Application value of frozen-thawed mouse round spermatids in in vitro fertilization / 中华男科学杂志

Objective@#To investigate the application value of the frozen-thawed round spermatids of the mouse in in vitro fertilization (IVF).@*METHODS@#Haploid spermatids of the mouse obtained in vitro were divided into a frozen-thawed and a fresh group and oocytes were collected from 6－8 weeks old female mice. After diamidino-phenyl-indole (DAPI) staining, the oocytes were subjected to intracytoplasmic round spermatid injection (ROSI), 259 in the frozen-thawed and 238 in the fresh group. Comparisons were made between the two groups in the capacities of fertilization and embryonic development.@*RESULTS@#The survival rate of the frozen-thawed haploid spermatids was (75.9 ± 2.3) %. No statistically significant differences were observed between the frozen-thawed and fresh groups after ROSI in the rates of fertilization (51.9 vs 55.7%, P >0.05), 2-cell embryos (51.0 vs 62.2%, P >0.05), 4－8-cell embryos (41.8 vs 42.9%, P >0.05), or morula-blastocysts (12.2 vs 21.4%, P >0.05).@*CONCLUSIONS@#Frozen-thawed round spermatids of the mouse are similar to fresh ones in their capacities of fertilization and embryonic development.

Ultrastructural observation of dormant mouse embryos cultured in vitro after freezing-thawing / 中国实验动物学报

Objective The aim of this study was to investigate the differences of the cell ultrastucture of normal mouse hatched blastocysts and their dormant ones cultured in vitro after freezing-thawing, and to explore whether the dor-mant embryos have a better anti-freezing shock property than the normal hatched mouse embryos .Methods By transmis-sion electron microscopy , the ultrastructure of these two types of mouse embryos was observed and analyzed .Results By comparative analysis of their ultrastructure , the results showed that the dormant embryos before freezing are being austerity and with lower energy metabolism at a ‘ground state ’ .After freezing-thawing and culture , their cellular structure seemed to be similar to that of the normal embryos cultured in vitro before freezing.However, after freezing-thawing and culture, the number of mitochondria decreased , the nuclei were loose , and their heterochromatin also increased .Conclusions From the ultrastructural observation , compared with the normal mouse hatched embryos , the cellular state of dormant mouse em-bryos after freezing-thawing is more favorable for material storage and energy metabolism , thus, indicating that they have a better anti-freezing property than normal hatched embryos .

The Effect of Freezing-thawing Activated Platelet Rich Plasmas (PRP) on the Proliferations of Bacteria / 대한수혈학회지

BACKGROUND: Fresh platelet rich plasma (PRP) gel has been reported to have anti-bacterial properties. We evaluated the anti-bacterial effects of liquid type activated PRP (tAPRP) using thrombin and heparin treatment after a freezing-thawing (F-T) procedure, using a disk diffusion method. METHODS: PRP and platelet poor plasma (PPP) were prepared from CPDA-1 anticoagulated blood received from 20 donors. PRP was concentrated to 8 times the base platelet counts of donors for the first trial and to 11 times the base platelet counts of donors for the second trial. Both F-T PRP and F-T PPP were divided into a nonactivated group and an activated (tA) group, which was then treated with bovine thrombin and CaCl2, and heparin was added to prevent gel formation. The anti-bacterial effects of F-T PRP, F-T PPP, F-T tAPRP with heparin and F-T tAPPP with heparin on S. aureus and P. aeruginosa were evaluated using a disk-diffusion and direct dropping method. All experiments were duplicated. RESULTS: The inhibited diameters resulting for S. aureus and P. aeruginosa, using the disk-diffusion and direct dropping method, were zero for all 20 sets of results for F-T PRP, F-T PPP, F-T tAPRP with heparin and F-T tAPPP with heparin. CONCLUSION: No anti-bacterial effects were detected for S. aureus or P. aeruginosa in the F-T PRP, F-T PPP, F-T tAPRP with heparin and F-T tAPPP with heparin. This negative result may be due to the F-T treatment and/or because liquid instead of gel form of PRP was used. The use of the disk diffusion method for the determination of anti-bacterial ability of PRP may also be a factor in the negative study results.

Light and Electron Microscopic Observation in the Frozen-thawed Mouse Testicular Tissues / 대한불임학회지

OBJECTIVE: The aim of this study was to investigate the morphological aspects of testicular tissue before and after freezing-thawing by light and transmission electron microscopy. METHODS: Tissue biopsies were carried out on mouse testis for freezing. Samples in medium containing 20% glycerol were frozen by computer-controlled freezing program. The effect of freezing-thawing on the structural change of testicular tissues were examined by light and electron microscopy. RESULTS: The freezing-thawing procedure had no significant effect on tubular diameter. However, it caused folding of the lamina propria, and notable damage to Sertoli cells, spermatogonia and spermatocytes. The cells were detached, desquamated from the basal lamina and had increased vacuolization. Round spermatids, elongated spermatids and spermatozoa were less affected, and most of them maintained their normal structure. CONCLUSIONS: The structure of spermatogonia, spermatocyte and basal compartments in seminiferous epithelium was significantly altered by freezing-thawing procedure of mouse testicular tissues. Thus, we need to develop a more reliable method for the cryopreservation of testicular tissues.

Light and Electron Microscopic Observation in the Frozen-thawed Mouse Testicular Tissues / 대한불임학회지

OBJECTIVE: The aim of this study was to investigate the morphological aspects of testicular tissue before and after freezing-thawing by light and transmission electron microscopy. METHODS: Tissue biopsies were carried out on mouse testis for freezing. Samples in medium containing 20% glycerol were frozen by computer-controlled freezing program. The effect of freezing-thawing on the structural change of testicular tissues were examined by light and electron microscopy. RESULTS: The freezing-thawing procedure had no significant effect on tubular diameter. However, it caused folding of the lamina propria, and notable damage to Sertoli cells, spermatogonia and spermatocytes. The cells were detached, desquamated from the basal lamina and had increased vacuolization. Round spermatids, elongated spermatids and spermatozoa were less affected, and most of them maintained their normal structure. CONCLUSIONS: The structure of spermatogonia, spermatocyte and basal compartments in seminiferous epithelium was significantly altered by freezing-thawing procedure of mouse testicular tissues. Thus, we need to develop a more reliable method for the cryopreservation of testicular tissues.

Freezing and Thawing Conditions of Rat Hepatocytes / 대한간학회지

BACKGROUND/AIMS: During cryopreservation of hepatocytes, a dramatic loss in cell number, viability and differentiated cell function is usually inevitable because hepatocytes are very sensitive to stress during freezing and thawing. We tried to investigate the optimal cryopreservation conditions of hepatocytes including the constituents of the freezing medium and freezing rate. METHODS: Isolated hepatocytes were cryopreserved in media containing 10% glycerol or dimethyl sulfoxide (DMSO) of variable concentration. Different freezing procedures (stepwise, rapid, and programmed with or without shock cooling) were used and they were stored in a liquid nitrogen tank. After rapid thawing at 39degrees C, followed by dilution and removal of the cryopreservative, the ability of the hepatocytes to exclude trypan blue dye (TB) was evaluated. Hepatocytes were fractionated through a Nycodenz density gradient centrifugation (DGC) to eliminate dead cells. Cells were plated on dishes coated with type I collagen. RESULTS: Cell viability of hepatocytes recovered from cryopreservation was maintained better using 10, 15, and 20% DMSO as a cryopreservative and programmed cell freezer with shock cooling. After Nycodenz DGC a hepatocyte fraction highly enriched in viable cells could be taken between 11% and 30%. In culture, cryopreserved hepatocytes exhibited a morphology with epithelial characteristics. CONCLUSIONS: These results suggest that rate-adjusted programmed freezing with shock cooling and 10, 15 and 20% DMSO increased the viability of cryopreserved hepatocytes. The hepatocyte fraction highly enriched in viable cells could be taken using Nycodenz DGC. In order to establish a bank of hepatocytes for hepatocyte transplantations and artificial livers a more improved method is nevertheless necessary to increase the viability of hepatocytes after cryopreservation.