Skin and sclera are two of the most difficult tissues to homogenize. Neither rotor stators nor bead beaters are effective at disrupting this tissue. Cryogenic grinding is effective as the cold makes the samples brittle so they can be cracked and pulverized. Unfortunately, grinding is manual so high throughput processing of sclera and skin is not yet practical.
To cryogenically homogenize samples with a CryoGrinder™, samples should be 100 mg or less, and preferably smaller. Skin and sclera are tough, thus thorough grinding will require some extra effort.
CryoGrinder™ can be decontaminated with rinse solutions (e.g., RNase Away) and by autoclaving. Dry heat sterilization can damage the pestles.
If using a CryoCooler™, charge the reservoir with liquid nitrogen (about 1 L). If using a pan or foam cooler, pour in liquid nitrogen to a depth of approximately 1.5 inches. For skin and sclera, keep the CryoGrinder and samples as cold as possible. Even at slightly elevated temperatures, the collagen in the tissues becomes very rubbery.
Place the pestle inside the CryoGrinder™ well and place in an insulated pan or by lying on the rack within the reservoir of the CryoCooler™. Allow the CryoGrinder to chill for 15-20 min. If necessary, replenish the liquid nitrogen, but don't expose the well and pestle tip to the liquid nitrogen which may contain significant contamination (see link).
The sample, which must be less than 100 mg for best results, is placed in the mortar's well. Wait 5 minutes for the sample to drop in temperature. Recharge the liquid nitrogen if necessary during the cool down wait.
Wear gloves to protect from the cold. Attach the pestle to the cordless wrench. Hold the mortar firmly while pressing down on the sample with the pestle while the wrench is moving. Move the pestle over the sample to ensure all the pieces become pulverized. If necessary, return the sample to the liquid nitrogen reservoir to keep it cold. Repeated steps of chill then grind may be necessary.
When the sample is homogenized, remove the pestle from the wrench and tap in the well to remove any residual sample.
Using a pre-chilled thin tipped spatula, gently loosen the ground tissue from the walls and bottom of the mortar. Place a large microfuge tube or larger tube over the well, invert, and tap the mortar to transfer the ground sample to the tube.
For RNA isolation, add extraction buffer (e.g., Trizol or Tri Reagent) immediately before the sample thaws. The tube can also be placed into the liquid nitrogen so it remains frozen.
Allow the CryoGrinder mortar and pestle to warm up before decontamination by autoclaving and cleaning with a biologically safe detergent and water.