Gholami Keykavos, Pourmand Gholamreza, Koruji Morteza, Sadighigilani Mohammadali, Navid Shadan, Izadyar Fariborz, Abbasi Mehdi
Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Urology Research Center, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Reprod Biol. 2018 Dec;18(4):397-403. doi: 10.1016/j.repbio.2018.09.006. Epub 2018 Oct 2.
Optimization of in vitro culture system for the expansion and the maturation of male germ cells to post meiotic stages is a valuable tool for studies exploring spermatogenesis regulation and the management of male infertility. Several studies have reported promising results of mouse spermatogonial stem cells culture in three-dimensional (3D) culture systems and a subsequent production of sperm. In the present study, we investigated the capacity of a three-dimensional soft agar culture system (SACS) supplemented with Knockout Serum Replacement (KSR) in colony formation and inducing human germ cells to reach post-meiotic stages. Testicular cells from testes of brain -dead donors were first cultured for three weeks in proliferation medium. The cells were subsequently cultured in the upper layer of the SACS (3D group) in a medium supplemented with KSR and hormones, and the results were compared with that of a two-dimensional (2D) culture system. We found that the number and diameter of colonies and the levels of expression of Scp3 and Integrin α6 in the 3D culture group were significantly higher than in the 2D group. Our findings indicate that SACS can reconstruct a microenvironment capable of regulating both proliferation and differentiation of cell colonies.
优化体外培养系统以促进雄性生殖细胞扩增并使其成熟至减数分裂后阶段，是研究精子发生调控和男性不育症治疗的一项重要工具。多项研究报道了在三维（3D）培养系统中培养小鼠精原干细胞并随后产生精子的可喜成果。在本研究中，我们探究了添加敲除血清替代物（KSR）的三维软琼脂培养系统（SACS）在集落形成以及诱导人类生殖细胞达到减数分裂后阶段方面的能力。首先将脑死亡供体睾丸中的睾丸细胞在增殖培养基中培养三周。随后将这些细胞在添加了KSR和激素的培养基中培养于SACS的上层（3D组），并将结果与二维（2D）培养系统进行比较。我们发现3D培养组中的集落数量和直径以及Scp3和整合素α6的表达水平均显著高于2D组。我们的研究结果表明，SACS能够重建一个能够调节细胞集落增殖和分化的微环境。
