Bacterial culture and growth conditions

A multidrug-resistant clinical strain of V. cholerae stored in the collection of the Bacteriology Department of Tarbiat Modares University, Tehran, Iran, was used. The strain was resistant to tetracycline, ciprofloxacin, chloramphenicol, cotrimoxazole, and trimethoprim. The bacterial culture was performed in 1mL brain heart infusion (BHI) broth (Merck, Germany) at 37C until reaching the log phase. The bacterial suspension concentration was determined by measuring the absorbance at 540nm and comparing it with the standard 0.5 McFarland optical density (OD). According to the growth curve, the bacterial suspension with an OD of 1:0 (~108CFU/mL) was used21.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) and Caco-2 cells were purchased from the Iranian Biological Resource Center and Pasteur Institute of Iran, respectively. BM-MSCs were confirmed by assaying the differentiation of the cells into osteoblasts and adipocytes using an immunohistochemistry (IHC) assay. BM-MSCs were also characterized using the flow cytometry method for CD34, CD45, CD44, and CD73 markers18.

BM-MSCs were cultured in lowglucose Dulbecco's modified Eagles medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) at 37C in a humidified atmosphere containing 5% CO2. The medium was replaced after every 2days. A total of 5105 cells were seeded in a T75 flask (SPL, Korea) containing 15mL of DMEM supplemented with 10% FBS. When the confluency of the cells was near 90% at passage 2, the medium was replaced with serum-free DMEM. Subsequently, MSC CM was collected and centrifuged at 4000rpm for 30min and finally stored at 80C until use22. According to our previous study, chitosan nanoparticles were synthesized, characterized, and loaded with the supernatant of mesenchymal stem cells23. At all stages of this study, mesenchymal stem cell conditioned media (MSC CM; 1000g), chitosan nanoparticles incorporated with mesenchymal stem cell conditioned media (MSC CM-CS NPs; 1000g+0.05%), and chitosan nanoparticles (CS NPs; 0.05%) were used.

Vibrio cholerae cell suspensions were inoculated (1:100 dilution) into 1mL BHI broth medium containing 0.05% sucrose. The bacterial suspension was inoculated with MSC CM, MSC CM-CS NPs, and CS NPs overnight at 37C to evaluate the expression of biofilm genes. After this time, each well was washed three times with PBS, and adherent cells were harvested to evaluate the expression of biofilm-related genes. PBS and V. cholerae without exposure to the compounds were used as negative and positive controls, respectively. Each assay was performed in triplicate.

Caco-2 cells were cultured in DMEM supplemented with 10% FBS, 1% l glutamine (DNA Biotech, Iran), and 1% penicillin/streptomycin and incubated at 37C with 5% CO2. The culture medium was changed every two days, and when the confluency reached 80%, the cells were passaged. Since cells in monolayer culture with full confluency can form polarized cells while maintaining cell surface molecules, we explored cells at 85% confluency for all experiments24.

Caco-2 cell viability was estimated by the conventional MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. This was tested to evaluate the viability of Caco-2 cells after the exposure time to bacteria, MSC CM, MSC CM-CS NPs, and CS N. Briefly, 2({10}^{4}) Caco-2 cells per well were seeded into a 96-well plate and cultured for 24h at 37C. The medium was removed, and the cells were exposed to bacterial suspension (10 bacteria per epithelial cell; MOI: 10), MSC CM, MSC CM-CS NPs, and CS NPs separately for 24h. To assess cytotoxicity, a separate test was also performed for 72h. After these times, the medium was removed, and MTT solution was added for 3h at 37C. Then, the formazan crystals formed in cells were dissolved in 100L of dimethyl sulfoxide (Sigma Aldrich, USA). The resulting purple solution was measured using an ELISA reader (OD 540nm) (800 TS, BioTek, Winooski, Vermont, USA). Each assay was performed in triplicate25.

For this purpose, five groups were designed: (i) untreated Caco-2 cells (negative control); (ii) Caco-2 cells incubated with V. cholerae (MOI: 10-positive control); (iii) Caco-2 cells+V. cholerae+MSC CM; (iv) Caco-2 cells+V. cholerae+CS NPs; and (v) Caco-2 cells V. cholerae+MSC CM-CS NPs. Caco-2 cells were cultured in 96-well microplates until they reached 80% confluence. Before treatment, cells were washed three times with PBS. Then, the cells were infected with V. cholerae for 1h, and the extracellular bacteria and medium was removed and replaced with DMEM-free compounds, including 100L of MSC CM, MSC CM-CS NPs, and CS NPs, for 18h at 37C. After this time, the supernatant were removed, and then Caco-2 cells were used for total RNA extraction. Each test was performed in triplicate.

Total RNA from bacteria and Caco-2 cells was extracted using an RNA Miniprep Super Kit (Bio Basic, Canada) according to the manufacturers recommendations. The RNA was assayed by absorbance at OD260/280. Samples with a ratio of 1.82.0 were used for cDNA synthesis using Yekta Tajhiz Azma, Iran. According to the protocol, template RNA (5L), random primer (0.5L), and DEPC-treated water (7.5L) were mixed, centrifuged briefly, and incubated for 5min at 70C. Then, 5 first strand buffer (4L), dNTPs (1L), RNase 40U/L (0.5L), and M_MLV (1L) were mixed and incubated for 60min at 42C, and the reaction was terminated by heating for 5min at 70C. The cDNA samples were stored at 20C until use in the following experiment.

Conventional SYBR Green-based real-time PCR was used for target gene quantification. Real-time PCR was performed by using 10L 5 Real-time PCR Master Mix (Biomake, Houston, TX, USA), 1L of each primer (Table 1), 2L of cDNA, and 6L of distilled water in a total reaction volume of 20L in Stratagene Mx3000P real-time PCR system (Stratagene, La Jolla, CA). 16S rRNA was used as an endogenous control to normalize the expression levels of target genes of V. cholerae. Beta-actin was also utilized as an internal control to normalize the expression levels in RNA samples from Caco-2 cells. The CT of each sample was measured (CT targetCT reference). We used Caco-2 cells not treated as a calibrator, and the CT method was used to determine the difference between treated cells and the control. The fold change of gene expression level was calculated using the comparative CT (2CT).

The data were analyzed by using GraphPad Prism version 6 using one-way ANOVA and Bonferroni post hoc test. P value<0.05 was accepted as significant. The results of replications were also evaluated as the meanstandard deviation (SD).

The study was reviewed and approved by the Medical Ethics Committee of Tarbiat Modares University (Code: IR.MODARES.REC.1398.060). All methods were also carried out in accordance with the guidelines and regulations related to the committee.

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Effects of chitosan nanoparticles loaded with mesenchymal stem cell conditioned media on gene expression in Vibrio cholerae and Caco-2 cells |...

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