RLI Mouse Vitrification Media Kit

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Aubrie Arnold
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1 RLI Mouse Vitrification Media Kit Product Description RLI Vitrification Media Kit (Catalog#: RLI Vitri-Cooling 01, RLI Vitri-Warming 01, RLI Vitri Complete Kit 01) enables ultra-rapid cooling and recovery of mouse embryos, which eliminates ice crystal formation, resulting in increased mouse blastocyst development and survival rates. It is specifically designed for vitrification of mouse embryos from 4-cell to blastocyst stage. A high blastocyst development and survival rate is guaranteed. This medium is developed with Renova Life Inc. (RLI) standardized recipes that have been proven to be perfectly suitable for vitrification of mouse embryos in peer-reviewed publications. Years of our research and experience have been put into the development of this medium, offering you the best quality and performance for vitrification. It is specifically tested by mouse embryo assay. RLI Vitrification Media Kit vitrified and warmed embryos have a survival rate of 98.1% to 99.6%; developmental potentials of %. After embryo transfer of vitrified ICR embryos from 4- celled, 16-celled, morulae and blastocyst stage, % of the embryos develop into live offspring (An L et al. Cryobiology; 2015, RLI Vitri-Cooling media includes, Base, Holding and Vitrification solution. solution is 7.5% fetal bovine serum in DPBS supplemented with Pen-Strep and phenol red. Base solution is 15% FBS in D-PBS supplemented with Pen-Strep and phenol red. Holding solution is basal solution supplemented with 1 M dimethylsulphozide (DMSO) and 1.2 M ethylene glycol (EG). Vitrification solution is basal solution supplemented with 2.2 M DMSO and 2.6 M EG and sucrose, with ph at 7.8. RLI Vitri-Warming media includes Warming, Rehydration, Base and solution. Warming solution is base solution supplemented with 0.28 M sucrose. Rehydration solution is base solution supplemented with 0.18 M sucrose. Base and solutions are the same with the ones in RLI Vitri-Cooling media. RLI Vitrification Media Kit is a constant high quality product, ideal for mouse embryo vitrification. It is prepared for commercial and research use. RLI Vitrification Media Kit has been prepackaged as follows: RLI Vitri-Cooling 01:, 30 ml/bottle; Base, 4 ml/vial, 2 vials; Holding, 4 ml/vial, 4 vials; Vitrification 4 ml/vial, 2 vials. RLI Vitri-Warming 01, Warming, 30 ml/bottle; Rehy, 4 ml/vial, 2 vials; Base, 4 ml/vial, 2 vials; 4 ml/vial, 4 vials. Cat # RLI Vitri Complete Kit 01 Includes Cat # RLI Vitri-Cooling 01 and Cat # RLI Vitri-Warming 01. Each kit is good for 10 reactions of vitrification and 10 reactions of warming. Storage temperature: Store Holding and vitrification solution at -20 C upon arrival. Store, Base, Rehy and Warm at 2-8 C upon arrival. Expiration: 2 months after production date.

Protocol and Procedures for Vitrification of Mouse Embryos with Modified Droplet Vitrification (MDV) A. Materials Required but not Included for Vitrification and warming 1.

2 Protocol and Procedures for Vitrification of Mouse Embryos with Modified Droplet Vitrification (MDV) A. Materials Required but not Included for Vitrification and warming 1. Sterile Petri Dishes (60 x 15 mm, Falcon , 35 x 10 mm, Falcon or equivalent) and 4-well multidishes (1 ml wells, Nunc or equivalent) 2. Cryovials (from Corning, 2 ml vials) and cryocanes including one cryocane bended above the bottom first grid 3. Disposable gloves 4. Transfer pipettes (pulled glass pipette with micro-pipette tips fit the embryos 5. One ultra-point tips tweezers, two hemostatic forceps (6.5 long). 6. Timers 7. Liquid Nitrogen Reservoir (stainless steel, 5 I.D., 5 depth; a half open inner cover with ¾ depth from top, ¼ height to form a working surface for MDV method) sitting is a styrofoam box (3 depth) 8. Liquid Nitrogen (sufficient volume to achieve 5 inch depth in reservoir, and some extra to add into the reservoir when Liquid Nitrogen in the working surface and in reservoir evaporates)

3 B. Vitrification Mouse Embryo Vitrification and Warming Vitrification Package LN2 Metal cube Storage LN 2 Metal cube Selected Code 1 embryos LN 2 Warming Selection and culture Warming solution LN 2 Metal cube 1. Warm Vitri-cooling media to 37 C at least 30 min prior to vitrification on a heating plate at 38 C. 2. Determine the number of embryos to be vitrified. 3. Label cryovials (with #18 gage injection needle, poke 4 holes on the side of round cryovial underneath the cap thread. Make sure the holes are made in case of gas explosion during sealing with cap, LN2 gas can leak out from cryo-vial through small holes) and cryocanes with necessary information. 4. Make sure the contents of each vial of warmed Vitri-cooling media are well mixed by gentle inversion several times before use. 5. Using pipetman p1000 add 0.75 ml of solution into each well of a 4-well dish. Label each well as. Using pipetman p1000 add 0.75 ml of Base, Holding and Vitrification solution into each well of a 4-well dish. Label each well as Base, Hold, Hold and Vitri. Vitrification Medium 0.75 ml/well Solution 0.75 ml/well Base Hold Hold Vitri 4-well dish-1 4-well dish-2 6. Take the culture dish with the embryo(s) out of the incubator and check the quality of the embryos. Use only the best quality embryo(s) for vitrification.

7. Carefully transfer the embryo(s) with a minimal volume of culture medium to solution, sit for 5 min, keep the 4-well dish on the 38 C heating plate. 3-4 times.

Fill the liquid nitrogen reservoir with liquid nitrogen to a sufficient depth to touch the bottom of the inner cover and place near to the heating plate and the microscope.

4 7. Carefully transfer the embryo(s) with a minimal volume of culture medium to solution, sit for 5 min, keep the 4-well dish on the 38 C heating plate. 3-4 times. Transfer the embryo(s) to Base solution, sit for 5 min on the 38 C heating plate. 8. Fill the liquid nitrogen reservoir with liquid nitrogen to a sufficient depth to touch the bottom of the inner cover and place near to the heating plate and the microscope. Put the straight cryocane(s) and a bended cryocane into the liquid nitrogen. 9. Attach a cryovial to the bottom of the bended cryocane and submerge in the liquid nitrogen in preparation for storage of the vitrified embryo(s). Transfer the embryo(s) from Base solution to Holding solution on the 38 C heating plate, sit for 1.5 min till embryos deposit to the bottom. Meanwhile, make 6 small drops (around 20 µl/drop) of Vitrification solution in 60 mm 1007 petri dish, and sit on the 38 C heating plate. 10. Transfer 4-5 embryo(s) from Holding solution to the first drop of Vitrification solution ( petri dish) within 10 seconds. 11. Quickly transfer the embryo(s) from the first drop to the second drop of Vitrification solution ( petri dish) within 10 seconds. 12. Finally, from the third drop, take 3 µl vitrification medium containing 4-5 embryos (about 1µl Vitrification solution, air bubble, and then 2 µl vitrification solution containing four to five embryos. Blow all the whole content out the fine glass pipet to form a small drop (total 3 µl) at the tip of the glass pipet.

One hand holds the glass pipet tip toward the top layer of the LN 2 container (about 10 cm above the LN2 layer, the other hand hits the hand holding the glass pipet.

If more embryo(s) are to be vitrified, repeat steps 7 through 12 above.

5 One hand holds the glass pipet tip toward the top layer of the LN 2 container (about 10 cm above the LN2 layer, the other hand hits the hand holding the glass pipet. The tipping action makes the 3 µl drop fall into LN 2 layer in LN2 container. After a while, the glass-like ball will sink to the bottom of LN cover container. 13. If more embryo(s) are to be vitrified, repeat steps 7 through 12 above. After all the drops are made, put the cryovial onto the bended can, open the cryovial, make it close to the edge of the top layer of the LN 2 container, pick up drops one by one and put all drops into labelled cryovial, using a ultra-point tips tweezers. 14. Screw closely the cap of the cryovial with two hemostatic forceps. 15. Put the tube on a previously labeled cryocan, store the can in LN 2 tank, label the tank. Vitrification of Mouse Embryos with Cryo-Loop and Cryo-Tech If other carrier devices to be used, follow the instructions for the carrier devices. Follow the step 1-11.

6 For CryoLoop and CryoTech vitrification, after equilibrating (step1-12), embryos in 3 μl vitrification solution are loaded on the tip of CryoLoop (CVM1, FP10NS, Cyologic, Inc, Australia) or CryoTech (Cryotech, Vitrification Kit 101, CryoTech, Japan) and plunged into liquid nitrogen immediately. This will make a glass-like ball. Vitrification by Cryo-Loop and Cryo-Tech Straw Seal Straw Cryo-Loop Straw Seal Straw Cryo-Tech 14. CryoLoop or CryoTech embryo samples are packed in labeled canes and stored in liquid nitrogen. C. Warming 1. Warm Vitri-warming media to 37 C at least 30 min prior to warming on a heating plate at 38 C. 2. Fill the liquid nitrogen container with LN 2 to a sufficient depth, and completely submerge a cryocan containing the cryovial, or other carrier device such as CryoLoop or CryoTech. 3. Remove the cryocanes with the cryovials containing the vitrified embryo(s) and quickly transfer them to the LN 2 container, keeping the cryovials under LN 2 at all times. 4. Make sure the contents of each vial of Warming, Rehydration, Base and solution are well mixed by gentle inversion several times before use. 5. Label a sterile petri dish (351008) with necessary information. Add 3 ml Warming solution into the petri-dish. 6. Label a 4-well dish with Rehy, Base,, and, add 0.75 ml of Rehydration, Base,, and solution into each well respectively. Warming Medium Warm 3 ml/dish Solution 0.75 ml/well Rehy Base Warm mm dish 4-well dish

7 7. Pour the vitrified balls (embryos) onto the top layer of the LN2 container which containing sufficient LN2 to submerge the vitrified embryos. 8. Pick up one ball with ultra-point tweezers and transfer immediately into 3 ml warming solution (at 38 C), mix the thawed solution with warming solution thoroughly for 1.5 min. 9. Transfer the embryos into Rehydration solution, and incubate on 38 C warming plate for 4 min. 10. Transfer the embryos into Base, and then Rise solution for 4 min on 38 C warming plate, respectively.

11. Finally, load embryos in ET straws for embryo transfer. Or culture warmed embryos in KSOM supplemented with 3mg/ml BSA at 37 C in 5% CO2, 5% O2, and 90% N2. 12.

For Cryo-Loop and Cryo-tech, directly drop the straw containing vitrified embryo ball into 1008 35 mm dish with 3 ml warming medium, then wash embryos from Rehy, Base and medium,

8 11. Finally, load embryos in ET straws for embryo transfer. Or culture warmed embryos in KSOM supplemented with 3mg/ml BSA at 37 C in 5% CO2, 5% O2, and 90% N If more embryos are to be warmed, repeat steps 7 through 11 above. For Cryo-Loop and Cryo-tech, directly drop the straw containing vitrified embryo ball into mm dish with 3 ml warming medium, then wash embryos from Rehy, Base and medium, respectively, each step for 4 min. After culture in KOSM+BSA under tri-gas condition, the embryos will recover and develop into blastocyst stage, or live babies will be born at embryo transfer at extremely efficiency. Please check the figure below, or check our newly published paper at Cryobiology, 2015.

References An L, Chang S, Hu Y, Xu B, Zhang F, Yang L, Presicce G and Du F*. Efficient Cryopreservation of Mouse Embryos by Modified Droplet Vitrification (MDV). Cryobiology; 2015, http://dx.doi.

9 References An L, Chang S, Hu Y, Xu B, Zhang F, Yang L, Presicce G and Du F*. Efficient Cryopreservation of Mouse Embryos by Modified Droplet Vitrification (MDV). Cryobiology; 2015, Ordering Information Cat # RLI Vitri-Cooling, 30 ml/bottle; Base, 4 ml/vial, 2 vials; Holding, 4 ml/vial, 4 vials; Vitrification 4 ml/vial, 2 vials. $ plus S&H Cat # RLI Vitri-Warming Warming, 30 ml/bottle; Rehy, 4 ml/vial, 2 vials; Base, 4 ml/vial, 2 vials; 4 ml/vial, 4 vials. $ plus S&H Cat # RLI Vitri Complete Kit 01 Includes Cat # RLI Vitri-Cooling 01 and RLI Vitri-Warming 01 $ plus S&H Each kit is good for 10 reactions of vitrification and 10 reactions of warming. Each reaction is good for vitrifying/warming more than 100 embryos. Therefore, one Vitri Complete Kit 01 (cooling + warming) can be used for vitrification of 1000 mouse embryos. Other packaging and bulk ordering is available upon request.

CRYOTOP SAFETY KIT Protocol. Cryotop Method

CRYOTOP SAFETY KIT Protocol. Cryotop Method CRYOTOP SAFETY KIT Protocol Cryotop Method R Vitrification PART Materials Required Cryotop Safety Kit-Vitrification No.0 Basic Solution (BS): 1 X 1.5ml vial (Only for Oocyte Vitrification) No.1 Equilibration