TITLE: Tissue Procurement: Cryopreservation with OCT Compound
AUTHORS: Kerry Wiles

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Specimens are frozen to provide a hardened matrix for sectioning and to preserve the morphological, biochemical, and immunological properties of cells and tissues. Freezing tissue specimens has the potential to eliminate many problems associated with standard practices of chemical fixation and paraffin or resin embedding. In practice however, freezing can dramatically alter the physical and chemical structure of cells and tissues and cause the formation of ice crystals. Cryoprotectant agents control the rate of cooling in order to limit the formation of ice crystals. Optimal Cutting Temperature (OCT) is a routinely used water-soluble glycol and resin cryoprotectant agent that provides an excellent specimen matrix for cryostat sectioning at temperatures of -10°C and below.</div></div>

[STEPS]
?. [Tissue Procurement: Cryopreservation]
Procure anatomic site and tissue type per request, but sizes should be no larger than 0.4cm x 0.4cm x 0.3cm. OCT ratio should be 30:1 to properly infiltrate the tissue.
?. [Tissue Procurement: Cryopreservation]
Arrange the pre-­labeled, UV sterilized clamshells on a block of dry ice to cool. Make sure the clamshells are evenly laid on the dry ice to allow the OCT to be evenly aliquoted.
?. [Tissue Procurement: Cryopreservation]
Invert the OCT compound to ensure that no bubbles will be introduced in the media, as this will cause problems with the microtome (artifacts, shearing and samples not cutting cleaning or laying cleanly) and avoid uneven distribution.
?. [Tissue Procurement: Cryopreservation]
Fill each clamshell with OCT by slowly and carefully filling the mold with an even layer, so that the OCT just touches all sides of the clamshell, just enough to OCT to cover all tissue. DO NOT allow the OCT to freeze completely white, but with sterile forceps or needle, transfer the specimen to the OCT-­filled cryomold and gently submerge the tissue into media in the correct orientation and cover with additional OCT.
?. [Tissue Procurement: Cryopreservation]
Harden by placing back in the dry ice chest. DO NOT leave the specimen on dry ice for any longer than it takes to turn the completely white, as overcooling can cause cracking and brittleness, rendering the sample un-usable.
?. [Tissue Procurement: Cryopreservation]
On occasion, we may be requested to use the Liquid nitrogen method (holding the clamshell with forceps on top of the liquid nitrogen), and investigators’ should be advised that this procedure might cause samples to be incompletely processed, as a layer of gaseous nitrogen can form at the sample surface reducing the heat transfer considerably. CHTN VUMC will only procure using this method when requested.
?. [Tissue Procurement: Cryopreservation]
After the OCT has hardened, place the clamshell in the freezer and log the location in the donor IT system.
?. [Tissue Procurement: Cryopreservation]
Properly dispose of all biohazardous waste and other waste.
?. [Troubleshooting]
The following troubleshooting guide can provide you with some common problems that have been seen: