2 Materials
Except where otherwise stated, obtain all chemicals from Sigma
(Sigma Chemical Co., St Louis, MO, USA).
2.1 Media
for Spermatozoa
Preparation
1. Density gradient medium: GM501 Gradient 90% and GM501
Gradient 45% (Gynemed GmbH, Lensahn, Germany).
2. Basic medium: human tubal fluid (HTFs) [39] supplemented
with 1% Dextran Substitutive Supplement (DSS, Irvine Sci.,
Santa Ana, USA).
2.2 Medium
for Spermatozoa
Vitrification
1. Sucrose solution: 0.5 M sucrose in distilled water. Filter sterilize by passing through a 0.22 μm filter (Millipore, Darmstadt,
Germany). Store frozen until use (see Note 1).
2. Vitrification solution: mix equal volumes of sucrose solution
(0.5 M sucrose) and basic medium (HTF medium supplemented with 1% DSS), for obtaining a vitrification solution containing 0.25 M [7].
3. Cooling agent: liquid nitrogen and clean liquid air (see Note 2).
2.3 Reagents
for Spermatozoa
Assessment
1. PBS Dulbecco’s phosphate-buffered saline.
2. Viability assay: Dead/Live Spermatozoa Viability Staining Kit
(L-7011, Molecular Probes Inc., Eugene, USA).
3. Apoptosis assay: FITC Annexin V Apoptosis Detection Kit with
7-AAD (Biolegend, San Diego, USA).
2.4 Equipment
1. Makler counting chamber (0.01 mm
2 and 10 μm deep; SefiMedical Instruments Ltd., Haifa, Israel).
2. 15 mL tubes: polystyrene round-bottom tube for sperm
preparation.
3. 15 mL centrifuge tubes for sperm warming.
4. 1.5 mL microcentrifuge tubes.
5. Aseptic spoon for transferring droplets into cryotubes easily.
6. 1.8 mL cryotubes (Nunc, Roskilde, Denmark).
7. Micropipette and tips (10–100 μL) for generating drops.
8. A Styrofoam box.
3 Methods
3.1 Spermatozoa
Preparation
1. After informed consent, obtain ejaculates from patients with
normozoospermatozoaic characteristics (see Notes 3 and 4).
2. Prepare a sperm solution of 30 Â 10
6 spermatozoa/mL in basic
medium. Spermatozoa are preferentially collected via density
gradient centrifugation (DGC):
Vitrification of Human Spermatozoa in Sterile Liquid Air
429
Except where otherwise stated, obtain all chemicals from Sigma
(Sigma Chemical Co., St Louis, MO, USA).
2.1 Media
for Spermatozoa
Preparation
1. Density gradient medium: GM501 Gradient 90% and GM501
Gradient 45% (Gynemed GmbH, Lensahn, Germany).
2. Basic medium: human tubal fluid (HTFs) [39] supplemented
with 1% Dextran Substitutive Supplement (DSS, Irvine Sci.,
Santa Ana, USA).
2.2 Medium
for Spermatozoa
Vitrification
1. Sucrose solution: 0.5 M sucrose in distilled water. Filter sterilize by passing through a 0.22 μm filter (Millipore, Darmstadt,
Germany). Store frozen until use (see Note 1).
2. Vitrification solution: mix equal volumes of sucrose solution
(0.5 M sucrose) and basic medium (HTF medium supplemented with 1% DSS), for obtaining a vitrification solution containing 0.25 M [7].
3. Cooling agent: liquid nitrogen and clean liquid air (see Note 2).
2.3 Reagents
for Spermatozoa
Assessment
1. PBS Dulbecco’s phosphate-buffered saline.
2. Viability assay: Dead/Live Spermatozoa Viability Staining Kit
(L-7011, Molecular Probes Inc., Eugene, USA).
3. Apoptosis assay: FITC Annexin V Apoptosis Detection Kit with
7-AAD (Biolegend, San Diego, USA).
2.4 Equipment
1. Makler counting chamber (0.01 mm
2 and 10 μm deep; SefiMedical Instruments Ltd., Haifa, Israel).
2. 15 mL tubes: polystyrene round-bottom tube for sperm
preparation.
3. 15 mL centrifuge tubes for sperm warming.
4. 1.5 mL microcentrifuge tubes.
5. Aseptic spoon for transferring droplets into cryotubes easily.
6. 1.8 mL cryotubes (Nunc, Roskilde, Denmark).
7. Micropipette and tips (10–100 μL) for generating drops.
8. A Styrofoam box.
3 Methods
3.1 Spermatozoa
Preparation
1. After informed consent, obtain ejaculates from patients with
normozoospermatozoaic characteristics (see Notes 3 and 4).
2. Prepare a sperm solution of 30 Â 10
6 spermatozoa/mL in basic
medium. Spermatozoa are preferentially collected via density
gradient centrifugation (DGC):
Vitrification of Human Spermatozoa in Sterile Liquid Air
429