What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

RESEARCH QUESTION: From a clinical perspective, which parameters grant optimal cryopreservation of mouse testicular cell suspensions? DESIGN: We studied the effect of different cryopreservation rates, the addition of sugars, different vessels and the addition of an apoptotic inhibitor on the efficiency of testicular cell suspension cryopreservation. After thawing and warming, testicular cell suspensions were transplanted to recipient mice for further functional assay. After selecting the optimal cryopreservation procedure, a second experiment compared the transplantation efficiency between the selected freezing protocol and fresh testicular cell suspensions. RESULTS: Multiple- and single-step freezing did not differ significantly in terms of recovered viable cells (RVC) (33 ± 28% and 38 ± 25%). The addition of sucrose did not result in a higher RVC (33 ± 20%). Cells frozen in vials recovered better than those frozen in straws (52 ± 20% versus 33 ± 20%; P = 0.0049). The inclusion of an apoptosis inhibitor (z-VAD[Oe]-FMK) significantly increased the RVC after thawing (61 ± 18% versus 50 ± 17%; P = 0.0480). When comparing the optimal cryopreservation procedure with fresh testicular cell suspensions, a lower RVC (63 ± 11% versus 92 ± 4%; P < 0.0001) and number of donor-derived spermatogonial stem cell colonies per testis (34.04 ± 2.34 versus 16.78 ± 7.76; P = 0.0051) were observed. CONCLUSION: Upon freeze-thawing or vitrification-warming, and assessment of donor-derived spermatogenesis after transplantation, Dulbecco's modified Eagle's medium supplemented with 1.5M dimethyl-sulphoxide, 10% fetal calf serum and 60 µM of Z-VAD-(OMe)-FMK in vials at a freezing rate of -1°C/min was optimal.

Short-term storage of human testicular tissue: effect of storage temperature and tissue size.

During short-term storage, before cryopreservation, testicular tissue quality can be affected by storage medium, duration, temperature and tissue size. We previously established the best storage medium and time for short-term maintenance of tissue. In this study, three different storage temperatures (4°C, room temperature, 37°C) and four tissue sizes (~6 mm3; ~ 15 mm3; ~ 50 mm3 or ~80 mm3) were assessed over the course of a fixed period of 3 days. Storing human testicular tissue at 37°C caused a significant increase in the number of apoptotic cells per tubule (P = 0.002), compared with fresh control, but this was not the case at 4°C or room temperature. Temperature did not affect viability, tissue morphology or number of spermatogonia in samples. The morphology of the testicular tissue was optimally preserved when stored as large fragments (~50 mm3: P = 0.018; ~ 80 mm3: P = 0.018). Tissue size did not significantly affect viability, number of spermatogonia or apoptotic cells. Adult human testicular tissue can be preserved at 4°C or room temperature without altering tissue morphology, Sertoli cell morphology, number of spermatogonia or number of apoptotic cells. The tissue does not need to be extensively dissected as tissue morphology is better maintained in larger fragments.

In search of an improved injection technique for the clinical application of spermatogonial stem cell transplantation.

When fertility is impaired by anticancer treatment, spermatogonial stem cell transplantation (SSCT) could be used as a fertility restoration technique later on in life. Previously, we have demonstrated that a testicular cell suspension could be injected into a human cadaver testis, however, leakage to the interstitium was observed. In this study, injection of mouse testicular cells at an injection height of 50 cm (hydrostatic pressure) or via an automated injection pump (1400 µl, 2600 µl and 3000 µl) was evaluated. Significant difference in the filled radioactive volume was reached between the group in which 1400 µl was injected with an infusion pump and the groups in which 2600 µl (P = 0.019) or 3000 µl (P = 0.010) was injected. In all experimental groups green fluorescent protein positive (GFP+) cells were observed in the seminiferous tubules. In conclusion, a lower injection height did not resolve the leakage of the injected cells to the interstitium. Using the infusion pump resulted in more efficient filling of the seminiferous tubules with lower interexperimental variability. Although leakage to the interstitium was still observed, with further optimisation, the use of an infusion pump for clinical application is advantageous.

Short-term hypothermic preservation of human testicular tissue: the effect of storage medium and storage period.

OBJECTIVE: To optimize the storage medium and period during short-term preservation of human testicular tissue. DESIGN: First, human testicular tissue fragments from five patients were kept at 4°C for 3 days in different media (Dulbecco's modified Eagle's medium [DMEM]/F12, DMEM/F12 + 20% human serum albumin [HSA], DMEM/F12 + 50% HSA, and HSA). Secondly, fragments from four patients were kept in DMEM/F12 for 3, 5, or 8 days at 4°C. SETTING: Laboratory research environment. PATIENT(S): Adult human testicular tissue. INTERVENTION(S): Biopsy and short-term storage of human testicular tissue at different conditions. MAIN OUTCOME MEASURE(S): Viability, general tissue morphology, Sertoli cell morphology, number of spermatogonia, and apoptosis. The experimental conditions were compared with fresh control samples. RESULT(S): Storing human testicular tissue in DMEM/F12 did not alter any of the investigated parameters. In most conditions containing HSA, tissue morphology was altered, and in all of them the Sertoli cell morphology was affected. The number of spermatogonia was only affected when tissue was stored in 100% HSA. In the second part of the study, tissue morphology deteriorated significantly as of 5 days of hypothermic storage, and Sertoli cell morphology after 8 days. CONCLUSION(S): Human testicular tissue can be preserved for 3 days at 4°C in DMEM/F12 without altering tissue morphology, Sertoli cell morphology, number of spermatogonia, or number of apoptotic cells.

The Effect of a Unilateral Orchiectomy before Gonadotoxic Treatment on the Contralateral Testis in Adult and Prepubertal Rats.

PURPOSE: Previous studies have shown that the removal of one testis leads to a compensatory mechanism in the contralateral one, but this was species and age dependent. The aim of this study was to check whether this compensation would still occur after the combination of a unilateral orchiectomy and gonadotoxic treatment, since this resembles the clinical situation of patients who have to undergo highly toxic cancer treatment and therefore choose to cryopreserve a testicular biopsy for fertility restoration purposes. MATERIALS & METHODS: Sprague Dawley rats underwent either unilateral orchiectomy, gonadotoxic busulfan treatment, the combination of both or served as fertile control. A comparison of the compensatory effects was made between adult and prepubertal treated rats. Mating experiments were performed, testosterone levels were followed-up, testicular weight was recorded and histology was analysed. RESULTS: Adult treated rats were able to restore fertility spontaneously in all treatment groups. On the other hand, 30% of the rats that underwent a unilateral orchiectomy and gonadotoxic treatment at prepubertal age showed hampered spermatogenesis, low testosterone levels, decreased testicular weights and were not able to reproduce. CONCLUSION: This study emphasizes the need of fertility preservation strategies in prepubertal patients before gonadotoxic interventions.

Testicular cell transplantation into the human testes.

OBJECTIVE: To translate spermatogonial stem cell (SSC) transplantation towards a clinical application. DESIGN: Mouse green fluorescent protein (GFP)-positive testicular cells were labeled with (99m)technetium and microbubbles. These labeled cells were injected into the rete testis of isolated human testes under ultrasound guidance. Three different conditions were tested: 1) 800 µL of a 20 million cells/mL suspension; 2) 800 µL of a 10 million cells/mL suspension; and 3) 1,400 µL of a 10 million cells/mL suspension. After injection, the human cadaver testes were analyzed with the use of single-photon-emission computerized tomography (SPECT) imaging and histology. SETTING: Laboratory research environment. PATIENT(S): Cadaver testes, obtained from autopsies at the pathology department. INTERVENTION(S): Ultrasound-guided injection of mouse GFP-positive testicular cells. MAIN OUTCOME MEASURE(S): Presence of radioactive-labeled cells in the human cadaver testes and GFP-positive cells in the seminiferous tubules. RESULT(S): In all of the experimental groups, GFP-positive cells were observed in the seminiferous tubules, near and far from the rete testis, but also in the interstitium. On SPECT, significant difference was seen between the group injected with 800 µL of a 20 million cells/mL suspension (1,654.6 ± 907.6 mm³) and the group injected with 1,400 µL of a 10 million cells/mL suspension (3,614.9 ± 723.1 mm³). No significant difference was reached in the group injected with 800 µL of a 10 million cells/mL suspension. CONCLUSION(S): Injecting cells in the human cadaver testis is feasible, but further optimization is required.