5.6 g KCl, and 4.2 g NaHCO 3 , and make up to 1 L with water
to make a 5Â stock solution. Stir continuously during formulation; when the solution is clear of particles, it should be
filtered using 0.2 μm filters. We do not test the osmolality or
pH of 5Â EC. Dilute 1:5 with water to make 1Â EC and check
the pH (~7.4) and osmolality (365 Æ 5). Store at 4
C.
2.2 Preparation
of Vitrification
Solutions
1. Vitrification solutions (VS) are formulated by place a stir bar
and then each component, in the order that they appear in the
list below (see Table 2), in a graduated container. Stir continuously; when finished and is clear of particles, it should be
filtered. We use bottle top 0.2 μm filters, and it takes a long
time (hours) to filter due to the high viscosity of the solutions.
At the conclusion of preparation, check the final pH and make a
1:20 dilution for assessment of osmolality. Store at 4
C.
2.3 Preparation
of Addition
and Removal Solutions
1. Sequential vitrification solution addition and removal solutions
are used in certain protocols (see Note 1). The VS chemical
component concentration can be increased from 1/8, 1/4,
1/2, 3/4 to 100% of the final full-strength VS formulation
being employed. The diluted VS solutions are prepared by
addition of appropriate volumes of 1Â EC. The solutions
employed for removal are similar to those employed for addition except that 200–400 mM mannitol is added to the formulation as an osmotic buffer. For example, using seven steps, the
VS CPAs plus mannitol are decreased from full strength to 0 M,
plain EC, and finally Dulbecco’s Modified Eagle Medium
(DMEM).
Table 2
VS formulation, quantities
VS55
VS83
5Â EuroCollins (mL)
200
200
HEPES buffer (g)
2.39
2.39
Propylene glycol
a (g)
168.38
252.57
Formamide
a (g)
139.56
209.34
Me 2 SO
b (g)
242.14
363.21
Water (mL)
to 1 L
to 1 L
pH (units)
7.9–8.1
7.9–8.1
Osmolality (mOsm/kg)
460 Æ 5
670 Æ 5
a
Liquids measured by weight
b
20Â dilution ¼ 50 μL in 950 μL of distilled water
598
Kelvin G. M. Brockbank et al.
to make a 5Â stock solution. Stir continuously during formulation; when the solution is clear of particles, it should be
filtered using 0.2 μm filters. We do not test the osmolality or
pH of 5Â EC. Dilute 1:5 with water to make 1Â EC and check
the pH (~7.4) and osmolality (365 Æ 5). Store at 4
C.
2.2 Preparation
of Vitrification
Solutions
1. Vitrification solutions (VS) are formulated by place a stir bar
and then each component, in the order that they appear in the
list below (see Table 2), in a graduated container. Stir continuously; when finished and is clear of particles, it should be
filtered. We use bottle top 0.2 μm filters, and it takes a long
time (hours) to filter due to the high viscosity of the solutions.
At the conclusion of preparation, check the final pH and make a
1:20 dilution for assessment of osmolality. Store at 4
C.
2.3 Preparation
of Addition
and Removal Solutions
1. Sequential vitrification solution addition and removal solutions
are used in certain protocols (see Note 1). The VS chemical
component concentration can be increased from 1/8, 1/4,
1/2, 3/4 to 100% of the final full-strength VS formulation
being employed. The diluted VS solutions are prepared by
addition of appropriate volumes of 1Â EC. The solutions
employed for removal are similar to those employed for addition except that 200–400 mM mannitol is added to the formulation as an osmotic buffer. For example, using seven steps, the
VS CPAs plus mannitol are decreased from full strength to 0 M,
plain EC, and finally Dulbecco’s Modified Eagle Medium
(DMEM).
Table 2
VS formulation, quantities
VS55
VS83
5Â EuroCollins (mL)
200
200
HEPES buffer (g)
2.39
2.39
Propylene glycol
a (g)
168.38
252.57
Formamide
a (g)
139.56
209.34
Me 2 SO
b (g)
242.14
363.21
Water (mL)
to 1 L
to 1 L
pH (units)
7.9–8.1
7.9–8.1
Osmolality (mOsm/kg)
460 Æ 5
670 Æ 5
a
Liquids measured by weight
b
20Â dilution ¼ 50 μL in 950 μL of distilled water
598
Kelvin G. M. Brockbank et al.