Tissue Freezing Techniques


Modern technology advancements in proteomics and genomics have enabled the identification of disease mechanisms at molecular levels. Research data can be obtained through animal models and cell lines. However, to translate knowledge into clinical applications, numerous well documented high-quality human tissues are required. For these investigations, tissue collections from pathology institutes are necessary. For diagnosis, tissue samples can be formalin-fixed paraffin-embedded or frozen using the cooling chamber of a cryostat or solid carbon dioxide. 

Frozen Section Technique

Frozen section technique is a tool that can rapidly prepare slides from donor tissues for microscopic interpretation. This technique is used in various research and clinical settings. Frozen sections are commonly used in surgical pathology for rapid intra-operative diagnosis as it can be a valuable guiding tool for surgeons. For example, in Mohs micrographic surgery, the frozen sections help the surgeon determine the extent of excision required to remove a skin tumor. There are many research applications that depend on the frozen section technique in the preparation of microscopic slides that use molecular, immunohistochemical, and morphologic methods. The preparation of frozen section slides can be a complex process that requires an understanding of pathology, microanatomy, histology along with refined technical skills. Regardless of the use of the frozen section, the results depend on the quality of the specimen. The training for frozen tissue preparation may vary depending on the different practitioners. It is part of the curriculum in pathologist assistant and formal histology programs. However, there is also a lot of hands-on technique that is learned and passed along through experience at the workbench. Just like many research applications and pathology residency programs, most of the training is accomplished on the job. 

In intraoperative sections, tissue samples can be placed onto a metal chuck that has the optimal cutting temperature (OCT) compound. Slow freezing of the tissues can cause artifacts because of the accumulation of water molecules that become ice crystals. This issue can be resolved using liquid nitrogen. Due to the low temperature of liquid nitrogen, tissues that are extremely soft such as lymph node, brain, or spleen become rigid, brittle, and difficult to cut. Additionally, liquid nitrogen also evaporates rapidly, especially when it is in a small container. This means that it has to be refilled multiple times a day. 

Since there is limited FFPE tissue of quality, there are additional tissue pieces that are frozen for diagnostic and research purposes in various pathology institutes. However, the protocols for freezing tissues are not standardized and commonly differ. When it comes to tissue banking, specimens that are not needed can be frozen in cooled isopentane or liquid nitrogen. There are different strategies to preserve tissue specimens. Cold resistant cryomolds and cryovials are usually used as it does not require much space and can be labeled easily. To protect the tissue from desiccation, a freezing medium can be added or wrapped in aluminum foil or plastic bags. These methods can be disadvantageous as the tissues become harder to handle when further processing is required. Snap-frozen tissues can be transferred into plastic bags that are then placed into a box that contains other samples. However, as the number of samples in the cryo-container continues to grow, it may cause issues when trying to determine the specific location of the tissue. As the collection grows, it can become unmanageable. 

Examples of Freezing Methods

As previously mentioned, there are many freezing methods. Some examples include:

•    Dry ice – A block of dry ice can be placed in a Styrofoam container. This gives an estimated -70°C. The filled cryomold is then placed on the block to freeze it. This method is safe and simple but does not freeze the tissue as fast when compared to immersing it in a freezing medium. 

•    Pellet – Another method is to use dry ice that is in the pellet form. This method requires the placement of a small bowl that is stainless steel, pyrex, or a polypropylene beaker in a Styrofoam container and filling the surrounding space with dry ice pellets. Some pellets should also be placed in the bowl and isopentane or acetone added. This should be done in a fume hood. Care should also be taken as it is easily flammable. Once the pellets have stopped bubbling, fill the mold and orientate the tissue before immersing it in the liquid for freezing. 

•    Liquid nitrogen – Place the liquid nitrogen in a Dewar flask or Styrofoam container. Lower a container of isopentane into the liquid nitrogen using a pair of tongs. The isopentane will then begin to appear opaque once it nears freezing. Once ready, take the isopentane out and freeze the specimen. The isopentane can be chilled for subsequent uses. This method is advantageous due to its rapid freezing time. 

•    Fresh tissue freezing – The tissue is placed in OCT and flash frozen.

•    4% Paraformaldehyde (PFA) – This method uses 4% PFA and sucrose as a cryo-protectant. The tissue is placed in OCT and frozen using dry ice or flash frozen. 

•    Enzyme study – This method is often used for fresh muscle tissue. 

It is important to note that using just liquid nitrogen can cause cracking of tissue and OCT due to the unpredictable freezing pattern. Liquid nitrogen also boils and creates a vapor barrier that can cause the freezing to occur in a slow and unpredictable pattern. 


1)    Emge DJ. Tissue freezing methods for cryostat sectioning. Accessed 6/20/2019. https://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezing.pdf

2)    Peters S. A method for preparation of frozen sections. IHC World. Accessed 6/20/2019. http://www.ihcworld.com/_protocols/histology/frozen_section_technique_4.htm

3)    Freezing tissues for cryosectioning. UAB Research. Accessed 6/20/2019. https://www.uab.edu/research/administration/offices/ARP/ComparativePathology/Pathology/Histopathology/TissueSubmission/Pages/Freezing-Tissues-for-Cryosectioning.aspx

4)    Steu S, Baucamp M, von Dach G, et al. A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology. Virchows Arch. 2008; 452: 305-312.

5)    Peters SR. A practical guide to frozen section technique. 2010.