细胞培养系统：昆虫细胞

2018-01-17 18:14:00    infors3319 分享

导语：All the Infors range can be supplied in an alternative configuration to provide an excellent bioreactor. The following adaptations are made:

Cell Culture System for: Insect cells
Insect cell culture –bioreactor

All the Infors range can be supplied in an alternative configuration to provide an excellent bioreactor. The following adaptations are made: 

* The added security from contamination using magnetic drive and slow speed motor 
* Jacketed with temperature control by water circulation with either an electric element or steam pocket/exchanger 
* Special accessories such as Marine and "cell lift" type impellors 
* Spin filter 
* Sterile sampling system 
* Gas mix,including spearate addition of CO2 into the sinter sparger or head space.

Vessels: 
All vessels are dished (optional hemispherical base) with top axial magnetic drive (easier cleaning, with no crevices) and water circulation for temperature control. The top plate is equipped with 10mm, PG13.5 and rounded -thread 19mm ports ( numbers depending on type). These can be fitted with a septum and retaining collar to allow flaming of the port during inoculation etc. The base has 25mm Ingold ports (numbers depending on size and type).

The vessel is sterilized in situ (usually with PBS) with the reagent bottles, needles etc sterilized separately an autoclave. Re-sterilizable ports are an option, depending on the system.

Stirring
For stirring of insect cell cultures, a one or more marine-type impellors are used as standard (options available), with a brushless motor with integral speed controller for speeds up to 20 - 300 rpm. 


x-DDC Control System:
All fermenter models except Minifors incorporate the x-DDC (eXtended DDC microprocessor control) which uses a high-performance digital system. With the use of almost identical instrumentation, the step from Bench-scale to larger equipment is made considerably easier.

The control panel is compact and is usually mounted above the base unit for clear visibility or within the instrument cabinet (Techfors). The panel can be removed if required. Programming of parameters is via a splashproof keypad with a rotary knob for navigation and an LCD display. Communication is via a robust data-cable. For each fermenter, process information such as parameter set points, PID values etc are stored automatically on an interchangeable "memory card" . This allows changes in fermentation processes by fitting an alternative card or downloading a recipe without re-programming and the chance of errors plus an unlimited number of recipes to be stored.

An "open frame" gas supply system with four magnetic valves allow addition of components for automatic mixing of gasses for oxygen supplementation. An optional mass flow control valve ensures a controlled, steady flow of gas into the vessel. Agitation & temperature control. Four high-accuracy digital peristaltic pumps supplied as standard with the option to add an integral analogue feed pump. Addition and removal of liquids is therefore possible with a standard system (using the antifoam pump for medium take-off). Accepts a conductive probe for automatic control of antifoam addition or media addition/removal in a vessel. Works together with the integral peristaltic pumps.
Set points and controller setup

Set points are entered into the controller prior to inoculation and stored on a memory card. The vessel was allowed to equilibrate prior to inoculation. The pO2 actual value stays above the set point up to (and some time after) inoculation.

Temperature： 28°C
pH： 6.3
DO： 50%
Agitation： 70 - 130 rpm
O2： (Gases) 4 Gas (3 gas is a minimum as CO2 control of pH may not be needed)

PH Control
The pH probe is calibrated prior to the autoclave cycle (refer to Instruction Manual). CO2 gas and liquid base is used to maintain the pH set point. pH control parameters are:

Base： Sodium bicarbonate, 7.5% solution
Acid： Supplied as CO2 gas, if needed (not usual for Insect cells) 
Transfer tubing ：Narrow bore silicone tubing with Marprene insert, as supplied (4mmOD)
Vessel inlet ：4mm fixed pipes in the vessel top plate

PID values: factory default setting should be used unless it is clear adjustment is needed (hunt or drift).

Dissolved Oxygen (pO2) Control
The dissolved oxygen electrode is normally calibrated after the autoclave cycle (refer.Instruction Manual) but can be calibrated in air beforehand for cell culture applications. Control of pO2 can be cascaded and should be set to GasMIx for optimum results The controller automatically maintains the pO2 setpoint using 2 (Minifors) or 3 gases (Air, O2, N2). The gas flow rate should be constant and set at a fairly low rate eg typically 0.05-0.3 vessel volumes per minute (VVM).

Continuous Feed
Feed pumps can be calibrated using the standard tubing to keep track of the liquid quantities entering and exiting the vessel. Either a digital pump with shot and delay dosing or an analogue, continuously-variable pump can be used for medium addition. Samples should be taken several times a day to measure glucose (GlucCell) and cell density (stain and count cells and/or nucleii). If, as is typical, cells are to be retained and just the medium exchanged, a spin filter is added to the vessel and cell-free medium removed at a rate adjusted to match the medium input. Iris v5 software can be used to control nutrient addition.

Example methodology

Medium
Eg. Graces medium or HyQ SFX-Insect medium (supplied by Hyclone in liquid form - serum-free) plus supplements. Serum-free medium overcomes the problem of expense, removal and foaming problems sometimes associated with the use of foetal calf serum (FCS). Use of antifoam in the medium can help suppress foaming and/or addition of reagents such as Pleuronic can help protect cells. Before sterilization, glass vessels are often coated with a barrier such as SigmaCote® to prevent attachment of cells to the walls.

Inoculum 
A Hi-5 or SF9 insect cell inoculum can be prepared in an Infors Shaker (Multitron or Minitron) or in a 
spinner flask (1000ml, filled to 600ml, stirred at 80-120rpm at 27-28oC. Approximately 500ml at a density of 2 x106 cells/ml are needed from this culture.

Method
Electrodes should have been calibrated before autoclaving (100% in air for pO2).
• Sterilize the vessel with a little eg. (10-20ml) phosphate buffer solution (PBS) or water for 60 minutes
• If necessary, remove any remaining liquid from the vessel.
• Add eg.1.45 -1.95L of insect cell culture media to a 3.6LTV vessel.
• Inoculate eg. 50 ml of concentrated insect cell suspension to provide a starting cell count of ~5 x 106 cells/ml, either by pouring under a laminar flow hood or from a flask equipped with an inoculation needle or from a syringe.

After several days of normal batch culture eg 3, introduction of baculovirus at multiplicities of infection (MOI) of approximately 0.9-1 can be made, with the culture proceeding for another 5-7 days. During this time, the required protein is expressed. Use of a spin filter can allow perfusion for medium replenishment and removal of supernatant liquid. . 

Additional Ideas for further Optimisation:
1) Use of Pluronic at 0.1-0.4% (BASF/GIBCO) to protect cells plus use an antifoam such as Sigma O-30 at 0.1% with addition via pump if necessary when foaming is a significant problem. The pluronic would allow a slightly faster gas flow rate by preventing cells and bubbles from being in direct contact.
2) Use of low flow rate of 0.01-0.05 VVM with micro-sparger (eg as low as 0.5 micron) for very small, slow bubbles. Pulsed sparging may also help (On/Off rate determined empirically and may change with increasing cell density i.e. longer On, shorter Off). A ring sparger with a low gas flow rate can work in some circumstances and would be an inexpensive, rapid first step.
3) Use of perfusion culture will certainly enhance cell growth. Accurate control of glucose and glutamate addition will produce higher yields by matching growth to available nutrient and keeping build-up of lactate/ammonia to a minimum. Glucose additions as low as 0.1g/l have been used but this tends to aid product formation - higher concentrations (up to 4g/l) enhance cell growth. Fed-batch would be a good first step to see if improvement is possible with just neutralisation of lactate by pH control.
4) Cell numbers can be estimated from rate of glucose consumption (sample) or inferred from the respiratory quotient RQ. A mass spectrometer or exit O2/CO2 analyser will be necessary for this technique and rates of CO2 evolution/O2 uptake are only likely to be measurable in actively growing, relatively dense cultures. Glucose feed could be optimised by holding RQ to a particular value eg. 1.7.

IRIS SEQUENCE FOR RQ CACLUALTION:

#0 Definitions for simple RQ calculation
DEF WV=2.0
DEF O2=(20.95-ExitO2.v)
DEF CO2=(ExitCO2.v-0.03)
DEF CER=CO2*Flow.v/WV
DEF OUR=O2*Flow.v/WV
RQ.v=CER/OUR

//where WV=working volume of culture 
//O2=amount of oxygen consumed
// CO2=amount of carbon dioxide produced. 
// Exit O2 and Exit CO2 values from mass spec (channels 17 & 18)
//MS inlet in fermenter settings matched to MS outlet allocation.

For control fo a feed pump add these lines:

Feed Pump.sp=IF(RQ.v>1.15){Feed Pump.sp-1}ELSE{Feed Pump.sp}
Feed Pump.sp=IF(RQ.v<0.95){Feed Pump.sp+1}ELSE{Feed Pump.sp}
Feed Pump.sp=IF(Feed Pump.sp<0){Feed Pump.sp=0}ELSE{Feed Pump.sp}
Feed Pump.sp=IF(Feed Pump.sp>100){Feed Pump.sp=100}ELSE{Feed Pump.sp}

//Feed Pump.sp is for inbuilt pump 0-100% On
//Flow rate could be calculated as pump secs x calibration factor
// This could then be used to update working volume to allow for increase over time

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