3 Â 2 mm biopsies are freshly examined before cryopreservation,
and the remaining three are frozen separately as thawing samples, at
the freezing process of the cortex strips for (re)transplantation and
for later thawing and assessment using the same viability assay (e.g.,
calcein staining) [1, 3, 23] (see Notes 5–7):
1. Prepare medium 1 day before use. Equilibrate AIM-V serumfree culture medium (per sample, 600 μL/well in a four-well
dish) overnight with following incubation conditions: 6% CO 2
and 37
C.
2. Put three biopsies with 2 mm mean diameter from different
cortex areas for 2–3 h in pre-equilibrated culture medium in a
Nunc four-well dish (the maximum culturing time should be
not longer than 2 days/48 h).
3. Prepare a solution with 2 μL calcein AM +998 μL pre-warmed
DPBS solution and 6–8 mg collagenase type I A in a 1.5 mL
Eppendorf reaction tube (1 1 mL approach of calcein
AM/DPBS/collagenase type I A solution can be used for
two assays/patients).
4. Pipette 500 μL of this solution in one well of the Nunc fourwell plate, and transfer the 3 Â 2 mm biopsies into the calcein
AM/DPBS/collagenase type I A solution.
5. The incubation time is 1.5–2 h at 5% CO 2 and 37
C.
6. After 1 h the lysate should be carefully homogenized using a
1000 μL pipette; this step is repeated after 2 h.
7. Terminate the reaction by taking the samples out of the incubator, placing them at room temperature, and storing the
specimens (i.e., culture well) in a light-protected environment.
8. Conduct a quantitative evaluation of the number of healthy
follicles (green fluorescent) via inverted fluorescence microscopy (using a tenfold magnification). Divide the determined
value by 3 to obtain an approximate number of follicles per
2 mm biopsy.
4 Notes
1. Ideally, the preparation should be carried out in a separate
laboratory, accessible via a moving sluice, with a
contamination-free environment in which only the ovarian
tissue is prepared under sterile and cooled conditions.
2. Usually it is recommended to remove approximately one- to
two-thirds of an ovary. This will cause an earlier occurrence of
the natural menopause by 1–2 years earlier, provided that the
remaining ovaries still remain active [3, 32].
3. In the case of ovarian tissue cryopreservation, the immediate
histological examination of a reference sample of the harvested
496
Jana Liebenthron and Markus Montag
and the remaining three are frozen separately as thawing samples, at
the freezing process of the cortex strips for (re)transplantation and
for later thawing and assessment using the same viability assay (e.g.,
calcein staining) [1, 3, 23] (see Notes 5–7):
1. Prepare medium 1 day before use. Equilibrate AIM-V serumfree culture medium (per sample, 600 μL/well in a four-well
dish) overnight with following incubation conditions: 6% CO 2
and 37
C.
2. Put three biopsies with 2 mm mean diameter from different
cortex areas for 2–3 h in pre-equilibrated culture medium in a
Nunc four-well dish (the maximum culturing time should be
not longer than 2 days/48 h).
3. Prepare a solution with 2 μL calcein AM +998 μL pre-warmed
DPBS solution and 6–8 mg collagenase type I A in a 1.5 mL
Eppendorf reaction tube (1 1 mL approach of calcein
AM/DPBS/collagenase type I A solution can be used for
two assays/patients).
4. Pipette 500 μL of this solution in one well of the Nunc fourwell plate, and transfer the 3 Â 2 mm biopsies into the calcein
AM/DPBS/collagenase type I A solution.
5. The incubation time is 1.5–2 h at 5% CO 2 and 37
C.
6. After 1 h the lysate should be carefully homogenized using a
1000 μL pipette; this step is repeated after 2 h.
7. Terminate the reaction by taking the samples out of the incubator, placing them at room temperature, and storing the
specimens (i.e., culture well) in a light-protected environment.
8. Conduct a quantitative evaluation of the number of healthy
follicles (green fluorescent) via inverted fluorescence microscopy (using a tenfold magnification). Divide the determined
value by 3 to obtain an approximate number of follicles per
2 mm biopsy.
4 Notes
1. Ideally, the preparation should be carried out in a separate
laboratory, accessible via a moving sluice, with a
contamination-free environment in which only the ovarian
tissue is prepared under sterile and cooled conditions.
2. Usually it is recommended to remove approximately one- to
two-thirds of an ovary. This will cause an earlier occurrence of
the natural menopause by 1–2 years earlier, provided that the
remaining ovaries still remain active [3, 32].
3. In the case of ovarian tissue cryopreservation, the immediate
histological examination of a reference sample of the harvested
496
Jana Liebenthron and Markus Montag