experimental data suggest that chromosomal integrity of freezedried sperm is compromised when stored at ambient temperature
for more than 1 month [46, 47]. It is likely that the sugar- and
protein-packed sperm nucleus is stabilized in a glass-like vitrified
state upon removal of water by freeze-drying. However, this may
not be sufficient for its long-term preservation at ambient temperature without stabilizing its surrounding in a vitrified state as well.
Indeed, improved DNA integrity has been reported after adding
trehalose, a good glass former, to freeze-drying solution [30, 48–
50]. Taken together, further comprehensive research is needed to
achieve long-term preservation of mammalian sperm at ambient
temperature.
In the following, we present a freeze-drying protocol based on
utilization of a manifold-type freeze-dryer. With some minor modifications, it should be adaptable to freeze-drying of mammalian
sperm using a shelf freeze-dryer.
2 Materials
In addition to standard laboratory equipment and supplies, the
following items are required for freeze-drying:
1. Manifold-type freeze-dryer such as Flexi-Dry (FTS Systems,
Inc. Stoner Ridge, NY 12484) and FreeZone 77530-00 (Labconco Corporation, Kansas City, MO, USA) (see Note 1).
2. Long-necked borosilicate glass ampules (e.g., W651506,
Wheaton, Millville, NJ, USA). These glass ampules can be
autoclaved or heat sterilized in advance (see Note 2).
3. Air/gas torch for flame sealing.
4. Tris-buffered freeze-drying solution containing a calcium chelator: The two most commonly used freeze-drying solutions
are (a) 10 mM Tris–HCl containing 1 mM EDTA (pH 8) and
(b) 10 mM Tris–HCl containing 50 mM NaCl and 50 mM
EGTA (pH 8). Rosmarinic acid (RA, Sigma-Aldrich) can also
be added to these freeze-drying solutions at 105 μM based on
its proven beneficial effect. We suggest selecting one of these
solutions and preparing its concentrated 2Â stock solution in
ultrapure water. Next, its pH can be adjusted to 8 and then
diluted to its 1Â solution by adding ultrapure water. The
resulting final solution is stable at ambient temperature or
4
C once sterilized using a 0.22 μm filter.
726
Levent Keskintepe and Ali Eroglu
for more than 1 month [46, 47]. It is likely that the sugar- and
protein-packed sperm nucleus is stabilized in a glass-like vitrified
state upon removal of water by freeze-drying. However, this may
not be sufficient for its long-term preservation at ambient temperature without stabilizing its surrounding in a vitrified state as well.
Indeed, improved DNA integrity has been reported after adding
trehalose, a good glass former, to freeze-drying solution [30, 48–
50]. Taken together, further comprehensive research is needed to
achieve long-term preservation of mammalian sperm at ambient
temperature.
In the following, we present a freeze-drying protocol based on
utilization of a manifold-type freeze-dryer. With some minor modifications, it should be adaptable to freeze-drying of mammalian
sperm using a shelf freeze-dryer.
2 Materials
In addition to standard laboratory equipment and supplies, the
following items are required for freeze-drying:
1. Manifold-type freeze-dryer such as Flexi-Dry (FTS Systems,
Inc. Stoner Ridge, NY 12484) and FreeZone 77530-00 (Labconco Corporation, Kansas City, MO, USA) (see Note 1).
2. Long-necked borosilicate glass ampules (e.g., W651506,
Wheaton, Millville, NJ, USA). These glass ampules can be
autoclaved or heat sterilized in advance (see Note 2).
3. Air/gas torch for flame sealing.
4. Tris-buffered freeze-drying solution containing a calcium chelator: The two most commonly used freeze-drying solutions
are (a) 10 mM Tris–HCl containing 1 mM EDTA (pH 8) and
(b) 10 mM Tris–HCl containing 50 mM NaCl and 50 mM
EGTA (pH 8). Rosmarinic acid (RA, Sigma-Aldrich) can also
be added to these freeze-drying solutions at 105 μM based on
its proven beneficial effect. We suggest selecting one of these
solutions and preparing its concentrated 2Â stock solution in
ultrapure water. Next, its pH can be adjusted to 8 and then
diluted to its 1Â solution by adding ultrapure water. The
resulting final solution is stable at ambient temperature or
4
C once sterilized using a 0.22 μm filter.
726
Levent Keskintepe and Ali Eroglu