Freezing Virus in CoolRack on Dry Ice - BioCision

<strong>Virus</strong> Cryopreservation Protocol 1002<strong>Virus</strong> Cryopreservation ProtocolIntroductionSnap freez<strong>in</strong>g, or flash freez<strong>in</strong>g, is theprocess by which samples are lowered totemperatures below 70°C very rapidly us<strong>in</strong>gdry ice or liquid nitrogen. Snap freez<strong>in</strong>gachieves the same endpo<strong>in</strong>t as slow ratecontrolledfreez<strong>in</strong>g, but at approximate rateof10 to 1000°C/m<strong>in</strong>, compared to -1°C/m<strong>in</strong>.Snap freez<strong>in</strong>g with a <strong>CoolRack</strong> module willprovide sample vessel stability, organizationand consistent freez<strong>in</strong>g parameters, rapidhands-free sample process<strong>in</strong>g while avoid<strong>in</strong>glost or contam<strong>in</strong>ated samples. Snap freez<strong>in</strong>gis performed on a pre-cooled <strong>CoolRack</strong>module, which ensures fast heat transfer.This method can provide excellent specimen<strong>in</strong>tegrity and a wide array of options foranalysis, <strong>in</strong>clud<strong>in</strong>g extraction of prote<strong>in</strong>s,DNA and RNA for use <strong>in</strong> research anddiagnostics.This procedure is <strong>in</strong>tended to ensure thatvirus samples collected will be frozen <strong>in</strong> asafe and efficient manner while elim<strong>in</strong>at<strong>in</strong>gthe risks of contam<strong>in</strong>ation and variation <strong>in</strong>molecular <strong>in</strong>tegrity. The follow<strong>in</strong>g protocoldescribes a general procedure for long-termstorage of viruses. For each specific virusspecie or stra<strong>in</strong>, always refer to laboratorySOP.Materials• <strong>Virus</strong> preparation• Cryoprotective agent (special cases only)• CoolBox TM CFT30 ice-free cool<strong>in</strong>g station• <strong>CoolRack</strong> ® CFT30 module• Cryolabels and/or cryomarkers• ThermalTray LP platform (optional)• 50ml Reagent Reservoirs• CoolS<strong>in</strong>k ® LX55 (optional)• CoolBox TM XT (optional)• TruCool ® cryogenic vials• TruCool h<strong>in</strong>ged cryostorage box• 37°C Waterbath• -80°C Freezer and Dry Ice<strong>Virus</strong> PreparationFollow the laboratory protocol for viral growthand/or purification. Refer to Centers forDisease Control and Prevention (CDC)guidel<strong>in</strong>es for utilization of pathogens <strong>in</strong>specific Biosafety Level (BSL). Pathogens are<strong>in</strong>fectious agents and should always bemanipulated under a biosafety cab<strong>in</strong>et withlam<strong>in</strong>ar flow.<strong>Virus</strong> <strong>Freez<strong>in</strong>g</strong>Non-enveloped viruses, some DNA virusesand virus-like particles can be ma<strong>in</strong>ta<strong>in</strong>edstably at 4°C for a relatively long period oftime. However, RNA and most envelopedviruses are extremely heat labile and need tobe snap-frozen (frozen rapidly) and stored at-80°C for long-term storage. Please note thatmost viruses will suffer damage if storagetemperatures exceed greater than -60°C.1. As a general rule, ma<strong>in</strong>ta<strong>in</strong> the viralpreparation at 4°C by plac<strong>in</strong>g it <strong>in</strong> areagent reservoir and place the reservoiron a thermo-conductive CoolS<strong>in</strong>k LX55module for uniform and stable cool<strong>in</strong>g.Rest the CoolS<strong>in</strong>k LX55 on ice tom<strong>in</strong>imize ice contact with the reservoirand it’s contents.2. Dispense 1 mL of the virus preparation*(or desired amount) <strong>in</strong> a pre-labeledTruCool cryogenic vial. To avoid titerreduction, ma<strong>in</strong>ta<strong>in</strong> the vials at 4 o C <strong>in</strong> aCoolBox CFT30 ice-free cool<strong>in</strong>g system.The <strong>CoolRack</strong> CFT30 cryogenic vialmodule <strong>in</strong>side the CoolBox standardizesvial temperature and reducescontam<strong>in</strong>ation and spill accidents byallow<strong>in</strong>g one-handed open<strong>in</strong>g and clos<strong>in</strong>gof the cryogenic vials while seated <strong>in</strong> the<strong>CoolRack</strong> CFT30 module.*Most virus preparations consist of virussupernatants from <strong>in</strong>fected cells cleared of celldebris and therefore conta<strong>in</strong> enough serum toact as cryopreservants. However, some virusesBioCision, LLC • 12 E. Sir Francis Drake Boulevard, Unit B, Larkspur, CA 94939 USA • <strong>in</strong>fo@biocision.com • www.biocision.com

need an extra cryopreservant agent such as a50% sucrose. Please refer to your laboratorySOP or the literature to assess whether you needto add a special media or cryopreservant to theviral supernatant or preparation.3. While virus samples are kept cold at 4°C<strong>in</strong> the CoolBox CFT30, place a second<strong>CoolRack</strong> CFT30 module on dry-ice andlet it equilibrate to -78 o C, which takesapproximately five to ten m<strong>in</strong>utes. Note:with this protocol, there is no need tomake a dry-ice/ethanol slurry. Place thecryogenic vials directly <strong>in</strong> the wells of thepre-equilibrated <strong>CoolRack</strong> CFT30 moduleto snap-freeze the samples. This will takeapproximately three to five m<strong>in</strong>utes.4. Transfer the frozen samples to a TruCoolh<strong>in</strong>ged cryostorage box and place it <strong>in</strong> the-80°C freezer for long-term storage.<strong>Virus</strong> Thaw<strong>in</strong>g1. Transfer the cryogenic vials from the -80°C freezer <strong>in</strong>to a pre-equilibratedCoolBox CFT30 conta<strong>in</strong><strong>in</strong>g a greenfreez<strong>in</strong>g cartridge <strong>in</strong>side it. This will keepthe vial frozen and allow transport of thevials <strong>in</strong> a safe manner.2. Place the vials directly <strong>in</strong> a 37°C waterbath, and slowly manually agitate thevials to enable the thaw<strong>in</strong>g process. Rightbefore the whole liquid is completelythawed remove the vial from the 37°Cwater bath and place it on a <strong>CoolRack</strong>CFT30 module which has been previouslyequilibrated on ice. Samples are nowready for desired experimentalprocedures or titer assessment (TCID 50 ,plaque assay, etc.).BioCision, LLC • 12 E. Sir Francis Drake Boulevard, Unit B, Larkspur, CA 94939 USA • <strong>in</strong>fo@biocision.com • www.biocision.com