Explants are transferred to a base of gelled collagen, then overlaid with a cover of gelling collagen. Explants are manipulated and positioned appropriately as the top cushion sets.
1. Prepare a desired volume (100-500 |L) of 90% collagen and 10% of 10X DMEM (8) in an Eppendorf tube. Vortex. Add enough 0.8 M NaHCO3 to make the solution turn straw yellow after vortexing. For each batch of collagen, the NaHCO3 must be titered: typically 0.7-1.2 |L NaHCO3 is added to 100 |L of collagen and 10X DMEM (9). Collagen can be used within 10-15 min if kept on ice. At room temperature, it will start to set within minutes.
2. In a four-well multidish (10), prepare a collagen cushion by pipeting 20-25 ||L of collagen mix onto the bottom of the dish. Stir around to spread and flatten the drop (11), and let it set at room temperature for 20 min.
3. Transfer explant(s) from the medium onto the cushion, using either a Pasteur pipet or a fine-drawn capillary tube with a mouth attachment (12).
4. Remove excess medium with a fine-drawn 25-|L Drummond micro-dispenser capillary tube with a mouth attachment. Leave on a tiny amount of medium so that the explants do not dry out.
5. Overlay with 25-30 |L of gelling collagen (13) prepared in the same way as the bottom cushion, position explants using an electrophoretically sharpened tungsten needle (14) (with blunted tip), and allow to set for 30-60 min at room temperature (see Fig. 1). Cover collagen moulds with approx 0.4-0.5 mL medium (15), and transfer to incubator for the desired time of incubation (16).
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