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- Challenges and Chances: A Review of the 1st Stem Cell Community Day
- Summertime, and the Livin’ Is Easy…
- Follow-on-Biologics – More than Simple Generics
- Bacteria Versus Body Cells: A 1:1 Tie
- Behind the Crime Scene: How Biological Traces Can Help to Convict Offenders
- Every 3 Seconds Someone in the World Is Affected by Alzheimer's
- HIV – It’s Still Not Under Control…
- How Many Will Be Convicted This Time?
- Malaria – the Battle is Not Lost
- Physicians on Standby: The Annual Flu Season Can Be Serious
- At the Forefront in Fighting Cancer
- Molecular Motors: Think Small and yet Smaller Again…
- Liquid Biopsy: Novel Methods May Ease Cancer Detection and Therapy
- They Are Invisible, Sneaky and Disgusting – But Today It’s Their Special Day!
- How Many Cells Are in Your Body? Probably More Than You Think!
- What You Need to Know about Antibiotic Resistance – Findings, Facts and Good Intentions
- Why Do Old Men Have Big Ears?
- The Condemned Live Longer: A Potential Paradigm Shift in Genetics
- From Research to Commerce
- Chronobiology – How the Cold Seasons Influence Our Biorhythms
- Taskforce Microbots: Targeted Treatment from Inside the Body
- Eyes on Cancer Therapy
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Bioprocess Operation Modes: Batch, Fed-batch, and Continuous Culture
Overview
Feed automation
Perfusion
The addition of nutrients and removal of by-products to and from the culture medium, respectively will affect the cell density and viability, the bioprocess duration, and as a result, the product titer, yield, and cost. This is true for cell culture in bioreactors as well as microbial cultures and is why optimizing the nutrient supply is key in bioprocess development . To help with this, different bioprocess operation modes have been established with varied substrate feeding strategies suited towards particular bioreactor cultures. The main bioprocess operation modes include batch culture, fed-batch culture, continuous culture, and perfusion culture – a type of continuous culture. Here you can find guidance on batch, fed-batch, and continuous culture operation modes, including the advantages and disadvantages, and how to set up the respective bioprocesses.
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What are the differences between batch, fed-batch, and continuous culture?
Read more about the differences between batch, fed-batch, continuous, and perfusion culture and learn more about advantages and disandvantages of the different bioprocess modes.
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Batch culture
In a batch culture, substrate is administered at the start of the bioprocess, with no further additions. The feed is gradually consumed by the culture and there is an accumulation of by-products over time. When the cells enter the stationary growth phase, the culture is harvested.
Advantages
- Simple operation and low risk of contamination
- Suitable for the early stages of experimental design
- Relatively short duration
- Relatively low cell densities and yields
- Toxic by-products accumulate over time
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Fed-batch culture
In a fed-batch culture, batch bioprocessing is conducted for a certain period of time. Then, to counter any decline in cell growth or cell densities, additional substrate is added in increments throughout the rest of the bioprocess.
Advantages
- The most widely used bioprocess mode
- Compared to batch fermentation:
- A higher cell density can be achieved
- Extended duration of the cell culture
- Less substrate overfeeding is required at the start of the culture, to decrease the accumulation of toxic by-products and improve cell growth
- Incremental feeding following the batch fermentation period increases the risk of contamination
- Harvesting is only carried out at the end of the process and so there is a lack of continuous by-product removal
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Continuous culture
In a continuous culture, substrate is continuously added to the culture, with the continuous harvesting of cells and culture medium. Like this, nutrients are replenished and by-products removed. The culture volume remains constant when feeding and harvesting are carried out at the same rate. This allows for a steady state to be achieved and maintains a constant environment and rate of cell growth.
Advantages
- A constant growth environment can be maintained, which can positively influence the product quality
- Steady state cultures can be maintained for up to several months to reduce the culture downtime
- Process operation is more challenging than in batch or fed-batch processes
- Increased risk of contamination due to the extended duration and continuous feeding and harvesting
- Limited traceability, as the culture suspensions aren’t harvested in batches
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Perfusion culture
Perfusion culture is a type of continuous culture where cells are retained or recycled back into the bioreactor. Similar to other types of continuous culture, nutrients are replenished, and toxic by-products are removed to keep the culture in a steady state.
Advantages
- Steady state cultures can be maintained for up to several months to reduce the culture downtime
- High cell densities - and subsequently high yields and volumetric productivity can be achieved
- Due to higher volumetric productivity the working volume and therefore equipment footprint can be reduced, compared to batch or fed-batch processes
- Process operation is more challenging than in batch or fed-batch processes
- Increased risk of contamination due to the extended duration and continuous feeding and harvesting
- Limited traceability, as the culture suspensions aren’t harvested in batches
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Which bioprocess mode is best suited for your application?
You would like to cultivate your cells or microorganisms in a bioreactor but are unsure which process mode to use? In our “Beginners’s Guides Bioprocess Modes” we explain the differences between batch, fed-batch, and perfusion bioprocesses and how to select the right process mode for your needs. We guide you through a complete bioprocess, including inoculum preparation, bioreactor setup, sensor and pump calibration, culture feeding, and sample analysis throughout the run.
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How can feed automation improve bioprocesses? And what is required to establish automated feeding?
Sensor integration
Feed automation can have several benefits in upstream bioprocessing. It reduces manual workload, prevents nutrient depletion, creates a more stable culture environment, and improves yields and standardization. To do so, the metabolic status of the culture and/or nutrient concentrations in the culture medium need to be continuously monitored in real-time. In-process monitors are implemented into bioreactor control systems for the calculated and automated adjustment in feed rate, via the use of feed pumps. Learn more about the integration of sensors with bioprocess control systems.
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Strategies for feed automation
There are different strategies to automize culture feeding. In a time-based strategy a defined volume of feed solution is added to the culture per time increment. In a sensor-based feeding strategy, a feed solution is added based on a sensor reading. Feed automation can be achieved through monitoring of several different parameters, including the concentration of the substrate (e.g., glucose) itself or the dissolved oxygen (DO) concentration or the respiratory quotient (RQ), which are influenced by the metabolic state of the culture. The Eppendorf bioprocess control software packages DASware® control and BioCommand® facilitate the implementation of tailored feedback control loops for feed automation. Learn more about Bioprocess Feed Automation Enabled through Software Scripts in our ebook.
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Perfusion cell culture: What are cell retention techniques for perfusion culture and which devices can be used for these?
Perfusion processes can deliver more product for a given bioreactor volume as they can maintain higher viable cell densities than traditional fed-batch processes. Smaller bioreactors can be used, saving precious lab space. Cell retention is an essential aspect of perfusion culture. It is used to maintain high cell densities while harvesting media and removing by-products, and can be achieved using several different techniques, including:
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Filtration using spin filters
Filtration using spin filters
Filters are used to remove liquid and small molecules from the culture, while retaining cells. Spin filters are the simplest cell retention filter. Porous spin tubes are placed in bioreactors to retain cells on the inside of the tube while allowing other molecules and liquids to pass through. Spin filters can be used in suspension cell as well as microcarrier cultures. They are relatively cheap and suitable for sensitive cell cultures. Clogging can be an issue with time passing, even if it is to a certain extend removed by the spinning motion of the filter. Many Eppendorf glass and stainless-steel bioreactors in a volume range 1 L to 32 L can be used with spin filters.
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Get more information on how a spin filter functions.
Learn, how anchorage-dependent Vero cells were cultivated in perfusion using a microcarrier spin-filter as retention device.
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Tangential flow filtration (ATF and TFF)
Filters are used to remove liquid and small molecules from the culture, while retaining cells.
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Tangential flow filtration (TFF)
These filters are commonly used in bioreactors. With this method, the culture flows in parallel to the filter, rather than perpendicularly to help reduce the risk of clogging.
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Alternating tangential flow (ATF) filtration
Like TFF devices, these filters are also commonly used in bioreactors. ATF filters operate similarly to TFF filters, although the direction of the flow is reversed once in each cycle. This helps to further reduce the risk of clogging and reduce the shear forces on the cells.
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Perfusion process development at small scale
During process development small scale perfusion systems are desired to save resources and reduce costs, especially for culture media, which are a main cost driver in cell culture bioprocessing. ATF devices have been integrated with Eppendorf small scale bioreactor systems, which facilitated perfusion process development in working volumes as low as 230 mL. Read more in our application notes.
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- Using the DASbox® Mini Bioreactor System , ATF perfusion cultivation of CHO cells and HEK293 cells in a working volume as low as 230 mL was established.
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- Using a DASGIP® Parallel Bioreactor System , CHO cells were cultivated in perfusion in a working volume of 1 L.
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Packed-bed bioreactors
Packed-bed bioreactors are filled with a solid growth support on or in which the cells are immobilized. One such support are Fibra-Cel® discs . Fibra-Cel is a porous meshwork of polyester and polypropylene that provides a growth surface for adherent cells and entraps suspended cells. Immobilization of cells in the matrix facilitates the easy harvest of cell-free medium from the bioreactor. Furthermore, Fibra-Cel® offers a high surface to volume ratio and protects cells from damaging shear forces.
Learn, how packed-bed bioreactors were used for the cultivation of anchorage-dependent Vero cells in perfusion.
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Cell-Lift impeller with decanter column
Cell-Lift impellers with decanter columns can be used for perfusion cultivation of cells grown on microcarriers. Cell-Lift impeller rotation creates a negative pressure in the hollow impeller tube causing medium to circulate uniformly in a closed loop. Cells and microcarriers are separated from the medium in the decanter column. The medium is harvested and the cells and microcarriers are transferred back to the bioreactor. Advantages of this system include reduced shear force, high mass transfer of nutrients, and a high oxygen transfer rate.
Many glass and stainless-steel bioreactors from Eppendorf can be equipped with Cell-Lift impellers and decanter columns, including benchtop BioFlo® 320 bioreactors and the CelliGen 510 sterilize-in-place bioprocess system .
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Learn more about the functioning of Cell-Lift impellers with decanter column and how it was used for cultivating Vero cells on microcarriers in perfusion.
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