In situ Raman imaging of osteoblastic mineralization

Hydroxyapatite (HA) is synthesized at early stages of bone formation by osteoblasts. Nondestructive observation of early stages of osteoblastic mineralization provides crucial information for biological mechanism of bone formation. Raman microscopy serves as an ideal tool to observe the osteoblastic mineralization process because it shows the chemical information of the sample at a minimally invasive level. In addition, HA is a marker for osteoblastic mineralization, and HA Raman signal is strong enough to identify mineralized spots in osteoblasts. In this research, we visualized the distribution of HA in cultured mouse osteoblasts by Raman imaging and observed the location of the mineralized spots in the culture. We monitored HA Raman signal from osteoblast culture for 3 days after administrating the osteogenic differentiation medium and observed Raman signal associated with HA. We identified mineralized spots of KUSA‐A1 by Raman imaging constructed from the distribution of HA Raman signal. We successfully visualized the distribution of the mineralized spots in the culture of KUSA‐A1. We compared our Raman images with Alizarin red S staining assay, which was a conventional method to evaluate the mineralization process. Raman imaging of the KUSA‐A1 culture visualized the mineralized spots more accurately than Alizarin red S staining assay. Raman imaging of HA serves as a powerful tool to identify the mineralized spots in an in vitro culture of osteogenic lineage cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Monodispersed β-Glycerophosphate-Decorated Bioactive Glass Nanoparticles Reinforce Osteogenic Differentiation of Adipose Stem Cells and Bone Regeneration In Vivo

Design and development of highly bioactive nanoscale biomaterials with enhanced osteogenic differentiation on adipose stem cells is rather important for bone regeneration and attracting much attention. Herein, monodispersed glycerophosphate-decorated bioactive glass nanoparticles (BGN@GP) are designed and their effect is investigated on the osteogenic differentiation of adipose mesenchymal stem cells (ADMSCs) and in vivo bone regeneration. The surface-modified BGN@GP can be efficiently taken by ADMSCs and shows negligible cytotoxicity. The in vitro results reveal that BGN@GP significantly enhances the alkaline phosphatase activity and calcium biominerialization of ADMSCs either under normal or osteoinductive medium as compared to BGNs. Further studies find that the osteogenic genes and proteins including Runx2 and Bsp in ADMSCs are significantly improved by BGN@GP even under normal culture medium. The in vivo animal experiment confirms that BGN@GP significantly promotes the new bone formation in a rat skull defect model. This study suggests that bioactive small molecule decorating is an efficient strategy to improve the osteogenesis capacity of inorganic ceramics nanomaterials.     

The pertinence of expression of heat shock proteins (HSPs) to the efficacy of cryopreservation in HELAs

HELAs (Hela cells, passed cells of human cervical carcinoma) were heat or cold treated (named heat or cold shock) and then resumed normal culture for 2, 4 or 8 hours respectively. The expressions of heat shock protein 70 (HSP70) and 90 (HSP90) of the HELAs were measured by Northern and Western blotting. HELAs after 4-hour culture were exposed to or cryopreserved with different concentration of dimethyl sulfoxide (Me2SO, 2.5%, 5%, 10%, 15% and 20% respectively, V/V). Meanwhile, the HELAs after different culture time (2, 4 and 6 hours of culture) were cryopreserved with 5% Me2SO. After exposure or cryopreservation, the number of live HELAs was counted and the survival rate was calculated. The results showed that heat shock increased the expression of HSP70 and HSP90 of HELAs, while cold shock decreased the expression of the two proteins. When the concentrations of Me2SO were 10%, 15% and 20%, the survival rates of HELAs after exposure to Me2SO or cryopreservation were much lower than those when the concentrations were small. The survival rates of the heat shocked HELAs were significantly higher than those of the cold shocked and control HELAs. After cryopreservation with 5% Me2SO, the survival rate of heat shocked HELAs group with 2 hours culture time was the lowest among all the groups of HELAs with different cultural time. From the results of this study, we conclude that the expressions of HSP70 and HSP90 in HELAs increased significantly after heat shock, while cold shock decreased the expressions of these two proteins. The over-expressions of HSPs in the heat shocked HELAs could protect the cells from both injury caused by potential toxicity of high concentrations of Me2SO and cryoinjury caused by the freeze-thawing/cryopreservation procedure.     

Cryopreservation of goldfish caudal fin explants using glycerol as a cryoprotecant

The objective of this study was to investigate the effect of glycerol on the cryopreservation fin explants of goldfish, Carassius auratus. Four different concentrations, 5, 10, 15, and 20% (v/v) of glycerol and a control were tested. These were prepared in Dulbecco's modified Eagle's medium with 20% (v/v) Fetal Bovine Serum. Attachment and outgrowing rates were monitored from day 3 to day 14. Results showed that fin explants cryopreserved in 20% concentration of glycerol was significantly higher (P < 0.05) with a 100% attachment rate compared to 5, 10, and 15% concentrations with 36.67, 84.19 and 86.51% attachment rate, respectively. Fin explants cryopreserved in 20% glycerol concentration also had significantly higher (P < 0.05) outgrowth of cells (73%) than the other three concentrations on day 3. Moreover, a 100% outgrowth of cells in all concentrations was achieved after 14 days of culture. No attachment and out growth of cells were observed in control group. Goldfish caudal fin explants cryopreserved in glycerol can produce live cells efficiently, regardless of concentration.     

Intracellular sugars improve survival of human red blood cells cryopreserved at -80 degrees C in the presence of polyvinyl pyrrolidone and human serum albumin

Cryopreservation with impermeable protectants has great significance on storage of human red blood cells. It has become feasible to use glycerol free cryopreservation for human red blood cells. This study focuses on the effect of intracellular trehalose or glucose on human red blood cells cryopreserved in the presence of polymer. Red blood cells were cryopreserved for 48 h-72 h at -80 degrees C. The data showed that the loading efficiency of glucose was significantly higher than that of trehalose, but trehalose loading process induced more hemolysis than glucose loading process. Compared with the other groups, the combination of intracellular glucose, PVP, and human serum albumin can significantly decrease the percent hemolysis after cryopreservation (P<0.01). However, the percent hemolysis induced by intracellular trehalose was less than that induced by extracellular trehalose, but the difference was not significant (P<0.05). The adenosine 5'-triphosphate (ATP) level and 2,3-diphosphoglycerate (2,3-DPG) level of cryopreserved red blood cells were significantly less than those of fresh red blood cells. However, sugars can provide certain protection for ATP and 2, 3-DPG compared with red blood cells cryopreserved in the absence of sugars. The protection of glucose on the metabolic function was more than that of trehalose. Cryopreservation can increase the percentage of cells with exposed phosphatidylserine (PS), but the ability of trehalose to maintain PS normal distribution is higher than that of glucose. Furthermore, intracellular sugars can protect membrane integrity of cryopreserved red blood cells, although a small portion of cells appeared spherocytic or echinocytic shape. Finally, most membrane proteins of cryopreserved red blood cells were similar to the membrane proteins of fresh red blood cells, but trehalose can result in loss of glyceraldehyde phosphate dehydrogenase (GAPD) and peroxiredoxin 2. In conclusion, it is feasible to cryopreserve red blood cells using polymer, human albumin and sugars as main protectants. The cryoprotective effect of glucose may be better than that of trehalose in the presence of PVP and human serum albumin, because sugar loading process causes more cell injuries in case of trehalose compared to glucose, and these injuries in turn manifest themselves during subsequent cryopreservation and thawing. In the future, finding an approach to decrease the injuries during trehalose loading process still is critical.     

Importance of a three-stage cooling regime and induced ice nucleation during cryopreservation on colony-forming potential and differentiation in mesenchymal stem progenitor cells from human fetal liver

Colony-forming activity, as well as osteogenic and adipogenic capacities of primary human fetal liver (HFL) mesenchymal stem or progenitor cells (MSCs) were compared before and after cryopreservation using a standard three-step cooling protocol (Cryo3-S) or the same protocol with induced ice nucleation (Cryo3-IIN) and 5% and 10% w/v dimethyl sulphoxide (Me?SO). Cell viability, using the Cryo3-S protocol with 5 % and 10 % Me?SO, was about 60 to 70 % as assessed by the trypan blue staining method, but the ability to undergo growth in culture and form colonies was completely lost. Cryopreservation using Cryo3-IIN resulted in conservation of colony-forming MSCs. Colony-forming efficiency (CFE) of the cell samples cryopreserved with Cryo3-IIN and 5 % Me?SO was on average 0.4 +/- 0.1 colonies per 10? cells, whereas with 10% Me?SO 1.6 +/- 0.7 colonies were obtained. HFL MSCs recovered after cryopreservation in the both groups demonstrated capacity to be expanded and induced into either osteogenic or adipogenic differentiation.     

The biological characteristics of different generation mesenchymal stem cells cultured in vitro and cocultured with vascular scaffold

Mesenchymal stem cells (MSCs) were used widely as seed cells in tissue engineering blood vessel construction. However, the biological characteristics difference of different generation MSCs in vitro culture is unknown, which laid a foundation for appropriate generation seeded cells selection for tissue engineering blood vessel construction. In this report, MSCs were isolated from SD rat bone marrow and identified by flow cytometry; cell growth curve test, cell surface antigen expression rate detection, cryopreservation resuscitation rate test, CD31 expression rate test, cell cycle analysis, and adhesion difference on vascular scaffold test were performed. The research results indicated that the MSCs shape was spindle and uniform with vigorous growth. CD105 and CD90 factor expression rate reached 82.5 and 84.9%, respectively, and the expression rate of CD45 was only 7.3%. The proliferation capacity of the fourth generation MSCs were more exuberant, with proliferation index as 20.3%; the cell proliferation index of the eighth generation decreased to only 9.1%. The cryopreservation resuscitation rate of the second generation and fourth generation MSCs were both higher than 80%, and the cryopreservation resuscitation rate of the eighth generation MSCs was only about 60%. After the induction for 5 days, MSCs had weak CD31 expression, and with the prolonged induction time, expression increased. All generation MSCs expressed CD31 after being induced for 10 days; however, the CD31 positive expression rate of the second generation, fourth generation, and sixth generation MSCs had significant difference with the eighth generation MSCs. Adhesion rate of MSCs before sixth generation was around 40%, but the adhesion rate of eighth generation MSCs was only about 27%. In all, biological characteristics of different generation MSCs existed certain differences, and especially the eighth generation MSCs aged seriously, whose cell activity decreased significantly. The researchers believed that the MSCs before the sixth generation can maintain excellent properties of MSCs, and can be used as seed cells for vascular tissue engineering.     

Comparison of Telomere Length of Vocal Folds With Different Tissues: A Physiological Measurement of Vocal Senescence

The objective of this article is to determine telomere length, a measure of biological age, in true vocal fold (TVF), false vocal fold (FVF), and five other tissue types, to ascertain whether there is tissue-specific telomere shortening. The study design is that of a prospective, basic science study. Tissue samples were obtained from the TVF, FVF, skin from the back of hand, skin from thigh, aorta, blood, and bone marrow from 12 patients ages 54 to 76 years. Genomic DNA was isolated from each sample, and telomere lengths were calculated with real-time polymerase chain reaction. In our small age group, age was not significantly associated with telomere length across tissue types, nor were there any linear correlations within tissue types and age. Controlling for age, significant differences were found between the following tissues: aorta and blood (P < 0.000), aorta and bone marrow (P = 0.033), aorta and FVF (P = 0.015), aorta and hand skin (P = 0.004), blood and thigh skin (P = 0.012), and blood and TVF (P = 0.048). A significant linear correlation between telomere length and tissue type without considering donor age was established between bone marrow and hand skin (P < 0.05, R2 = 0.766), thigh skin and hand skin (P < 0.01, R2 = 0.926), TVF and blood (P < 0.01, R2 = 0.836), and thigh skin and TVF (P < 0.05, R2 = 0.624). Our findings indicate that surrogate tissue for measurement of telomere length of TVF includes FVF, bone marrow, skin, and aorta. These findings have implications for understanding vocal fold aging at the cellular level.     

Alendronate decorated nano hydroxyapatite in mesoporous silica: Cytotoxicity and osteogenic properties

Mesoporous silica is a promising drug delivery vehicle due to its large surface area and order porous structure. Hydroxyapatite-modified mesoporous silica materials (MSH) have been developed, and the cytotoxicity of MSH and unmodified mesoporous silica (HMS) has also been studied in this work. The results indicated that MSH exhibited lower cytotoxicity than HMS. The drug release property of MSH was also investigated in this paper. Alendronate (AL) was laden into MSH and HMS, respectively. MSH exhibited long release period lasting over 30 days with a weak burst release in the first 5 days; however, the AL release period of HMS was just 5 days with a remarkable burst release. In addition, the osteogenic commitment induced in human marrow mesenchymal stem cells (MSCs) by MSH-alendronate (MSH-AL) was also investigated, and the osteogenesis of MSCs was evaluated by alkaline phosphatase (ALP) assay. The osteogenesis of MSCs induced by MSH-AL is comparable to that induced by the osteogenic medium. Taken together, MSH can be severed as potential bone repair materials with lower cytotoxicity.     

Monodispersed Bioactive Glass Nanoparticles Enhance the Osteogenic Differentiation of Adipose‐Derived Stem Cells through Activating TGF‐Beta/Smad3 Signaling Pathway

It is important to understand the interaction mechanisms between nanomaterials and adipose‐derived stem cells for biomedical application. Nanoscale bioactive glass has positive effects on guiding osteoblasts differentiation and bone regeneration. However, the effects and molecular mechanism of monodispersed bioactive glass nanoparticles on the osteogenic differentiation of adipose‐derived stem cells are still not clear up to now. In this study, the effects and underlying molecular mechanism of monodispersed bioactive glass nanoparticles on the osteogenic differentiation of adipose‐derived stem cells are investigated in minute detail. The results show that nanoparticles (100–200 nm) can be absorbed by stem cells and is distributed in cytoplasm and nucleus. In both culture conditions (normal and osteoinductive), nanoparticles (80 µg mL⁻¹) can significantly enhance the osteogenic differentiation of stem cells through upregulating the alkaline phosphatase activity, osteogenic genes and protein expressions, as well as calcium deposition. Further study suggests that the activation of transforming growth factor‐beta/Smad3 signaling pathway plays an important role in the osteogenic differentiation of adipose‐derived stem cells enhanced by monodispersed nanoparticles. This study may have important implications for better understanding of stem cells fate induced by monodispersed nanoparticles and provide a promising approach toward stem cells‐based bone regeneration.     

Facile Synthesis of Mg2+‐Doped Carbon Dots as Novel Biomaterial Inducing Cell Osteoblastic Differentiation

Carbon dots (CDs), as an emerging fluorescent nanomaterial with low toxicity, has been widely applied in various bio‐related fields. However, investigations on their capabilities in guiding osteogenic differentiation are rarely seen, which has great significance in osteoporosis therapy and bone regeneration. Herein, for the first time, a new kind of Mg²⁺‐doped CDs is facilely synthesized through a one‐step hydrothermal method from metal gluconate salts. The CDs can serve as nanocarrier of Mg²⁺ ions entering into cells, and the bioessential metal ions subsequently stimulate osteoblastic differentiation by improving alkaline phosphatase (ALP) activity and upregulation related mRNA expression. Noteworthy, the raw material has almost negligible performance on osteoblastic differentiation compared to Mg‐CDs, which is due to the ultrasmall sizes of CDs and the efficient uptake by cells. Moreover, benefitting from the fluorescence properties, Mg‐CDs can also be applied as cell labeling agents. This work proposes a new strategy to synthesize multifunctional metal ion‐doped CDs, which might had great potential in serving as promising nanodrugs for bone loss therapy.     

Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30 mW/cm² is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30 mW/cm² are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30 mW/cm² and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30 mW/cm² but also 2 and 15 mW/cm², modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30 mW/cm², alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2 mW/cm². As the intensity increased to 15 and 30 mW/cm², the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30 mW/cm² continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.     

Cryopreservation of human skeletal muscle impairs mitochondrial function

Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity of oxidative phosphorylation was significantly (P < 0.05) reduced in cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P < 0.05). Addition of cytochrome c revealed an increase in respiration indicating cytochrome c loss from the mitochondria. The results from this study demonstrate that normal mitochondrial functionality is not maintained in cryopreserved human skeletal muscle samples.     

Developing cryopreservation for Picea sitchensis (sitka spruce) somatic embryos: a comparison of vitrification protocols

Two vitrification-based cryopreservation protocols, encapsulation/dehydration and PVS2 were applied to Stage 2 (globular) and Stage 4 (torpedo) somatic embryos (SE) from Picea sitchensis. Two recovery responses: partially differentiated embryogenic suspensor masses (ESM) and dedifferentiated non-embryogenic masses (NEM) were observed following exposure to LN. All genotypes tested, proliferated NEM, approximately 10 to 100% of the total SE cryopreserved. A General Linear Model applied to NEM recovery data demonstrated several different factors (developmental state and genotype, treatment, culture age) interacted at a significant level (P less than 0.05) to influence proliferation. One genotype was capable of proliferating ESM after cryopreservation using encapsulation-dehydration, this response was achieved for Stage 4 embryos derived from the youngest ESM tissue.     

Reduced osteoblastic population and defective mineralization in osteopetrotic (op/op) mice

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization.     

Studies on cryopreservation of luc gene transfected bluegill sunfish fibroblast cell line

The effect of cryopreservation on the survival of luc gene transfected bluegill sunfish fibroblast (BF-2) cells was investigated. Propane-1,2-diol was found to be the least toxic cryoprotectant when compared with DMSO and methanol. Both propane-1,2-diol and DMSO are effective in protecting cells from freezing damage. Whilst there were no significant differences in cell survival between cryoprotectant concentration (10 or 15%) and culture age used in this study, 7-day old culture appeared to be more resistant to freezing without cryoprotectant when compared with 3- and 14-day old culture. The highest cell survival values obtained with propane-1,2-diol (10%) and DMSO (10%) protection were 96.2 1.2% and 94.0 3.1% respectively. Initial subsequent cell growth after cryopreservation was slower than their non-frozen controls. The survival of transfected BF-2 cells (BF-2/luc1) after cryopreservation were very similar to those obtained with wild type cells being: 94.0 3.1% and 95.2 1.5% respectively with 10% DMSO protection. These results suggested that genetically modified fish cell lines may be equally amenable to cryopreservation as the wild type.     

Monodisperse Branched Molybdenum‐Based Bioactive Nanoparticles Significantly Promote Osteogenic Differentiation of Adipose‐Derived Stem Cells

Adipose‐derived stem cells (ADSCs) are considered to be ideal stem cell sources for bone‐tissue regeneration owing to their ease of collection and high activity. However, the regulation of osteogenic differentiation of ADSCs using biomaterials without adding growth factors is still not satisfactory. For the first time, molybdenum‐doped bioactive glass nanoparticles with a radial porous morphology (Mo‐rBGNs) are reported and their role in the osteogenic differentiation of ADSCs is investigated. The results show that Mo‐rBGNs exhibit radially porous and spherical morphology, relatively homogeneous particle size (200–400 nm), and excellent apatite‐forming bioactivity. They do not affect the proliferation of ADSCs, but significantly regulate their osteogenic differentiation and biomineralization. 5% Mo‐rBGNs significantly enhance the alkaline phosphatase activity and biomineralization ability and promote the osteogenic gene expressions of collagen I secretion and bone sialo protein in ADSCs. A reasonable and promising strategy for designing nanoscale bioactive materials with the excellent osteogenic ability for stem cell–based bone tissue regeneration is provided.     

Improvement of cryopreservation results in garlic using low temperature preculture and high-quality in vitro plantlets

The efficiency of garlic cryopreservation is, amongst other factors, depending on the origin of the donor explants. So far, in vitro grown material has always been the least responding one with respect of the regrowth rates. On the other side, the possibility to produce virus-free material via meristem culture and to keep these clones then under isolated conditions in a clean culture induced studies to increase the efficiency of cryopreservation using this kind of material. Experiments have been performed to use various materials and cultivation temperatures for a vitrification protocol. Best results (up to 70 percent regrowth) were obtained with cultures grown for only 10 months under in vitro conditions including a cold preculture of two months either at alternating or at permanently low temperatures. The conclusion was drawn that the quality of the explants and temperature conditions play a major role for the efficiency of cryopreservation using in vitro plantlets.     

Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments

Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution.