1. FIGURE 19.1 Freezing Curve. Record of the fall in temperature in an ampoule containing medium, clipped with five other ampoules on an aluminum cane, enclosed in a cardboard tube, placed withina polyurea-foam tube (see Fig. 19.2), and placed in a freezer at −70 C. ◦

2. FIGURE 19.2 Ampoules on Cane with Insulation for Slow Cooling. Plastic ampoules are clipped onto an aluminum cane (bottom), enclosed in a cardboard tube (middle), and placed inside an insulating foam tube (top). The insulating tube is plugged at either end with cotton or another suitable insulating material before placing it to cool slowly in a −70 C or −80 C freezer. ◦◦

3. FIGURE 19.3 Neck Plug Cooler. Modified neck plug for narrownecked freezers, allowing controlled cooling at different rates (Taylor Wharton). Shown is the section of the freezer neck with the modified neck plug in place. The retaining ring is used to set the height of the ampoules within the neck of the freezer. The lower the height, the faster the cooling.

4. FIGURE 19.4 Nunc Cooler. Plastic holder with fluid-filled base. The specific heat of the coolant in the base insulates the container and gives a cooling rate of about 1 C/min in the ampoules. ◦

5. FIGURE 19.5 Programmable Freezer. Ampoules are placed in an insulated chamber, and the cooling rate is regulated by injecting liquid nitrogen into the chamber at a rate determined by a sensor on the rack with the ampoules and a preset program in the console unit (Planer Biomed). (a) Control unit and freezing chamber (lid open). (b) Close-up of a freezing chamber with four ampoules, one with a probe in it.

6. FIGURE 19.6 Liquid-Nitrogen Freezers. (a) Narrow-necked freezer with storage on canes in canisters. (b) Interior of the narrow-necked freezer, looking down on canes in canister, positioned in center as it would be for withdrawal. Normal storage position is under shoulder of freezer, just visible top right. (c) Wide-necked freezer with storage in triangular drawers. (d) Narrow-necked freezer with storage in square drawers (see also Fig. 19.7). (e) High-capacity freezer with offset access port open, revealing canes in canisters. (f ) High-capacity freezers in a cell bank. Nearest freezer shows connections for monitoring and automatic filling. (e, f, photos courtesy of ATCC.)

7. FIGURE 19.7 Nitrogen Freezer Design. Four main types of nitrogen freezers: (a) Narrow-necked with ampoules on canes in canisters (high capacity, low boil-off rate). (b) Narrow-necked with ampoules in square racks (moderate capacity, low boil-off rate). (c) Wide-necked with ampoules in triangular racks (high capacity, high boil-off rate). (d) Wide-necked with storage in drawers; piped liquid nitrogen perfused through freezer wall and level controlled automatically by high- and low-level sensors (top left).

8. FIGURE 19.8 Freezing Cells. Trypsinized cells, in medium with cryoprotectant, aliquoted into ampoules, which are then clipped on to an aluminum cane, inserted into a cardboard tube, and inserted into an insulated tube. The tube and contents are placed at −70 C or −80 C for 4 h or overnight, before transferring the cardboard tube containing the ampoules to a liquid nitrogen freezer with canister storage (see Fig. 19.7a). ◦ ◦

9. FIGURE 19.9 Thawing Cells. Ampoules are removed from the freezer and thawed rapidly in warm water, under cover, to avoid the risk of explosion if the ampoules have been stored in the liquid phase (right-hand side of figure). Ampoules, which have not been submerged in liquid nitrogen, can be thawed in a water bath (left-hand side), with the water level kept below the cap and neck.

10. FIGURE 19.10 Neck Plug Cooler. Vitrification. Colonies of hES cells are cut into small pieces, transferred through vitrification solutions in a 4-well plate, drawn into a vitrification straw on a pipettor tip, snap-frozen in liquid nitrogen, and transferred in a predrilled 4.5-mL cryovial to a liquid nitrogen freezer (see Protocol 19.3). ( [After Cooke & Minger, 2007].)

11. FIGURE 19.11 Cell Banking. A newly acquired cell line, importedor subcultured from a primary, is frozen as soon as enough cellsare available for a few ampoules. The stock is then grown up, characterized, shown to be free of contamination, and frozen as a validated seed stock. One ampoule of the seed stock is thawed, amplified, and revalidated and then frozen as distribution stock. Ampoules distributed to users can be used to generate user stock for the duration of a project but must then be discarded and not passed on to another user.

12. FIGURE 19.12 Serial Culture Replacement. An ampoule is thawed and the cells grown up to the desired bulk for regular use and maintained for 3 months. Three months after the first thaw, a fresh ampoule is thawed, grown up, and, after checking characteristics against current stock, used to replace the current stock, which is then discarded. The cycle is repeated every 3 months, so that no culture remains in use for more than 3 months.

13. FIGURE 19.13 Transportation Containers for Cells. Cells shipped as an ampoule of frozen cells or as a growing culture. (a) Rigid cardboard box with insulating expanded polystyrene lining containing dry ice with the ampoule, sealed in a tube with absorbent wadding and the tube sealed in a ziplock bag, placed in the center of the dry ice. (b) Double-skin plastic container with flask trapped in inner compartment by inflating the outer chamber (Air Box, Air Packaging Technologies).