Recommendations on how to safely freeze and thaw cell cultures culture s in TPP cryotubes Information from Biochrom AG, May 23, 2011 Cryopreservation can be used to store cell cultures for a virtually indefinite period of time. Freezing and thawing do, however, represent exhausting processes for cells. In order to protect cells during these processes, they are normally being frozen in suspension along with protective substances such as serum, with antifreeze agents, such as glycerine or dimethyl sulfoxide (DMSO), or with specific cryoprotective media. After this conditioning, cells are given into sterile vials or cryotubes, respectively. TPP cryotubes are suitable for freezing and storing cell cultures. In order to freeze a broad range of cell suspension volumes, TPP offers cryotubes in different sizes: 1.2 ml, 2.0 ml, 3.8 ml, and 4.5 ml. Cryotubes are made of polypropylene (abbreviated: PP), a heat- and coldresistant type of plastics. This is why TPP cryotubes are approved for the use under conditions of up to -190 °C. The TPP cryotubes containing the cell suspension are slowly cooled until they are frozen; then they can be stored in freezers. If the cells are to be stored in liquid nitrogen, users should take special precautions; protective clothing, gloves and glasses should be worn. TPP recommends storing samples in the nitrogen’s gas phase instead of the liquid phase. For reasons of safety, users should always guarantee that the tubes are kept tightly closed and adhere to the specified filling volumes of the cryotubes. The following table shows ideal filling volumes for TPP cryotubes. In addition, we provide an exemplary protocol on how to freeze and thaw cells, as well as further tips in the FAQ section in point 4. 1 Features of TPP cryotubes TPP cryotubes feature an external thread, which allows for a smooth inner tube surface. The combination of external thread and caps with sealing lips guarantee a tight closure that cannot be over-tightened. The tubes are free-standing, with the star-base of the tube snapping in the rack (cat. no. P 99016) like a gear. This allows for one-handed operation, as the user is able to easily open and close the tubes with one hand. The user may click inserts in different colours into the tubes’ cap for an easy identification. All TPP cryotubes are delivered in resealable bags. Biochrom AG Leonorenstraße 2-6 12247 Berlin Germany internet: www.biochrom.de e-mail: info@biochrom.de phone: +49 (30) 77 99 06-0 fax: +49 (30) 771 00 12 page 1 of 6 Extra equipment for using TPP cryotubes: storage boxes (material: PP) with low cap (for cat. no. P 89040 and cat. no. P 89050) and high cap (for cat. no. P 89012 and cat. no. P 89020) for storing 81 cryotubes each cryo racks (cat. no. P 99016; material: polycarbonate, PC): for 40 cryotubes each, suitable for all sizes coloured inserts for the caps, allowing for an easier identification, in green, white, purple, red, blue, and yellow The TPP cryotubes family: P 89012 1.2 ml Biochrom AG Leonorenstraße 2-6 12247 Berlin Germany internet: www.biochrom.de e-mail: info@biochrom.de phone.: +49 (30) 77 99 06-0 fax: +49 (30) 771 00 12 P 89020 2.0 ml P 89040 3.8 ml P89050 4.5 ml page 2 of 6 2 Ideal filling volume for TPP cryotubes The upper marking on the scaling of the tubes corresponds to the ideal filling volume (the picture shows P 89020 with an ideal filling volume of 1.5 ml). tab.: recommended volumes for TPP cryotubes and further product details cat. no. tube volume ideal filling volume diameter length material pieces/bag pieces/case P 89012 1.20 ml 0.90 ml 12 mm 36 mm PP 100 800 P 89020 2.00 ml 1.50 ml 12 mm 48 mm PP 100 800 P 89040 3.80 ml 3.50 ml 12 mm 75 mm PP 100 400 P 89050 4.50 ml 4.00 ml 12 mm 90 mm PP 100 400 3 Protocol on how to freeze and thaw cell cultures in TPP cryotubes The following instructions on how to freeze and thaw cell cultures include the freezing medium Biofreeze as an example. Biofreeze is a chemically defined, serum- and DMSO-free freezing medium offered by Biochrom AG for the cryopreservation of cell cultures. It can be used for freezing a broad range of cell lines, replacing thus also traditional freezing media. 3.1 Freezing of adherent cells (1x106 cells/ml) 1. Remove culture medium from the cell culture flask (e.g. 75 cm2). 2. Wash cell layer carefully with PBS (10 ml) without Ca2+ and Mg2+ (cat. no. L1825). 3. Isolate cells with suitable protease, such as trypsin/EDTA (cat. no. L 2143). Biochrom AG Leonorenstraße 2-6 12247 Berlin Germany internet: www.biochrom.de e-mail: info@biochrom.de phone.: +49 (30) 77 99 06-0 fax: +49 (30) 771 00 12 page 3 of 6 4. Transfer cell suspension into a centrifuge tube which contains either medium with FBS or medium with trypsin inhibitor for inactivating the protease. 5. Centrifugation (300 g). 6. Resuspend cells in freezing medium, such as Biofreeze, (1 part cell pellet + 9 parts freezing medium). 7. Fill cells in quantities of 1.5 ml into labelled cryotubes (cat. no. P 89020) and freeze them (temperature reduction of 1 °C per minute to -70 °C); store immediately in a nitrogen tank. 3.2 Freezing of suspension cells (5x106 cells/ml) 1. Centrifuge suspension, resuspend cell pellets in freezing medium, such as Biofreeze, (1 part pellet + 9 parts freezing medium). 2. Freeze cells in quantities of 1.5 ml (temperature reduction of 1 °C per minute to -70 °C) and immediately store in a nitrogen tank. 3.3 Thawing of cells Thaw cells as quickly as possible: 1. Remove the frozen culture from the nitrogen tank and warm it in a water bath or incubator at 37 °C. 2. Place thawed cell suspension in 10 ml culture medium. 3. Afterwards, centrifuge cells (300 g) and place the pellet in the culture medium. 4. Transfer cell suspension in culture vessels and perform a medium change after 24 hours. Biochrom AG Leonorenstraße 2-6 12247 Berlin Germany internet: www.biochrom.de e-mail: info@biochrom.de phone.: +49 (30) 77 99 06-0 fax: +49 (30) 771 00 12 page 4 of 6 4 Frequently asked questions about freezing and thawing 1. What happens in the cell during the freezing process? Cryopreservation is an exhausting process for all cells. During the freezing freezing process, ice crystals form temperature 20 °C temperature -10 °C in the ambient medium as well as in the cells. In order to prevent such damaging crystallisation, antifreezes, such as glycerine or DMSO, are being used. Serum may be additionally used, as some ice crystals in medium cells in medium of its components feature a hypertonic: isotonic: protective effect. same solute concentration inside and outside Biochrom AG’s freezing medium no water flow Biofreeze does intentionally not contain any animal-derived components, such as serum, and cells may be frozen in it without DMSO. high extracellular solute concentration temperature -20 °C cells shrink reduction of the transition temperature water flows out The following is a detailed illustration of the freezing process: 2. What should be considered when freezing cell cultures? Cell suspensions should be transferred into suitable vials or cryotubes, respectively. Cells should then be cooled by reducing the temperature in increments of 1 °C per minute to reach -70 °C. In practice, the following procedure is possible: cryotubes are being isolated within a styrofoam box before being placed in a freezer of -80 °C. After two hours, the samples can be transferred quickly into a nitrogen tank. Alternatively, freezing machines or chest freezers may be used for the cooling process. 3. In which growth phase should the cells be when starting the freezing process? Cells should preferably be in the exponential growth phase. In addition, check the cell culture for contamination before starting the freezing process. 4. What should be considered when selecting cryotubes? Only vessels should be used that qualify for freezing conditions. Tubes should be made of temperature-resistant material (polypropylene, PP, is most suitable for cell cultures). Cryotubes should be well closed and tight in order to prevent a potential permeation of liquid nitrogen. There are tubes with external or internal threads. Biochrom AG offers cryotubes with external thread (TPP). Despite an unavailable silicone seal, the tube’s external thread as well as the nature of the cap guarantee safe and tight closure. When storing samples in liquid nitrogen, however, there is no guarantee for any kind of locking mechanism; appropriate safety precautions should thus be taken. 5. What should be considered when filling the cryotubes? It is important that the tubes are not being filled completely, as the liquid may burst the tubes due to the volume expansion caused by the freezing process. Some laboratories follow the Biochrom AG Leonorenstraße 2-6 12247 Berlin Germany internet: www.biochrom.de e-mail: info@biochrom.de phone.: +49 (30) 77 99 06-0 fax: +49 (30) 771 00 12 page 5 of 6 rule that the tube should always be filled up to two thirds. The ideal filling volume corresponds to the upper marking on the scaling of the TPP tubes. 6. What should be considered when storing samples in liquid nitrogen? When dealing with liquid nitrogen, the following safety precautions should absolutely be taken: wear protective clothing, including protective glasses and gloves. If not being closed tightly enough, liquid nitrogen might enter the tubes. This may lead to an explosion of the tube during the thawing process due to the quick expansion of nitrogen. In order to prevent this, cultures should either be stored in the gas phase or they should be frozen and thawed inside an additional protective covering. If the tubes should be removed from the liquid nitrogen without any protective covering, users should store them for 24 hours in the gas phase and then be removed safely. 7. Is it possible to store cryotubes only in the gas phase or is it necessary to store them in the liquid phase of the liquid nitrogen? The temperature in the liquid phase is -196 °C, while it is -155 °C in the gas phase. Since no chemical reactions occur within the cells at temperatures of less than -130 °C, storage in the gas phase is not only possible but also recommended for security reasons.1 8. Are there any alternative methods for the storage in liquid nitrogen? Cell cultures may also be stored in cryogenic chest freezers (approx. -140 °C). They are safer and less expensive than nitrogen vessels. 9. What should be considered when thawing the tubes? Users should wear protective clothing when handling the tubes. It is important that the cryotubes are being filled up to the identification mark, before being tightly closed. The tubes can then be thawed as explained in the different freezing protocols. If the TPP cryotubes had been stored in the liquid phase of nitrogen, TPP recommends the use of additional coverings. It is also possible to store the tubes in the gas phase of the liquid nitrogen for 24 hours before the removal. More information: further details on cryotubes on our homepage: http://www.biochrom.de/en/products/plastic-for-cell-culture/cryo-tubes/ full information about the freezing medium Biofreeze on our homepage: http://www.biochrom.de/en/products/cell-culture-media/freezing-medium-new/ our tip: use our new disposable counting chamber C-Chip for an easy and quick check of the cell count. Please contact info@biochrom.de to receive a free sample. 1 Mazur P.; Am J Physiol; 247 (3 Pt 1): C125-42 [1984] Biochrom AG Leonorenstraße 2-6 12247 Berlin Germany internet: www.biochrom.de e-mail: info@biochrom.de phone.: +49 (30) 77 99 06-0 fax: +49 (30) 771 00 12 page 6 of 6