Cell cultures need oxygen to grow. In low density cultures, such as cell culture growth in a flask, oxygen is delivered from the head space alone. The head space is defined as the area between the top of the container and the surface of the culture medium. However, where cultures are supplemented or are grown in a large volume, it is difficult for oxygen to be delivered throughout the culture medium by headspace alone.
Several systems have been developed for improving oxygen delivery to the cell cultures. One such system principally employs a "sparging ring." A sparging ring is a tube formed in a circle. The tube has many tiny holes. The tube is placed at the bottom of the culture vessel and extends outside to an oxygen supply. As oxygen is pumped into the sparging ring, air bubbles are introduced to the cell cultures at the bottom of the culture vessel. As the air bubbles rise, oxygen is delivered to the cell cultures throughout the culture vessel.
Oxygen delivery systems of this type, however, have several disadvantages. First, oxygen delivery via air bubbles may cause physical disruption of the cell membranes especially at higher impeller speeds. Additionally, air bubbles cause the formation of foam which may trap cells, degrade product and plug exit filters.
Another type of oxygen delivery system uses gas permeable tubing. In this system, pliable silicon tubing is wrapped around the inside of the culture vessel. The silicon tubing, unlike the sparging ring, has no holes. The gas permeable nature of the silicon allows oxygen to diffuse through the tubing and into the cell culture medium without the introduction of air bubbles.
Oxygen delivery systems of this type, however, also have several disadvantages. First, because of the quantity of tubing necessary to maintain satisfactory oxygen delivery, it is not practical to use gas permeable tubing for industry use. Second, to insure the absence of pin holes, the silicon tubing needs to be replaced routinely, a chore in itself.
Another type of oxygen delivery system involves emulsifying a liquid chemical and suspending it in the cell culture medium. The liquid chemical has the capacity of binding or collecting oxygen in an oxygen rich environment and liberating oxygen in an oxygen deficient environment. Some liquid chemicals also have a reciprocal relationship to CO2. In other words, the liquid chemicals will absorb CO2 in a CO2 rich environment and liberate CO2 in a CO2 deficient environment. One common chemical used in such a system is perfluorocarbon (PFC) of which a number of varieties exist. The exact chemical is not essential as long as the chosen liquid chemical absorbs oxygen in an oxygen rich environment and gives it up in a deficient environment.
Oxygen delivery systems of this type, however, also have disadvantages. First, emulsion systems require high concentrations of emulsion in order to provide the necessary oxygen transfer. High concentrations of emulsion increases the risk of chemical contamination to the cell culture due to the various detergents, etc. required. Second, the oxygen delivery process is limited to internal re-oxygenation; the emulsion receives the oxygen from the sparging ring and the head space only, and thereafter delivers it to the cell cultures. This process is not as practical as external oxygenation where the regeneration process can be manipulated as need be. Third, emulsified PFC cannot be readily recycled or retained as in perfusion experiments. As such, the PFC must be continuously replaced at a very high cost.
Another type of oxygen delivery system involves the introduction of a non-emulsified PFC. In systems of this type, oxygenated PFC is introduced to the inside of the cell culture where it liberates oxygen and collects CO2. Thereafter, the de-oxygenated PFC is transported to the outside where it is "recharged" with oxygen. In essence, the PFC is recycled. In this system, the PFC is sprayed by a nozzle into the head space. Because the PFC is a hydrophobic substance with densities higher than water, it does not mix with water. As such, the PFC falls to the bottom of the culture vessel. A tube at the bottom of the culture vessel is provided to remove the PFC for recycling.
Oxygen delivery systems of this type, however, also have disadvantages. First, the PFC is sprayed throughout the culture medium, and as such, collects at various points on the bottom of the culture vessel. Collection of the PFC with a tube thus becomes difficult, requiring a different configuration for each different bioreactor. Second, spraying of the PFC may cause damage to the cell membranes or generate foam. Third, of necessity, cells must directly contact the PFC which may not always be desirable.
USPTO Backgrounds
USPTO Backgrounds