effect of FBS concentration on cryopreservation of isolated spermatogonial cells from neonatal mouse by MACS method

The purpose of this study was the separation of spermatogonial cells from prepubertal NMRI mouse testis by magnetic activating cell sorting [MACS] method and assessment of FBS concentration on surviving these cells after cryopreservation. Testes from 6-days-old mouse removed and digested in two enzymatic mediums, first 8-9 min at medium containing collagenase, trypsin and DNase and second 10-12 min in medium containing collagenase, trypsin, DNase, hyalloronidase and EDTA and then the spermatogonial cells were isolated by [MACS] method. In the freezing step, the cells were frozen in three medium containing DMEM/F12 medium, 10% DMSO and 50%, 60% and 70% FBS serum, named group I, II.III. The viable cells obtained after enzymatic disaggregation were 91.66% +/- 0.60% and after being isolated by MACS method were 95.25% +/- 0.33%. The purity of the isolated cells was 93.79% +/- 2.20%. The cells frozen in group I, II and III had 39.09% +/- 0.15, 85.55% +/- 6.98 and 90/29 +/- 1/38% viability, respectively. according to the obtained results, the increase of temperature at digestion step reduces the time of testicular tissue disaggregation and consequently increase viable cells. Higher viable cells and purity can be attained by using of a6 integrin and magnetic beads. The results show that spermatogonial cells in freezing media containing 60% - 70% FBS have the highest viability after thawing

[Ultrastructural changes of sheep oocytes follow vitrification by different methods and in vitro maturation]

Our aim was determination of the sheep oocytes ultrastructural changes follow vitrification and in vitro maturation. Good quality isolated cumulus-oocyte complexes [COCs] were randomly divided into non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In the conventional and cryotop methods the vitrified COCs were plunged directly into liquid nitrogen [LN2], whereas in the solid surface group the vitrified COCs were cooled before plunging into LN2. Fresh and vitrified-warmed healthy COCs were matured in vitro and then their ultrastructural changes were evaluated. The results indicated that vitrification by cryotop and solid surface methods preserved the total arrangement of the ooplasm, whereas conventional straw vitrification disturbed the ooplasm organization. Additionally, the number of vacuoles in the ooplasm increased after vitrification, some of these vacuoles were filled partially or completely with lipids and some had filamentous scaffolding. Also, in the mature oocytes, the amount and the density of cortical granules decreased after conventional straw and solid surface vitrification. Cryotop group compared with other vitrification methods could preserve oocyte ultrastructure properly and create a condition the same as like as the control group

[Effects of lithium chloride on in vitro proliferation rate of rat marrow-derived mesenchymal stem cells]

To evaluate the effects of lithium chloride on MSCs in vitro expansion rate. In this experimental study, bone marrow from 8 rats was plated at 5x105-cells/cm2 in the presence of 1,2,5,7 and 10 mM Lithium chloride and expanded through 3 passages. Twelve days after initiatial culture, the cells of different groups were stained with crystal violet in order to compare the number and diameter of the colonies. Also the cells from different groups were compared in terms of the population doubling [PD] during the 1-3 passages. Different groups of growth curves were plotted for the third passage cells. At the end of cultivation period, the cells were examined wheatear they could differentiate into bone and adipose cells. The Number and diameter of the colonies in primary cultures treated with 5mM lithium chloride were significantly higher than those of control and other groups [P<0.001]. The cell population in the culture with 5 mM lithium chloride was doubled in average 12.02 +/- 0.04 times during 1-3 passages that was significantly higher than other groups. Compared to other groups, the cells from 5 mM group were reached platue in a short time [4.9 days] [P<0.001]. Alizarin red staining for bone and oil red for adipose cells indicated that the cells in different studied groups preserved their differentiation potential. Finally, it seems that the presence of 5 mM litium chloride in the cultures of rat bone marrow cells enhances the MSC in vitro expansion rate while maintaining their bone and adipose differentiation potential

[In vitro cell cycle synchronization of sheep granulosa cells]

To investigate the effect of different methods of synchronization on sheep granulosa cell cycle. Granulosa cells were aspirated from ovarian follicles and plated in a DMEM medium containing 15% FBS. Upon 70-80% confluency, the medium of the primary-cultured as well as the passaged-5 cells were replaced with the medium containing either 0.5% FBS for 24, 48 and 72 h or 0.5 mg mimosine for 24 h. In the last group the cells were cultured in a base medium for further 4 days. In the present investigation, for each culture system, the cells were examined in terms of their cell cycle stage using flow cytometry. Moreover, the cultures were investigated with respect to their apoptotic as well as the proliferating cell contents by using Brdu labeling and TUNEL staining. At primary as well as passaged-5 cultures subjected to serum starvation for 24 h, the frequency of GO/G1, proliferating as well as apoptotic cells were similar to those of control group. At culture with 48 and 72h serum starvation, the percentage of G0/G1 cells tended to increase significantly to 83% and 85% at primary culture and 89% and 90% at passage-5 culture respectively. Moreover, treating the cultures with mimosine caused the G0/G1 cell to increase. The percentages of apoptotic cells in cultures with either serum starvation [for 24 and 48 h] or with mimosine did not increase compared to those of control cultures. According to our results, 72 h after serum starvation, frequency of the apoptotic cells appeared to increase significantly

Investigating the effect of laser assisted hatching on the development and quality of vitrified-warmed 4-celI stage mouse embryos

The aim of this study was to investigate the effect of laser assisted hatching on the development and quality of vitrified-warmed 4-cell stage mouse embryos. The vitrified-warmed 4-cell mouse embryos were divided into two groups: control group [without laser assisted hatching] and experiment group [with laser assisted hatching]. All embryos in both groups were cultured in sequential media containing G1TMver3 and G2TMver3. Afterward, all expanded blastocysts were randomly selected and stained with differential [for cellularity] and TUNEL [for cell death] methods. On day 1[24hrs] of culture, the difference between the control and the experimental groups was insignificant in the rate of blastocyst formation. But on day 2[48hrs] of culture, 87.61% of embryos in the experimental group reached the blastocyst stage. This rate did not increase significantly as compared to the control group [78.14%]. Finally on day 3 [72 hrs], the rate of blastocyst formation reached 94.40% and 81.75%, respectively, in both the experimental and control groups. The difference between these two groups were significant [p<0.05]. The number of blastomeres and apoptotic cells were similar in the experimental and control groups. The laser assisted hatching has no decreasing effect on cellularity, but it has increasing effect on incidence of cell death. In addition, the assisted hatching significantly increases the blastocyst formation rate of intact vitrified-warmed 4-cell stage mouse embryos

[Ultra structure of human uterine and oviduct epithelial cells cultivated under the same ulture condition]

Introduction: Extra cellular matrix [ECM] as an important component of cellular microenvironment has a key role in maintaining the differentiated state of cells. Effects of ECM on morphologic differentiation of epithelial cells including those from uterus and oviduct has been shown in past studies in which cellular and hormonal factors have been used in addition to ECM to maintain epithelial cell differentiation. Not much attention has been paid, in these studies; about the ultra structure of cultured cells specially those from oviduct

Objective: The purpose of present study is to cultivate the human uterine and oviduct epithelial cells under the same microenvironment [ECM Gel and DMEM/Ham's F12 medium] and to observe and compare ultra structural characteristics of the cultured cells by transmission electron microscopy [TEM]

Materials and Methods: For this purpose, uterine and oviduct tissue were obtained from patients undergoing total hysterectomy in Emam Khomeini Hospital. Epithelial cells, after being isolated, were cultured on plastic surfaces and the epithelial nature of the cells was confirmed using immunocytochemistry. Cells with epithelial nature were trypsinized and cultured on ECM gel. At the end ultra structure of cells in parallel with tissue were prepared for TEM

Results: Our results showed that the plastic cultured cells have no signs of differentiation and appeared as elongated spindle cell in sections, whereas those cultured on ECM gel had highly differentiated structure and observed as columnar in shape. In this term they were very similar to epithelial cells from tissue fragment. Epithelial cells of oviduct, cultured on ECM gel, were noticed ultra structurally very similar to that from uterus. The main structural difference existed in vivo state [the presence of abundance cilia on apical surface of oviduct epithelial cells] were not observed in vitro

Conclusion: As a conclusion, it seems that ECM gel by itself is enough to induce morphologic differentiation and structural polarization of epithelial cells. Ultra structurally different cells grows and acquires the same structure when being cultured under the same microenvironment