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Alternative Culture Systems

Cook Book Volume 12

Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition

Cell Culture Scale-up Systems

Most tissue culture is performed on a small scale where relatively small numbers of cells are required for experiments. At this scale cells are usually grown in T flasks ranging from 25cm2 to 175cm2. Typical cell yields from a T175 flask range from 1x107 for an attached line to 1x108 for a suspension line. However, exact yields will vary depending on the cell line. It is not practical to produce much larger quantities of cells using standard T flasks, due to the amount of time required for repeated passaging of the cells, demand on incubator space and cost.

When considering scaling up a cell culture process there are a whole range of parameters to consider which will need to be developed and optimised if scale-up is to be successful. These include problems associated with nutrient depletion, gaseous exchange, particularly oxygen depletion, and the build up of toxic by-products such as ammonia and lactic acid. To optimise such a process for quantities beyond 1L volumes it is best left to expert process development scientists. Note, in these cases often a “scaledown” approach is adopted to allow many parameters to be evaluated on many replicates.

However, there are many commercially available systems that attempt to provide a "half-way house" solution to scale-up which do not necessarily require expert process development services. A selected list of some of the systems available along with a brief summary of their potential yields, advantages and disadvantages is provided in Table 4.

Table 4. ‘Half-Way House’ Solutions to Scale-up - without attempting to adapt cells or the process.

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Scale-up Solutions

The image in Figure 4 shows a BIOSTAT® A Plus autoclavable bioreactor from Sartorius Stedim that can be used for cell culture with a working volume of 1L to 5L. Please refer to the following sub-sections for a variety of alternative scale-up solutions.

A word of caution – although the systems listed in Table 4 are often described as “off-the-shelf” solutions to scale-up they are not universally applicable to all cell types and often require a period of familiarisation and optimisation.

Figure 4 Biostat® A plus bioreactor from Sartorius Stedim Biotech

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Roller Bottle Culture

This is the method most commonly used for initial scale-up of attached cells also known as anchorage dependent cell lines. Roller bottles are cylindrical vessels that revolve slowly (between 5 and 300 revolutions per hour) which bathe the cells that are attached to the inner surface with medium. Roller bottles are available typically with surface areas of 1050cm2. The size of some of the roller bottles presents problems since they are difficult to handle in the confi ned space of a microbiological safety cabinet. Roller bottles with expanded inner surfaces have become available which has made handling large surface area bottles more manageable, but repeated manipulations and subculture with roller bottles should be avoided if possible. A further problem with roller bottles is with the attachment of cells since some cells lines do not attach evenly. This is a particular problem with epithelial cells. This may be partially overcome by optimising the speed of rotation, generally by decreasing the speed, during the period of attachment for cells with low attachment efficiency. The RC40 from Cellon provides a semi-automated multiple roller bottle platform.

Figure 7 Roller Deck

Figure 8 Roller Bottles

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Multilayer Vessels

A variety of disposable multi-layer vessels are now available for simple and rapid scale-up of anchorage dependent cells with little or no process optimisation. These include triple layer flasks which are useful for maximising incubator space, however it is vessels such as CellStacks and Hyperflasks that offer the greatest advantages. CellStacks™ provide multiples of 1, 2, 5, 10 and 40 layers, each layer offering 636cm2 (therefore a 10 layer Cellstack™ provides 6,360cm2 for cell growth in a single vessel). Cell stacks are in effect giant cell culture flasks but with two vented caps for filling, harvest and gas exchange rather than a single cap. Some familiarisation, validation of cell growth and care and attention to manual handling is required as these vessels can only be practically employed if pouring techniques are used for filling and harvesting (although filling connectors can be exchanged for the caps) but in essence most cell growth in T flasks can be directly translated to CellStacks™. Forty layer CellStacks™ are too large to be handled manually and require specialised trolleys for manipulations. Originally designed for robotic systems Hyperflasks™ are another multilayer system, however, rather more revolutionary in their design. Consisting of 10 multiple “flasklets” each with the same approximate footprint as a T175 flask the HyperFlask™ is entirely filled with medium and cell inoculum. Gaseous exchange in this case is achieved by diffusion of gases directly through the thin surfaces of the flasklets.

Figure 5 Hyperflask (left) & T175 Flask (right) from Corning

Figure 6 Shake flasks in a Certomat CT plus from Sartorius Stedim Biotech

Figure 9 Spinner Flasks

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Disposable solutions for anchorage independent (suspension) cells

In the last few years many new disposable systems for growth of cells in suspension have emerged. These include disposable stirrer vessels, single use disposable bioreactors and Wave/Cultibag (GE Healthcare®/ Sartorius Stedim®) technology. The Wave™ /Cultibag™ systems allow cell growth in large (10s of litres) sterile, disposable bags which are filled with cells, medium and an air/gas headspace and then gently agitated on a temperature controlled rocking platform. These bags are either “off the shelf” or custom made with most of the connectors for seed, harvest and sampling built in. Recent advances in disposable sensors has now also meant that pH and dissolved oxygen sensors can be built into the bags making them effi cient bioreactors suitable for GMP production and seed vessels for larger bioreactors.

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Spinner Flask Culture

This is the method of choice for suspension lines including hybridomas and attached lines that have been adapted to growth in suspension e.g. HeLa S3. Spinner flasks are either plastic or glass bottles with a central magnetic stirrer shaft and side arms for the addition and removal of cells and medium, and gassing with CO2 enriched air. Inoculated spinner flasks are placed on a stirrer and incubated under the culture conditions appropriate for the cell line. Cultures should be stirred at 100-250 revolutions per minute.

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Other Scale-up Options

The next stage of scale-up for both suspension and attached cell lines is the bioreactor that is used for large culture volumes (in the range 100-10,000 litres). For suspension cell lines the cells are kept in suspension by either a propeller in the base of the chamber vessel or by air bubbling through the culture vessel. However, both of these methods of agitation give rise to mechanical stresses. A further problem with suspension lines is that the density obtained is relatively low e.g. 2x106 cells/ml.

For attached cell lines the cell densities obtained are increased by the addition of micro-carrier beads. These small beads are 30-100μm in diameter and can be made of dextran, cellulose, gelatin, glass or silica, and increase the surface area available for cell attachment considerably. The range of micro-carriers available means that it is possible to grow most cell types in this system. A recent advance has been the development of porous micro-carriers which has increased the surface area available for cell attachment by a further 10-100 fold. The surface area on 2g of beads is equivalent to 15 small roller bottles.

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