Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro.

Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro. Syst Biol Reprod Med. 2017 Dec 01;:1-12 Authors: Gat I, Maghen L, Filice M, Kenigsberg S, Wyse B, Zohni K, Saraz P, Fisher AG, Librach C Abstract Spermatogonial Stem Cell (SSC) expansion in vitro 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 afte...
Source: Systems Biology in Reproductive Medicine - Category: Reproduction Medicine Authors: Tags: Syst Biol Reprod Med Source Type: research