Determination of Medium Condition Effective to Cryopreservation of Primary Spermatogonial Stem Cells Derived from Porcine Neonatal Testes.

BACKGROUND: The superior genetic resources of breeding pigs have been preserved for use through freezing the sperm or semen. However, because there is no way to collect their sperm or semen after depletion, the generation of sperm via the differentiation of porcine spermatogonial stem cells (SSCs) can be an alternative. To date, there have been no reports of techniques customized to in-vitro culture and differentiation into sperm in porcine SSCs. Accordingly, it is important to preserve porcine SSCs with outstanding genetic backgrounds until these technologies are developed. Unfortunately, a protocol for the long-term preservation of porcine SSCs has yet to be reported. OBJECTIVE: We tried to develop a cryopreservation medium to preserve the characteristics of undifferentiated porcine SSCs for long-term cryopreservation. MATERIALS AND METHODS: SSCs retrieved from porcine testes were freeze-cryopreserved in StemPro-34 medium supplemented with various concentrations of fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and trehalose; then, after 7 days, the viability and alkaline phosphatase (AP) activity was measured in thawed porcine SSCs. Additionally, we investigated the use of hypotaurine and/or glutathione as antioxidants in the optimized freezing medium for maintaining the viability and AP activity of porcine SSCs during the freezing-cryopreservation-thawing process. RESULTS: Porcine SSCs frozen-cryopreserved-thawed in StemPro-34 medium supplemented with 10% (v/v) FBS, 10% (v/v) DMSO, 200 mM trehalose, 5 mM hypotaurine, and 5 mM glutathione showed the highest viability and AP activity. CONCLUSION: We optimized a cryopreservation medium that inhibits the loss of viability and the increases differentiation post-thawing of the frozen porcine SSCs.

Generation of embryonic stem-like cells from in vivo-derived porcine blastocysts at a low concentration of basic fibroblast growth factor.

Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self-renewal and pluripotency, its high cost has limited previous studies, and the development of a low-cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo-derived porcine blastocysts on 5.0 × 105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium-based medium for primary culture, on 2.5 × 105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5 × 105 MEF feeder cells in DMEM/Ham's F10-based medium for the post-2nd subpassage could support the establishment and maintenance of porcine ES-like cells at the low concentration of bFGF. The established porcine ES-like cells showed ES cell-specific characteristics such as self-renewal and pluripotency. We confirmed that porcine ES-like cells could be generated from in vivo-derived porcine blastocysts at a low concentration of bFGF.

Determination of feeder cell-based cellular niches supporting the colonization and maintenance of spermatogonial stem cells from prepubertal domestic cat testes.

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long-term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC-specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome-shaped morphology, AP activity and expression of SSC-specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.

Determination of size, concentration and elemental composition of colloids with laser-induced breakdown detection/spectroscopy (LIBD/S).

The application of laser-induced breakdown detection (LIBD) as a new and powerful particle-analyzing technique for the determination of solubility data by monitoring initial colloid generation, when the metal ion concentration just exceeds the solubility at a given pH value, is investigated. Laser-induced breakdown spectroscopy (LIBS) is used for selective analysis of an aqueous suspension of lanthanide oxide particles in the presence of the respective lanthanide aquo ion. The detection limit for aquo ion and oxide particle is determined. On the basis of the different detection limits, the LIBS technique is used to study the formation of hydroxide colloids in aqueous solution by varying the pH value until the solubility limit is exceeded. LIBS enables both qualitative and quantitative monitoring of particle formation without artifacts arising from other contaminants. LIBD and LIBS are described and compared. The advantages and disadvantages of the methods for the determination of solubility data are discussed.