Spermatogonial stem cells (SSCs) are very sensitive to chemotherapy and radiotherapy, so male infertility is a great challenge for prepubertal cancer survivors. Cryoconservation of testicular cells before cancer treatment can preserve SSCs from treatment side effects. Different two-dimensional (2D) and three-dimensional (3D) culture systems of SSCs have been used in many species as a useful technique to in vitro spermatogenesis. We evaluated the proliferation of SSCs in 2D and 3D culture systems of platelet-rich plasma (PRP). testicular cells of four brain-dead patients cultivated in 2D pre-culture system, characterization of SSCs performed by RT-PCR, flow cytometry, immunocytochemistry and their functionality assessed by xenotransplantation to azoospermia mice. PRP prepared and dosimetry carried out to determine the optimized dose of PRP. After preparation of PRP scaffold, cytotoxic and histological evaluation performed and SSCs cultivated into three groups: control, 2D culture by optimized dose of PRP and PRP scaffold. The diameter and number of colonies measured and relative expression of GFRa1 and c-KIT evaluated by real-time PCR.

Results indicated the expression of PLZF, VASA, OCT4, GFRa1 and vimentin in colonies after 2D pre-culture, xenotransplantation demonstrated proliferated SSCs have proper functionality to homing in mouse testes. The relative expression of c-KIT showed a significant increase as compared to the control group (*: p < 0.05) in PRP- 2D group, expression of GFRa1 and c-KIT in PRP scaffold group revealed a significant increase as compared to other groups (***: p < 0.001). The number and diameter of colonies in the PRP-2D group showed a considerable increase (p < 0.01) as compared to the control group. In PRP- scaffold group, a significant increase (p < 0.01) was seen only in the number of colonies related to the control group.

Our results suggested that PRP scaffold can reconstruct a suitable structure to the in vitro proliferation of SSCs.

精子原干细胞（SSCs）对化疗和放疗非常敏感，因此男性不育对青春期前癌症幸存者来说是一个巨大的挑战。在癌症治疗前对睾丸细胞进行冷冻保存可以保护 SSC 免受治疗副作用的影响。SSC 的不同二维 (2D) 和三维 (3D) 培养系统已在许多物种中用作体外精子发生的有用技术。我们评估了 SSC 在富血小板血浆 (PRP) 的 2D 和 3D 培养系统中的增殖。在 2D 预培养系统中培养的四名脑死亡患者的睾丸细胞，通过 RT-PCR、流式细胞术、免疫细胞化学进行的 SSC 表征及其通过异种移植到无精子症小鼠的功能评估。制备 PRP 并进行剂量测定以确定 PRP 的最佳剂量。在制备 PRP 支架后, 进行细胞毒性和组织学评估, SSCs 培养成三组: 控制组、通过优化剂量的 PRP 和 PRP 支架进行二维培养。测量的集落直径和数量以及通过实时 PCR 评估 GFRa1 和 c-KIT 的相对表达。

结果表明 PLZF、VASA、OCT4、GFRa1 和波形蛋白在 2D 预培养后在菌落中的表达，异种移植证明增殖的 SSCs 具有适当的功能以在小鼠睾丸中归巢。PRP-2D组c-KIT的相对表达较对照组显着增加（*：p<0.05），PRP支架组GFRa1和c-KIT的表达较其他组显着增加(***: p < 0.001)。与对照组相比，PRP-2D 组的菌落数量和直径显着增加（p < 0.01）。在 PRP-支架组中，仅在与对照组相关的集落数量中观察到显着增加（p < 0.01）。

我们的结果表明，PRP 支架可以重建适合 SSC 体外增殖的结构。