Alternative Cell Culture Systems

1.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 practicable 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 is a whole range of parameters to consider which will need to be developed and optimized 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 optimize such a process for quantities beyond 1L volumes is best left to expert process development scientists.

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.

2.Scale-up Solutions

The example shown is a Cell-Pharm 2000 hollow fiber based bioreactor from Biovest International Inc. and produces grams scale of antibody per month. These reactors are in routine use under cGMP manufacturing conditions at Sigma Aldrich Immunochemical Production facility in Rehovot, Israel.

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 for the user to adapt to the system as well as the cells!

3.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 60 revolutions per hour) which bathes the cells that are attached to the inner surface with medium. Roller bottles are available typically with surface areas of 1050cm2 (Prod. No. Z352969). The size of some of the roller bottles presents problems since they are difficult to handle in the confined space of a microbiological safety cabinet. Recently 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 as some cells lines do not attach evenly. This is a particular problem with epithelial cells. This may be partially overcome a little by optimizing the speed of rotation, generally by decreasing the speed, during the period of attachment for cells with low attachment efficiency.

4.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. Spinner flask systems designed to handle culture volumes of 1-12 liters are available from Techne, Sigma, and Bellco, e.g. (Prod. No’s. Z380482-3L capacity and Z380474-1L capacity).

5.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 liters). 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 (Prod. No. C4853 (220v) or C4728 (110v)). 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; in the order of 2x106 cells/ml.

For attached cell lines the cell densities obtained are increased by the addition of micro-carrier beads. These small beads are 30-1005m 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.