Gat, Itai Maghen, Leila Filice, Melissa Kenigsberg, Shlomit Wyse, Brandon Zohni, Khaled Saraz, Peter Fisher, Andrée Gauthier Librach, Clifford Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion <i>in vitro</i> <p>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><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>.</p> <p><b>Abbreviations</b>: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting </p> Differential plating;fertility preservation;GFRα1;gonadotoxic treatments;prebubertal boys;spermatogonial stem cells 2017-12-01
    https://tandf.figshare.com/articles/dataset/Initial_germ_cell_to_somatic_cell_ratio_impacts_the_efficiency_of_SSC_expansion_i_in_vitro_i_/5660473
10.6084/m9.figshare.5660473.v1