The Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong
Peer-reviewed,author-submitted protocol Published in November 2009 (p.55) DOI: 10.2144/000113258
Assemble of vitrification spatula (VS)
1. A pair of fine forceps with a region of ~1 mm in width is used to hold the thin end of an autoclaved gel-loading tip in a perpendicular position (Figure 1).
2. With the wide end of the loading tip pointing to the bench at a narrow angle (~10° from the vertical; Figure 1), the very tip of the fine forceps is heated with a Bunsen flame until the tip of the gel-loading tip melts.
ATTENTION: The melting of the loading tip is indicated by the movement of the wide end toward the vertical position (Figure 1).
3. The fine forceps are immediately removed from the flame and the gel-loading tip released.
4. A pair of forceps are heated in the flame for 1 s and used to seal the distal edge of the newly formed spatula.
5. The wide end of the gel-loading-tip is cut away at the midpoint.
6. The cut end is heat-melted by direct flaming, and the spatula is attached to the underside of the cryogenic vial cap.
7. Monitoring under dissecting microscope confirms that the distal edge of the spatula is sealed properly.
Collection of preimplantation embryos
8. Serum gonadotrophin and chorulon gonadotrophin is administered to 4-week-old C57BL/J6/CBAF1 females sequentially, 45–48 h apart.
9. Mating is set with at a 1:1 female to male ratio with the formation of a vaginal plug confirmed the next day.
10. Zygotes are harvested at the same day of vaginal plug checking [0.5 days post-coitum (dpc)]
11. Zygotes are isolated from the ampullae of the oviducts and cleared from the cumulus cells with 0.3 μg/mL hyaluronidase in M2.
12. Morulae are harvested 2 days after vaginal plug checking (2.5 dpc) by flushing the oviducts and uteri from the infundibulum to the proximal uterine horn with M2.
13. Blastocysts are harvested 3 days after vaginal plug checking (3.5 dpc) by flushing the uteri from the cervix to the distal ends of the uterine horns with M2.
14. Embryos are kept in M16 micro-drop under light mineral oil in a humidified 37°C incubator with 5% CO2 until the cryopreservation steps commence.Vitrification of embryos
15. On a cover of a 35-mm dish, a drop of M2 (20 μL per drop), a drop of pre-vitrification solution, and a drop of vitrification solution is loaded.
16. Using a mouth-controlled micropipet, the embryos from the M16 micro-drop are transferred to the drop of M2.
17. The body of a cryogenic vial is pre-cooled with the lower half of the vial kept in liquid nitrogen. The spatula-carrying vial cap is set horizontally on the stage of a dissecting microscope for convenient positioning of the spatula into the field of microscopic observation.
18. Under the dissecting microscope, the embryos are transferred with a micropipet (prefilled with pre-vitrification solution) from the M2 drop to the drop of pre-vitrification solution. Incubation in the last solution for
30 s follows.
ATTENTION: The embryos were loaded at different positions at the bottom of the drop and are allowed to float to disperse the leftover of M2. No stirring is required. For handling a large amount of embryos (i.e., 50), the first embryo loaded into the first solution should be picked up and transferred to the next solution right after the last embryo has been loaded into the first solution. The entire transfer takes a couple of minutes and so the incubation time will exceed 30 s.
19. The embryos are transferred with a micropipet prefilled with vitrification solution from the pre-vitrification solution drop to the vitrification solution drop, followed by a 30-s incubation.
20. The embryos are then loaded onto one side of the vitrification spatula with the same micropipet prefilled with vitrification solution.
21. The tip is immediately dipped into a fresh liquid nitrogen supply.
ATTENTION: Do not dip the tip into the liquid nitrogen of the vessel for cell storage.
22. After 5 s of rapid freezing, the VS is inserted in the cryopreservation vial with the cap tightly screwed.
ATTENTION: The VS must be inserted and screwed to the body of the vial immediately. Hardening of the rubber ring on the cap, due to the cooling by the liquid nitrogen vapor, will prevent a complete sealing of the vial. Infiltration of liquid nitrogen from the cell storage vessel will occur and may cause contamination.
23. The vial is then transferred to the liquid nitrogen vessel for cell or embryo storage.Thawing of vitrified embryos
24. The VS-containing vial is removed from the cell storage vessel and kept in liquid nitrogen at all times until the VS leaves the vial.
25. A 35-mm dish containing 2 mL of 0.5 M sucrose in M2 is prepared.
26. The tip of the VS containing embryos is dipped into the 0.5 M sucrose solution. The falling of embryos from the VS to the bottom of the dish is monitored under a dissecting microscope.
27. The embryos are transferred to a new drop (20 μL) of 0.5 M sucrose followed by an incubation of 2 min.
28. The embryos are transferred to a drop (20 μL) of 0.25 M sucrose and the embryos are incubated for another 2 min.
29. The embryos are transferred to a drop (20 μL) of M2. They are incubated for 1 min.
30. The embryos are washed twice with M2 and transferred to M16 under oil in a 37°C humidified incubator with 5% CO2 before further manipulation takes place.
31. Thawed zygotes are transferred to the oviduct of a 0.5-dpc pseudo-pregnant female, 10 embryos at a time.
32. Thawed morulae are cultured for one night first and are transferred to the uterus of 2.5-dpc pseudo-pregnant females, 10 embryos at a time.
33. Thawed blastocysts are transferred to the uteri of 2.5-dpc pseudo-pregnant females, 10 embryos at a time.
• FS (10 mL) Ficoll PM70 3.0 g Sucrose 1.7 g in PBS (pH 7.4), filter sterilized
• Pre-vitrification solution (100 μL, freshly prepared) Ethylene glycol 10 μL (final: 10%) DMSO 10 μL (final: 10%) M2 80 μL (final: 80%)
• Vitrification solution (100 μL, freshly prepared) Ethylene glycol 15 μL (final: 15%) DMSO 15 μL (final: 15%) FS 60 μL (final: 60%) M2 10 μL (final: 10%)
• 0.5 M sucrose (100 mL) 34 g sucrose in M2, filter-sterilized Stored as 1.5-mL aliquots at -20°C
• 0.25 M sucrose (10 mL) 1.7 g sucrose in M2, filter-sterilized Stored as 100-μL aliquots at -20°C
• fiM2 medium (Specialty Media, Phillipsburg, PA, USA)
• M16 medium (Specialty Media)
• Light mineral oil (embryo-tested; Specialty Media)
• Serum gonadotrophin (Folligon; Intervet, Milton Keynes, UK)
• Chorionic gonadotrophin (Chorulon; Intervet)
• Hyaluronidase (Cat. no. H4272; Sigma-Aldrich, St. Louis, MO, USA)
• Ethylene glycol (Cat. no. 102466; Sigma-Aldrich)
• DMSO (Cat. no. D2650; Sigma-Aldrich)
• Ficoll PM70 (Cat. no. F2878; Sigma-Aldrich)
• Sucrose (Cat. no. S9378; Sigma-Aldrich)
• Mouth-controlled micropipet system, with fire-polished micropipets with openings of ~200-μm i.d. (Cat. no. G150T-4, Warner Instrument, Hamden, NJ, USA)
• Fine forceps (N4, Regine, Switzerland)
• Bunsen burner (Model no. F4003, R & L Enterprises, Bramley, Leeds, UK)
• Dissecting microscope (Model no. EMZ-5, Meiji Techno, Saitama, Japan)
• Gel-loading tips (Cat. no. 1022–000, USA Scientific, Ocala, FL, USA)
• Cryogenic vial (Cat. no. 430488, Corning, NY, USA)
• 35-mm Petri dish (Cat. No. 353001, BD Bioscience, NJ, USA)
Address correspondence to King L. Chow, The Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, email: [email protected]; or Wai Hung Tsang, The Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, email: [email protected]
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