Dr. Manfred Schleyer ,1999 July 07.

Equilibration of oocytes/pronuclear stages with freezing media

Into the wells of a 4-well-dish (NUNC) are pipetted 1 ml each of the four solutions of a FREEZE-KIT (REF 6120 Scandinavian IVF Science AB, Vitrolife AB, Molndalsvagen 30, S-412 63 Gothenburg, Sweden) and warmed at 37 °C in air for at least 1 hour.

Oocytes/PN-stages are transferred with a 50 microliter Eppendorf pipette to the first well (and float to surface) and incubated at 37 °C protected from light. After 10 min they are transferred into the second well (float to the surface) and incubated at room temperature. After transfer into well 3 follows incubation for ten minutes, and transfer into well 4. Then, the oocytes/PN-stages are sucked into freezing straws ( ref no 19040-0018, MINITÜB Abfüll- und Labortechnik, Hauptstr. 41, D-84184 Tiefenbach, Germany) in the following way: first about 1 cm column length of medium (for finally wetting the self-sealing powder between the cotton plugs on the upper end), about 1 cm of air, about 2 cm of medium followed by the oocytes in another cm of liquid, then air until the first medium-column seals the upper end. The lower open end is closed with a stopper stick (ref. no 98390030.87) labelled with patient's data.

Freezing

Freezing programme for Planer KRYO 10:

start temperature 22.0 °C

ramp 1 rate -1.00 °C/min temp -6.7 °C

ramp 2 rate -0.00 °C/min hold 05m 00s

ramp 3 rate -0.50 °C/min temp -80.0 °C

Manual seeding, temperature -6.7 °C, soak time 5m 00s.

Activate freezing programme NORFOLK. When "AT START TEMPERATURE, LOAD SAMPLES": insert straw with stopper first, fix column with oocytes/PN-stages suspended in air completely.

Press RUN/HOLD to start ramp 1.

Preparations for seedinq

When freezer has reached -6.7 °C, during soaking time of five minutes: Pour liquid-N2 in a dewar, immerse wooden stick with cotton swap, position next to Planer. After soaking, at -6,7 °C: take out strawholder/ touches end of column of oocytes shortly but firmly with cold cotton swap soaked in liquid N2. Insert straws immediately back into Planer. Press RUN/HOLD to continue programme. At end of last ramp: "RUN FINISHED". Take straw out of Planer and transfer into the dewar with liquid N2, then into a storage tank. Press RUN/HOLD and wait for completion of warm-up before switching off.