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Hollow fiber systems come in a variety of sizes from those designed for large-scale production to small systems that may be appropriate for research into the dynamic control of protein secretion. The systems can be devised to retain or pass proteins of interest by using different molecular weight cutoff membranes for the filters and different configurations of cell growth and medium circulation. Figure 11.4 shows a hollow fiber setup.
Special Substrates For Cell Culture
Growth in Semisolid Media
Cells may be grown in semisolid media to prevent attachment. The ability for single cells to grow in semisolid medium is often used as an indicator of transformation from a normal phenotype to a tumorigenic phenotype. There are, however, some normal cells that
Hollow fiber systems for cell culture (Millipore setup).
can grow, especially if plated in small aggregates, in semisolid media. Here, the lack of attachment maintains a more rounded 3-dimensional shape in vitro that can help maintain differentiated cell function in some cell types (Halban et al., 1987; Verhoeven et al., 1986).
1. Difco Noble agar
2. 2x growth medium
3. Purified water
4. Water bath, set at 45jaC
5. 15-ml conical tubes
6. 125-ml glass bottle with screw cap
7. 60-mm plastic petri dishes Procedure
1. Add 1 g agar to 100 ml purified water in a glass bottle. Loosely cap. This is the 1% agar solution.
2. Add 0.5 g agar to 100 ml purified water in a glass bottle. Loosely cap. This is the 0.5% agar solution.
3. Autoclave for 30 min at 120jaC.
4. Transfer the bottles to a 45jaC water bath.
5. Prepare the 0.5% bottom agar layer by adding 2 ml of 1% agar to 2 ml 2 x growth medium in a 15-ml conical tube.
6. Resuspend and add to a 60-mm plate, taking care not to introduce air bubbles that might create holes in the agar. Set aside and allow time to gel at room temperature (about 30 min).
7. Trypsinize, neutralize, and count cells to be plated. Prepare the cell suspension so that the seeding density/dish is in a 100-÷1 volume.
8. Prepare the 0.25% top agar layer by adding 1.5 ml of 1% agar to 1.5 ml 2x growth medium in a 15-ml conical tube.
9. Add 100 ÷1 of the cell suspension to each 15-ml conical tube of cooled (40jaC) agar.
10. Resuspend and add to each previously prepared dish.
11. Swirl the dish gently to allow even spreading.
12. Place in a 37jaC, 5% CO2 humidified incubator and leave for 7"C10 days.
Collagen gels also can be used to maintain cells in a more rounded, less spread configuration. While these are often more expensive and difficult to prepare than semisolid media, they do provide a more physiologically relevant substrate and therefore may improve cell function. The gel also may be detached from the surface of the culture dish, allowing a floating raft to form for the cells to grow on. In this configuration, the cells receive their nutrients from the medium on their basal surface and are close to the oxygen-rich atmosphere on the top. The raft can also contract and change configuration with the cells. Cells, such as normal mammary cells (Stampfer and Yaswen, 1994) and endothelial cells (Nishida et al., 1993), can re-create complex threedimensional structures similar to their cell"Ccell association in vivo in these cultures.
1. Rat tail collagen (30 mg, Boehringer-Mannheim)
2. 0.2 M acetic acid
3. Neutralizing buffers:
-A. 12 mg/ml NaHCO3 in 0.1 N NaOH
B. 1.3 M NaCl in 0.2 M Na2HPO4
4. 24-well, 12-well, or 6-well plates
5. 50-ml conical tubes
6. Trypsinized, neutralized cell suspensions Procedure
1. Reconstitute collagen in 12 ml 0.2 N acetic acid. Store at 4jaC.
2. On ice, make a solution mixing 800 ÷1 reconstituted collagen, with 100 ÷1 neutralizing buffer A and 100 ÷1 neutralizing buffer B, for each 1 ml of collagen solution. Mix well.
3. Add 250 ÷1 to each well of a 24-well plate, 500 ÷1 to each well of a 12-well plate, or 1 ml to each well of a 6-well plate. Allow to gel in a 37jaC incubator (this takes around 20"C30 min).
4. When the gel has set, add at least 1 x 105 cells/ml to the as yet ungelled collagen solution on ice. Resuspend well and add an equal volume of this solution to the collagen in the well.
5. If it is neither desired nor necessary to incorporate the cells into a gel, the cells may be resuspended in 1 x growth medium and added on top of the set gel. In this case the liquid volumes should be 500 ^l/well for 24-well plates, 1 ml/well for 12-well plates, and 2 ml/well for 6-well plates.
Collagen rafts can be made by suspending the cells in collagen at the time of plating. Then, once set, carefully detach the gel with a small sterile spatula. Lay the gel on a stainless steel grid that has been bent along the edge to keep the gel off the surface of the dish.
Small 1-mm or 2-mm-thick "rings" of silicon tubing can be cut and placed under the grid as well. Stainless steel sieves (in graduated mesh sizes) work well, can be sterilized, and are thin enough to cut to size if necessary.