Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC50 values. The IC50 values were then used to predict the LD50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays.

Biological Synthesis of Low Cytotoxicity Silver Nanoparticles (AgNPs) by the Fungus Chaetomium thermophilum-Sustainable Nanotechnology.

Fungal biotechnology research has rapidly increased as a result of the growing awareness of sustainable development and the pressing need to explore eco-friendly options. In the nanotechnology field, silver nanoparticles (AgNPs) are currently being studied for application in cancer therapy, tumour detection, drug delivery, and elsewhere. Therefore, synthesising nanoparticles (NPs) with low toxicity has become essential in the biomedical area. The fungus Chaetomium thermophilum (C. thermophilum) was here investigated-to the best of our knowledge, for the first time-for application in the production of AgNPs. Transmission electronic microscopy (TEM) images demonstrated a spherical AgNP shape, with an average size of 8.93 nm. Energy-dispersive X-ray spectrometry (EDX) confirmed the presence of elemental silver. A neutral red uptake (NRU) test evaluated the cytotoxicity of the AgNPs at different inhibitory concentrations (ICs). A half-maximal concentration (IC50 = 119.69 µg/mL) was used to predict a half-maximal lethal dose (LD50 = 624.31 mg/kg), indicating a Global Harmonized System of Classification and Labelling of Chemicals (GHS) acute toxicity estimate (ATE) classification category of 4. The fungus extract showed a non-toxic profile at the IC tested. Additionally, the interaction between the AgNPs and the Balb/c 3T3 NIH cells at an ultrastructural level resulted in preserved cells structures at non-toxic concentrations (IC20 = 91.77 µg/mL), demonstrating their potential as sustainable substitutes for physical and chemically made AgNPs. Nonetheless, at the IC50, the cytoplasm of the cells was damaged and mitochondrial morphological alteration was evident. This fact highlights the fact that dose-dependent phenomena are involved, as well as emphasising the importance of investigating NPs' effects on mitochondria, as disruption to this organelle can impact health.

Using inhibition of the adipogenesis of adipose-derived stem cells in vitro for toxicity prediction.

In vitro stem cell models are used as alternatives to animal models and are important tools for cytotoxicity studies. Researchers can determine the effects of test substances on human cells by evaluating cell viability and differentiation. Here, we describe an in vitro model to quantify adipogenesis based on the Nile red staining of specific lipid droplets and the emission of basic lipids from human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) in the presence of test substances. This assay allows for the prediction of toxicity based on the inhibition of adipogenesis in vitro in a 96-well format. The differentiation of a progenitor cell into a specialized cell, the adipocyte, is easy to monitor and quantify, making this a simple assay. The fluorescence staining of nuclei and lipid droplets is measured after 14 days of cell differentiation to determine cell number and assess cell differentiation using high-content imaging analysis, thus allowing for the identification of chemicals that impact differentiation. We also describe a protocol to assess adipocyte differentiation by fluorescence intensity using a multiplate reader.•Researchers can utilize the protocol described here for many purposes to evaluate in vitro adipogenesis.•With this method, it is possible to reduce the use of animals.

Dose-dependent cell necrosis induced by silica nanoparticles.

In recent years, much attention has been given to nanoparticles (NPs) due to their many possible applications, and as research has progressed, these NPs have become valuable tools for medical purposes. Among many different types of NPs, silica nanoparticles (SiO2NPs) have been specifically evaluated for medical purposes and have also been used in many different types of products. Although SiO2NPs have already been applied and are believed to be nontoxic, there is still a concern regarding possible adverse effects that may be triggered after SiO2NP exposure. Therefore, in the present study, we employed a recommended cell line (BALB/c 3T3) for the toxicity evaluation to investigate the cytotoxic effects of SiO2NPs produced by chemical synthesis at a laboratory scale. First, we employed OECD guideline 129 in order to evaluate cytotoxicity effects and also estimate the starting doses for acute oral systemic toxicity tests. We evaluated the cytotoxic effects of two types of SiO2NPs (nonfluorescent and fluorescent) and found that they were not significantly different (IC50 = 1986.39 ± 237 µg/mL and IC50 = 1861.13 ± 186.72 µg/mL, respectively). Then, we used the predicted LD50 of both types of SiO2NPs to suggest that they could be categorized as GHS category 4 substances. By ultrastructural evaluation, we found that SiO2NPs are internalized by 3 T3 cells and are located in vacuole-like structures with no other significant changes in cell structure. We also found that SiO2NPs lead to cell necrosis in a dose-dependent manner.

Dose-dependent cytotoxicity of bismuth nanoparticles produced by LASiS in a reference mammalian cell line BALB/c 3T3.

Nanoparticles (NPs) have emerged as new potential tools for many applications in previous years. Among all types of NPs, bismuth NPs (BiNPs) have a very low cost and potential for many applications, ranging from medicine to industry. Although the toxic effects of bismuth have been studied, little is known about its toxicity at the nanoscale level. Therefore, in this study, we aimed to investigate the cytotoxic effects of BiNPs produced by laser ablation synthesis in solution (LASiS) in a reference mammalian cell line to evaluate their cytotoxicity (BALB/c 3 T3 cells). We also stabilized BiNPs in two different solutions: culture medium supplemented with fetal bovine serum (FBS) and bovine serum albumin (BSA). The cytotoxicity of BiNPs in culture medium (IC50:28.51 ±â€¯9.96 µg/ml) and in BSA (IC50:25.54 ±â€¯8.37 µg/ml) was assessed, and they were not significantly different. Second, the LD50 was predicted, and BiNPs were estimated as GHS class 4. We also found that cell death occurs due to apoptosis. By evaluating the interaction between BiNPs and cells at ultrastructural level, we suggest that cell death occurs once BiNPs are internalized. Additionally, we suggest that BiNPs cause cell damage because myelin figures were found inside cells that had internalized BiNPs. To date, this is the first study to assess the cytotoxicity of BiNPs produced by LASiS and to predict the possible LD50 and GHS class of BiNPs.