Long-Term In Vitro Maintenance of Piglet Testicular Tissue: Effects of Tissue Fragment Size, Preparation Method, and Serum Source

Abstract

Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum—FBS—or knockout serum replacement—KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks.

Optimized Recovery of Immature Germ Cells after Prepubertal Testicular Tissue Digestion and Multi-Step Differential Plating: A Step towards Fertility Restoration with Cancer-Cell-Contaminated Tissue

“…Our findings, together with other advantages of using porcine material for research on fertility restoration methods, e.g., easy access to castration-derived testes in large quantities providing a reliable and constant source of testicular material [ 36 ], piglets having an extended prepubertal period by contrast with rodent models [ 37 ] and data on reproductive processes in pig [ 38 , 39 , 40 ] highlight that piglet testicular tissue can be considered an appropriate alternative experimental material that is highly representative of human prepubertal testicular tissue.…”

Effects of Growth Factors on In Vitro Culture of Neonatal Piglet Testicular Tissue Fragments

“…Testes were collected from neonatal (~1-wk-old) Yorkshire-cross piglets (Camborough-22 × Line 65; PIC Canada, Winnipeg, MB, Canada) through aseptic castration at a university-affiliated swine center. Collected testes were transported to the lab and prepared using previously reported protocols with minor modifications [ 6 ]. Briefly, the testis parenchyma was cut into small fragments of ~2 mg, following the removal of tunica albuginea, rete-testis, and excess connective tissue.…”

“…10828028; Thermo Fisher Scientific, Carlsbad, CA, USA), and 1% w/v antibiotics along with individual and/or combined concentrations of GDNF, bFGF, SCF, and/or EGF ( Table 1 ) for 8 weeks. The control group had testicular tissue fragments cultured in basic media (DMEM + 10% KSR) without any growth factors [ 6 ]. During the 8 weeks of study, culture media were changed every 2–3 days, and the cultures were maintained under standard culture conditions (at 37 °C in 5% CO 2 ) in a humidified incubator.…”

“…The long-term culture of rodent SSCs is relatively well established [ 2 ], while this knowledge is largely lacking in domestic animals [ 3 ]. However, the short-term culture of SSCs has been reported in different farm animals, including pigs [ 4 , 5 , 6 ], goats [ 7 , 8 , 9 , 10 ], buffalo [ 11 , 12 , 13 ], and sheep [ 14 ].…”

“…The expression of GDNF was then observed in different organs and tissues, including the intestine, kidney, muscle, cartilage, and lungs, suggesting that GDNF also has physiological roles in non-nervous systems [ 21 , 22 ]. The crucial role of GDNF in regulating SSC numbers was shown when mice with impaired GDNF signaling showed a gradual loss of SSCs, while its pan-ectopic overexpression promoted germ cell hyperplasia and ultimately tumor formations [ 6 ]. Subsequently, it was reported that supplementation of GDNF promotes short-term and/or long-term in vitro expansion of mouse SSCs [ 23 , 24 ].…”