material
Material, cell-seeding-density are set in 0.2~0.5 × 106Cell/mL can embody the difference between different vaccination density, use
In influence of the assessment different vaccination density to product yield and quality.
Cell growth and the appraisal procedure of output and quality mostly use feed supplement batch training method and carry out at present, if volume of culture
Larger, with the extension (12~18 days) of cultivation cycle, volume of culture increases, and can increase the machine of cross contamination between culture hole
Rate, later stage will appear cell aggregation vigor and decline situation, mask the true growing state of cell；And the culture of smaller size smaller,
The volatilization that can cause culture medium because of the problems such as control of humidity, causes the change of culture medium nutrient concentrations, and then affect
The normal growth of cell.Therefore, larger or smaller volume of culture can introduce a large amount of uncertain factors, influence cell growth and production
Measure the authenticity of quality condition assessment.For the present invention using 24 orifice plates as culture materials, volume of culture is set as 0.6~1.2mL,
Be not in not only cross-contamination phenomena, but also can preferably suspend under shaking speed of the present invention, make cell growth and
Cell viability can maintain higher level.
The difference of shaking speed can influence cell and the exchange rate of nutriment and ambient atmos in culture medium, in turn
The metabolism for affecting cell shows as cell number and the difference of cell viability.Shaking speed is too low, and cell cannot preferably hang
It is floating, it is unfavorable for cell growth, and shaking speed is excessively high, and the cell culture fluid in Nostoc commune Vanch plate can be made to overflow, and causes to intersect dirty
Dye.Shaking speed of the present invention is set as 115~200rpm, is adapted to the fostering requirement of different cell strains.
Temperature, humidity, CO2Concentration etc. is all to influence the factor of cell growth, and general method is in the prior art for use
Can, general cultivation temperature is set as 36~37 DEG C, CO2Be both cell metabolite and maintain culture solution pH it is related, one
As need the cell to be placed in 95% air and add 5%CO2Mixed-gas environment in.Cell culture is also required to maintain centainly wet
Degree, the too low evaporation that can lead to cell culture fluid of humidity make osmotic pressure change, influence the growth of cell.Used in the present invention
24 well culture plate volume of culture it is relatively small, the volatilization of culture medium cell growth is influenced it is huge, so general setting is opposite
Humidity is 75%~95%, to maintain the normal growth of cell.
Advantageous effect
The present invention carries out the cell strain high flux screening that shake culture reaches early stage using common 24 orifice plate for the first time, not only solves
Determined bio-pharmaceutical early stage development phase high flux screening the problem of, and with using 24 hole deep-well plates method compared with, due to
The usage amount for significantly reducing culture medium significantly reduces R＆D costs under the premise of not influencing screening effect, and is follow-up
More culture mediums are saved when mass cell culture, the process of colony screening is accelerated, shortens the R＆D cycle, can be accelerated
The development rate of drug.
Description of the drawings
Fig. 1：Under the conditions of identical inoculum density and shaking speed, the growth curve comparison diagram of cell under different volume of culture；
Fig. 2：Under the conditions of identical inoculum density and shaking speed, cell viability comparison diagram under different volume of culture；
Fig. 3：Under the conditions of identical inoculum density and shaking speed, cell expression quantity comparison diagram under different volume of culture；
Fig. 4：Under the conditions of identical volume of culture and shaking speed, cell growth curve comparison diagram under different vaccination density；
Fig. 5：Under the conditions of identical inoculum density and volume of culture, cell growth curve comparison diagram under different shaking speeds；
Fig. 6：B1 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Fig. 7：B2 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Fig. 8：B3 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Fig. 9：B4 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 10：B5 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 11：B6 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 12：B7 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 13：B8 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 14：B9 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 15：B10 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 16：B11 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 17：B12 cell strains growth curve comparison diagram under condition of culture of the present invention and under shake flask culture conditions；
Figure 18：Area (IVCD) under different cell strain viable count lines under condition of culture and shake flask culture conditions of the present invention
Figure；
Figure 19：Different cell strain expression quantity comparison diagrams under condition of culture and shake flask culture conditions of the present invention.
Specific implementation mode
It is further illustrated the present invention below by the specific embodiment for preparing, it should be understood, however, that, these embodiments are only
It is only for specifically describing in more detail and be used, and be not to be construed as limiting the present invention in any form.
Medium component used in the embodiment of the present invention, culture plate, cell strain etc. purchased in market can obtain.
24 well culture plates：COSTAR 3524 is purchased from Corning companies, is 3.4mL per pore volume.
Infors shaking tables：Inspire confidence in gloomy biotechnology (China) Co., Ltd, model Multitron Pro purchased from her.
Clonpix2、CloneSelect Image：Purchased from Molecular Devices companies.
CloneMatrix, CloneDetect and Reagent XL：Purchased from Molecular Devices companies.
CHO-S cells：Purchased from Life Technology companies.
Basal medium used is XH001, is prepared to be autonomous, the XH001 culture mediums specifically comprise：4- ethoxys
Piperazine ethanesulfonic acid (HEPES) 5g/L, glucose 8g/L, Sodium Pyruvate 0.2g/L, sodium chloride 3g/L, potassium chloride 0.4g/L, sub- selenium
Sour sodium 0.000005g/L, manganese sulfate 0.05g/L, ethanol amine 10 μ g/L, ironic citrate 5mg/L, zinc sulfate 0.1g/L, copper sulphate
0.02g/L, glutathione 0.05g/L, magnesium chloride 0.1g/L, sodium dihydrogen phosphate 0.2g/L, sodium bicarbonate 2.1g/L, alanine
0.015g/L, asparagine 0.035g/L, arginine 0.25g/L, aspartic acid 0.2g/L, cystine 0.41g/L, cysteine
0.11g/L, glutamic acid 0.08g/L, glycine 0.01g/L, histidine 0.15g/L, isoleucine 0.25g/L, leucine
0.25g/L, lysine 0.2g/L, methionine 0.11g/L, phenylalanine 0.065g/L, proline 0.045g/L, serine
0.2g/L, threonine 0.15g/L, tryptophan 0.02g/L, tyrosine 0.2g/L, valine 0.33g/L, biotin 0.006mg/
L, calcium pantothenate 0.0023g/L, choline chloride 0.061g/L, folic acid 0.003g/L, inositol 0.015g/L, niacinamide 0.003g/L,
Vitamin B6 0.0023g/L, vitamin B2 0.0002-0.0006g/L, vitamin B1 0.002-0.006g/L, vitamin
B12 0.000009g/L, linoleic acid 0.001g/L, blocked polyethers F-68 (Pluronic F-68) 1.02g/L.
Embodiment 1：Influence the screening of high-flux cell suspension culture principal element of the present invention
Keeping 36.5 DEG C of temperature, relative humidity 80%, CO2Under conditions of concentration 5% (v/v), 24 well culture plates are consolidated
It is scheduled in moisture preservation box, waterlogged absorbent cotton or sponge is reinforced around, to ensure the stabilization of moisture preservation box humidity environment, and every 2
It replaces once, to prevent the growth of the microorganisms such as bacterium, mould within~3 days.It is thin to influencing using CHO blanc cells as cell strain
The key parameter of intracellular growth such as inoculum density, volume of culture, shaking speed are screened.Two parallel studies are arranged in every group of experiment
Group, parameter level setting such as table 1：
1 influence factor of table and level design
(1) volume of culture research
With 0.3 × 106Chinese hamster ovary celI is inoculated into suspend in 24 well culture plates and cultivate by cell/mL cell densities, volume of culture
Respectively 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.5mL, it is 36.5 DEG C to keep cultivation temperature, humidity 80%, CO2It is a concentration of
5%, shaking speed 115rpm carry out dyeing counting to living cells using trypan blue staining in every 2~3 days, draw cell life
Long curve graph (Fig. 1), cell viability comparison diagram (Fig. 2), cell expression quantity comparison diagram (Fig. 3).
Fixed inoculum density (0.3 × 106Cell/mL) and shaking speed (115rpm) under the premise of, to cell growth status
It is assessed.Fig. 1's the result shows that, 24 well culture plate cells reached highest, about 12 × 10 in the 7th day density6Cell/mL,
Reflect the normal growth rule of cell.1.5mL volume of culture experimental group cell growths are slow, and peak cell density is only 3 ×
106Cell/mL, and cellular morphology is poor, under the conditions of inventor rule of thumb speculates current rotating speed, excessive bulk culture makes cell
It can not suspend, to affect utilization of the cell to nutriment, cause the slow phenomenon of cell growth.
Fig. 2's the result shows that, in 24 well culture plate cultures, cell viability all maintains high bit, no significant difference.
Fig. 3's the result shows that, the yield of cell and the height of cell density are directly positively correlated, at the same also with volume of culture
It is linear.Wherein, cell yield highest in shaking flask reaches 1.1g/L (the 9th day) Zuo You；Followed by volume of culture is 0.6
~1.2mL experimental groups, expression quantity is about 800mg/L within the 9th day；Be finally volume of culture be 1.5mL, since cell density is too low,
Its yield is also minimum, about 300mg/L.
According to the growing state of cell, in conjunction with the above experimental result, the training of 24 well culture plate suspension culture methods of the invention
It is 0.6~1.2mL to support volume.
(2) inoculum density is studied
Fixed volume of culture (1mL) and shaking speed (115rpm), respectively with 0.2 × 106Cell/mL, 0.3 × 106Carefully
Born of the same parents/mL, 0.4 × 106Cell/mL, 0.5 × 106Chinese hamster ovary celI is inoculated in 24 well culture plates by cell/mL cell densities, is kept
Cultivation temperature is 36.5 DEG C, humidity 80%, CO2A concentration of 5%, living cells is carried out using trypan blue staining within every 2~3 days
Dyeing counting draws cell growth curve figure, and the results are shown in Figure 4.
Fig. 4's the result shows that, under different vaccination density conditions, cell growth curve trend is almost the same, but with inoculation
The difference of density, cell number slightly have difference.Wherein, 0.5 × 106Cell density peak value is most under the conditions of cell/mL cell densities
Height, with the reduction of inoculum density, cell density peak value declines successively, and 0.2 × 106Cell/mL cell-seeding-density groups are lived
Cell number is relatively minimal.
And from fig. 4, it can be seen that under different vaccination density conditions, it is close that the growth curve of cell can embody different vaccination
Difference between degree, therefore, the inoculum density of 24 well culture plate suspension culture methods of the invention is 0.2~0.5 × 106Cell/mL.
(3) shaking speed research：
Fixed cell-seeding-density (0.5 × 106Cell/mL) under the premise of, with culture volume be 0.6mL, 0.8mL,
It is 115rpm, 150rpm and 250rpm that shaking speed, which is set separately, in tri- groups of 1.0mL, and the cultivation temperature of 24 well culture plates of holding is
36.5 DEG C, humidity 80%, CO2A concentration of 5%, dyeing counting is carried out to living cells using trypan blue staining within every 2~3 days,
Cell growth curve is drawn, the results are shown in Figure 5.
Fig. 5's the result shows that, the growth tendency of cell is almost the same under the conditions of different shaking speeds, and different volume of culture are real
Test the cell growth no significant difference of group.Therefore, the shaking speed of 24 well culture plate suspension culture methods be set as 115~
200rpm。
Embodiment 2：Recombination anti-vegf R2 antibody cell strains are carried out using the present invention to screen
(1) structure of anti-vegf R2 antibody recombinant plasmid
By the direct synthetic antibody complete sequence (bibliography of gene chemical synthesis company：WO03075840A2, WHO Drug
Information Vol 23, No.3,2009), according to Life Technology companies Freedom CHO-S Kit kits
Heavy chain, light chain gene are cloned into expression plasmid pCHO 1.0 by specification, after sequence verification is correct, using in plasmid
Extraction reagent kit NucleoBond Xtra Midi (are purchased from Macherey-Nagel companies, article No.：740410.50) extracting plasmid,
It is quantified using the micro ultraviolet detection of nucleic acids instrument of Denovix DS-11, -80 DEG C store for future use.
The specific building process of recombinant plasmid is as follows：Synthesize anti-vegf R2 monoclonal antibodies heavy chain and light chain DNA sequences, 5 ' and 3 ' ends point
It Han You not EcoRV/PacI and AvrII/BstZ171 restriction enzyme sites.It is anti-using restriction enzyme EcoRV/PacI double digestions
VEGFR2 monoclonal antibody heavy chain gene sequences, are recycled.Heavy chain gene is cloned into through the bis- enzymes of restriction enzyme EcoRV/PacI
On the carrier for expression of eukaryon pCHO 1.0 cut, recombinant plasmid containing heavy chain is built, pCHO 1.0-H are denoted as.Utilize restriction enzyme
AvrII/BstZ171 double digestion anti-vegf R2 monoclonal antibody light chain gene sequences, are recycled, then be cloned into through restriction enzyme
On the carrier for expression of eukaryon pCHO 1.0-H of AvrII/BstZ171 double digestions, structure is denoted as containing heavy chain, light chain recombinant plasmid
pCHO 1.0-H-L.Sequence verification is carried out to the recombinant plasmid of structure, is sequenced in correct rear progress and is carried, is quantitative, it is spare.
(2) cell recovery and shaking flask culture：
Anti-Clumping Agent (volume ratio 0.1~1%), L-Glutamine are added into XH001 culture mediums
(0.8~8mM), and preheat about 30min under the conditions of 37 DEG C；CHO-S cells of the present invention are taken out from liquid nitrogen, are put into 37 rapidly
DEG C water-bath quick-thawing, and be added in prior 37 DEG C preheated XH001 culture mediums in super-clean bench, CountStar cells
Calculating instrument is counted, and inoculum density is controlled 0.5~1.0 × 106Cell/mL, in 37 DEG C, 85% humidity, 5%CO2, 110~
Shaking flask culture is carried out in 150rpm shaking tables；
(3) cell transfecting
First 24 hours of transfection, is inoculated with cell, inoculum density is：0.5~0.6 × 106Cell/mL, condition of culture
It is 37 DEG C, 5%CO2, 110~150rpm, which suspends, to be cultivated.By taking out cell in incubator before transfection, 1000rpm centrifuges 3min,
Cell is collected, supernatant is abandoned, it is 1 × 10 that the XH001 culture mediums preheated with 37 DEG C, which prepare density,7The cell suspension of cell/mL.It takes
In 800 μ L cell suspensions to 1.5mL centrifuge tubes, 40 μ g plasmids are added in 800 μ L cell suspensions, are sufficiently mixed, plasmid-is thin
Born of the same parents' mixture is added in electric revolving cup.Output voltage values are set on electroporation, number of shocks 1 time, time 17ms, shock by electricity cup 4mm,
Start electric shock.
(4) cell strain pressurization screening
Electric shock is after 48 hours, and to cell bank application screening pressure in T75 bottle, MTX a concentration of 10~100nM are during which every
It was counted every 4-5 days, and the XH001 culture mediums containing identical MTX concentration is replaced to cell, cell bank is preferably minimized in vigor after a week
20%, by one week low vitality stage, cell restored, and first stage pressurization screening cell bank restores 20 days total.Take recovery
First stage pressurization screening cell bank count, by cell bank according to 3 × 105The density of cell/mL is inoculated with 125mL shaking flasks
In, it is inoculated with volume 20mL, carries out batch culture studies, the 8th day sample detection expression quantity.
After cell bank adapts to first stage pressurization screening, by cell bank further by screening pressure MTX in 125mL shaking flasks
Concentration is improved to 0.5~1 μM, is counted per 4-5 days, and replace the XH001 culture mediums containing identical MTX concentration, cell bank to cell
It is preferably minimized in vigor after a week.By one week low vitality stage, cell restored, and second stage pressurization screening cell bank restores
It is 20 days total.The cell bank that second stage pressurization screening restores is taken to count, by cell bank according to 0.3 × 106The density of cell/mL
It is inoculated with shaking flask, is inoculated with volume 20mL, carries out batch culture studies, the 8th day sample detection expression quantity.
(5) monoclonal cell strain is screened
Cell bank is obtained to above-mentioned pressurization and carries out monoclonal screening, detailed process is as follows：By pressure cell strain culture 2-3
It, waits for that cell density reaches 2-3 × 106When cell/mL, in 6 orifice plates according to 500 cells/mL inoculation 2mL CloneMedia half
Solid medium prepares 100mL semisolid cell liquid according to 2 system of table：
2 semisolid culturemedium system of table
6 orifice plates are observed again after being placed on incubator static gas wave refrigerator 4~5 days after inoculating cell, are utilized after 10~15 days
ClonePix2 equipment carries out monoclonal cell strain and selects.Six orifice plates being inoculated with are put into ClonePix2 and screen monoclonal,
96 orifice plates containing 100 μ L fresh cultures are put into ClonePix2 simultaneously, it is thin according to monoclonal in six orifice plate of fluorescence intensity pair
Born of the same parents classify, and select and are cultivated in fluorescence intensity high monoclonal to 96 orifice plates, and screening quantity is in 2000~3000 cells.
The cell filtered out from ClonePix2 is put into incubator culture, and every 3~5 days cells change liquid, wait for that cell covers with, the phase
Between using CSI scan cell growth status.After cell covers with, supernatant is taken to detect expression quantity by Octet, screened 500 plants thin
Born of the same parents spread cultivation into 24 orifice plates, and every 3~5 days cells change liquid (50%-90% changes liquid), are put into shaking table 90rpm cultures, wait for that cell covers with
Inoculation is screened afterwards.The cell strain filtered out from 24 orifice plates spreads cultivation after cell covers with to 6 orifice plates, and every 3~5 days cells change
Liquid spreads cultivation after cell covers with and is cultivated into shaking flask.12 plants of monoclonal cell strains are finally obtained, it is numbered, are compiled
Number for B1, B2 ... B12.
(6) 24 orifice plate of monoclonal cell strain screens
Will the above number be B1, B2 ... 12 plants of cell inoculation 125mL shaking flasks of B12, inoculum density are 0.5 × 106Carefully
Born of the same parents/mL, used medium XH001, volume of culture 20mL, shaking speed 115rpm carry out culture experiment, setting culture
Temperature is 36.5 DEG C, humidity 80%, CO2A concentration of 5%.Then, 2mL culture solutions are taken from shaking flask, then respectively with 1mL/
Hole is inoculated with 2 24 orifice plate culture holes, volume of culture 1.0mL, in same rotational speed, temperature, humidity, CO2It is carried out under concentration conditions
Culture experiment.Dyeing counting is carried out to living cells using trypan blue staining within every 2~3 days, draws cell growth curve, as a result such as
Shown in Fig. 6~17.
From Fig. 6~17 as can be seen that cell growth trend of the different cell strains in 24 well culture plates and shake flask results table
Now consistent, cell number is less than shaking flask condition.
It is found by calculating area (IVCD) under viable count line, the IVCD values between different cell strains and shaking flask conditional trends
Performance is consistent, and the cell growth significant difference between different subclones, shows 24 well culture plate method sensitivitys and accuracy and shakes
Bottle studies almost the same (result is as shown in figure 18).
The cell expression quantity and shaking flask conditional trends of 24 well culture plates are almost the same, and the volume variance between different cell strains can
Apparent performance (result is as shown in figure 19), further demonstrates the sensitivity and accuracy of this method.
Above as can be seen that the 24 well culture plate cell suspension cultures methods established using the present invention, can be used successfully to thin
The screening of born of the same parents and nutrient media components etc., not only accuracy rate and sensitivity are almost the same with shaking flask, and only need per hole 0.6~
1.2mL greatly reduces cost, achieves good technique effect.
Claims (8)
1. a kind of high-flux cell suspension culture method, which is characterized in that using 24 well culture plates as culture materials, cell connects
Kind density is 0.2~0.5 × 106Cell/mL, volume of culture be 0.6~1.2mL, use shaking speed for 115~200rpm into
Row shaking table culture.
2. cultural method according to claim 1, which is characterized in that the volume of culture is 0.8~1.0mL.
3. cultural method according to claim 1 or 2, which is characterized in that the volume of culture is 1.0mL.
4. cultural method according to claim 1, which is characterized in that the cell-seeding-density is 0.3 × 106Cell/
mL。
5. cultural method according to claim 1, which is characterized in that the shaking speed is 115rpm.
6. cultural method according to claim 1, which is characterized in that cultivation temperature is 36~37 DEG C, relative humidity 75%
~95%, CO2Concentration 5% (v/v).
7. cultural method according to claim 1, which is characterized in that 24 well culture plates are 3.4mL per pore volume.
8. cultural method described in claim 1 cell strain screening, nutrient media components screening, process conditions sieve in bio-pharmaceuticals
The application chosen.
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| Publication number | Priority date | Publication date | Assignee | Title |
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