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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.

SUBMITTER: Lee YA

PROVIDER: S-EPMC3551902 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Lee Yong-An YA Kim Yong-Hee YH Kim Bang-Jin BJ Kim Byung-Gak BG Kim Ki-Jung KJ Auh Joong-Hyuck JH Schmidt Jonathan A JA Ryu Buom-Yong BY

PloS one 20130122 1

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 ...[more]

PMID: 23349986

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Project description:Treatment of cancer in children is increasingly successful but leaves many prepubertal boys suffering from infertility or subfertility later in life. A current strategy to preserve fertility in these boys is to cryopreserve a testicular biopsy prior to treatment with the expectation of future technologies allowing for the reintroduction of stem cells and restoration of spermatogenesis. Spermatogonial stem cells (SSCs) form the basis of male reproduction, differentiating into all germ cell types, including mature spermatozoa and can regenerate spermatogenesis following transplantation into an infertile testis. Here, we demonstrate that rat SSCs frozen for more than 20 years can be transplanted into recipient mice and produce all differentiating germ cell types. However, compared with freshly isolated cells or those frozen for a short period of time, long-frozen cells do not colonize efficiently and showed reduced production of spermatids. Single-cell RNA sequencing revealed similar profiles of gene expression changes between short- and long-frozen cells as compared with fresh immediately after thawing. Conversely, following transplantation, long-frozen samples showed enhanced stem cell signaling in the undifferentiated spermatogonia compartment, consistent with self-renewal and a lack of differentiation. In addition, long-frozen samples showed fewer round spermatids with detectable protamine expression, suggesting a partial block of spermatogenesis after meiosis resulting in a lack of elongating spermatids. These findings strongly suggest that prolonged cryopreservation can impact the success of transplantation to produce spermatogenesis, which may not be revealed by analysis of the cells immediately after thawing. Our analysis uncovered persistent effects of long-term freezing not found in other cryopreservation studies that lacked functional regeneration of the tissue and this phenomenon must be accounted for any future therapeutic application.

Dataset Information

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Publications

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

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