Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells

Abstract

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Figure 1. Effects of trehalose on viability and proliferation of SSC enriched testis cells after thawing.

(A) Percentage of viable cells after thawing. (B) Percentage of proliferation capacity of frozen germ cells in SSC culture for 1 week after thawing. Figure bars: White: DMSO control group; Light gray: 50 mM trehalose group; Dark gray: 100 mM trehalose group; Black: 200 mM trehalose group. Each treatment group was thawed at 1 week, 1 month, and 3 months post-freezing. Values are means ± SEM (n = 5). Bars within a group with different letters are significantly different (P<0.05).

Figure 2. Effects of trehalose on apoptosis of SSC enriched testis cells 12 hours after thawing.

Percentage of annexin V binding PI excluding apoptosis positive GFP positive SSC enriched testis cells 12 hours after thawing. Figure bars: White: DMSO control group; Light gray: 50 mM trehalose group; Dark gray: 100 mM trehalose group; Black: 200 mM trehalose group. Each treatment group was thawed at 1 week, 1 month, and 3 months post-freezing. Values are means ± SEM (n = 3). Bars within a group with different letters are significantly different (P<0.05).

Figure 3. Effects of trehalose on proliferation of SSC enriched germ cells after extended freezing times.

Percentage of non-frozen germ cell proliferation capacity of frozen germ cells in SSC culture for 1 week after thawing. No significant differences were observed between proliferation percent after 3, 6, and 12 months of freezing within each treatment group. Figure symbols: Black circle: DMSO control group; White circle: 50 mM trehalose group; Black triangle: 100 mM trehalose group; White triangle: 200 mM trehalose group. Each treatment group was thawed at 1 week, 1 month, 3 months 6 months, and 12 months post-freezing. Values are means ± SEM (n = 5). Different letters indicate significant difference (P<0.05) among groups within each freezing period.

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.

Figure 5. SSCs frozen with 200 mM trehalose have the capacity to generate offspring.

(A) Offspring from a C57BL/6 female crossed with a W recipient male (surgery number 685) that was transplanted with SSCs frozen with 200 mM trehalose. (B) The same offspring under UV exposure. Transplanted germ cells are heterozygous for GFP which results in the generation of non-GFP pups from donor cells in addition to GFP+ pups.