Figure 4. Effects of trehalose on stem cell activity after thawing.

(A–B) Bright-field (A) and dark-field fluorescence (B) images of germ cells from GFP positive donors frozen for 3 months in 200 mM trehalose after thawing and 7 days in vitro. (C) Dark-field fluorescence images are over-layed on the bright field image of the same section of a recipient testis transplanted with germ cells from GFP positive donors frozen for 3 months in 200 mM trehalose after thawing and 7 days in vitro culture. Colonies of donor spermatogenesis are distinct green regions of the recipient seminiferous tubules. The number of colonies is directly proportional to the number of SSCs in the transplanted cell population. (D) Cryosections of donor derived germ cell colonies. Green image showing multiple layers of green donor germ cells is over-layed on the bright field image of the same section. Presence of sperm (arrow) in the lumen of the seminiferous tubules indicates complete spermatogenesis. (E) The number of colonies per 10⁵ transplanted cells. (F) The number of colonies per total number of cells recovered after freeze, thaw, and culture. Fresh: non-cryopreserved cells (n = 3 samples; 9 total mice and 16 total testes were transplanted); Trehalose: donor cells frozen with 200 mM trehalose (n = 3 samples; 9 total mice and 16 total testes were transplanted); DMSO: donor cells frozen in control basal freezing media (DMSO only; n = 3 samples; 8 total mice and 14 total testes were transplanted). Values are means ± SEM. Points with different superscripts are significantly different (P<0.05). Scale Bars: (A, B)  = 100 µm; (C)  = 2 mm; (D)  = 200 µm.