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and practical problems when the number of cells ro be cultured becomes very high (>10" cells). These problems include incubator space (Figure 1 j, time and support personnel required for cell manipulations, and high risk of microbial contaminations. In this latter respect, cell culture bags represent an improvement over flasks by providing a closed system from the time the cells are initially collected from a patient/donor, cultured and then reinfused into the patient. Moreover, the bags are single-use vessels, thus preventing any possibility of patient sample cross-contamination, and can be easily disposed of after use. More importantly, they greatly facilitate the handling of large-scale cultures since a single technician can easily manage up to 50-100 I of tissue culture at a time.

The two companies producing commercial cell

Figure 1 Large-scale expansion of leukocytes in T-175 polystyrene flasks. A standard-size incubator can hold an average of 64 flasks containing a maximum of 150 ml of cell culture each (total capacity per incubator - 9.6 I of cell culture and about 10'° total cells).

Figure 1 Large-scale expansion of leukocytes in T-175 polystyrene flasks. A standard-size incubator can hold an average of 64 flasks containing a maximum of 150 ml of cell culture each (total capacity per incubator - 9.6 I of cell culture and about 10'° total cells).

Applications

LAK cells and TILs LAK cells and TILs LAK cells

LAK cells and TILs Hematopoietic progenitors CD34' progenitors Hematopoietic progenitors

Gibco, Grand Island, NY BioWhittaker, Walkersville, MD DuPont, Wilmington, DE Ventrex, Portland, ME BioWhittaker, Walkersville, MD Life Science Tech., Gaithersburg, MD Quality Biologicals, Gaithersburg, MD

culture bags are Baxter Healthcare Corp. (Immunotherapy Division, Irvine, CA) and American Fluoroseal Corp. (Silver Spring, MD). Baxter cell culture bags are sold under the trade names of I.ifecell and Cryocyte. American Fluoroseal provides the Teflon cell culture bags. They come in different sizes ranging from 50 to 3000 ml maximum bag capacity (with culture volume ranging from 100 to 1500 ml, respectively). Importantly, Lifecell culture bags can be used in conjunction with the Lifecell solution transfer pump (Figure 2), thus allowing a drastic

Figure 2 Large-scale expansion of leukocytes in gas-permeable cell culture bags. Bags can be filled up manually (A) using a gravity transfer set (arrows) or using a solution transfer pump (B). An incubator can hold an average of 30 bags containing a maximum of 1500 ml of cell culture each (total capacity per incubator =451 of cell culture and about 2.5 x 1011 cells), b, gas-permeable cell culture bag; m, medium.

reduction in the risk of cell contamination, biohazard exposure, feeding time and occupied incubator space. Finally, culture harvesting is also facilitated by direct centrifugation of the cells in the bags using specially designed bag holders that fit into large centrifuge cups, such as the ones used in Sorvall centrifuges.

A number of factors need to be considered in the use of plastic cell culture bags:

1. The kinetics of cell growth varies with each cell type, creating specific requirements for culture medium components and/or oxygen and carbon dioxide.

2. Bag surface properties will affect growth. Adherent cells cannot adhere to gas-permeable plastic bags so they cannot be used to grow, for instance, dendritic cells.

3. Gas permeability is a critical factor. Oxygen limitation can occur in culture bags in which the material is not sufficiently gas permeable and the proper feeding schedule is not maintained. Currently, a limited number of bag materials are available that provide the appropriate necessary gas exchange for cell culture. However, depending upon the cell being cultured, an increase in cell proliferation can be achieved by simply altering the incubator gas conditions (10% carbon dioxide and 30% atmospheric oxygen have been shown to be the best gas conditions for LAK cells and TILs grown in Lifecell bags).

4. Appropriate volumes and cell surface ratio should be used. Using the optimal culture medium depth is important for gas diffusion and delivery of oxygen to the cells. In this respect, one should consider that the nominal bag size is usually much greater than the optimal culture volume. Bag cell cultures can be initiated at low-volumes and fed during the culture period by simply adding more culture medium and recombinant growth factors to the bags.

Finally, a disadvantage of cell culture in gas-permeable culture bags is the impossibility of visually checking the growth and viability of the cells while they are inside the bag (Table 2). Periodic sampling of the cultures is therefore needed to monitor their growth.

Figure 2 Large-scale expansion of leukocytes in gas-permeable cell culture bags. Bags can be filled up manually (A) using a gravity transfer set (arrows) or using a solution transfer pump (B). An incubator can hold an average of 30 bags containing a maximum of 1500 ml of cell culture each (total capacity per incubator =451 of cell culture and about 2.5 x 1011 cells), b, gas-permeable cell culture bag; m, medium.

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