How to Pulverize Biological Tissue in Liquid Nitrogen

Cryogenic pulverization involves snap-freezing biological tissue in liquid nitrogen and mechanically grinding/crushing it into a fine powder. This preserves RNA integrity, prevents protease activity, and improves extraction efficiency for downstream molecular analysis.

This step-by-step video demonstrates the Cellcrusher workflow.  The protocol is as follows;

      1. Lower the device into an insulated container of liquid nitrogen.  The temperature transition typically takes three minutes.
      2. The sample is then chilled in liquid nitrogen, in a small container, such as a weigh boat.  Once chilled, it is placed in the Cellcrusher.
      3. The piston is replaced, and struck sharply two or three times with the provided mallet.  The piston is lifted with the handling pliers and examined to see if pulverization is complete.
      4. The chilled recovery spoon is used to remove the sample the vast majority is recovered at the first attempt but even minute samples can be effectively recovered.
      5. Afterwards the Cellrusher can be cleaned at low temperatures without water, with the aid of the recovery spoon and laboratory paper.
      6. The cool device is again lowered into liquid nitrogen. On this occasion the temperature transition is much shorter, within one minute the Cellcrusher is ready for processing the next sample.

Required equipment:

  • Cellcrusher kit (Cellcrusher, handling pliers, mallet, recovery spoon)
  • An insulated container for liquid nitrogen
  • Smaller, disposable containers for chilling samples
  • Suitable PPE, i.e. eye protection, gloves, laboratory coat.

Notes on cleaning
The standard Cellcrusher can be cleaned at low temperatures without water, as demonstrated in the video on our website. This saves time and liquid nitrogen, but is only suitable for situations in which low levels (~0.1%) carryover or cross-contamination is not a problem. Otherwise, cleaning procedures should be judged by the user, whether to use certain detergents, RNAses, etc.
Particularly intense cleaning, for example to remove prions, can be achieved by soaking in room temperature 1N sodium hydroxide. Do NOT use bleach, as it is not compatible with the type of stainless steel used.

 

What are the advantages of using the Cellcrusher

The curved surfaces of the device allow for effective sample recovery and cleaning at liquid nitrogen temperatures.  The Cellcrusher is can be used to pulverize a wide sample size range (20 mg to 5 g), which is  possible because of these curved surfaces.  This shape is particularly important when recovering small samples.  The flat and right angled surfaces of generic tissue pulverizers can make sample recovery challenging.

As can be seen in the video, when using the Cellcrusher, samples can be recovered as frozen powder and quickly and easily transferred to dissolution buffer.  Subsequently, the device can be easily cleaned dry at liquid nitrogen temperatures and is ready for the next sample in approximately one minute.

This workflow demonstration can be watched directly on YouTube (Tissue Pulverizer).

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