Comparative evaluation of five different storage media and temperature effect on human periodontal ligament fibroblast viability

Introduction: Following dental avulsion, the immediate recommended treatment is tooth replantation to avoid adverse effects. Therefore, the tooth must be stored in a physiological storage medium to preserve the viability of the periodontal ligament fibroblast (PDLF) cells during transportation to dental office. The aim of this study was to determine the effect of several storage media in preserving the viability of human PDLF cells at different times and temperatures. Materials & Methods: In this experimental study, the human PDL cells were obtained from the healthy extracted third molars and cultured in Dulbecco's Modified Eagle Medium (DMEM). The studied media were DMEM (10% FBS + 1% penicillin G Na (10000 IU) + 1% streptomycin (10 mg)), tap water, sterilized whole milk, zero fat milk and soy milk. After the cells had reached sufficient density in the plate, they were added to the experimental media and kept at 1, 2, 4 and 24 hours at 4° and 37oCentigrade. After incubation, the cell viability was determined by tetrazolium salt-based colorimetric (MTT) assay. The results were statistically analyzed using Kruskal–Wallis and post hoc Tests. Results: Whole milk and DMEM showed significantly higher protective effect than other media. The viability of PDL cells had significant difference at 4oC compared to 37oC at 4 and 24 h in DMEM group and at 24 h in whole milk group (p≤0.05). Conclusion: The results have suggested that the whole milk like DMEM have enough essential nutrients for PDLFs and have confirmed the hypothesis that the milk similar to HBSS or DMEM might be effective in preserving the PDLF cells

Systemic effects of starved fibroblasts culture supernatant on immunosuppressed rats treated with cancer stem cells

Background: The present study aimed to investigate and compare the effect of starved fibroblast culture supernatant (SFS), DMEM and normal saline alone or along with LA7 on dexamethasone-treated immunosuppressed Wistar rats. Methods: After the isolation of fibroblasts from the fresh foreskin of children, it was cultured in serum-free DMEM, and the supernatant collected after 16 hours (16h-SFS). This solution and the other treatments were injected subcutaneously into the rats from each group once daily for 14 days. The liver, intestine and lung histology along with blood cellular and biochemical characteristics were studied. Results: The results showed that dexamethasone as immunosuppressant reduced the body weight. The histological change in the liver was mild fibrosis induced by LA7+16h-SFS. Also, among the different blood cellular and biochemical indices measured,  the eosinophil percentage in the 16h-SFS treated rats ,  glucose levels in the 16h-SFS+LA7 group and triglyceride concentrations  in the 16h-SFS group were changed (p<0.05). Conclusion: This study showed that the secretions of starved fibroblasts especially that combined with LA7 cancer stem cells could induce some minor histological and biochemical changes in immunosuppressed rats, and also it opened a new window for subsequent investigations on unknown mechanisms related to this work

Anticancer properties of chitosan on osteocarcinoma , breast cancer and cervical cancer cell lines

Background: Cancer refers to the abnormal growth of cells and is still the most common cause of morbidity in world. The purpose of this study was to determine cytotoxicity effect of high molecular weight (HMWC) and low molecular weight of chitosan (LMWC) on three cancerous cell lines MCF-7, HeLa and Saos-2 with different histological origin. Methods: The anticancer property of two types of chitosan on three cancerous cell lines and human fibroblast as normal cell was evaluated by cytotoxic activity and apoptosis induction .The cells were treated by different concentration of chitosan and viability was determined by MTT assay after 24, 48 and 72 h .Mode of death was determined by Annexin V staining assay for apoptosis and analyzed by flow cytometry. Results: While both types of chitosan were more efficient in inhibiting cell proliferation of three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan .Viability of cells was reduced concentration-dependently to 70-90 of the untreated cells as control. There were no significant differences between the effect of both types of chitosan on all cell lines. Flow cytometry analysis showed necrosis more observable with MCF7 while the apoptosis pattern of death was more in Saos-2 and HeLa. Also higher viability with both types of chitosan was seen in fibroblast as normal cells. Conclusion: While chitosan is compatible with normal diploid fibroblast cells, it shows anticancerous effect against 3 cancerous cell lines. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective: To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status. Methods: Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h. MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern. Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%. Results: The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78.8 kDa. These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells. Moreover, we found another seven small size (10–19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts. Most of these proteins expression were down regulated following fibroblast activation. This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death. Conclusions: Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation. Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay

Influence of Bilirubin, Hemin, Zinc Protoporphyrin, Glutathione, Curcumin, and Their Combinations as a Supplement to Support the Viability and Functionality of Pancreas and Liver Cells

Background: Previous experiments have shown different responses of pancreas and liver cells to the culture medium. It has been revealed that the most important step in preserving primary pancreas and liver cells is selecting the appropriate supplements to support the viability and functionality of these cells. Materials and Methods: Cultivation supplements were prepared by adding bilirubin, hemin, zinc protoporphyrin, glutathione, curcumin, and their combination in the pancreas and liver cell culture at a concentration of 1, 3, and 5 μM. Then, the survival rate and function of the pancreas and liver cells were evaluated. The function of pancreas cells was evaluated based on producing insulin and the function of liver cells was based on liver enzymes, including transaminases. Results: We found that bilirubin, hemin, zinc protoporphyrin, curcumin, glutathione, and their combination as supplements can dose-dependently maintain pancreas and liver cells viability and functionality proven by increasing insulin secretion levels and transaminase enzyme activity. The strength of effects is displayed in the following order: bilirubin > combination of all compounds > hemin > zinc protoporphyrin > curcumin > glutathione. Conclusion: This study shows that these compounds are suitable supplements with special biochemical properties. They provide optimal supplements for the culture media of pancreas and liver cells. They may fulfill a function in the antioxidant protection of pancreas and liver cells

Peripheral blood lymphocytes are able to maintain their viability and basic function in normal urine

Background: Similar to inflammatory cells, peripheral blood mononuclear cells (PBMCs) can also infiltrate in to kidney and urinary tracts and subsequently excreted by urine. In this study we determined the viability rate and response to phytohemagglutinin-A (PHA) of human PBMCs in normal urine. Methods: A number of 1&times;106 ficoll-hypaque isolated PBMCs were dispensed in 1 ml normal urine and 6 molar urea and RPMI-1640+FBS10 % were considered as negative and positive control, respectively. After 20, 60 and 120 minutes the viability of these cells was measured by trypan blue dye exclusion assay. 1&times;105 of PBMCs were isolated from urine and cultured as triplicate in RPMI-1640`supplemented with FBS 10% and&nbsp; PHA for 96hr. MTT assay was performed to determine the PBMCs response to PHA. These experiments were repeated three times independently. Results: There was no significant difference between the viability rates of the PBMCs incubated in urine and positive control after 20, 60 and 120 minutes while after 60 minutes they exhibited 75.6% of reactivity to PHA versus positive control. Overall, there was a significant difference in trends of viability rate across the three groups (p<0.05). Conclusion: Our results showed that not only PBMCs remained alive in urine after 120 minutes, but can also respond to PHA up to 60 minutes at a remarkable level. These data open a new avenue in the designation for cell culture-based techniques in urine cell analysis

Time dependent of epigenetic effect of disulfiram on tumor suppressor gene of RASSF1A in Hela cancer cell line

Introduction: Cervical cancer is the third most common tumor among women. Surgery, radiotherapy, and chemotherapy are common treatments, however high stage tumors have frequently poor prognosis. Nowadays, the epigenetic reversion introduced as an efficient strategy of treatment of cervical cancer. In the process, inhibitors of DNA methyltransferase (DNMT) induce re-expression of tumor suppressor genes. Among these inhibitors, disulfiram (DSF) has been suggested as non-nucleoside analogous. In this research, we evaluated the epigenetic effect of DSF on demethylation of the tumor suppressor gene, RASSF1A, in Hela cell line. Materials and methods: Hela cells were cultured and treated with different doses from 2.5 to 37.5µM during 24, 48 and 72 hours. MTT assay was carried out to find half maximal inhibitory concentration (IC50). The methylation specific PCR (MSP) assay was applied to evaluate methylation pattern. Results: The IC50 of DSF was determined at the 2.5, 12.5, and 15µM after72 hours. The MSP results showed partial demethylation at mentioned concentrations after 72h but unmethylated band was not observed after 24h. Conclusion: Our findings indicated that, IC50 of DSF exerted a biphasic effect in Hela cell line and at least 72 hours treatment is needed for the epigenetic reversion of DSF on RASSF1Ain Hela cell line

The efficacy of melatonin against radiotoxicity of iodine-131 and its response to treatment in hyperthyroid patients: a randomized controlled trial

Background: Since melatonin is a non-toxic compound with proven radioprotective effects, we aimed to investigate its efficacy in an in-vivo setting in hyperthyroid patients who are treated with iodine-131. This double-blind placebo-controlled study was conducted on hyperthyroid patients referred to nuclear medicine centers in Babol, Iran. We excluded patients suffering from hypertension treated with warfarin, autoimmune diseases, genetic diseases, cancers, smokers, chemical wounded, radiology and radiotherapy workers, and those who were treated with chemotherapy agents. Patients were randomly assigned to receive a capsule containing 300 mg of melatonin powder or a placebo. Just before receiving iodine-131, blood samples were taken from individuals. All 52 female patients received 10 to 20 mCi iodine-131 for treating hyperthyroidism. A second blood sample was taken one hour after the administration of iodine-131. Material and methods: To determine the chromosomal damages before and after receiving radioiodine, we performed the cytokinesis- block micronucleus assay. Also, at phase 2, 6 months follow-up was performed, in which patients’ positive responses to treatment were compared. Results: The findings of this study indicate that the difference in micronucleus formation between the placebo and melatonin groups is not significant. However, a significant difference in the 6 months follow-up revealed that 61.5% and 85.7% of patients had a positive response to treatment in the placebo and melatonin groups, respectively. Conclusions: As one of the first studies dealing with the human in-vivo assessment on the radioprotective effects of melatonin, it was concluded that melatonin has a non-significant positive impact on reducing the rate of chromosomal damages in hyperthyroid patients treated with iodine-131. Nevertheless, the outcome of treatment was significantly higher by melatonin compared to the placebo group