Alternative High-Yield Cell Culture Systems

ECACC Laboratory Handbook 4th Edition

Scaling-up Cell Culture 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 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, oxygen depletion, and the build-up of toxic by-products such as ammonia and lactic acid. Optimization of 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 below.

Table 1. Methods for high yield cell culture scale-up.

Technology	Suspension	Attached	Vol (ml)	S/A (cm2)	Max Cells (Suspension)	Max Cells (Attached)	Advantages	Disadvantages
T-Flasks	Yes	Yes	150	225	1.5x108	~107	Cheap, disposable, no cleaning/ sterilization required	Small scale. Multiples required for larger batches
Triple Flask	Yes	Yes	150	525	1.5x108	3x107	Cheap, disposable, no cleaning/ sterilization required. Save space	Difficult to harvest attached cells. Multiples required for larger batches
Millicell® HY Multilayer Flasks	Yes	Yes	180, 300	600, 1000	NA	2x108	Disposable flasks in a footprint similar to traditional T-flasks. Consistent high cell yield with uniform cell health across all layers.
Hyper-Flask™	No	Yes	560	1720	NA	2x108	Disposable, Small footprint	Revolutionary design
Cell-Stack™	No	Yes	150	NA	NA	1.5X1010	Disposable single batch manufacture.	Require additional equipment (vessels etc. may require cleaning). Difficult to harvest cells
Roller Bottle	Yes	Yes	1000	1700	1x109	1x108	Cheap, disposable, no cleaning/ sterilization required. Automated systems available.	Require “decks” to turn. Multiples required for larger batches. Automation very costly
Shaker Flasks	Yes	No	1000	NA	1x109	NA	Some disposables available.	Suspension only. Glass vessels to be cleaned & sterilized. Requires shaker incubator.
Spinner Flasks	Yes	No	1000	NA	1x109	NA	Some semi-disposables available	Suspension only. Glass vessels to be cleaned & sterilized. Requires stirrer base and incubator.

Multilayer Cell Culture Vessels

A variety of disposable multi-layer vessels are now available for simple and rapid scaleup of anchorage dependent cells with little or no process optimization. These include triple layer flasks which are useful for maximizing incubator space. 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). CellStacks™ are in effect giant cell culture flasks but with two vented caps for filling, harvest and gas exchange rather than a single cap. Some familiarization, 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 specialized trolleys for manipulations. Originally designed for robotic systems, HyperFlasks™ are another multilayer. 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.

Disposable solutions for anchorage independent (suspension) cells

In the last few years many new disposable systems for growth of cells in suspension have emerged in bioreactors. 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 means that pH and dissolved oxygen sensors can be built into the bags making them efficient bioreactors suitable for GMP production and seed vessels for larger bioreactors.

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, such as 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.

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, 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 methods of agitation give rise to mechanical stresses. A further problem with suspension lines is that the maximum achievable density of about 2x10⁶ cells/mL is relatively low.

For attached cell lines, cell densities obtained are increased by the addition of microcarrier 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 microcarriers 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 an additional 10-100 fold. The surface area on 2g of beads is equivalent to 15 small roller bottles.

Figure 1. Scaled-up alternative cell culture methods include using roller bottles and spinner flasks which are designed to grow large quantities of cells.