Testicular Tissue Cryopreservation

Abstract

Loss of spermatogonial stem cells can be associated to ageing or can be part of genetic disorders such as 47,XXY Klinefelter syndrome. However, cytotoxic therapies and/or irradiation is the main cause of germ cell loss and can disrupt spermatogenesis temporarily or permanently. These therapies are not only used to treat malignant disorders but may also be used for benign haematological conditions that need bone-marrow transplantation.

Since survival rates after cancer treatment are increasing, preservation of the reproductive potential has become an important quality of life issue for both adults but also for childhood cancer survivors. Adult men who cannot bank ejaculate spermatozoa can opt to cryopreserve surgically retrieved spermatozoa, i.e. testicular sperm. Prepubertal boys cannot benefit from sperm banking, but testicular stem cell banking is being introduced into more and more clinics. This strategy should still be regarded as experimental given the lack of any report on successful transplantation and the limited safety data of this method.

Appendix

9.1.1 Protocol for Cryopreservation of Human Testicular Tissue (Containing Spermatozoa) in View of Fertility Preservation

Cryopreservation Medium

ready for use sperm freezing medium based on glycerol, commercially available from different companies.

Collection Medium for Testicular Tissue

Hepes-buffered HTF or sperm buffer supplemented with HSA, commercially available from different companies.

Preparation and Freezing of Testicular Tissue

• 1. Place the excised biopsies in a sterile petridish or tube filled with 5 ml of Hepes-buffered collection medium at room temperature and transport it to the laboratory

• 2. Unravel and mince the biopsies with the help of sterile pincettes, scissors or bended needles under the stereomicroscope in a laminar flow

• 3. Control the presence of spermatozoa in the suspension under the inverted microscope at 200× or 400× magnification

• 4. Put the suspension in a 10 ml Facon tube and centrifuge at 750 × g for 5 min

• 5. Discard the supernatant and resuspend the pellet in a volume which depends on the volume of the tissue

• 6. Add cryopreservation medium which was brought at room temperature dropwise, the volume to add depends on the volume of the final suspension and on the volume: volume ratio according to the manufacturer’s instructions.

• 7. Aspirate the mixture in high-security straws, which are heat sealed.

• 8. Freezing is carried out in a programmable freezer with a programme for ejaculated sperm.

• 9. At the end of the programme, plunge the straws into LN2 and transport to and store in the LN2 container (vapour phase or liquid phase).

9.1.2 Protocol for Cryopreservation of Prepubertal Human Testicular Tissue (Spermatogonial Stem Cells) in View of Fertility Preservation

Components of the medium	Company and product N°	Storage
Hepes-buffered DMEM/F12	Gibco 31330-095	4 °C
HSA (human serum albumin)	Vitrolife 10064	4 °C
Sucrose	Sigma S1888-500G	RT
DMSO	WAK Chemie WAK-DMSO-10	RT

Fresh Preparation of the Cryopreservation Medium

• 2.55 g sucrose (0.15 M) + 40 ml DMEM/F12

• → in a sterile container, wait until sucrose dissolves, filter it

• +5 ml HAS 10 %)

• +5 ml sterile DMSO (1.5 M)

• Keep on melting ice (4 °C) prior to use

Preliminary Preparation of the Cryopreservation Medium

• 2.55 g sucrose (0.15 M) + 40 ml DMEM/F12

• → in a sterile sperm container, wait until sucrose dissolves, filter it

• → aliquot 8 ml in Falcon tubes

• → store at −20 °C

Day of Use

• Thaw one Falcon tube sucrose solution

• +1 ml HSA (10 %) per Falcon tube

• +1 ml sterile DMSO (1.5 M)

• Keep on melting ice (4 °C) prior to use

Washing Medium

• Hepes-buffered DMEM at 4 °C

Preparation and Freezing of Testicular Tissue

• 1. Clean the laminar flow, work as aseptic as possible.

• 2. The tissue is collected and transported in a sterile container with 0.9 % NaCl on ice. Take the tissue out of the collecting container.

  Transfer the tissue to a large sterile petridish containing DMEM. Work on ice water or use a cooling element.

  Rinse the tissue well.

• 3. Transfer the tissue to a second petridish containing DMEM on ice water/cooling element.

• 4. Cut the tissue in fragments of 6 mm³ using a sterile scissor and pincet.

  Remove the tunica, if present.

• 5. Rinse the fragments in a third petridish containing DMEM on ice water/cooling element.

• 6. Reserve one of the fragments for histological analysis.

  Transfer this fragment to a vial with 1.5 ml DMEM. Keep on ice.

• 7. Put the cryo vials in a cooling rack or on ice (absolutely avoid getting water or ice inside the vial!), and add 1.5 ml of cryopreservation medium (4 °C)

• 8. Put the tissue fragments two by two in the cryo vials. Close the vials.

• 9. Leave the vials on ice water/cooling element for 15 min.

• 10. Put the vials in a cooled container with propanodial.

  Transfer the propanodial container to a −80 °C freezer. Leave it there overnight.

• 11. For sterility control: Collect a fraction of the transport medium, of DMEM of the last washing dish (petridish 3) and of the cryopreservation medium, and put it in tubes for sterility testing.

Next day: Transfer the vials from the −80 °C freezer to the LN2 container. Freeze in the vapour phase in case of screw-cap vials.