Cryopreservation of Human Testicular Tissue by Isopropyl-Controlled Slow Freezing

Abstract

Tissue cryopreservation uses very low temperatures to preserve structurally intact living cells in their natural microenvironment. Cell survival is strongly influenced by the biophysical effects of ice during both the freezing and the subsequent thawing. These effects can be controlled by optimizing the fragment size, type of cryoprotectant, and cooling rate. The challenge is to determine cryopreservation parameters that suit all cell types present in the tissue. Here we describe a quick and convenient protocol for the cryopreservation of testicular tissue using an isopropyl-insulated freezing device, which was validated in both a mouse and a human model.

Setting Up a Cryopreservation Programme for Immature Testicular Tissue: Lessons Learned After More Than 15 Years of Experience

“…After testicular tissue sampling, the major part (>90%) of the testicular tissue biopsy is cut into small fragments of approximately 6 mm³, cryopreserved, and stored for potential later fertility restoration purposes. 50,53 Testicular tissue is cryopreserved by slow freezing in cryopreservation medium consisting of Dulbecco’s modified Eagle’s medium (DMEM/F12; 31330-038; Invitrogen, Merelbeke, Belgium) supplemented with 1.4 M dimethylsulfoxide (DMSO; D2650; Sigma, Bornem, Belgium), 0.15 M sucrose (10274; BDH Laboratory Supplies, Poole, UK), and 10% human serum albumin (HSA; 10064; Vitrolife, Gothenburg, Sweden). Two testicular tissue pieces are placed per vial.…”

“…Consecutive slides are stained for immunohistochemistry for melanoma-associated antigen 4 (MAGE-A4) (mouse monoclonal antibody, kindly provided by Giulio Spagnoli, University of Basel, Switzerland) as a positive marker and VIMENTIN (mouse monoclonal antibody clone V9; M0725; Dako, Heverlee, Belgium) as a negative marker for germ cells. 53 After deparaffinization and rehydration, slides are washed in phosphate-buffered saline (PBS; 70011-036; Life-technologies, Gent, Belgium) for 5 minutes. Endogenous peroxidases are blocked by incubation in 0.3% hydrogen peroxide (H 2 O 2 ) for 30 minutes.…”

“…After testicular tissue sampling, the major part (>90%) of the testicular tissue biopsy is cut into small fragments of approximately 6 mm³, cryopreserved, and stored for potential later fertility restoration purposes. 50,53 (Figure 1) to prevent cross-contamination between the cryopreserved samples.…”

“…In the next 25 years, methods for cryopreservation of SSCs and testicular tissue have been optimized [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] and translated to the human. [46][47][48][49][50][51][52][53][54] Protocols for transplantation of SSCs and testicular tissue need further adaptation to become a clinical application. 55,56 In 2002, the Universitair Ziekenhuis (UZ) Brussel was the first hospital worldwide initiating a clinical fertility preservation programme for young boys at high risk of losing their SSCs.…”

A review of methods for preserving male fertility

“…Preserving testicular tissue by freezing samples before chemotherapy and re-grafting them after treatment is a proposed therapeutic approach to maintain fertility in children with cancer. In this method, the ultimate goal of treatment is essentially to prevent infertility of the cancer patient (Pukazhenthi et al, 2015;Baert et al, 2018). Schlatt et al (2002) followed transplantation of testis tissue in mice and hamsters through to producing a new animal generation.…”