3 Methods
3.1 Preparation
of Tyrode’s Lactate
HEPES (TL-HEPES)
Medium
TL-HEPES medium is modified from Tyrode’s Lactate medium
[41], and part of the bicarbonate is replaced with HEPES.
TL-HEPES is used for washing OT, holding before cryopreservation, base solution for making freezing solution, and removing
cryoprotectant after thawing and dilution (see Note 1).
1. Add embryo-tested Milli-Q water (~80% of the final volume)
to an appropriate size Griffin beaker, and then add the components in gram as listed in Table 1. All the recipes are for 1 L final
volume, so adjust the volume of the media for your needs.
2. Solubilize the CaCl 2 .2H 2 O and MgCl 2 .6H 2 O separately from
the other reagents before combining the solutions.
3. Add 4.0 g/1 L bovine serum albumin fraction V (Sigma
A7638) into the stock solution, and let it dissolve slowly without using a stir bar.
4. After all components are completely dissolved, transfer the
contents to an appropriate size volumetric flask, and bring to
95% of the desired volume by rinsing out the Griffin beaker
with embryo-tested Milli-Q water and adding it to the volumetric flask.
5. Adjust the pH to 7.4 before bringing to the final volume.
Table 1
Composition of HEPES-buffered Tyrode’s albumin, lactate, and pyruvate medium (TALP-HEPES)
Components
Company
Add per L
Concentration (mM)
a CaCl 2 .2H 2 O
Sigma C7902
0.300 g
2.0
a MgCl 2 .6H 2 O
Sigma M2393
0.100 g
0.5
KCl
Sigma P5405
0.240 g
3.2
NaCl
Sigma S5886
6.66 g
114.0
NaHCO3
Sigma S5761
0.168 g
2
NaH 2 PO 4 .2H 2 O
Sigma S8282
0.056 g
0.4
Sodium lactate (60% syrup)
Sigma L7900
1.740 mL
11.77
Sodium pyruvate
Sigma P4562
0.036 g
0.2
Penicillin G potassium salt
Sigma P7794
0.06 g
N/a
Streptomycin sulfate
Sigma S1277
0.0125 g
N/a
HEPES
Sigma H6147
2.400 g
20
Phenol red (0.5%)
Sigma P0290
0.66 mL
N/a
a
Add last after all other reagents are in solution, may precipitate
Rodent Ovarian Tissue Cryopreservation
473
3.1 Preparation
of Tyrode’s Lactate
HEPES (TL-HEPES)
Medium
TL-HEPES medium is modified from Tyrode’s Lactate medium
[41], and part of the bicarbonate is replaced with HEPES.
TL-HEPES is used for washing OT, holding before cryopreservation, base solution for making freezing solution, and removing
cryoprotectant after thawing and dilution (see Note 1).
1. Add embryo-tested Milli-Q water (~80% of the final volume)
to an appropriate size Griffin beaker, and then add the components in gram as listed in Table 1. All the recipes are for 1 L final
volume, so adjust the volume of the media for your needs.
2. Solubilize the CaCl 2 .2H 2 O and MgCl 2 .6H 2 O separately from
the other reagents before combining the solutions.
3. Add 4.0 g/1 L bovine serum albumin fraction V (Sigma
A7638) into the stock solution, and let it dissolve slowly without using a stir bar.
4. After all components are completely dissolved, transfer the
contents to an appropriate size volumetric flask, and bring to
95% of the desired volume by rinsing out the Griffin beaker
with embryo-tested Milli-Q water and adding it to the volumetric flask.
5. Adjust the pH to 7.4 before bringing to the final volume.
Table 1
Composition of HEPES-buffered Tyrode’s albumin, lactate, and pyruvate medium (TALP-HEPES)
Components
Company
Add per L
Concentration (mM)
a CaCl 2 .2H 2 O
Sigma C7902
0.300 g
2.0
a MgCl 2 .6H 2 O
Sigma M2393
0.100 g
0.5
KCl
Sigma P5405
0.240 g
3.2
NaCl
Sigma S5886
6.66 g
114.0
NaHCO3
Sigma S5761
0.168 g
2
NaH 2 PO 4 .2H 2 O
Sigma S8282
0.056 g
0.4
Sodium lactate (60% syrup)
Sigma L7900
1.740 mL
11.77
Sodium pyruvate
Sigma P4562
0.036 g
0.2
Penicillin G potassium salt
Sigma P7794
0.06 g
N/a
Streptomycin sulfate
Sigma S1277
0.0125 g
N/a
HEPES
Sigma H6147
2.400 g
20
Phenol red (0.5%)
Sigma P0290
0.66 mL
N/a
a
Add last after all other reagents are in solution, may precipitate
Rodent Ovarian Tissue Cryopreservation
473