media is too large, it can still be reduced before plunging in
LN 2 by “drawing down” the droplet to flatten the blastocysts
slightly while removing all surplus vitrification solution.
6. To make sure that the cells are loaded on the carrier, perform
the loading process under a light microscope. Check the number of loaded cells.
7. After sealing the carrier, submerge the carrier loaded with the
cells directly in liquid nitrogen by passing fast through the
vapor phase (nitrogen gas).
8. Store cryo-cane in a pre-chilled PVC cryo-sleeve sitting in the
goblet in the Dewar. It is essential to maintain exposure of the
HSV to LN 2 at all times to eliminate risk of warming and
devitrification.
9. Before moving the carrier fast from the nitrogen in the warming solution, pull a glass pipette or have a stripper tip ready. Fill
the pipette with a small amount of the first warming solution
(TS). Using the HSV as carrier, rinse the open edge of straw
after placing in the pre-warmed (37
C) 1.0 M sucrose, or
because the droplet is so small it warms immediately, it is
essential to pull/stir the blastocysts off the carrier surface
directly as soon as possible to avoid any toxic effect of the
VS. A stirring motion is recommended when plunging into
the warm TS to agitate the cell off the carrier surface without
the need to remove it actively.
10. In general, during vitrification and warming, the LN 2 styrofoam box needs to be as close as possible to the working area
minimizing lag in cooling and warming rates.
11. Even when switching the cells between different concentrations of warming solutions, fill up the pipette with the next
lower concentration of warming solution before picking up the
cells for moving in the following concentration.
12. Be aware of the expiration dates of the vitrification and warming media; once opened, shelf life is 6 weeks (according to the
manufacture).
Acknowledgments
The author wants to thank the Physicians at the Fertility Centers of
Illinois (FCI) and the Embryologists at the FCI IVF Laboratory
River North (Sara Sanchez-Julias, BS; Rebecca Brohammer, BS;
Marianna Pantos, MS; Laura Baranyi, BS; Ivana Elzy, BS; Lyn
Claussen, MS; Cynthia Macias, BS; Alina Diamond, BS; Janna
Schwab, MS; Emily Spolarich, BS; and Jennifer Santiago, BS, for
514
Juergen Liebermann
LN 2 by “drawing down” the droplet to flatten the blastocysts
slightly while removing all surplus vitrification solution.
6. To make sure that the cells are loaded on the carrier, perform
the loading process under a light microscope. Check the number of loaded cells.
7. After sealing the carrier, submerge the carrier loaded with the
cells directly in liquid nitrogen by passing fast through the
vapor phase (nitrogen gas).
8. Store cryo-cane in a pre-chilled PVC cryo-sleeve sitting in the
goblet in the Dewar. It is essential to maintain exposure of the
HSV to LN 2 at all times to eliminate risk of warming and
devitrification.
9. Before moving the carrier fast from the nitrogen in the warming solution, pull a glass pipette or have a stripper tip ready. Fill
the pipette with a small amount of the first warming solution
(TS). Using the HSV as carrier, rinse the open edge of straw
after placing in the pre-warmed (37
C) 1.0 M sucrose, or
because the droplet is so small it warms immediately, it is
essential to pull/stir the blastocysts off the carrier surface
directly as soon as possible to avoid any toxic effect of the
VS. A stirring motion is recommended when plunging into
the warm TS to agitate the cell off the carrier surface without
the need to remove it actively.
10. In general, during vitrification and warming, the LN 2 styrofoam box needs to be as close as possible to the working area
minimizing lag in cooling and warming rates.
11. Even when switching the cells between different concentrations of warming solutions, fill up the pipette with the next
lower concentration of warming solution before picking up the
cells for moving in the following concentration.
12. Be aware of the expiration dates of the vitrification and warming media; once opened, shelf life is 6 weeks (according to the
manufacture).
Acknowledgments
The author wants to thank the Physicians at the Fertility Centers of
Illinois (FCI) and the Embryologists at the FCI IVF Laboratory
River North (Sara Sanchez-Julias, BS; Rebecca Brohammer, BS;
Marianna Pantos, MS; Laura Baranyi, BS; Ivana Elzy, BS; Lyn
Claussen, MS; Cynthia Macias, BS; Alina Diamond, BS; Janna
Schwab, MS; Emily Spolarich, BS; and Jennifer Santiago, BS, for
514
Juergen Liebermann