Cell immobilization is a standard biochemical engineering technique for encapsulating cells which are then used in reactors. Cells are needed in manufacturing for the production of secondary metabolites such as antibiotics, cancer drugs, amino acids or indeed any other product. These immobilized cells are also ingested as probiotics in foods such as beverages and yogurts with all the health benefits that follows.
Issues With Free Cell Cultures
The issues with free cell cultures are numerous. For example, any cell fermentations involved in secondary metabolite production incur a high production cost. This is usually due to the slow growth of cells with quite often low product yields, a fair degree of genetic instability of the fermented selected cells and in many examples, intracellular accumulation of the product. Cell immobilization appears to overcome some of these issues although the technique is not without its own problems.
The Basic Principles Behind Cell Immobilization
The principal objective is to prevent the cells from being free roaming as it were, so they are constrained or imprisoned in a matrix or membrane. This confines the cells to a defined volume which constitutes the reactor. Their immobilization prevents their movement into the mobile phase which contains the substrates, nutrients and of course any product which is made by the cells.
The matrix these cells are contained in are always polymers which must be robust enough to withstand the rigours of a reactor. These polymers can come from a variety of sources and include sodium and calcium alginate, polystyrene, collagen, agar, starch and cellulose derivatives. We look at this in a bit more detail later. The key difference is that when the cells are immobilized they are literally no longer free to divide as they normally would. The cells are also protected within the matrix.
When designing an immobilised cell reactor, there are two stages to their production. In the first stage, the conditions are optimised for biomass production usually as a suspension culture in readiness for immobilization. In the second stage the conditions are altered so they are optimal for creating the bioreactor using the immobilized cells.
Simple History Behind Cell Immobilization
In the 1950s researchers seeking to understand cell behaviour started exploring their metabolism when fixed to surfaces. This developed through the 1960s with a better understanding of behaviour at various interfaces. These were mainly liquid-solid and gas-liquid interfaces. Very little was known about growth and how the metabolic activity of such bacteria might alter when they moved from a free living state such as when they were suspended in a liquid medium to one where they might be living at a liquid-solid interface. Studies at the Institute for Agricultural Research in Tohoku University, Sendai, Japan by Hattori and Furusaka which were published right at the beginning of the 1960s revealed how the metabolic behaviour of the bacteria Escherichia coli altered when it was bound to a resin.
In the 1970s, the concept of cell immobilization exploded in considerable excitement as the technology became an extremely viable alternative to traditional production methods. The development through the 80s to the present day has broadened out to include plant cells and mammalian cell systems with extraordinary benefits in combating disease for example.
The Advantages Of Immobilized Cell Culture
(1) The cells are retained for much longer in an immobilized cell system than when freely mobile as it were, which means that they can be used for a much longer period. The cell density in an immobilized cell bioreactor is substantially increased often by 2 to 4 times that of a suspension culture where the latter has an average cell density is between 10 and 30 g/litre. This also implies that smaller reactors can be used because of the raised cell density. As a result the cost of the medium can be lower as is the subsequent cost of the equipment needed and the subsequent degree of downstream processing. By separating the cells from the medium, the downstream processing requirements are usually simplified especially if the desired product (hopefully) is extracellular.
(2) Keeping the cells in a matrix protects them from shear forces such as those produced by an impellar or stirrer. It also means a simpler reactor design can be employed. Air lift fermenters are classic examples of a fermentation arrangement which can be exploited in this situation.
(3) Non-dividing cells are often generated or stabilised in the situation with immobilization because they become less prone to genetic changes and so produce a more uniform, more stable production rate.
(4) The growth of the cells and product formation are decoupled from each other. That permits product optimization without affecting the growth of the cells.
(5) Cell suspensions often increase the viscosity of the medium in which they are fermenting so immobilization keeps fluid viscosity to a minimum. It means that mixing and aeration issues are also kept to a minimum.
(6) Immobilization can also encourage secondary metabolite formation. Immobilization maintains viable cells over an extended period of time. This implies that the bulk of the product is released into the extracellular medium in a stable form. It also reduces the cost of phytochemical productions for example or keeps inhibitors away from the site of cellular catalysis.
Materials Used For Cell Immobilization
A vast range of methods have been explored for immobilizing microbial cells. They include both ionic and covalent binding to water insoluble ion exchange materials, adsorption onto solid surfaces and entrapment into a polymeric matrix. Most processors nowadays seem to prefer the entrapment method in a gel or matrix. Unlike their immobilized enzyme counterparts, the methods to generate immmobilized cells have been too damaging to preserve cellular integrity so more refined techniques have been exploited.
One of the most useful and robust are the alginates of calcium or sodium. These are especially popular where they might be ingested as with probiotics because they are often food-grade. One of the reasons is that alginate can be polymerized at room temperature using Ca2+ ions.
Polyacrylamide gel is a robust, water insoluble polymer which holds onto cells extremely well. We also inlcude agar, agarose and carrageenan. Polyurethane foam is also popular for immobilizing plant cells. In some cases plant cells are entrapped by inclusion in membrane reactors.
Very early immobilization studies were studied using Dowex 1, X-4 in its chloride form which has a mesh size of 100-200 (Hattori & Furusaka, 1960). The material has largely fallen out of favour because it doesn’t hold onto cells as others might do.
Hydrogels were introduced in the 90s help with mammalian cell immobilization (Jen et al., 1996). These have significant material benefits including selective permeability to gases and nutrients and suitable levels of chemical and mechanical stability. They are flexible enough for them to allow for uniform cell distribution with defined mechanical strength and membrane thickness. The hydrogels are so versatile that they are ideal for tissue engineering and analytical applications as well as developing immobilized cells for disease therapy.
Preparation Of An Immobilized Cell System
The preparation of an immobilized cell system is usually quite straightforward. A cell broth is usually fermented to a cell concentration level of 1010 to 1011. The cell suspension is mixed with an appropriate amount of resin or gel and held at a temperature optimal for binding for 30 minutes. The resin needs to be washed a number of times until the wash concentration contains 106 cells/ml.
Bioreactors are designed to accommodate different immobilized cell systems. The optimal design depends on the method of immobilization. In many ways cells are can be trapped in a permeable gel or entrapped between a semi-permeable membrane. A semi-permeable membrane is ideal for immobilized plant cells. Membrane configurations are important in processes that exploit their semi-permeable nature.
The semi-permeable membrane system allows for bound cells to come into contact with the recirculating medium. These cells can be held in a system at a very high cell density under very mild conditions. There are a number of options where the circulating medium can flow in a single plane over the cells or have a double sided flow so that the bound cells are exposed as much as possible. In some cases the membrane reactor system can be a multi-membrane situation.
Hattori, T., Furusaka, C. (1960) Chemical Activities of E. Coli Adsorbed On A Resin. J. Biochem. (Jpn). 48 (6) pp. 831
Jen, A.C., Conley Wake, M., Mikos, A.G. (1996) Review: Hydrogels for cell immobilization. Biotech. Bioeng. 50(4) pp. 357-364 (Article)