Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion <i>in vitro</i>

Spermatogonial Stem Cell (SSC) expansion <i>in vitro</i> remains a major challenge in efforts to preserve fertility among pubertal cancer survivor boys. The current study focused on innovative approaches to optimize SSC expansion. Six- to eight-week-old CD-1 murine testicular samples were harvested by mechanical and enzymatic digestion. Cell suspensions were incubated for differential plating (DP). After DP, we established two experiments comparing single vs. repetitive DP (S-DP and R-DP, respectively) until passage 2 (P2) completion. Each experiment included a set of cultures consisting of 5 floating-to-attached cell ratios (5, 10, 15, 20, and 25) and control cultures containing floating cells only. We found similar cell and colony count drops during P0 in both S- and R-DP. During P2, counts increased in S-DP in middle ratios (10, 15, and especially 20) relative to low and high ratios (5 and 25, respectively). Counts dropped extensively in R-DP after passage 2. The superiority of intermediate ratios was demonstrated by enrichment of GFRα1 by qPCR. The optimal ratio of 20 in S-DP contained significantly increased proportions of GFRα1-positive cells (25.8±5.8%) as measured by flow cytometry compared to after DP (1.9±0.7%, <i>p</i>&lt;0.0001), as well as positive immunostaining for GFRα1 and UTF1, with rare Sox9-positive cells. This is the first report of the impact of initial floating-to-attached cell ratios on SSC proliferation <i>in vitro</i>. <b>Abbreviations</b>: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting

精原干细胞（Spermatogonial Stem Cell, SSC）的体外（in vitro）扩增，仍是青春期癌症存活男孩群体生育力保存研究中的核心挑战。本研究聚焦于优化SSC体外扩增的创新策略。

研究获取了6至8周龄的CD-1小鼠睾丸样本，通过机械解离与酶消化法完成样本制备。将细胞悬液进行差速贴壁培养（differential plating, DP）。完成差速贴壁培养后，本研究设置两组对照实验，分别比较单次差速贴壁与重复差速贴壁（分别记为S-DP与R-DP）直至第2代（passage 2, P2）培养完成的扩增效果。

每组实验均包含5组不同的悬浮-贴壁细胞比例（5、10、15、20与25）的培养体系，以及仅含悬浮细胞的对照组培养体系。

研究发现，在P0培养阶段，S-DP与R-DP组的细胞与集落数量下降趋势相似。在P2阶段，S-DP组的中比例组（10、15，尤其是20）的细胞与集落数量，相较于低比例（5）与高比例（25）组出现显著提升；而R-DP组在P2阶段后细胞数量出现大幅下降。

通过qPCR检测GFRα1的基因富集水平，证实了中间比例组的扩增优势。相较于差速贴壁培养后（1.9±0.7%），S-DP组中最优比例20的培养体系中，经流式细胞术检测的GFRα1阳性细胞比例显著升高（25.8±5.8%，*p*<0.0001），同时该体系中细胞呈现GFRα1与UTF1免疫染色阳性，且Sox9阳性细胞极为罕见。

本研究首次报道了初始悬浮-贴壁细胞比例对体外SSC增殖的影响。

**缩略词**：SSC：精原干细胞（Spermatogonial Stem Cells）；DP：差速贴壁培养（Differential Plating）；NOA：非梗阻性无精子症（Non-obstructive Azoospermia）；MACS：磁激活细胞分选（Magnetic-activated Cell Sorting）；FACS：荧光激活细胞分选（Fluorescence-activated Cell Sorting）