The Applications Of Alginate Microcapsules In STO Cells And Sheep Spermatogonial Stem Cells Cryopreservation

Spermatogonia stem cell is the only diploid cell which can completely pass the genetic information to the second generation. With the development of science and technology, stem cell research combined with regenerative medicine research is becoming a new field for the sake of applying the stem cell and its potential to practice, providing a research direction and providing the platform for the medical-related research. But this kind of research is stopped taking the next step because of the low efficiency of spermatogonial stem cell cryopreservation. So, lots of researchers have begun to study an efficient cryopreservation program in different ways. Our study based on the introduction of new biomaterials created/modified a micro-environment for cell survival in vitro, in which cryopreservation of cells can be manipulated to improve the efficiency of spermatogonial stem cells.1. The method of making the alginic acid microcapsulesAlginic acid microcapsules are formed for the feature that alginate solution encounter a variety of divalent cations can cross-linked into gel. The methods of forming alginic acid microcapsules including high voltage electrostatic microcapsule generating device production method, capillary crushing production method, and gas blown production method, etc. This study use high voltage electrostatic microcapsule generating device production method, for its advantages including easy operation, high efficiency, uniform microcapsule size etc. It generates alginate-Ca2+microcapsules with uniform size, morphological integrity, and diameter300μm. Resulting microcapsules can be used to cells entrapped and further research.2. Research on alginate microcapsules replacing DMSO and FBS on the cryopreservation of STO cellsObejective:To improve cryopreservation method and to establish an efficient cryopreservation system without DMSO and FBS, in order to provide high-quality cells for cell-therapy and other clinical applications. Methods:STO cells were embedded into and cryopreserved in freezing solution (alginate microcapsules D-F-). Four groups of cell suspension were set up as control:one group with10%DMSO and20%FBS (D+F+), one group only with10%DMSO (D+F-), one group only with20%FBS (D-F+), and one group without DMSO and FBS (D-F-). Cell number and the survival rate of STO cells were counted by trypan blue staining, cell morphology and the survival were assayed by double staining using EthD and Calcein-AM. Cell proliferation rate and growth activity was evaluated by using Methabenzthiazuron (MTT) assay. Results:For30days cryopreservation, then thawed the STO cells and analyzed cell number, cell viability and growth activity of five groups. The results showed that there was no significant difference between the group of alginate microcapsules embedded cells and the group with DMSO and FBS after cryopreserving and thawing. Conclusion:Alginate microcapsules replacing DMSO and FBS in the cryopreservation of STO cells can effectively maintaining cell morphology, cell number, viability and growth activity were not afifected. An efficient cryopreservation system without DMSO and FBS was established.3. The applications of alginate microcapsules in sheep spermatogonial stem cells cryopreservationCryopreservation of cells is an essential part of cell biology study. To improve efficacy of sheep spermatogonial stem cells (SSCs) cryopreservation, alginate microcapsules were used to cryopreserve the sheep SSCs in the present study. Cell viability and proliferation rate were evaluated and compared. Five SSC-specific genes, including CDH1, PLZF, GFRα1, OCT-4, Thy-1were examined by immunofluorescence after stored in liquid nitrogen for1day. The results indicated that alginate microcapsules could increase sheep SSCs viability to78.7%±1.3%, while stored in DMSO was58.4%±1.4%. After four days culture, the proliferation rate of alginate microcapsules group(2.1×105cells/hole) was significantly higher than the cell suspension cryopreserved group(1.1×105cells/hole). Immunofluorescence showed that CDH1, PLZF, GFRα1, OCT-4, Thy-1were expressed after cryopreservation. Cryopreserved in liquid nitrogen10days, survival rate of sheep SSCs in alginate microcapsules group remained at78.0%±1.5%, the survival rate is not reduction significantly compare with cryopreserved in liquid nitrogen by1day(78.7%±1.3%) and before cryopreserved(82.2%±1.9%). But in the cell suspension cryopreservation group, survival rate of sheep SSCs have significant decline from82.2%±1.9%(before cryopreserved) to58.4%±1.4%(cryopreserved in liquid nitrogen by1day) and48.1%±0.8%(Cryopreserved in liquid nitrogen by 10days). In other words, cryopreservation of alginate microcapsules embedded sheep SSCs, the survival was not significantly decreased with increasing time in liquid nitrogen cryopreservation. While, sheep SSCs frozen10days still maintained a rapid growth ability, also expressed the specific marker genes of sheep SSCs. In conclusions, alginate microcapsules significantly improved the efficacy of sheep SSCs cryopreservation, and maintained the expression of sheep SSC-specific genes after thawing. This study indicates a new method for the cryopreservation of animal resources by using the alginate microcapsule, especially for the livestock and those endangered species.