Vit Kit - Thaw. Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage)

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1 Vit Kit - Thaw Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage) Catalog No DSO Includes: Thawing Solution - TS (yellow cap) 4 x 1 ml Vials Dilution Solution - DS (orange cap) 1 x 1 ml Vials Washing Solution - WS (red cap) 1 x 2 ml Vials For assisted reproductive procedures.

3 Caution: Federal law restricts this device to sale by or on the order of a physician or a practitioner trained in its use. Caution: The user should read and understand the Directions for Use, Warnings and Precautions, and be trained in the correct procedure before using the Irvine Kits for vitrification of human embryos. INTENDED USE Vit Kit - Thaw is intended for use in the warming of pronuclear (PN) zygotes through blastocyst stage embryos, which were vitrified using Irvine Scientific s Vitrification Freeze Kit (Vit Kit - Freeze, Catalog No DSO) for embryos. PRECAUTIONS AND WARNINGS This device is intended to be used by staff trained in assisted reproductive procedures that include the indicated application for which the device is intended. Do not use any vial of solution that shows evidence of particulate matter or cloudiness. To avoid problems with contamination, handle using aseptic techniques. Vitrification Thaw Kit Solutions contain the antibiotic gentamicin sulfate. Appropriate precautions should be taken to ensure that the patient is not sensitized to this antibiotic. The long term safety of embryo vitrification on children born following this method of embryo cryopreservation is unknown. *Human source materials used in the manufacture of this product have been tested by FDA licensed kits, and found to be non-reactive to the antibodies for Hepatitis B surface antigen (HbsAg), antibodies to Hepatitis C (HCV) and antibodies to Human Immunodeficiency Virus, (HIV). Donors of the source material have also been screened for CJD. However, no test method offers complete assurance that products derived from human sources are noninfectious. Handle all human source material as if it is capable of transmitting infection, using universal precautions. PRODUCT DESCRIPTION Thawing Solution-TS is a HEPES buffered solution of Medium-199 containing gentamicin sulfate (35 µg/ml), 1.0 M sucrose and 20% (v/v) Dextran Serum Supplement*. Dilution Solution-DS is a HEPES buffered solution of Medium-199 containing gentamicin sulfate (35 µg/ml), 0.5M sucrose and 20% (v/v) Dextran Serum Supplement*. 1/7

4 Washing Solution-WS is a HEPES buffered solution of Medium-199 containing gentamicin sulfate (35 µg/ml) and 20% (v/v) Dextran Serum Supplement*. These three solutions are to be used in sequence according to the step-wise microdrop warming protocol. QUALITY ASSURANCE The solutions in Vit Kit-Thaw are membrane filtered and aseptically processed according to manufacturing procedures which have been validated to meet a sterility assurance level (SAL) of Each lot of Vit Kit- Thaw receives the following tests: Solutions: Endotoxin by LAL methodology Biocompatibility by mouse embryo assay (one-cell) Sterility by the current USP Sterility Test <71> Albumin Test All results are reported on a lot-specific Certificate of Analysis, which is available upon request. FOR WARMING CRYOTIP AS THE STORAGE DEVICE: MATERIALS REQUIRED BUT NOT INCLUDED Irvine Scientific Connector (Catalog No ) or adaptor Sterile Petri Dishes (50 X 9 mm, Falcon or equivalent) Disposable gloves Hamilton GASTIGHT Syringe (50 µl) catalog #80901 Transfer pipettes (pulled glass pipettes or micropipette tips with an inner tip diameter of ~200 µm) Tweezers Stopwatch or timer Liquid Nitrogen Reservoir (Dewar or Styrofoam container with lid, 1-2 L volume) Liquid Nitrogen (sufficient volume to achieve 4 inch depth in reservoir) Sharp scissors (sterile) 37 C waterbath Culture Medium (with 20% protein) appropriate for develop mental stage of specimen to be recovered. Prepare and pre-equilibrate dish with culture medium to 37 C in CO 2 incubator prior to thawing specimens. DIRECTIONS FOR USE Vit Kit-Thaw components (per application): 50 µl of Thawing Solution TS 50 µl of Dilution Solution-DS 100 µl of Washing Solution-WS 1 Connector WARMING PROTOCOL NOTE: Procedures are to be done at room temperature (20-27º C). DO NOT use heated microscope stage for the following procedures. CAUTION: Minimize exposure of specimen to light during manipulations through warming solutions. 2/7

5 1. Bring the quantity to be used of TS, DS and WS to room temperature (20-27 C) prior to warming vitrified specimens. NOTE: Avoid bringing the entire vials of TS, DS and WS to room temperature repeatedly when a small quantity of the solution is needed each time. It is better to aliquot the quantity to be used and return the vials to 2-8 C right after aliquoting. 2. Fill the liquid nitrogen reservoir with liquid nitrogen (~80 % full) and place close to the LN 2 freezer containing the specimens to be warmed. 3. Remove the canes with goblets containing the CryoTips with vitrified specimens from liquid nitrogen storage and transfer them into the liquid nitrogen filled reservoir. CAUTION: Make sure that CryoTips remain submerged in LN 2 (in goblet) during transfer from storage to LN 2 reservior to prevent uncontrolled thawing of specimens. Place the reservoir close to the microscope for rapid manipulation. 4. Label a sterile petri dish (or lid) with necessary information. 5. Gently invert each vial of TS, DS and WS twice to mix contents before use. 6. Prepare the dish with droplets of solutions for warming protocol as follows: Aseptically dispense a sequence of 2 microdrops on an inverted lid of sterile Petri dish as shown in Figure 1, and place the dish on the microscope stage: one-50 µl drop of TS one-50 µl drops of DS (Two drops of WS will be set up later at step 13) Figure 1. TS DS 50 µl Drops 7. Place the 37 C waterbath close to the microscope. Have the following nearby: a transfer pipet and tips, sterile sharp scissors, Hamilton syringe and sterile wipes. 3/7

6 8. Using tweezers (or tongs), retrieve the specific CryoTip from the cane in liquid nitrogen, quickly immerse the CryoTip into the 37 C waterbath (>500mL) and swirl gently for 3 seconds to warm (see Figure 2) at + 24,000 C/min. NOTE: Pull out the plunger about 0.5 inches before attaching the syringe to the Connector and the CryoTip. Figure C H C/min LN 2 (3 sec) Waterbath >500mL 9. Quickly dispense contents of CryoTip as follows (see Figure 3): Quickly wipe the CryoTip dry with a sterile wipe Remove the metal cover sleeve Cut the seal on the wide end of the CryoTip at Mark #4 Attach wide end of the CryoTip securely to Hamilton syringe or appropriate aspiration tool using a connector or adaptor. Gently wipe the fine tip dry with a sterile wipe. With the CryoTip positioned over the prepared thawing dish, quickly cut the seal on the Mark #2 of the fine tip end and dispense the contents of the CryoTip as a small drop (~ 1 µl) onto a dry region of the dish above the TS drop (see Figure 4). NOTE: AVOID BUBBLES WHILE DISPENSING THE CONTENTS. Figure 3. Mark #4 Seal #2 Cut #1 Mark #3 37 C After Warming Mark #2 Mark #1 Seal #1 Cut #2 4/7

7 Figure 4. CryoTip Contents (~1 µl) TS 1 min DS 50 µl Drops 4 min WS1 4 min each WS2 Transfer to Culture Medium with 20% (v/v) protein for Recovery 10. Then, merge the TS drop with the CryoTip content and allow gradual mixing for 1 minute (see Figure 4). NOTE: the specimens will shrink and float to the top of the drop. NOTE: After each transfer of the specimen(s), blow out any remaining fluid in the transfer pipet and draw up some solution from the next drop prior to the next manipulation. Avoid creating bubbles during the transfers. 11. Draw up some DS into the transfer pipet and transfer the specimen(s) from the drop of TS with minimal volume drop of DS for 4 minutes. NOTE: The specimen will remain shrunken during exposure to DS. DURING THIS TIME SET UP THE TWO-50 µl DROPS OF WS (WS1, WS2), AS SHOWN IN FIGURE Transfer the specimen(s) to the drop of WS (WS1) for 4 minutes. NOTE: The specimen(s) should re-expand to the original size within 2-3 minutes in WS. 13. Then transfer the specimen(s) to the second drop of WS (WS2) for 4 minutes. 14. Finally, transfer the specimen to a pre-equilibrated culture dish containing the appropriate culture medium (with 20% protein). Incubate the specimens accordingly in a 37ºC incubator with CO 2 to complete recovery prior to further manipulations (e.g. transfer or 5/7

8 extended culture of embryos). FOR WARMING OTHER CRYOSTORAGE DEVICES: MATERIAL REQUIRED BUT NOT INCLUDED Sterile 4-well dish (Nunc , or equivalent), or organ culture dish (BD Falcon ) Disposable gloves Transfer pipettes Tweezers Stopwatch or timer Liquid nitrogen resevoir Liquid Nitrogen Scissors Culture Medium with 20% protein, pre-equilibrated to 37 C in CO 2 incubator prior to thawing procedure. 37 C incubator without CO 2, or heating stage DIRECTIONS FOR USE Vit Kit Thaw components (per application) 250 µl of Thawing Solution TS 50µL of Dilution Solution - DS 50 µl of Washing Solution WS WARMING PROTOCOL NOTE: The warming steps include plunging the device into the 37 C TS and subsequent diluting and washing in DS and WS at room temperature 1. Set up warming dishes (as shown in Figure 5): At 37 C: Aseptically dispense 250 µl of TS into a sterile 4-well dish or an organ culture dish and place it in a 37 C incubator without CO 2 or on a heating stage at least 30 minutes prior to thawing procedure 2. Identify cryostorage device(s) to be thawed from LN 2 storage and quickly transfer to LN 2 filled reservoir in preparation for warming procedure. 3. Place LN 2 reservoir close to microscope for subsequent rapid manipulation. 4. Remove TS dish from 37 C incubator or heating stage and place it under focus on top of the microscope stage. 5. Warm the selected cryostorage device and dispense specimen(s) into 37 C TS solution according to manufacturer s instructions. 6. Leave specimen(s) in TS for 1 minute. Steps 7-10 must be performed at room temperature At room temperature: Aseptically dispense one (1) 50 µl drop of DS on a sterile Petri dish 7. Aspirate some DS into the transfer pipette and transfer the specimen(s) from the TS dish with minimal volume to the drop of DS for 4 minutes. NOTE: The specimen will remain shrunken during 6/7

9 exposure to DS. DURING THIS TIME SET UP THE TWO-50 µl DROPS OF WS (WS1, WS2), AS SHOWN IN DIAGRAM. 8. Transfer the specimen(s) to the drop of WS (WS1) for 4 minutes. NOTE: The specimen(s) should re-expand to the original size within 2-3 minutes in WS. 9. Then transfer the specimen(s) to the second drop of WS (WS2) for 4 minutes. 10. Finally, transfer the specimen to a pre-equilibrated culture dish containing the appropriate culture medium (with 20% protein). Incubate the specimens accordingly in a 37ºC incubator with CO 2 for 3-4 hours to complete recovery prior to further manipulations (e.g. transfer or extended culture of embryos). For additional details on the use of these products, each laboratory should consult its own laboratory procedures and protocols which have been specifically developed and optimized for your individual medical program. STORAGE INSTRUCTIONS AND STABILITY Store the unopened vials of solutions refrigerated at 2 C to 8 C. When stored as directed, Vitrification Thaw Kit solutions are stable until the expiration date shown on the vial label. Do not use media for more than eight (8) weeks once containers have been opened. As human source material is present in the product it may develop some particulate matter during storage. This type of particulate matter is not known to have an effect on product performance. GASTIGHT is a Registered Trademark of Hamilton Co. 7/7

10 REFERENCES 1. Kuwayama M, Vajta G, Kato O, Liebo SP. Highly efficient vitrification of human oocytes. RBM Online 11: , Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. RBM Online 11:53-57, Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 84:88-92, Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. RBM Online 11: , Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. RBM Online 6: , Isanchenko V, Selman H, Isachenko E, Montag M, El-Danasouri I, and Nawroth F. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reproductive BioMedicine Online 7, , Van den Abbeel E, Camus M, Van Waesberghe L, Devroey P, Van Steirteghem AC. A randomized comparison of the cryopreservation of one-cell human embryos with a slow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen. Hum. Reprod. 12: , Shee-Uan Chen, M.D., Yih-Ron Lien, M.D., Kuang-Han Charo, M.D., Hsin-Fen Lu, M.D., Hong-Nerng Ho, M.D., an Yu-Shih Yang, M.D., Ph.D.,, Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws, Fertility and Sterility, vol. 74(4), , Michel Camus, M.D., Etienne Van den Abbeel, B.Sc., Linda Van Waesberghe, M. Sci., Arjoko Wisanto, M.D., Paul Devroey, M.D., and Andre C. Van Steirteghem, M.D., PhD., Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant, Fertility and Sterility, Vol. 51 (3), , Balaban, et. al., A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation, Human Reproduction, Vol. 23, No. 9, pp , Yoko Kumasako, Mami Kumon, Takafumi Utsunomiya, and Yasuhisa Araki, Successful Pregnancy after the Vitrification of Zygotes Using Commercial Vitrification Solutions and Conventional Straws to Protect Against Infections of Liquid Nitrogen, Journal of Assisted Reproduction and Genetics, Vol. 22, No. 1, January S. Hashimoto, Y. Murata, M. Kikkawa, M. Sonoda, H. Oku, T. Murata, K. Sugihara, F. Nagata, Y. Nakaoka, A. Fukuda, and Y. Morimoto, Successful delivery after the transfer of twice-vitrified embryos derived from in vitro matures oocytes: A Case Report, Human Reproduction, Vol. 22, No. 1, pp , Safaa Al-Hasani, Batuhan Ozmen, Nikoleta Koutlaki, Beate Schoepper, Klaus Diedrich, Askan Schultz-Mosgau, Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?, RMB Online, Vol. 14, No. 3, pp , Meghan B. Oakes, M.D., Claudia Messias Gomes, M.D., Joyce Fioravanti, B.S., Paulo Serafini, M.D., Ph.D., Eduardo L.A. Motta, M.D., Ph.D., and Gary D. Smith, Ph.D., A case of oocyte and embryo vitrification resulting in clinical pregnancy, Fertility and sterility, Vol. 90, No. 5, November Yoko Kumasako, B. E., Eiko Otsu,.En., Takafumi Utsunomiya, M.D., and Yasuhisa Araki, Ph.D., The efficacy of transfer of twice frozen-thawed embryos with the vitrification method, Fertility and Sterility, Vol. 91, No. 2, pp , February Nina Desai, Heather Blackmon, Julia Szeptycki, James Goldfarb, Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births, RBM Online, Vol. 14, No. 2, pp , A-J Kartberg, F Hambiliki, T Arvidsson, A Stavreus-Evers, P Svalander., Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO. RBM online, Vol 17, No. 3, pp , 2008.

12 Symbols: Catalog Number Lot Number Sterilized using aseptic processing techniques (filtration) Expiration: Year - Month - Day Caution: See instructions for use 2 C 8 C Storage Temperature Manufacturer 2511 Daimler Street, Santa Ana, California Telephone: Fax: PN Rev. 8

Vit Kit - Thaw. Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage)

Vit Kit - Thaw. Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage) Vit Kit - Thaw Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage) Catalog No. 90137DSO Includes: Thawing Solution - TS (yellow cap) 4 x 2 ml Vials Dilution Solution - DS (orange cap) 1 x