and last step are depending on the way distance of the laboratory for preparation and the room with the freezing machine).
10. Tissue pieces in Nunc cryo vials are cooled at a rate of À2
C/
min, and automatic seeding is initiated as soon as the medium
in the sample vial is reaching the temperature of À6
C in the
freezing machine. After successful seeding the temperature is
on hold for another 5–8 min and then cooled down at
À0.3
C/min to À40
C followed by fast cooling at
À10
C/min to À140
C.
11. Nunc cryo vials will then be plunged into liquid nitrogen and
transferred into storage boxes. Storage is performed in a vaporphase storage tanks, e.g., MVE 1500 Series at À190
C (Chart
MVE BioMedical Industries, Inc., Garfield Heights, USA).
3.5 Thawing
Procedure
Cryopreserved ovarian tissue is thawed during (re)transplantation
of the ovarian cortex in patients who have had ovarian cortex
cryopreserved for fertility protection prior to gonadotoxic therapy.
If a (re)transplantation of cryopreserved ovarian cortex tissue is
planned, it is first necessary first to determine the cortex potential
and tissue vitality in the thawing samples (3 Â 2 mm cortex biopsies
from different areas of the prepared/cryopreserved ovarian cortex
tissue) by means of a viability assay. The thawing protocol is modified from published procedures [26, 28–31]. The cryo vials are
thawed in a water bath at 37
C. The samples are then gradually
converted into thawing solutions with decreasing sucrose concentrations and 10% HSA for gradual removal of the cryoprotectants.
After two washes in a DPBS/HSA medium, the cortex pieces are
transported to the operation room and transplanted as quickly as
possible.
3.5.1 Preparations
of Warming/Thawing
Solutions
1. One day before thawing, prepare three sucrose solutions, of
0.75 M, 0.375 M, and 0.125 M); for the gradual removal of
cryoprotectants from the tissue:
(a) Prepare 50 mL DPBS/HSA solution, consisting of 45 mL
DPBS and 5 mL HSA. Prepare in a sterile Oosafe sample
container.
(b) Weigh and transfer sucrose in each of three sterile Oosafe
sample containers (2.65 g, 1.33 g, 0.45 g), and mark these
containers with “thawing solution 1–3.”
(c) Add DPBS/HSA (10 mL) to each of the differently
weighed sucrose quantities.
(d) Mix the sucrose/DPBS/HSA solution on an orbital
shaker for 30 min.
(e) Make a sterile filtration of the sucrose/DPBS/HSA solutions via passing the sucrose/DPBS/HSA solution
through a 0.20 μm filter, using a 20 mL syringe.
Cryopreservation and Thawing of Human Ovarian Tissue
493
10. Tissue pieces in Nunc cryo vials are cooled at a rate of À2
C/
min, and automatic seeding is initiated as soon as the medium
in the sample vial is reaching the temperature of À6
C in the
freezing machine. After successful seeding the temperature is
on hold for another 5–8 min and then cooled down at
À0.3
C/min to À40
C followed by fast cooling at
À10
C/min to À140
C.
11. Nunc cryo vials will then be plunged into liquid nitrogen and
transferred into storage boxes. Storage is performed in a vaporphase storage tanks, e.g., MVE 1500 Series at À190
C (Chart
MVE BioMedical Industries, Inc., Garfield Heights, USA).
3.5 Thawing
Procedure
Cryopreserved ovarian tissue is thawed during (re)transplantation
of the ovarian cortex in patients who have had ovarian cortex
cryopreserved for fertility protection prior to gonadotoxic therapy.
If a (re)transplantation of cryopreserved ovarian cortex tissue is
planned, it is first necessary first to determine the cortex potential
and tissue vitality in the thawing samples (3 Â 2 mm cortex biopsies
from different areas of the prepared/cryopreserved ovarian cortex
tissue) by means of a viability assay. The thawing protocol is modified from published procedures [26, 28–31]. The cryo vials are
thawed in a water bath at 37
C. The samples are then gradually
converted into thawing solutions with decreasing sucrose concentrations and 10% HSA for gradual removal of the cryoprotectants.
After two washes in a DPBS/HSA medium, the cortex pieces are
transported to the operation room and transplanted as quickly as
possible.
3.5.1 Preparations
of Warming/Thawing
Solutions
1. One day before thawing, prepare three sucrose solutions, of
0.75 M, 0.375 M, and 0.125 M); for the gradual removal of
cryoprotectants from the tissue:
(a) Prepare 50 mL DPBS/HSA solution, consisting of 45 mL
DPBS and 5 mL HSA. Prepare in a sterile Oosafe sample
container.
(b) Weigh and transfer sucrose in each of three sterile Oosafe
sample containers (2.65 g, 1.33 g, 0.45 g), and mark these
containers with “thawing solution 1–3.”
(c) Add DPBS/HSA (10 mL) to each of the differently
weighed sucrose quantities.
(d) Mix the sucrose/DPBS/HSA solution on an orbital
shaker for 30 min.
(e) Make a sterile filtration of the sucrose/DPBS/HSA solutions via passing the sucrose/DPBS/HSA solution
through a 0.20 μm filter, using a 20 mL syringe.
Cryopreservation and Thawing of Human Ovarian Tissue
493