Vitrification of mouse embryos in straw

Transcription

1 Vitrification of mouse embryos in straw Based on a method originally published by Nakagata et al (1997) 1. Materials 1.1. Media and solutions PB1 as basic solution M DMSO solution in PB DAP213 vitrification solution M Sucrose in PB1 as warming solution KSOM +AA for embryo recovery from vitrification and in vitro culture 1.2. Equipment mm culture dishes (Falcon, Cat. No ) mm culture dishes (Falcon Cat. No ) Vitrification plate for straw (Elim Springs Biotech Ltd) Base and lid form a clear polystyrene box (Part No ; Azpack Limited) ml French Straws (Cat. No. FZA201; Planer Plc) µl, 200µl and 1.0ml pipette, plus tips (e.g. Gilson) Mouth pipette Microscope Liquid nitrogen Straw rods Scissors 2. Vitrification procedure minutes before the vitrification, fill a polystyrene box with crushed ice; 1

2 2.2. Put the vitrification plate on the ice for 5min before starting vitrification Push the cotton/polyvinyl alcohol plug into straw, from position A to position B, using a metal rod with a stop Place the straws on a Perspex support. Using a permanent marker pen, make two calibration marks using the guidelines on the plate to obtain the correct distances. The calibration marks should be placed at 11mm and 25mm intervals from the end of straw (opposite the PVA plug end), see blew: 2.5. Place the straws on the pre-cooled vitrification plate and place DAP213, 0.25M Sucrose solution on ice. 2

3 2.6. Place a 60mm dish on the vitrification plate marked with DAP213. Then place a few drops of 45μl DAP213 in the dish (the number of drop depends on how many straws of embryos will be vitrified.) 2.7. Filter the 1M DMSO and place 4-6 drops (~100µl) of 1M DMSO into the base of 60mm culture dish. One is to wash the embryos, while the others are to hold the embryos. 3

4 2.8. Transfer the embryos from the culture medium into one of the 4 drops, then divide the embryos into groups and transfer them to the other drops Place the plate on the vitrification plate for 5min Using a wedge-shaped 10µl pipette tip (Axygen Inc; T-400) transfer 5μl 1M DMSO and embryos into the top of one of the 45μl DAP213 drops. Set a countdown timer for 5mins After 1min attach a 1ml syringe to the labelled end of the straw. Using the syringe, aspirate 0.25M sucrose (in PB1 medium) until the meniscus reaches Mark 1; Aspirate air, to move the sucrose meniscus from Mark1 to Mark 2; Aspirate DAP213 and the embryos until the meniscus reaches Mark 2; Aspirate air until the sucrose meniscus reaches the polyvinyl alcohol, half way along the cotton plug. Then put back the straws on vitrification plate for the rest of the 5mins During the equilibration, seal the labelled end of the straws After the 5mins have elapsed, plunge the straws directly into liquid nitrogen (LN 2 ). Transfer the straws to a secure LN 2 location as soon as you can. 3. Warming vitrified embryos 3.1. Place a tube of 0.25M sucrose warming solution (1ml) in incubator for at least 5min before warming; 3.2. Add 0.9ml 0.25M sucrose (pre-warmed to 37 C) into a 60mm culture dish, and place the dish on a 37 C warmed stage; 3.3. Remove the required straw from the storage tanks and place in a flask of LN Using forceps take the straw from LN 2 and place it in a C water bath. Once the DAP213 fraction has melted remove the straw immediately from water bath Wipe the straw with a tissue, then holding the straw firmly and horizontally, cut through middle of the cotton/pva plug; 3.6. Cut off the heat sealed end of the straw, then holding the straw vertically over the drop of 0.9ml 0.25M sucrose, use a metal rod to push down the remaining 4

5 cotton/pva plug, expelling the contents of the straw into the drop. Do not allow the tip of straw to touch the drop Gently rotate the dish to mix the solution Set a countdown timer for 3mins. Collect the embryos from the solution, and transfer them into a drop of KSOM +AA, then keep them in an incubator (37 C, 5%CO 2 in air) for 10mins Wash the embryos through another two KSOM +AA drops before the embryos are assessed for damage After the final wash the embryos are ready to be transferred into the recipient female. Follow the surgical embryo transfer procedures described by Nagy et al If you are not ready to set up the embryos transfers the embryos should be kept in KSOM +AA drop and held in the incubator until required. 4. Reagents and solutions Composition of PB1 medium. Osmolality should be mOsm. Reagent Name mg/100ml Vendor Catalogue Number NaCl 800 Sigma S-5886 KCl 20 Sigma P-5405 CaCl 2 12 Sigma C-5670 KH 2 PO 4 20 Sigma P-5655 MgCl 2 6H 2 O 10 Sigma M-2393 Na 2 HPO Sigma S 5136 Na-Pyruvate 3.6 Sigma P-4562 Glucose 100 Sigma G-6152 Penicillin 7.5 Sigma P-4687 Streptomycin 5.0 Sigma S-1277 BSA 300 Sigma A-4378 This medium can be purchased commercially from Cosmo Bio Co., Ltd ( as PB1 5

6 Composition of DAP213 vitrification solution Solution A Reagent Name ml/10ml Vendor Catalogue Number PB DMSO 3.13 Sigma D-2650 Propylene glycol 4.56 Sigma P-1009 Solution B Reagent Name mg/10ml in PB1 Vendor Catalogue Number Acetamide Sigma A-0500 NOTES: a) Solutions A and Solution B need to be prepared separately b) Equal volumes of A and B are then mixed to form DAP213. c) The solution may become cloudy when DMSO is added. This medium can be purchased commercially from Cosmo Bio Co., Ltd ( as DAP Prepare the 0.25M sucrose solution by adding 1.711g of sucrose to 20ml of PB1 medium. Filter the solution through a 0.22µm syringe end filter. This solution has a shelf life of 7 days at 4 C. 2. Reference Nakao, K., Nakagata, N., and Katsuki, M Simple and efficient vitrification procedure for cryopreservation of mouse embryos. Experimental animals / Japanese Association for Laboratory Animal Science 46: Nagy A., G.M., Vintersten K., Behringer R Manipulating the Mouse Embryo - A laboratory manual (3th Edition). Cold Spring Harbor Laboratory press, New York. 6