5S and 5W2H Tools Applied to Research Laboratories: Experience from Instituto Carlos Chagas - FIOCRUZ/PR for Cell Culture Practices

Abstract Good cell culture practices are a set of technical and management tools recommended for application in research and service laboratories to guarantee the traceability and reproducibility of in vitro experiments. However, most research laboratories do not have a structured quality assurance system and have difficulties organizing their workflows or even priorities in implementing acceptable laboratory practices. In this study, we applied management and quality assurance tools to define the steps necessary to implement acceptable laboratory practices in the multiuser laboratory of cell culture and establish a cell bank at the Carlos Chagas Institute FIOCRUZ-PR. Our team applied the 5W2H and 5S tools for initial diagnosis and established an action plan to implement and manage the laboratory over two years. Thereby, we defined the scope of laboratory activities, including the demand for establishing a cell bank, the supply of cell lines to internal users, user training, and quality control tests. We also mapped the main activities, establishing their flows and all the necessary documentation to ensure traceability and reproducibility. Additionally, the laboratory was organized in compliance with the 5S principles. In conclusion, using management tools, such as the 5W2H and 5S methods, is a simple and affordable method to organize and manage a cell culture laboratory and can be applied to other research laboratories.

The terpenic diamine GIB24 inhibits the growth of Trypanosoma cruzi epimastigotes and intracellular amastigotes, with proteomic analysis of drug-resistant epimastigotes.

The effect of N-geranyl-ethane-1,2-diamine dihydochloride (GIB24), a synthetic diamine, was assayed against different developmental forms of the parasitic protozoan Trypanosoma cruzi (strain Dm28c). The compound was effective against culture epimastigote forms (IC50/24h = 5.64 µM; SI = 16.4) and intracellular amastigotes (IC50/24h = 12.89 µM; SI = 7.18), as detected by the MTT methodology and by cell counting, respectively. Incubation of epimastigotes for 6h with 6 µM GIB24 (IC50/24h value) resulted in significant dissipation of the mitochondrial membrane potential, prior to permeabilization of the plasma membrane. Rounded epimastigotes with cell size reduction were observed by scanning electron microscopy. These morpho-physiological changes induced by GIB24 suggest an incidental death process. Treatment of infected Vero cells did not prevent the intracellular amastigotes from completing the intracellular cycle. However, there was a decrease in the number of released parasites, increasing the ratio amastigotes/trypomastigotes. Proteomic analysis of 15 µM GIB24 resistant epimastigotes indicated that the compound acts mainly on mitochondrial components involved in the Krebs cycle and in maintaining the oxidative homeostasis of the parasites. Our data suggest that GIB24 is active against the main morphological forms of T. cruzi.

Biological activity of the azlactone derivative EPA-35 against Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, affects six to seven million people worldwide. Treatment is based on benznidazole, producing several side effects and debatable efficacy, highlighting the need for new alternative drugs. We investigated the activity of four C-4 functionalized azlactone derivatives (EPA-27, EPA-35, EPA-63 and EPA-91) as potential T. cruzi inhibitors. Screening with epimastigotes indicated EPA-35 as the best compound (IC50/24 h: 33 µM). This compound was 14.1 times more potent against intracellular amastigotes (IC50/24 h: 2.34 µM). Treatment of infected Vero cells for 72 h (up to 30 µM EPA-35) resulted in a dose-dependent decrease in number of trypomastigotes and amastigotes released in the supernatant, but the amastigote/trypomastigote ratio remained constant, indicating that amastigote growth was disturbed, but cell differentiation was unaffected. Analysis of treated epimastigotes by flow cytometry indicated that the plasma membrane remained intact, but there was a significant decrease in mitochondrial membrane potential. The pattern of cell distribution in the cell cycle stages (G1, G2, M) was unaltered in treated epimastigotes, indicating a trypanocidal rather than a trypanostatic activity. Scanning electron microscopy and flow cytometry showed epimastigotes with a round shape and decrease in cell size. Taken together, our data indicate that the EPA-35 is effective against T. cruzi. Synthetic transformation of EPA-35 into other derivatives may provide promising compounds for further evaluation against this parasite.

Biological activity of the essential oils from Cinnamodendron dinisii and Siparuna guianensis.

This study had analyzed the antibacterial, antifungal and trypanocidal activity of the essential oils from Cinnamodendron dinisii Schwacke (Canellaceae) and Siparuna guianensis Aublet (Siparunaceae). The essential oils were obtained from fresh leaves by hydrodistillation, using a modified Clevenger apparatus. Chemical analysis by gas-liquid chromatography coupled to mass spectrometry (GC-MS) showed that these essential oils are rich in monoterpene and sesquiterpene hydrocarbons. Activity against the pathogenic bacteria Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella choleraesuis and Staphylococcus aureus was evaluated with the agar cavity diffusion method, while activity on the filamentous fungi Aspergillus flavus , Aspergillus niger , Aspergillus carbonarius and Penicillium commune was evaluated by the disk diffusion technique. Trypanocidal activity was tested against Trypanosoma cruzi epimastigotes, using the Tetrazolium salt (MTT) colorimetric assay. Both essential oils exhibited low inhibitory effect towards bacteria, showing high MIC values (125-500 µg mL (-1) ), with Gram positive bacteria being more susceptible. Better inhibitory effect was obtained for the evaluated fungi, with lower MIC values (7.81-250 µg mL (-1) ), being A. flavus the most susceptible species. Both essential oils presented low trypanocidal activity, with IC 50 /24 h values of 209.30 µg mL (-1) for S. guianensis and 282.93 µg mL (-1) for C. dinisii . Thus, the high values observed for the MIC of evaluated bacteria and for IC 50 /24 h of T. cruzi , suggest that the essential oils have a low inhibitory activity against these microorganisms. In addition, the low MIC values observed for the tested fungi species indicate good inhibitory activity on these microorganisms's growth.

Biological activity of the essential oils from Cinnamodendron dinisii and Siparuna guianensis

This study had analyzed the antibacterial, antifungal and trypanocidal activity of the essential oils from Cinnamodendron dinisii Schwacke (Canellaceae) and Siparuna guianensis Aublet (Siparunaceae). The essential oils were obtained from fresh leaves by hydrodistillation, using a modified Clevenger apparatus. Chemical analysis by gas-liquid chromatography coupled to mass spectrometry (GC-MS) showed that these essential oils are rich in monoterpene and sesquiterpene hydrocarbons. Activity against the pathogenic bacteria Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella choleraesuis and Staphylococcus aureus was evaluated with the agar cavity diffusion method, while activity on the filamentous fungi Aspergillus flavus, Aspergillus niger, Aspergillus carbonarius and Penicillium commune was evaluated by the disk diffusion technique. Trypanocidal activity was tested against Trypanosoma cruzi epimastigotes, using the Tetrazolium salt (MTT) colorimetric assay. Both essential oils exhibited low inhibitory effect towards bacteria, showing high MIC values (125–500 μg mL−1), with Gram positive bacteria being more susceptible. Better inhibitory effect was obtained for the evaluated fungi, with lower MIC values (7.81–250 μg mL−1), being A. flavus the most susceptible species. Both essential oils presented low trypanocidal activity, with IC50/24 h values of 209.30 μg mL−1 for S. guianensis and 282.93 μg mL−1 for C. dinisii. Thus, the high values observed for the MIC of evaluated bacteria and for IC50/24 h of T. cruzi, suggest that the essential oils have a low inhibitory activity against these microorganisms. In addition, the low MIC values observed for the tested fungi species indicate good inhibitory activity on these microorganisms’s growth.

In vitro biological evaluation of eight different essential oils against Trypanosoma cruzi, with emphasis on Cinnamomum verum essential oil.

BACKGROUND: Essential oils (EOs) are complex mixtures of secondary metabolites from various plants. It has been shown that several EOs, or their constituents, have inhibitory activity against trypanosomatid protozoa. Thus, we analyzed the biological activity of different EOs on Trypanosoma cruzi, as well as their cytotoxicity on Vero cells. METHODS: The following EOs were evaluated on T. cruzi epimastigote forms: Cinnamomum verum, Citrus limon, Cymbopogon nardus, Corymbia citriodora, Eucalyptus globulus, Eugenia uniflora, Myrocarpus frondosus, and Rosmarinus officinalis. Inhibitory activity against T. cruzi (IC50/24 h) and cytotoxicity against Vero cells (CC50/24 h) were evaluated by the MTT assay. The EO of C. verum was selected for further evaluation against trypomastigotes and intracellular amastigotes, as well as on parasite metacyclogenesis. Constituents of C. verum EO were identified by GC-MS. One-way ANOVA statistical analysis was performed with GraphPad version 5.01. RESULTS: Cinnamomum verum EO was the most effective against T. cruzi epimastigotes (IC50/24 h = 24.13 µg/ml), followed by Myrocarpus frondosus (IC50/24 h = 60.87 µg/ml) and Eugenia uniflora (IC50/24 h = 70 µg/ml). The EOs of C. citriodora, E. globulus, and R. officinalis showed no activity at concentrations up to 300 µg/ml. Incubation of T. cruzi metacyclic trypomastigotes and intracellular amastigotes with C. verum EO resulted in IC50/24 h values of 5.05 µg/ml and 20 µg/ml, respectively. Therefore, trypomastigotes are more susceptible than epimastigotes, with selectivity index (SI) about 4.7-fold higher (9.78 and 2.05, respectively). Analysis of C. verum EO by GC-MS showed mainly (E)-cinnamaldehyde (81.52%) and eugenol (16.68%). CONCLUSIONS: C. verum essential oil is effective against T. cruzi (epimastigotes, trypomastigotes and amastigotes) and interferes with the parasite differentiation process in vitro. Thus, it represents a strong candidate for further studies to improve its activity on pathogenic trypanosomatids.