1. What advantages does the Askion C-line system offer over conventional systems?

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1 FAQ s Hermetic storage (HS) & work bench (WB) 1. What advantages does the Askion C-line system offer over conventional systems? - They feature a cryogenic workroom uninterrupted cooling chain, controlled environmental conditions samples that were not handled in an extremely cold working space, thawing at about -130 C at room temperature within 39sec (1 ml vial), they are therefore in the range in witch recrystallization processes deploy - Hood system self contained unit results in minimal icing - Possibility of regulation by reference sample sensor. - Semi and fully automated storage and retrieval processes in temperatures below -100 C - Excellent data management. Complete recording and storage of all system and tracking data. - Semi automated sample handling (motorized storage containers). - Individually configurable freezing system. - Modular and can be customized regarding sample size and storage capacity. - Interface to existing LIS / HIS. - Minimal errors due to SOP based / supported user guidance. 2. Which sample formats can be processed? - Vials of all sizes and from established manufactures (e.g Nalgene, Nunc, Sarstedt, Brand 1 5ml with internal or external thread). - Blood bags and cassettes from all established manufacturers (Miltenyi, Fresenius DF200, Macopharma, Baxter) up to a size of 200 mm edge length (equivalent to 100ml filling volume). - Straws (e.g. CryoBioSystem, Miltenyi) - Vials with integrated electronic memory chip "Icebreaker" from Perma Cryo technology ( 3. How many samples can be frozen and stored? Freezing in the WB2X0: WB 220 per basket WB 230 per basket : 300 x 0,5ml, 0,25ml 300 x 0,5ml, 0,25ml cassettes: 6 x ml, ml, ml - 18 x 10-30ml 6 x 10-30ml vials: 6 x 96 wells (1; 0,5ml) 6 x 96 wells (1; 0,5ml) 4 x 81 box - 6 x 18 (2ml) (tube rack) 6 x 18 (2ml) (tube holder) 1 / 6

2 Storage in the HS200: sample type volume numbers of samples / drawer drawer / rack numbers of samples / tower vial (Ø 9mm) 0,5ml ,360 vials vial (Ø 9mm) 0,75ml ,920 vials vial (Ø13mm) 1,8ml ,008 vials straw straw 0,5ml 0,25ml 351 goblets; each with goblets; each with goblets; each with goblets; each with wells 96x0,5ml wells cassette 100ml (192x142x13) cassettes 4. What are the benefits of storing samples in the gas phase of liquid nitrogen? - There is less stress for the sample containers because there are no aging effects caused by liquid nitrogen. - Lower consumption of liquid nitrogen. - The risk of possible cross-contamination is essentially eliminated (Tedder, 1995). - It prevents liquid nitrogen entering poorly sealed tubes. This can cause problems when thawing at a later date (Lindl 2008). - Most cryovials are not authorized or suitable for storage in liquid nitrogen ( Warning: Do not use CryoTubes in liquid nitrogen (liquid phase storage), unless correctly protected by CryoFlex tubing. (VWR)). Tedder R.S., Zuckermann M.A., Goldstone A.H., Hawkins A.E., Fielding A., Briggs E.M., Irwin D., Blair S., Gorman A.M., Patterson K.G., Linch D.C., Heptonstall J. and Brink N.S. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 346: , 1995 Lindl T., Gstraunthaler G. Zell- und Gewebekultur. Spektrum, What advantage does the temperature range of -150 C have? - When compared to storage at -80 C, the quality and survival rate of certain organic materials (e.g. cells) increase substantially when stored at -150 C (Leuthold 2001) C for long term storage is not suitable (Sputtek 1996 a) 2 / 6

3 - Above -130 C, there are still measurable recrystallization processes (Körber 1995, Sputtek 1996 b) which are not desired. Below -130 C, there are no measureable recrystalisation processes and, therefore, biological samples will not be damaged (Mazur 1984). - In conjunction with the cryogenic working space and a temperature of less than -150 C in the storage tank, the samples are never exposed to temperatures greater than -130 C during the storage and retrieval of other samples. Körber, Ch.; "Physikochemische Grundlagen der Kryokonservierung von Zellsuspensionen", Beitr. Infusionsther. Transfusionsmed. 22 Nr.1, (1995 ) Leuthold J., Untersuchungen zur Qualität von peripheren Blutstammzellpräparaten - ultrastrukturelle Studien zur Beurteilung der Kryoprotektion, 2001 Sputtek, A.: "Kryokonservierung von Blutzellen". in: Mueller-Eckhardt, C. (Hrsg.): Transfusionsmedizin, Grundlagen-Therapie- Methodik, 2. Auflage, S , Springer Verlag (1996 a) Sputtek, A.; Gutensohn, K.; Hummel, K.; Löliger, C.; Kühnl, P.: "Zur Kryokonservierung von Blutstammzellen". J. Lab. Med. 20 Nr.2, (1996 b) Sputtek, A.; Jetter, S.; Hummel, K.; Kühnl, P.: "Cryopreservation of periphal blood progenitor cells: Characteristics of suitable techniques". Beitr. Infusionsther. Transfusionsmed. 34, (1997 ) Sputtek A.; Horn E.P.; Schulte A.; Esch J.; Kühnl P.: "Kryokonservierung von Erythrozyten mit Hydroxyethylstärke (HES) - Vom Laborversuch zur klinischen Anwendung". Anaesthesiologie, Intensivmedizin, Notfallmedizin, Schmerztherapie : 36, (2001) 6. What does uninterrupted cooling chain mean? - From initial freezing, at no point do the samples come in to contact with environmental conditions within the Askion C-line devices (humidity, room temperature.) This means that ice formation is minimal and the frozen samples remain permanently frozen. - The temperature in all of the processing steps is below -100 C (adjustable between approx -120 C 10 C if necessary.) - A precooled transport vessel is used to maintain the uninterrupted cooling chain when moving between the freezing stage and the storage stage. 7. What is the consumption rate of liquid nitrogen in each device? - hermetic storage: stand by mode: approx 12 kg/day 1 L. Ca. 0,5-1,0 operation mode: approx 50 kg/day - work bench: operation approx 50 kg/day 8. Does the system have a medical license? - The Hermetic Storage and Workbench can be delivered as medical devices of Class 2A guideline 93/42. 3 / 6

4 9.. How can the C-line system be connected to an existing lab system? - In general, any component of the Askion C-line system can be integrated into an existing laboratory setup - The connection is via a data interface from the Askion C-line control. 10. What services are provided by Askion as a system supplier? - Consulting - Installation - Service - Support in the layout of your laboratory space and advice on what other requirements are necessary. - Customised manufacturing of certain parts. - Ordering extra parts for your system based on our expertise. 11. What is the temperature distribution inside of a rack of the Askion C-line hermetic storage? - At the uppermost storage position where the samples are located, a temperature of -150 C is guaranteed when the rack is in a resting position - The tank becomes increasingly colder when descending from the top to the bottom. The lowest temperature is at the very bottom where the source of liquid nitrogen is located. 12. How does the freezing process in the Askion C-line work bench work? - Liquid nitrogen is inserted into a tank at the bottom of the freezer. This causes a temperature gradient between the bottom and the top of the freezer (-160 C up to 8 C.) The samples to be frozen begin near the top of the freezer and are automatically driven towards the bottom according to a specified freezing curve. This process is regulated by a temperature sensor located in close proximity to the samples. A second sensor which is 4 / 6

5 placed directly inside one of the samples can also be used to regulate the freezing process more accurately. 5 / 6

6 13. What is the temperature distribution in the workbench? - From the top to the bottom of the freezing area, the temperature ranges between 8 C and -160 C. The temperatures are homogeneous over the horizontal cross sections of the freezing area. - The working space has a temperature below -100 C (adjustable between approx -120 C 10 C if necessary). 14. What freezing gradients can be achieved in the Askion C-line workbench? - This ranges from 0.1 K/min to 30 K/min. This is strongly dependent on the sample format (e.g Straws at 30 K/min, cassettes at 3 K/min) and the mass of what is being frozen. 15. What types of samples can be frozen and stored in the Askion C-line system? - All sample types can be accommodated. These include cell lines, tissue samples, stem cells, umbilical cord blood, sperm, embryos etc. - The system can be utilised in blood banks, reproduction establishments, pathology, medicine and many other areas of research. Askion GmbH Gewerbepark Keplerstraße D Gera Germany Tel. +49 (0) Fax +49 (0) cryo@askion.com 3-Dec / 6