Biopolym. Cell. 2018; 34(3):196-206.
Molecular and Cell Biotechnologies
Advancing recovery and cryopreservation of rat spermatogonia for germ stem cell banking
1Syvyk T. L., 1Djachenko L. S., 2Syvyk A. E.
  1. Bila Tserkva National Agrarian University
    8/1, Soborna Sq., Bila Tserkva, Ukraine, 09117
  2. Blinn College
    902 College Ave, Brenham, USA, 77833

Abstract

Aim. Optimisation of spermatogonial cryopreservation medium and spermatogonia recovery procedures essential for the male germ cell research and spermatogonia mediated transgenesis in rodents. Methods. PCR, LIVE/DEAD® Cell Viability Assays, Spermatogonial Colony Forming Assay, immunocytochemistry, cryopreservation and thawing spermatogonia cells. Results. Three Sleeping Beauty mutant spermatogonia stem cell lines were stored in liquid nitrogen, after 17–24 months of cryopreservation, were derived in parallel from recovered cryopreserved cultures via an established laminin selection procedure and by an alternative method termed as direct SG (Spermatogonia Growth) medium selection on MEFs (mouse embrionic fibroblasts). A spermatogenic potential of the cell lines derived by these two methods was verified by the spermatogonia transplantation in vivo and reestablishing the mutant animal lines. We optimized the concentration of DMSO in a freezing medium for spermatogonial cell lines. Conclusions. We developed and optimized recovery of spermatogonial stem cells, that could be consistently derived from the freshly thawed stocks of testicular cells cryopreserved in SG medium. Our data suggest that the 8% DMSO is the most effective concentration of the cryoprotectant in SG medium.
Keywords: spermatogonia stem cell, mutant lines, laminin selection, cryopreservation

References

[1] Wu X, Goodyear SM, Abramowitz LK, Bartolomei MS, Tobias JW, Avarbock MR, Brinster RL. Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years. Hum Reprod. 2012;27(5):1249-59.
[2] Liu S, Tang Z, Xiong T, Tang W. Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol. 2011;9:141.
[3] Syvyk T, Syvyk A. Customized transgenesis via modification of spermatogonial stem cells. J Microbiol Biotechnol Food Sci . 2018; 7(5):475-9.
[4] Mays-Hoopes LL, Bolen J, Riggs AD, Singer-Sam J. Preparation of spermatogonia, spermatocytes, and round spermatids for analysis of gene expression using fluorescence-activated cell sorting. Biol Reprod. 1995;53(5):1003-11.
[5] Gassei K, Ehmcke J, Schlatt S. Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting. Cell Tissue Res. 2009;337(1):177-83.
[6] Kanatsu-Shinohara M, Morimoto H, Shinohara T. Enrichment of Mouse Spermatogonial Stem Cells by the Stem Cell Dye CDy1. Biol Reprod. 2016;94(1):13.
[7] Kubota H, Brinster RL. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal. Methods Cell Biol. 2008;86:59-84.
[8] Kanatsu-Shinohara M, Inoue K, Ogonuki N, Morimoto H, Ogura A, Shinohara T. Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod. 2011;84(1):97-105.
[9] Wu Z, Falciatori I, Molyneux LA, Richardson TE, Chapman KM, Hamra FK. Spermatogonial culture medium: an effective and efficient nutrient mixture for culturing rat spermatogonial stem cells. Biol Reprod. 2009;81(1):77-86.
[10] Chapman KM, Saidley-Alsaadi D, Syvyk AE, Shirley JR, Thompson LM and Hamra FK. Spermatogonial Stem Cell Mediated Gene Transfer. Transgenic Technology, Eds. Shirley Springer Protocols Handbooks Springer Protocols Handbooks; 2011. 237-66 p.
[11] Ivics Z, Izsvák Z, Chapman KM, Hamra FK. Sleeping Beauty transposon mutagenesis of the rat genome in spermatogonial stem cells. Methods. 2011;53(4):356-65.
[12] Goodyear S, Brinster R. Spermatogonial Stem Cell Transplantation to the Testis. Cold Spring Harb Protoc. 2017;2017(4):pdb.prot094235.
[13] Syvyk A, Djachenko L, Syvyk T. Transgenesis through the spermatogonia stem cells in the animals and prospects of its application in Bilotserkiva NAU. Collection of scientific works of Bila Tserkva NAU. 2017; 1(134):5-12.
[14] Stacey GN, Masters JR. Cryopreservation and banking of mammalian cell lines. Nat Protoc. 2008;3(12):1981-9.
[15] Cronkhite JT, Norlander C, Furth JK, Levan G, Garbers DL, Hammer RE. Male and female germline specific expression of an EGFP reporter gene in a unique strain of transgenic rats. Dev Biol. 2005;284(1):171-83.
[16] Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2015;1257:3-19.
[17] Mirzapour T, Movahedin M, Tengku Ibrahim TA, Haron AW, Nowroozi MR. Evaluation of the effects of cryopreservation on viability, proliferation and colony formation of human spermatogonial stem cells in vitro culture. Andrologia. 2013;45(1):26-34.