CN104974933A - Device and method of continuously and repeatedly suspending transient transfection expression recombination protein on large scale - Google Patents
Device and method of continuously and repeatedly suspending transient transfection expression recombination protein on large scale Download PDFInfo
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Abstract
The invention discloses a device and method of continuously and repeatedly suspending transient transfection expression recombination protein on a large scale. The device and method are characterized in that cells are maintained in a relatively good growing status by using a perfusion technology so that the transient transfection process is carried out repeatedly (more than or equal to 2), moreover, the viability of cells of a repeatedly transient transfection group is not significantly reduced in a continuously repeated transient transfection process, the proliferation is not influenced, and therefore, the period of the whole process can be effectively prolonged, the yield of the recombinant protein is increased, and the production cost of the recombination protein is reduced.
Description
Technical field
The present invention relates to a kind of apparatus and method of bioengineering field, be specifically related to a kind of in continuous suspended biological reactor repeatedly transient transfection express the apparatus and method of recombinant protein for a long time.
Background technology
Transfection refers to that eukaryotic cell obtains the process of new genetic marker because foreign DNA mixes.Routine transfection technology can be divided into transient transfection and the large class of stable transfection two.Foreign DNA in transient transfection process/RNA unconformability is in host chromosome, therefore multiple copy number can be there is in a host cell, produce high-caliber expression, but usually only continue several days, after transfection 24 little in 96 hours harvested cell, its concrete time depends on the many factors such as cell type, vector construction.
In recent years, along with the development of transient transfection techniques and cell culture technology, by having carried out suspension culture to some clones generally used (such as HEK293 and Chinese hamster ovary celI), the extensive recombinant protein synthesis of transient transfection can be realized.Select transient expression can be time saving and energy saving, cost-saving in a suitable case.
Through finding the retrieval of existing document, C é line Raymond, Roseanne Tom, " A simplified polyethylenimine-mediated transfection process forlarge-scale and high-throughput applications " literary composition that the people .2011 such as Sylvie Perret delivers April on " Methods " reports a kind of large-scale transient transfection process of work simplification, have studied the process that the transient transfection of suspension cell in orifice plate and reactor expresses recombinant protein respectively.But the transient transfection process in bio-reactor only continue for one week and the output of recombinant protein is very limited.In article there is following defect in large-scale transient transfection:
The first, due to this characteristic of plasmid loss in transient transfection process, transient transfection techniques is only limitted to laboratory level, and industrialization promotion has a lot of limitation;
The second, continue 3-7 days common mass-producing transient transfection technique (as batch), the cycle is short, thus causes whole process costs high.
In sum, existing gene transient transfection express the method cycle of recombinant protein short, yield poorly, cost is high, be not suitable for the production of industrially scalable, be difficult to meet the demand of preclinical laboratory to recombinant protein.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, the apparatus and method that a kind of recombinant protein transient transfection that is continuous, bio-reactor scale is expressed are provided.It is long that the method has the cycle compared with traditional transient transfection method, and output is high, can the advantage such as automatization, therefore can provide in the shorter time than relatively large recombinant protein, for new drug development and screening, meet clinical before to recombinant protein demand.
The extensive continuous several times of a kind of bio-reactor scale provided by the invention suspends and turns the device of expression recombinant protein wink, comprise device for storing liquid, transfection composite mixing device, cell culture apparatus, central control unit, cell retention device, supernatant collection device, cell collection device, some liquid asepsis thrust units and some controlled fast liquid flow conduits, the mode of connection of above-mentioned each device is: device for storing liquid is connected with cell culture apparatus by one first controlled fast liquid flow conduits, the aseptic thrust unit of a first liquid is provided with between device for storing liquid and cell culture apparatus, transfection composite mixing device is connected with cell culture apparatus by one second controlled fast liquid flow conduits, is provided with the aseptic thrust unit of a second liquid between transfection composite mixing device and cell culture apparatus, cell culture apparatus transforms output line with central control unit by signal and is connected, one end of cell culture apparatus is connected with the fore portion that retains of cell retention device by one the 3rd controlled fast liquid flow conduits, the other end of cell culture apparatus by one the 4th controlled fast liquid flow conduits with dam after cell retention device be connected, between cell culture apparatus and the cell retention device after damming, be provided with one the 3rd liquid asepsis thrust unit, cell retention device retains front one end and is connected with supernatant collection device, is provided with one the 4th liquid asepsis thrust unit between supernatant collection device and the cell retention device before damming, cell retention device is retained the rear the other end and is connected with cell collection device by one the 5th controlled fast liquid flow conduits, is provided with one the 5th liquid asepsis thrust unit between cell collection device and the cell retention device after damming.
Described device for storing liquid is common cylinder or square body bottle structure or liquid storing bag, and in order to hold aseptic culture medium, its outlet is tightly connected with controlled fast pipeline system, and has interface on the apparatus and be communicated with air by sterilised membrane filter strainer; Described device for storing liquid is at least provided with three holes, wherein the nutrient solution after sterile filtration is injected described device for storing liquid by controlled fast liquid flow conduits by a hole, nutrient solution after sterile filtration pushes in cell culture apparatus by controlled fast liquid flow conduits by another hole, and another hole communicates with outside atmosphere by air filter; Described device for storing liquid is can high-temperature sterilization unit.
Described transfection composite mixing device has at least reagent to add entrance and mixture outlet, and has the function of automatic stirring mixing liquid, is embodied in possess to be uniformly mixed function and clocking capability, such as adjustable stir speed (S.S.) and setting churning time; The transfection composite of hatching end enters cell culture apparatus by mixture outlet through described second controlled fast liquid flow conduits.
Described cell culture apparatus is at least provided with nutrient solution entrance, nutrient solution outlet, mixture entrance, cell reflux inlet, gas (as air, oxygen, carbonic acid gas, nitrogen) entrance, cell sampling mouth, alkali entrance, cell inoculation mouth, probe insert port (as PH, dissolved oxygen, temperature), high-temperature sterilization, enclosed sterile can carry out cell cultures; Described cell culture apparatus controls the various parameters (as PH, dissolved oxygen, temperature, glucose concn etc.) in cell cultivation process by central control unit.
Described central control unit is the controller of the physical-chemical parameters (as PH, dissolved oxygen, temperature, glucose concn etc.) that at least can record and regulate in various culturing process, this central control unit can control the various parameters of cell cultivation process, makes the growth of optimum cell or obtains object product.
Described cell retention device (as biosep, cell settlement device, Hollow fiber units etc.) is at least provided with cell mixture entrance, the outlet of cell conditioned medium liquid, cell bypass outlet, retains outlet, wherein cell mixture entrance is connected with cell culture apparatus, for being retained by the cell in the cell mixture of discharging from cell culture apparatus, described 3rd controlled fast liquid flow conduits is arranged between described cell mixture entrance and cell culture apparatus; The outlet of cell conditioned medium liquid is connected with supernatant collection device through described 4th liquid asepsis thrust unit, makes the cell conditioned medium liquid in cell retention device be pushed in supernatant collection device by described 4th liquid asepsis thrust unit; Cell bypass outlet connects cell culture apparatus through described 3rd liquid asepsis thrust unit, is pushed in cell culture apparatus for the cell be retained down by cell retention device by the 3rd liquid asepsis thrust unit; Retain outlet and be connected to cell collection device through described 5th liquid asepsis thrust unit, be pushed in cell collection device by described 5th liquid asepsis thrust unit for the cell that cell retention device is retained down.
Described supernatant collection device is provided with bottle cap, described bottle cap is provided with liquid inlet, can sterilizing storing unit for what have two holes at least, wherein a hole is connected with cell retention device top (as cell conditioned medium liquid exports) by controlled fast liquid flow conduits, cell culture supernatant enters supernatant collection device by the pushing effect of described 4th liquid asepsis thrust unit, and another hole is communicated with outside atmosphere by air filter.
Described cell collection device be have two holes at least can sterilizing storing unit, wherein a hole is connected with cell retention device bottom (as retaining outlet) by described 5th controlled fast liquid flow conduits, cell after retaining enters cell collection device by the pushing effect of described 5th liquid asepsis thrust unit, and another hole is communicated with outside atmosphere by air filter.
Described controlled fast liquid flow conduits is the pipeline controlling nutrient solution flow velocity by Sliding Control valve or clip, and pipeline is such as silicone tube.
Described liquid asepsis thrust unit is the device that sterilely can promote the liquid-flow in pipeline, as peristaltic pump.
Above-mentioned extensive continuous several times provided by the invention suspends and turns the device of expression recombinant protein wink, its principle is as follows: a kind of cell is after transient transfection, only have part cell can accept external source recombinant plasmid and carry out the process that recombinant protein was transcribed, expressed to exogenous plasmid, because the metabolic rate (as specific growth rate, than glucose consumption rate, than cell yield etc.) successfully accepting the cell of plasmid will lower than the cell not accepting plasmid, the growing state thus not accepting the cell of plasmid under identical culture condition is better than the cell accepting plasmid.Cell in cell culture apparatus is by after cell retention device, the aseptic thrust unit (as peristaltic pump) of active cell device for trapping, the different cell of growth speed to be pushed uniformly in cell retention device and according to cell growth state needs, be partly refluxed in cell culture apparatus, collect detection and purifying that supernatant liquor carries out recombinant protein simultaneously.Due to the cell that do not accept plasmid and the difference of specific cell growth rate accepting plasmid, cell (the not accepting recombinant plasmid) quantity that in cell culture apparatus, specific growth rate is large can get more and more, simultaneously, accept the cell of exogenous plasmid because the characteristic of transient transfection plasmid loss, along with the growth of incubation time, also the cell not accepting exogenous plasmid can be become at any time, thus after for some time, cell in cell culture apparatus is provided with the ability accepting recombinant plasmid transient transfection once more, now then can carry out second time transient transfection.This technique of the present invention also uses perfusion technique (perfusion) and maintains cell and be stored in a reasonable growth conditions, the process of transient transfection is carried out repeatedly (>=2), thus the cycle of whole technique can effectively be extended, increase the output of recombinant protein, reduce the production cost of recombinant protein.
When extensive continuous several times of the present invention suspends and turns the device work expressing recombinant protein wink, nutrient solution in device for storing liquid is under the aseptic thrust unit effect of described first liquid, enter in cell culture apparatus by described first controlled fast liquid flow conduits, and central control unit controls the various parameters (such as but not limited to PH, dissolved oxygen, temperature etc.) of cell cultures, grow under the cell cultures parameters optimization condition that cell controls at central control unit.When completing first time transient transfection, nutrient solution in device for storing liquid is under the aseptic thrust unit effect of described first liquid, enter in cell culture apparatus by described first controlled fast liquid flow conduits, and central control unit controls the various parameters (such as but not limited to PH, dissolved oxygen, temperature etc.) of cell cultures, grow under the cell cultures parameters optimization condition that cell controls at central control unit; Meanwhile, the speed of the described 4th liquid asepsis thrust unit at the setting weight regulation and control supernatant collection device place that central control unit is claimed by continous pouring, makes the nutrient solution cumulative volume of cell culture apparatus remain unchanged.The travelling speed of the described 3rd liquid asepsis thrust unit (as peristaltic pump) between the cell bypass outlet of cell retention device and cell culture apparatus requires sets itself according to cell cultures, unsuitable excessive or too small (such as in cell retention device, the excessive cell injury that then refluxes of speed is excessive, the too small then rejection effect of speed is not good, causes comparatively many cells all to enter in supernatant liquor).When the cell density in cell culture apparatus is higher than the perfusion cell density set, central control unit then active cell device for trapping retain outlet with cell collection device between the 5th liquid asepsis thrust unit (as peristaltic pump), the part cell retained is pushed in cell collection device, when the cell density in cell culture apparatus is lower than the perfusion cell density set, then close the 5th liquid asepsis thrust unit (as peristaltic pump) retained between outlet and cell collection device of cell retention device, make cell density maintain setting density constant.By the control process of this intelligence, cell can maintain a reasonable cell state.Now can again add recombinant plasmid, transfection mediation reagent and cell transfecting liquid to transfection composite mixing device, start timing agitation function, prepare fresh transfection composite, under the aseptic thrust unit effect of described second liquid, transfection composite is pushed in cell culture apparatus, carries out secondary transfection.Secondary Transfected cells enters again the state similar to after a transfection, regulates and controls filling process to maintain the good growth of cell, expression status again by central control unit.The simultaneously supernatant collection device collecting cell culture supernatant that can continue, the recombinant protein in purified pool supernatant liquor carries out analyzing, detect and other preclinical pharmacology, toxicity detect.Extensive continuous several times suspension of the present invention turns the device of expressing recombinant protein wink and can carry out repeatedly transfection, and concrete transfection number of times can according to cell state, and the practical situation such as plasmid character and product are determined flexibly.
Extensive continuous several times of the present invention suspends and turns the method expressing recombinant protein wink and be mainly and make use of perfusion (perfusion) technology maintenance cell and be stored in a reasonable growth conditions, the process of transient transfection is carried out repeatedly (>=2 times), particularly, the method using above-mentioned extensive continuous several times suspension to turn the device of expressing recombinant protein wink can mainly comprise the following steps:
(1) install, connect above-mentioned extensive continuous several times and to suspend the complete assembly that turns in wink and express recombinant protein carry out high-temperature sterilization; High-temperature sterilization condition high-temperature sterilization can be 121 DEG C known by technician, 30min;
(2) in cell culture apparatus, a certain amount of aseptic culture fluid is pumped under aseptic condition, the amount of nutrient solution at least can there be detection probe as the probe of detected temperatures, dissolved oxygen, pH, carry out Sterility testing, such as, under 37 DEG C of conditions, carry out Sterility testing reach 48 hours;
(3) aseptically, that cultivate in advance with certain cell density inoculation in cell culture apparatus, in good condition cell to be transfected, and according to the best transfection conditions (plasmid concentration of different cell, different plasmid, transfection reagent and plasmid concentration ratio, incubation time), by DNA, transfection mediation reagent, cell transfecting liquid mixes in transfection composite mixing device and the transfection composite obtained after hatching joins in cell culture apparatus; Usually, the cell density to be transfected of inoculation can be cell density common in recombinant plasmid cell transfecting process known by the technical staff, as 0.05 ~ 1 × 10
7individual cell/mL(is according to inoculation volume computing);
(4) according to the growth characteristics of culturing cell and the working volume of cell culture apparatus, by every reason of the reactor of cell culture apparatus, life, change target setting in the top condition of cell cultures and protein expression, control perfusion parameters by central control unit simultaneously, set the perfusion parameters of this reactor, return velocity, the useless cell velocity of discharge etc.;
(5) according to the change of concrete protein expression level, after perfusion culture certain hour, again add appropriate transfection composite, according to the transfection conditions transfection again in above-mentioned steps (3), this process can repeat for several times;
(6) such as every day, collecting cell nutrient solution carried out detection and the process of subsequent recombination protein purification of protein content each work period, after collecting enough albumen, stopped cell cultures and protein expression process.
Described best transfection conditions (plasmid concentration, transfection reagent concentration, incubation time) can be: plasmid DNA concentration is 0.5-3 μ g/mL, DNA: transfection mediation ratio of reagents is 1:2-1:10, incubation time is 5-30 minute, to form stable DNA-transfection mediation reagent complex.Transfection mediation reagent can select conventional all ingredients, such as Lipofectamine2000,25KDPEI, PEI MAX etc. in the industry, but is not limited to only mentioned reagent.
In described step (4), the top condition of cell cultures and protein expression can the temperature set by each bio-reactor cell cultures known by technician, pH value, oxyty value etc.Can be such as: nutrient solution uses T39(CHO) or Free Style(293), pre-treatment to 37 DEG C ± 0.2 DEG C, pH value 7.1 ± 0.1, DO25% ~ 55%, even with the index of the velocity interpolation nutrient solution of 50 ~ 150 turns.
In described step (4), control perfusion parameters to carry out testing determining according to cell growth condition and protein expression process, its concrete numerical value can be determined through overtesting by persons skilled in the art, such as, can be that 0.05 bioreactor culture volume/sky is to 20 bioreactor culture volume/skies.
In described step (5), the determination repeating the multiplicity of transient transfection process is different according to the protein expression level under different cells, different recombinant plasmid conditions, to determine for the purpose of the enough target proteins of final results to reach, can be more than 2 times or carry out transfection procedure more frequently.
Compared with prior art, beneficial effect of the present invention is as follows:
It is long that the apparatus and method that extensive continuous several times suspension provided by the invention turns expression recombinant protein wink have the cycle compared with traditional transient transfection method, output is high, can the advantage such as automatization, therefore can provide than relatively large recombinant protein in the shorter time, for new drug development and screening, meet clinical front to recombinant protein demand.
Accompanying drawing explanation
Fig. 1 is that the extensive continuous several times of the present invention one example suspends and turns the schematic diagram of the device of expressing recombinant protein wink;
Fig. 2 is the variation diagram of cultivated days after cell density and transfection in the transfection process of the present invention one example;
Fig. 3 is the variation diagram of cultivated days after transfection efficiency and transfection in the transfection process of the present invention one example;
Fig. 4 is the total amount of the blood coagulation II factor of accumulation cell expressing in a transfection process cycle process of the present invention one example.
Embodiment
Be described in detail embodiments more of the present invention below in conjunction with accompanying drawing, to help understanding the present invention, but following detailed description is not intended to limit the scope of the invention.Above description describes content of the present invention haply, and specific embodiment below will more directly embody situation of the present invention, but it must be noted that, be only used to the object be illustrated at this example enumerated, the present invention is not limited thereto.
Refer to Fig. 1, turn the concrete structure schematic diagram of the device of expressing recombinant protein wink for a kind of extensive continuous several times provided by the invention suspends.As shown in the figure, this device comprises device for storing liquid 1, transfection composite mixing device 2, cell culture apparatus 3, central control unit 4, cell retention device 5, supernatant collection device 6, cell collection device 7, five liquid asepsis thrust units (8,9,10,11,12), multiple controlled fast liquid flow conduits; Its mode of connection is: device for storing liquid 1 is connected with cell culture apparatus 3 by one first controlled fast liquid flow conduits (as silicone tube), is provided with the aseptic thrust unit 9 of a first liquid between device for storing liquid and cell culture apparatus; Transfection composite mixing device 2 is connected with cell culture apparatus 3 by one second controlled fast liquid flow conduits, should be provided with the aseptic thrust unit 8 of a second liquid in the middle of both; Cell culture apparatus 3 transforms output line with central control unit 4 by signal and is connected; Cell culture apparatus 3 one end retains fore portion by one the 3rd controlled fast liquid flow conduits with cell retention device 5 and is connected, the other end by one the 4th controlled fast liquid flow conduits with dam after cell retention device 5 be connected, be provided with one the 3rd liquid asepsis thrust unit 12 between cell culture apparatus 3 and the cell retention device 5 after damming; Cell retention device 5 retains front one end and is connected with supernatant collection device 6, is provided with one the 4th liquid asepsis thrust unit 10 between supernatant collection device 6 and the cell retention device 5 before damming; Cell retention device 5 is retained the rear the other end and is connected with cell collection device 7 by one the 4th controlled fast liquid flow conduits, is provided with one the 5th liquid asepsis thrust unit 11 between cell collection device 7 and the cell retention device 5 after damming.
Wherein, cell retention device 5 is provided with cell mixture entrance, the outlet of cell conditioned medium liquid, cell bypass outlet, retains outlet, wherein cell mixture entrance is connected with cell culture apparatus 3 by described 3rd controlled fast liquid flow conduits, for being retained by the cell in the cell mixture of discharging from cell culture apparatus 3; The outlet of cell conditioned medium liquid is connected with supernatant collection device 6 through the 4th liquid asepsis thrust unit 10, makes the cell conditioned medium liquid in cell retention device be pushed in supernatant collection device 6 by the 4th liquid asepsis thrust unit 10; Cell bypass outlet connects cell culture apparatus 3 through the 3rd liquid asepsis thrust unit 12, is pushed in cell culture apparatus 3 for the cell be retained down by cell retention device 5 through cell bypass outlet by the 3rd liquid asepsis thrust unit 12; Retain outlet and be connected to cell collection device 7 through the 5th liquid asepsis thrust unit 11, be pushed in cell collection device 7 by the 5th liquid asepsis thrust unit 11 retaining exit for the cell that cell retention device 5 is retained down.
In following the present invention one specific embodiment, liquid asepsis thrust unit all adopts peristaltic pump.
To recombinate the blood coagulation II factor to use method transient transfection of the present invention to express people below, describe extensive continuous several times of the present invention in detail and to suspend the method turning in wink and express recombinant protein.
Example: bio-reactor continuous several times transient transfection is expressed people and to be recombinated the blood coagulation II factor
Cell: serum free suspension domestication HEK293 cell strain;
Nutrient solution: the Excell293 serum-free medium that U.S. Sigma produces;
Cell culture apparatus: the 3L zooblast reactor that Dutch Applicon company produces;
1) sterility of the system before inoculation detects and Detection of Stability
Design temperature 37 DEG C on the control tower that Dutch Applicon company produces, pH value 7.2, oxyty value 50%, by thermostatic water-jacket control temperature, the flow valve on control tower is control CO automatically
2, N
2, O
2the method of three tunnel air inlet bubbling oxygen supplys controls oxyty and molten gas concentration lwevel, control tower is connected with the peristaltic pump of 1mol/L NaOH and 1mol/L HCL with control PH, nutrient solution is added to set(ting)value according to the weighing system signal control of perfusion, the tank body of bio-reactor is connected with simultaneously the liquid inlet of supplementary fresh medium, and for cell backflow cell retention device, complete assembly has been connected and installed latter 121 DEG C, high pressure steam sterilization 30min.After cooling, be placed in sterilisable chamber, in system, pass into aseptic PBS damping fluid, wash cycles whole system.Subsequently, discharge PBS, add fresh medium, the temperature starting control tower controls and stirs, and continues 24h, checks whether microbiological contamination and system run all right.Check aseptic after, even with the index of the velocity interpolation nutrient solution of 80rpm.
2) the front cultivation of the transfection of cell and for the first time transient transfection
With 1 × 10
6the density of individual cell/mL prepares cell suspension, to above-mentioned 3L(working volume 2L) in bio-reactor inoculating cell suspension to final concentration 2.5 × 10
5individual cell/mL, batch cultivation, after 48 hours, treats that cell amplification is to 1 × 10
6first time transient transfection is carried out during the density of individual cell/mL, this example selects direct transfection method, (be cylindrical appliance in this example to transfection composite mixing device, there are a fluid inlet and a liquid outlet, seal with porous rubber plug, rubber consent inserts controlled speed, can timing agitation device) in add recombinant plasmid pGMAX-FII and cell transfecting liquid, 120rpm stirs 5min, then transfection mediation reagent PEI MAX is added, 120rpm stirs 5min, stirs and terminates rear stationary incubation 10min.Wherein plasmid pGMAX-FII amount is 2 μ g/10
6individual cell, DNA:PEI=1:2.5.Be pushed in cell culture apparatus by peristaltic pump by transfection composite, cell carries out transfection.Within after transfection 48 hours, open perfusion, set each desired value and be respectively temperature 37 DEG C, pH value 7.0, oxyty value 40%, glucose concn 1.5g/L, control automatically to regulate the fluid inlet of cell culture apparatus and the peristaltic pump rotating speed of liquid outlet by master control system, to maintain the Growth of Cells environment of setting, master control system is according to bio-reactor level electrode and temperature simultaneously, PH, dissolved oxygen, the rotating speed of peristaltic pump that in the signal auto-adjustment control device for storing liquid of molten carbon dioxide electrode, nutrient solution adds and control device, to ensure the supply of nutrient solution.Irrigation rate is set to 1 reactor tank body volume/sky, and useless cell rate of discharge is set to 1/3 reactor tank body volume/sky.Collect supernatant liquor every day and in-80 DEG C of storages, for carrying out next step blood coagulation II factor protein purifying, quality test and preparation set-up procedure.
3) cell repeatedly transient transfection
First time transient transfection after the 6th day, stop perfusion culture, abandon a part of cell and add fresh medium, regulate cell density to 1 × 10
6individual cell/mL, incubated overnight 24 hours, regulates cell to 1 × 10 again
6individual cell/mL carries out second time transient transfection.(be cylindrical appliance in this example to transfection composite mixing device, there are a fluid inlet and a liquid outlet, seal with porous rubber plug, rubber consent inserts controlled speed, can timing agitation device) in add recombinant plasmid pGMAX-FII and cell transfecting liquid, 120rpm stirs 5min, then add transfection mediation reagent PEI MAX, 120rpm stirs 5min, stirs and terminates rear stationary incubation 10min.Wherein plasmid pGMAX-FII amount is 1.5 μ g/10
6individual cell, DNA:PEI=1:2.5.Be pushed in cell culture apparatus by peristaltic pump by transfection composite, cell carries out transfection.Within after transfection 48 hours, open perfusion, perfusion culture is consistent with first time transient transfection culturing process.
First time transient transfection after the 13rd day, repeat above-mentioned steps 3) described in the treating processes of second time transient transfection, after first time transient transfection, within the 14th day, carry out third time transient transfection, after transfection conditions and transfection, culture condition is completely the same with second time transient transfection process.
In cell cultivation process, every day collects supernatant liquor and is placed in-80 DEG C of preservations, for carrying out next step blood coagulation II factor protein purifying, quality test and preparation set-up procedure.
4) produced
After cultivation terminates, off-response device cleaning experiment equipment.
The culturing process parameter of the present embodiment refers to Fig. 2 and Fig. 3.Wherein Figure 2 shows that the variation diagram of cultivated days after cell density and transfection in the transfection process of above-mentioned example.As seen from Figure 2, repeatedly the cell state of transient transfection group Cell viability in the transient transfection process of continuous three times is substantially steady in this example, later stage appearance declines slightly, and Cell viability does not significantly decrease in whole transfection period, and propagation is not affected.In whole culturing process, cell keeps good growth conditions, and Cell viability remains on more than 90%.
Shown in Figure 3, be the variation diagram of cultivated days after transfection efficiency in the transfection process of above-mentioned example and transfection.Visible in figure, in this example, after first time transient transfection the transfection efficiency of cell and blood coagulation II factor protein expression amount present first rise after downward trend, after second time transient transfection and third time transient transfection, significantly rising appears in the transfection efficiency of cell and the expression amount of blood coagulation II factor protein.Whole continuous several times transient transfection process cycle 21 days, the process cycle more traditional batch of transient transfection is cultivated and is obviously extended.
Refer to Fig. 4, it illustrates accumulation in whole process cycle process and calculate the total amount of the blood coagulation II factor of cell expressing, the transient transfection technique of this example of the present invention obtains blood coagulation II factor total amount and adds 97% than traditional transient transfection technique.
Example of the present invention is only above, under the instruction of the present invention and above-described embodiment, those skilled in the art are easy to predict, cited or each raw material that exemplifies of the present invention or its equivalent alterations, each working method or its equivalent alterations can realize the present invention, and the parameter bound value of each raw material and working method, interval value can realize the present invention, do not enumerate embodiment at this.
Claims (10)
1. an extensive continuous several times suspends and turns the device of expression recombinant protein wink, it is characterized in that, comprise: device for storing liquid, transfection composite mixing device, cell culture apparatus, central control unit, cell retention device, supernatant collection device, cell collection device, some liquid asepsis thrust units and some controlled fast liquid flow conduits, the mode of connection of above-mentioned each device is: device for storing liquid is connected with cell culture apparatus by one first controlled fast liquid flow conduits, the aseptic thrust unit of a first liquid is provided with between device for storing liquid and cell culture apparatus, transfection composite mixing device is connected with cell culture apparatus by one second controlled fast liquid flow conduits, is provided with the aseptic thrust unit of a second liquid between transfection composite mixing device and cell culture apparatus, cell culture apparatus transforms output line with central control unit by signal and is connected, one end of cell culture apparatus is connected with the fore portion that retains of cell retention device by one the 3rd controlled fast liquid flow conduits, the other end of cell culture apparatus by one the 4th controlled fast liquid flow conduits with dam after cell retention device be connected, between cell culture apparatus and the cell retention device after damming, be provided with one the 3rd liquid asepsis thrust unit, cell retention device retains front one end and is connected with supernatant collection device, is provided with one the 4th liquid asepsis thrust unit between supernatant collection device and the cell retention device before damming, cell retention device is retained the rear the other end and is connected with cell collection device by one the 5th controlled fast liquid flow conduits, is provided with one the 5th liquid asepsis thrust unit between cell collection device and the cell retention device after damming.
2. extensive continuous several times according to claim 1 suspends and turns the device of expression recombinant protein wink, it is characterized in that, described cell retention device is at least provided with cell mixture entrance, the outlet of cell conditioned medium liquid, cell bypass outlet, retains outlet, wherein cell mixture entrance is connected with cell culture apparatus, for being retained by the cell in the cell mixture of discharging from cell culture apparatus, described 3rd controlled fast liquid flow conduits is arranged between described cell mixture entrance and cell culture apparatus; The outlet of cell conditioned medium liquid is connected with supernatant collection device through described 4th liquid asepsis thrust unit, makes the cell conditioned medium liquid in cell retention device be pushed in supernatant collection device by described 4th liquid asepsis thrust unit; Cell bypass outlet connects cell culture apparatus through described 3rd liquid asepsis thrust unit, is pushed in cell culture apparatus for the cell be retained down by cell retention device by the 3rd liquid asepsis thrust unit; Retain outlet and be connected to cell collection device through described 5th liquid asepsis thrust unit, be pushed in cell collection device by described 5th liquid asepsis thrust unit for the cell that cell retention device is retained down.
3. extensive continuous several times according to claim 1 suspends the device turning in wink and express recombinant protein, and it is characterized in that, described transfection composite mixing device is at least provided with reagent and adds entrance and mixture outlet, and possesses and be uniformly mixed function and clocking capability.
4. extensive continuous several times suspends and turns a method for expression recombinant protein wink, it is characterized in that, mainly comprises the steps:
(1) provide the extensive continuous several times according to any one of a claim 1-3 to suspend and turn the device of expression recombinant protein wink, install, connect complete assembly and carry out high-temperature sterilization;
(2) in cell culture apparatus, pump into aseptic culture fluid under aseptic condition, the amount of nutrient solution at least can there be detection probe, then carries out Sterility testing;
(3) aseptically, prior that cultivate, the in good condition cell to be transfected of inoculation in cell culture apparatus, determine best transfection conditions according to different cell, different plasmid, under described best transfection conditions, DNA, transfection mediation reagent, cell transfecting liquid are hatched and joined in cell culture apparatus by the transfection composite obtained in transfection composite mixing device; Wherein, described best transfection conditions comprises plasmid concentration, transfection reagent and plasmid concentration ratio, incubation time;
(4) according to the growth characteristics of culturing cell and the working volume of cell culture apparatus, by every for the reactor of cell culture apparatus reason, life, change target setting in the top condition of cell cultures and protein expression, simultaneously by central control unit Controlling System perfusion parameters, described systemic perfusion parameter comprises perfusion, the return velocity of reactor, the cell velocity of discharge of giving up;
(5) according to the change of concrete protein expression level, after perfusion culture certain hour, again add appropriate transfection composite, according to the transfection conditions transfection again in above-mentioned steps (3), this process repeats at least one times;
(6) each work period collecting cell nutrient solution carries out detection and the process of subsequent recombination protein purification of protein content, after collecting enough albumen, stops cell cultures and protein expression process.
5. extensive continuous several times according to claim 4 suspends and turns the method for expression recombinant protein wink, and it is characterized in that, in described step (3), the inoculating cell density of cell to be transfected is 0.05 ~ 1 × 10
7individual cell/mL.
6. extensive continuous several times according to claim 4 suspends and turns the method for expression recombinant protein wink, it is characterized in that, in described step (3), best transfection conditions comprises: plasmid DNA concentration is 0.5-3 μ g/mL, plasmid DNA: transfection reagent ratio is 1:2-10, incubation time is 5-30 minute, to form stable DNA-transfection mediation reagent complex.
7. extensive continuous several times according to claim 4 suspends and turns the method for expression recombinant protein wink, it is characterized in that, in described step (4), the top condition of cell cultures and protein expression comprises: temperature is 37 ° of C ± 0.2 ° C, pH value is 7.1 ± 0.1, dissolved oxygen is 25% ~ 55%, and rotating speed is 50 ~ 150 revs/min.
8. extensive continuous several times according to claim 4 suspends and turns the method for expression recombinant protein wink, it is characterized in that, in described step (4), the perfusion of described reactor, return velocity, the useless cell velocity of discharge are 0.05-20 bioreactor culture volume/sky.
9. extensive continuous several times according to claim 4 suspends and turns the method for expression recombinant protein wink, and it is characterized in that, the multiplicity of described transfection process is more than 2 times.
10. extensive continuous several times suspends and turns a method for expression recombinant protein wink, it is characterized in that, utilizes perfusion technique to maintain cell and is stored in a reasonable growth conditions, the process of transient transfection is carried out repeatedly.
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| CN116769832A (en) * | 2023-05-23 | 2023-09-19 | 皓阳生物科技(上海)有限公司 | Transient transfection method of mammalian cells |
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| CA3205588A1 (en) * | 2020-12-21 | 2022-06-30 | Pfizer Inc. | Methods and systems for improved cell transfection |
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