Pathobiology Research
Year:2013 | Volume:16 | Issue:3|Papers Count:8

The provision of an adequate quantity of cells with proper function and purity is one of the main challenges of tissue engineering studies. Stem cells, with their self-renewal and differentiation capacity, are considered one of the main cell sources in the field of tissue engineering. Previously, the use of chemical factors seemed to be the only possible way for stem cell differentiation. However, scientists have recently realized that physiological processes of the human body are composed of chemical, mechanical and electrical signals. Mechanical stimulation is one of the current methods that produce cells with proper morphology and alignment in the scaffold. Specific differentiation, a higher rate of cell growth, proliferation and differentiation, and lower experiment costs can be achieved using mechanical stimulation. Different parameters such as the chemical environment, physical environment that surrounds the cell (including geometry, stiffness and topology of scaffold surface), amplitude, frequency, and duration of mechanical stimulation can affect the stem cell fate. In this study we have investigated the impact of all types of mechanical stimulations under different loading regimes on the fate of stem cells with respect to the target tissue. The result has been reflected in the design of a proper bioreactor.

More than the half of cancer patients undergo cancer treatments of chemotherapy and/or radiotherapy. Unfortunately treatment with invasive methods occasionally lead to severe side effects. Patients who undergo chemotherapy can be affected by premature ovarian failure, an important cause of infertility. Ovarian tissue cryopreservation is suggested as the only way for preservation of sex cells and fertility preservation in cases of prepubertal girls and women with sterility attributed to chemotherapy, radiotherapy, genetic disorders or specific diseases.

Objective: Growth differentiation factor 9B (GDF9B) is an oocyte-derived growth factor. This protein is essential for the development of ovarian follicles and acts mainly by binding to its receptor on the surface of granulosa cells. The effect of GDF9B on the growth of follicles in various developmental stages, particularly primordial and primary follicles, is unknown. Thus the aim of this study is to investigate these effects after mouse whole ovarian culture.Methods: Female NMRI mice (14 day-old) were sacrificed by cervical dislocation Subsequently their collected ovaries were cultured in α-MEM basic medium (control group) and medium supplemented with different doses of recombinant GDF9B (10, 20, 40 ng/ml) for seven days in 5% CO2 and 37oC. At the end of the culture period, serial sections of ovaries were prepared and stained with hematoxylin and eosin. The follicles were counted in the primordial, primary, preantral and antral stages and compared among the different groups.Results: In GDF9B supplemented groups the percentage of antral follicles significantly increased whereas the percentage of preantral follicle decreased when compared with the control group. However there were no significant differences between the percentage of primordial and primary follicles in all supplemented GDF9B (10, 20, 40 ng/ml) groups and the control.Conclusion: Overall, this study showed that GDF9B stimulated the growth of preantral follicles to the antral stage. However this factor did not have a remarkable effect on the growth of primordial and primary follicles.

Objective: Fusarium species are prevalent contaminants of foodstuffs and agricultural crops. They produce fumonisins, which are carcinogenic mycotoxins. The present study has evaluated maize and wheat samples from ten provinces in Iran that were contaminated with Fusarium species. Special attention was paid to the ability of the isolates to produce fumonisin B1 (FB1) as a public health hazard.Methods: We collected 32 maize and 15 wheat samples from ten provinces that were major cultivation areas. Samples surface disinfected with a 1% sodium hypochlorite solution for 2 minutes. Fusarium species were isolated by the flotation method on malachite green agar. Pure cultures on potato dextrose agar (PDA) were identified using a combination of macroscopic and microscopic morphological criteria. The ability of the isolates to produce FB1 was evaluated by thin layer chromatography (TLC) and the amounts of fumonisin B1 produced were assessed by high performance liquid chromatography (HPLC).Results: A total of 55 Fusarium isolates that belonged to five species were isolated. There were 27 of the 32 maize samples (84.4%) and 11 of 15 wheat samples (73.3%) that were contaminated with Fusarium species. Species consisted of F. verticillioides (23 isolates), F. proliferatum (22 isolates), F. subglutinans (5 isolates), F. nygamai (4 isolates) and F. redolens (1 isolate) based on morphological criteria. Twenty-two of the 55 (40%) Fusarium isolates produced FB1 in a total range from 230.4 to 9565 mg/ml. The highest amounts of FB1 production were related to toxigenic isolates of F. verticillioides and F. proliferatum.Conclusion: Results of the present work indicates a high degree of contamination of maize and wheat with Fusarium strains that belong to the Gibberella fujikuroi species complex و particularly F. verticillioides and F. proliferatum. This contamination is a potential public health threat due to food spoilage and subsequent production of high levels of carcinogenic FB1.

Objective: Despite availability of an effective vaccine against hepatitis B virus (HBV), the global prevalence of this virus infection has not diminished significantly. Contrary to numerous other human viruses, HBV does not have the ability to propagate in cell culture. However, infectious virus has been produced by transfection of human hepatoma cells with plasmids that contain full length HBV genome. Generation and optimization of appropriate cell culture systems can help us in demonstrating the quality of genome replication by PCR as well as expression of surface antigen secretion. Interferon stimulating genes (ISGs) are usually produced in response to interferon and can be determined as a measure of response to IFN-therapy. Therefore, in pharmacological studies, in addition to assessing the effects of a medicine on viral determinants of replication, its’ effects on stimulation of various ISGs, as indicators of innate immune responses, can be achieved.Methods: In this study, we transfected the Huh-7 hepatoma cell line with pCH-9/3091. HBsAg production and viral mRNA transcription were subsequently evaluated. In this system, by using ISGs-specific primers, the ISG mRNAs recognition method was optimized and utilized.Results: Huh-7 cells supported HBV replication. The peak HBsAg secretion was observed at 72 h post-transfection. By using designed primers for the S and pg/pC regions, transcription and genome replication of the virus was shown. RT-PCR results for ISG production by transfected cells showed no role for HBV in enhancement of ISGs levels in Huh-7 cells.Conclusion: The results indicated that this system can be used for functional studies of HBV-specific genes as well as assessment of the effects of new drugs or new vaccines. In addition, it may be used to study the mechanisms of drug resistance that have resulted in difficulties in response to HBV antivirals, including IFN-a.

Objective: Human retinal pigment epithelium (hRPE) is a cell monolayer located in the outer part of the retina that is in contact with photoreceptors. In many diseases RPE cells damage. One way for treating this disease is the implantation of intact instead of damaged cells. For this reason different types of substrates have been used for cell cultivation. This study has used alginate and a blend of alginate/gelatin (A/G) to study RPE cell growth.Methods: We prepared alginate solutions in concentrations of 1% and 2% (w/v) in water and DMEM/F12. The solutions were infused into each well of 6-well micro plates until a uniform culture substrate that had a 1 mm thickness was generated. Passage-4 hRPE cells were cultivated on the substrate and the cell characteristics studied. hRPE cells did not adhere to alginate in DMEM/F12 and did not exhibit interaction with alginate substrate. For this reason A/G solutions at concentrations of 1% and 2% (w/v) in water were prepared. We prepared A/G blends at weight ratios of: 20: 80, 30: 70, 40: 60, 50: 50, 60: 40, 70: 30, and 80: 20. These blends were infused into each well of 6-well plates until the appropriate 1 mm thickness of A/G was achieved. Isolated hRPE cells were cultured on synthetic substrate after which we studied the cells' characteristics.Result: hRPE cell generated adhesive colonies on the A/G substrate. In all studied combinations of A/G, the diffused hRPE cells formed a monolayer under the substrate sheets. However the A/G 20: 80 ratio had cell growth in the upper face of the substrate. hRPE survived indefinitely on A/G substrate. After the cells were re-cultured on polystyrene, they showed general morphological features of normal hRPE cells.Conclusion: The A/G blend at a 20: 80 ratio was chosen to be used for future studies.

Objectives: This study investigated the possible synergistic effect of simultaneous treatment of bone morphogenic protein (BMP) -4 as a chemical stimulator and static magnetic field (SMF) as a physical stimulator on viability percent and proliferation rate in rat bone marrow stem cells.Methods: Passage 5 cells were trypsinized, and a cell suspension prepared after which the cells were counted and cultured in 25 cm2 flasks. Cells were incubated for one day and washed with phosphate-buffered saline. We added BMP-4 at the optimum concentration of 25 ng/ml at different times (24, 48 and 96 h) into the medium. The cells were exposed at an optimum intensity of 4 mT of the SMF at different exposure times (24, 48, and 96 h). Subsequently cells were washed with phosphate-buffered saline, trypsinized, and separate cell suspensions were prepared from each flask. We investigated the viability and proliferation rates of treated cells by staining them with Trypan blue and performed cell counts with an optical microscope. The mean numbers of whole cells and living cells were considered to be the proliferation and survival rates, respectively.Results: Increased SMF exposure and BMP-4 increased the viability percent and change in proliferation rate in the treated groups compared with their corresponding controls. The maximum increased viability was observed in the group that was treated with BMP-4 for 96 h.Conclusion: Our results have supported the hypothesis that SMF alters the viability and proliferation rate of treated BMSCs, which was enhanced when the cells were treated simultaneously with SMF and BMP-4.

Objective: Mycoplasma salivarium (M. salivarium) is one of the most common contaminants present in cell culture laboratories that cause undesirable effects on cell cultures. Thus, the identification and rapid diagnosis in controlling and prevention of this contaminant are important. The aim of this study is the detection of Mycoplasma salivarium contamination in cell culture using polymerase chain reaction (PCR) method.Methods: A 16S rRNA-based Mycoplasma genus and specific primer PCR method for M. salivarium was developed. The sensitivity and specificity of this method were determined. The PCR test was used after we extracted DNA from the cultured isolates.Results: A total of 62 cell culture samples were sent to the Mycoplasma Reference Laboratory at Razi Institute, Karaj, Iran for detection of Mycoplasma contamination. A total of 42 (67.75%) out of 62 samples scored positive according to the Mycoplasma genus. From these 42 samples, 15 (35.72%) reacted positively with a clear band of 434 bp in the M. Salivarium-specific PCR method.Conclusion: Due to the high percentage of M. salivarium contamination in cell cultures, we recommend aseptic conditions be used in the laboratory when working with cell cultures. The PCR method is a suitable and valuable tool for the detection of M. salivarium contamination in cell cultures with appropriate and specific primers. This PCR method can be processed in less than one day.