The effects of topical sodium-hyaluronan on the stromal and endothelial healing were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were produced in one eye of each rabbit by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the central cornea for 60 seconds. Then the eyes were treated topically with either 1% Na-HA(the treatment group)or a phosphate buffered saline(PBS)(the con-trol group)4 times per day for 3 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The stromal healing was assessed by counting polymorphonuclear leukocytes(PMNs)and keratocytes in the central and marginal wounds areas. The stroma treated with Na-HA had less PMNs than that of the control group during the early healing period. The defect area of the endothelium was significantly smaller in the Na-HA treated group than in the control corneas. The present findings indicate that topically applied 1%Na-HA affects stromal and endothelial healing during the early repair process after corneal alkali wounds.
Alkalies* ; Cornea ; Endothelium ; Trypan Blue ; Wounds and Injuries*

PURPOSE: To recognize the anatomical positions of the superior oblique muscle in enucleated eyes using trypan blue. METHODS: Twenty-two surgically-enucleated eyes of 11 bodies were studied. The shortest distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon, the distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion, and the greatest width of superior oblique tendon insertion were measured by caliper 3 consecutive times. The average values in each of the above 3 points were calculated, and values prior to and after trypan blue staining were compared. RESULTS: Prior to staining with trypan blue, the average distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon was 4.97 mm and the average distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion was 7.57 mm; after staining with trypan blue, the average values were 5.09 mm and 7.65 mm, respectively. There was no statistically meaningful difference in values prior to and after staining (p > 0.05). Prior to staining, the average value of the greatest width of the superior oblique tendon was 10.32 mm, and after staining with trypan blue, the average value increased to 10.76 mm. There was a statistically meaningful difference between the values (p = 0.02). CONCLUSIONS: Trypan blue staining helped to recognize the location and the width of the superior oblique tendon more precisely.
Diminazene ; Eye ; Muscles ; Tendons ; Trypan Blue

BACKGROUND: The viability of cord blood is an important measure of product quality. Trypan blue (TB) stain is the most commonly and conveniently used method to measure the viability of the cord blood. Recently, cytometric analysis using 7-Aminoactinomycin D (7-AAD) was introduced. Staining with 7-AAD is more sensitive in detecting cellular damage than staining with TB. In addition to this, 7-AAD allows specific measurement of the viability of total nucleated cells (TNC), mononuclear cells (MNC) and CD34+ cells. In this study, we compared the viability of TNC between the TB and 7-AAD method, as well as analyzing the viability of each cell population. METHODS: From February to July 2010, 102 cord blood units were collected and assessed for the viability of TNC by the TB and 7-AAD methods. The viability of mononuclear cells (MNC) and CD34+ cells was assessed by 7-AAD method. RESULTS: The TB and 7-AAD methods were used to assess the viability of TNC, which was 90.1+/-5.7% and 68.4+/-8.0%, respectively. The viability of MNC and CD34+ cells measured by the 7-AAD method was 91.8+/-4.3% and 93.4+/-5.1%, respectively. CONCLUSION: The TNC viability of 7-AAD method was significantly lower than that of TB method. In 7-AAD method, the viabilities of MNC and CD34+ cells were significantly higher than that of TNC. As those are important prognostic factors and measures for successful engraftment after the transplantation, the measurement of the viabilities of MNC and CD34+ cells by 7-AAD method would be helpful to the quality control of the cord blood product.
Cell Survival ; Cryopreservation ; Dactinomycin ; Diminazene ; Fetal Blood ; Quality Control ; Transplants ; Trypan Blue ; Umbilical Cord

BACKGROUND: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. METHODS: Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining. RESULTS: Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8+/-4.5%, 78.4+/-7.8%, and 65.5+/-8.1%; mononuclear cells, 94.4+/-1.7%, 90.8+/-4.2%, and 84.2+/-6.7%; and CD34-positive cells, 92.4+/-3.0%, 90.7+/-4.7%, and 89.3+/-7.0%. The viability using trypan blue was more than 90% until 48 hr after collection. CONCLUSIONS: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study's data will provide useful information for the assessing the quality of cord blood products.
Annexin A5 ; Apoptosis ; Cell Survival* ; Female ; Fetal Blood* ; Humans ; Methods ; Pregnant Women ; Trypan Blue ; Umbilical Cord*

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 x 10(5) cells/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR: 64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (3.0 x 10(4) cells/ml) or 20% KSR (4.8 x 10(4) cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.
Animals ; Cell Proliferation ; Connective Tissue ; Diminazene ; Extracellular Matrix ; Fibroblasts ; Humans ; Trypan Blue

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.
Apoptosis ; Caspases* ; Catalytic Domain ; Cell Death* ; Cisplatin ; HEK293 Cells ; HeLa Cells ; Humans* ; Ribonucleoproteins ; Telomerase* ; Trypan Blue

PURPOSE: To evaluate the effect of mitomycin-C (MMC) on osteotomy site as an adjunctive therapy for dacryocystorhinostomy, the effect of MMC on cultured human osteoblasts was tested. METHODS: Cultured osteoblasts which was obtained from the human iliac crest, were treated with four different concentrations of MMC (0 mg/ml, 0.2 mg/ml, 0.02 mg/ml, 0.002 mg/ml) and cultured for 24hours. To observe the effect of exposed time dependency, cells were treated with MMC during 5, 30minutes, and 24hours and washed and changed with fresh osteogenic media, and then cultured for 24 hours. The effect of fibroblast growth factor (FGF) and transforming growth factor-beta(TGF-beta) on the MMC-treated cells was evaluated. Cell viability was measured using trypan blue staining method and MTT assay. RESULTS: As compared with control group, the lowest growth rate of osteoblasts was 6.8% in 0.2 mg/ml MMC-treated cells. There were no significant differences in the growth rate between 5 minutes and 30 minutes MMC treatment groups, but in case of 24 hours treatment group with MMC (0.2 mg/ml) the growth rate was suppressed to 77.5% of control group with statistical significance. Both growth factors had promotive effect on the growth of in 0.02 mg/ml MMC-treated osteoblasts, but not in 0.2 mg/ml MMC-treated cells. CONCLUSIONS: Osteoblasts which were treated for longer time and with higher concentration of MMC showed more suppression in growth rate. These results suggest that intraoperative application of MMC during dacryocystirhinostomy could have a positive effect of mucosal ostium with suppression of osteoblasts proliferation.
Cell Survival ; Dacryocystorhinostomy ; Fibroblast Growth Factors ; Humans* ; Intercellular Signaling Peptides and Proteins ; Mitomycin* ; Osteoblasts* ; Osteotomy ; Trypan Blue

PURPOSE: To evaluate the effects of trypan blue (TB) on the survival of cultured human trabecular meshwork cells (HTMCs). METHODS: Primarily cultured HTMCs were exposed to 0.05, 0.10 or 0.50% TB for 1, 5 or 30 min. Cellular survival was assessed using the MTT assay and degree of apoptosis was analyzed with flow cytometry using annexin-V/propidium iodide double staining. RESULTS: Long-term exposure or high concentration of TB decreased the survival of HTMCs (p < 0.05). In flow cytometric analysis, exposure to 0.50% TB for 30 min increased the degree of apoptosis (p < 0.05). Commercial TB decreased cell survival after exposure for 5 min and increased the degree of apoptosis after exposure for 30 min (p < 0.05). CONCLUSIONS: TB may cause cellular damage of cultured HTMCs and apoptosis could be the underlying mechanism. In TB-assisted cataract surgery, TB should be used for the shortest time possible and removed completely.
Apoptosis ; Cataract ; Cell Survival ; Flow Cytometry ; Humans ; Trabecular Meshwork* ; Trypan Blue*

BACKGROUND: Adipose tissue damage of cryopreserved fat after autologous fat transfer is inevitable in several processes of re-transplantation. This study aims to compare and analyze the survivability of adipocytes after thawing fat cryopreserved at -20degrees C by using thawing methods used in clinics. METHODS: The survival rates of adipocytes in the following thawing groups were measured: natural thawing at 25degrees C for 15 minutes; natural thawing at 25degrees C for 5 minutes, followed by rapid thawing at 37degrees C in a water bath for 5 minutes; and rapid thawing at 37degrees C for 10 minutes in a water bath. The survival rates of adipocytes were assessed by measuring the volume of the fat layer in the top layers separated after centrifugation, counting the number of live adipocytes after staining with trypan blue, and measuring the activity of mitochondria in the adipocytes. RESULTS: In the group with rapid thawing for 10 minutes in a water bath, it was observed that the cell count of live adipocytes and the activity of the adipocyte mitochondria were significantly higher than in the other two groups (P<0.05). The volume of the fat layer separated by centrifugation was also measured to be higher, which was, however, not statistically significant. CONCLUSIONS: It was shown that the survival rate of adipocytes was higher when the frozen fat tissue was thawed rapidly at 37degrees C. It can thus be concluded that if fats thawed with this method are re-transplanted, the survival rate of cryopreserved fats in transplantation will be improved, and thus, the effect of autologous fat transfer will increase.
Adipocytes ; Adipose Tissue ; Autografts ; Baths ; Cell Count ; Centrifugation ; Cryopreservation ; Fats ; Mitochondria ; Survival Rate ; Trypan Blue ; Water

The effects of topical dexamethasone on the endothelial healing and the change of aqueous composition were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were induced, then the eyes were treated topically with either 0.1%dexamethasone or abalanced salt solution[BSS]4 times per day for 8 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The concentrations of ascorbic acid, glucose, and the ions, Na, K , Ca2 and Mg2 , were measured in the aqueous humor. Endothelial healing in control corneas showed a biphasic pattern of healing:an initial short-term healing for the first week and then a late long-term healing following a secondary endothelial breakdown. Topical administration of 0.1%dexamethasone deterred endothelial healing during the early period and prevented secondary endothelial breakdown. Total repair process of endothelium was accelerated by the dexamethasone treatment. Among the various components of the aqueous humor examined, ascorbic acid seemed most sensitive to change caused by the alkali injury and dexametha-sone treatment. The present data indicate that dexamethasone may have a therapeutic potential in the management of endothelial healing after corneal alkali injury.
Administration, Topical ; Alkalies* ; Aqueous Humor ; Ascorbic Acid ; Cornea ; Dexamethasone ; Endothelium ; Glucose ; Ions ; Trypan Blue ; Wounds and Injuries*