In vitro spermatogenesis in two‐dimensionally spread mouse testis tissues

Abstract

Purpose Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. Methods Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr‐Gfp, which reflects the progression of spermatogenesis. Results Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. Conclusions The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control

“…Secondly, we applied silicone cover ceiling method, which used polydimethylsiloxane (PDMS) ceiling (PC) chip having 160 µm-depth square dent, to hold the testis tissue in the dent space making it thin and flat on the agarose gel (Fig. 2 C,D) 28 . Thirdly, we tested different concentrations of AlbuMAX from 10 to 40 mg/mL.…”

“…In cases that PC method was applied, single tissue was placed on a gel, and PC chip having 160 μm depth of dent was covered over the tissue. The PC was prepared based on our previous papers 28 , 53 . Briefly, PDMS prepolymer and curing reagent (Silpot 184; Dow Corning) were mixed at a 10:1 weight ratio.…”

Advanced bioengineering of male germ stem cells to preserve fertility

“… 63 In follow-up work in 2019, Komeya et al placed the immature mouse testis tissues on agarose gel blocks and forced them to be spread as a monolayer using a microfluidic ceiling system. They observed that the presence of the PDMS microfluidic device chip elevated the initiation and maintenance of spermatogenesis, following by increasing the number of meiotic germ cells, and enhancing the spermatogenesis up to round/elongating spermatids which were confirmed by immunohistochemical evaluation 171 ( Table 4 ).…”

Influence of hydrogel encapsulation during cryopreservation of ovarian tissues and impact of post-thawing in vitro culture systems in a research animal model

“…Our contradictory findings may have resulted from differences in the tissue culture system (with or without an agarose gel block), culture media flow rate (0.002 mL/min [ 37 ] vs. 0.00017 mL/min in our study), or the type of device fabrication (3D printing-PDMS or a modified conventional cell culture plate). Although gonadal tissue culture on agarose gel blocks has been attempted [ 17 , 38 ], recent evidence showed a diminished distribution of GFP protein markers related to spermatid development during long-term mouse testicular tissue culture using agarose gel [ 36 ]. With more than a 10-times lower flow rate [ 37 ], the oxygen and nutrients diffused through the agarose gel and the hydrogel encapsulation might have been insufficient [ 35 ].…”