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PATROLS SOP Handbook

The PATROLS project has generated a suite of more representative in vitro and in silico tools to better understand human & environmental hazards following ENM exposures. Some of these tools are summarised in the below table, which lists the methodological Standard Operating Procedures (SOPs) generated by the PATROLS project. The SOPs from PATROLS are open access and can be freely accessed through the links provided in the table.

Identification and ownershipPATROLS IDMethod CategoryDescription
Guidance Document for ENMs Aerosolization using VITROCELL® Cloud System 3601 Toxicology: In vitro Due to the constant increase in their production, exposure to engineered nanomaterials (ENM) poses an inevitable health risk to both humans and the environment through long-term, repetitive, low-dose exposures. The majority of the literature however, focuses on short-term, high-dose exposures. Hazard assessment of ENM, when applying alternative testing strategies to in vivo research, has previously engaged exposures under submerged conditions. Such exposures poorly mimic human exposures, and possess several limitations, such as issue with measuring a delivered dose, and interaction with exposure medium. Therefore, air-liquid interface exposure chambers allowing for single droplet deposition of ENM aerosols onto cell surface were developed. VITROCELL® Cloud system equipped with Aeroneb Pro nebulizer, and Quartz crystal microbalance for measuring online deposition of ENMs, is referred in this SOP.
Guidance Document for cell culture of lung epithelial cell-line (Calu-3) 3104 Toxicology: In vitro In order to evaluate lung toxicity of long-term, repeated, airborne particle exposure, we developed an air-liquid interface (ALI) model using the human bronchial epithelial cell line, Calu-3. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. This bronchial model is suitable for testing the effects of long-term, repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system.
Guidance Document for macrophage differentiation from THP-1 cells 3108 Toxicology: In vitro THP-1 differentiation; This document provides a step-by-step description of how to successfully culture monocyte THP-1 cells and differentiate using PMA to macrophage like cells (dTHP-1) to then be used as a component of a co-culture at the air-liquid interface.
Standard operating protocol: Nanomaterial pre-treatment with simulant fluids to mimic oral and inhalation exposures for Hazard assessment using 3D liver models in vitro 4202 Toxicology: In vitro This document includes a description of the setup and standard operation procedure devised to mimic the translocation of ENMs through the human body to the liver following either oral or inhalation exposure. This pre-treatment scheme provides a physiologically relevant ENM exposure regime, allowing for a more realistic evaluation of the human health hazards associated with hepatic ENM exposure. This SOP can be easily adapted to complement other organ systems that are known to be secondary sites of ENM deposition.
Guidance Document for ENMs lung dosing consideration based on In silico analysis for Dörntruper quartz (DQ12), barium sulphate (BaSO4), cerium oxide (CeO2), and titanium dioxide (TiO2), and multi-walled carbon nanotubes (MWCNT) 3105 Toxicology: In vitro The current document gives guidance which deposited doses to achieve in the in vitro cell systems to allow comparison with available in vivo data. Important note is that the in vitro models are a simplification of the real situation and might therefore respond differently. Especially when the in vitro models are based on cell lines, potentially higher deposited doses are needed compared to the in vivo studies to observe a similar effect. The main aim is to use more realistic exposure scenarios using repeated exposure compared to unrealistic single high exposures and correlate the results to in vivo data
SOP: 3D In Vitro HepG2 Spheroid Model 4104 Toxicology: In vitro This document summarises the cell culture and maintenance of the human cell lines Caco-2, HT29-MTX-E12 and THP-1 as well as their use for the establishment of a transwell-based triple culture model of the intestine in healthy state. In addition to the basics on the triple culture establishment, the SOP contains protocols for the analysis of several parameters to characterise the culture. The model is not suitable for the study of non-sedimenting nanomaterials.
Standard operating protocol: Triple culture of the inflamed-like intestine 4205 Toxicology: In vitro This document summarises the cell culture and maintenance of the human cell lines Caco-2, HT29-MTX-E12 and THP-1 as well as their use for the establishment of a transwell-based triple culture model of the intestine in inflamed state. In addition to the triple culture establishment, the SOP contains protocols for the analysis of several parameters to characterise the culture and confirm the induction of an inflamed-like condition. The model is not suitable for the study of non-sedimenting nanomaterials.
Guidance Document for co-culture of a lung epithelial cell-line (Calu-3), and macrophages derived from peripheral blood monocytes 3603 Toxicology: In vitro  
Guidance Document for co-culture of an lung epithelial cell-line (Calu-3), and macrophages derived from a monocytic cell line (dTHP-1) 3604 Toxicology: In vitro Calu-3 and dTHP-1 Cell Culture; This document provides a step-by-step description of how to successfully culture Calu-3 cells, differentiate THP-1 cells (dTHP-1) upon transwell-membrane inserts at the air-liquid interface and then complete specific nanomaterial exposures using the VitroCell exposure system.
Method of freeze-thaw sample preparation with dosimetry based on DG code and Vila et al 2017 1204 General toxicology  
SOP for AOP-informed Nano-QSAR 6204 General toxicology This document includes a description of the setup and standard operation procedure for developing the AOP-anchored Nano-QSAR model. The model has been used to predict transcriptomic pathway-level response for mice lung fibrosis exposed to different multiwalled carbon nanotubes (MWCNTs). The pathway “agranulocyte adhesion and diapedesis”, which is perturbed across the MWCNTs panel, shows dose-response (Benchmark dose, BMDs), and is anchored to the key events (KEs) identified in the lung fibrosis adverse outcome pathway (AOP173); is considered in the modeling. The developed Nano-QSAR model predicts the BMDL level based on the aspect ratio of MWCNTs. The model is additionally presented by using the QSAR model reporting format (QMFR).
DALI system: a description of the device and its characterisation 3202 Toxicology: In vitro  
SOP: Culture and characterisation of mono and multi-cellular models of the gastrointestinal system 4101 Toxicology: In vitro Intestinal epithelial cell culture models, such as Caco-2 cells, are commonly used to assess absorption of drugs and transcytosis of nanomaterials (NMs) across the intestinal mucosa. However, it is known that mucus strongly impacts NM mobility and that specialized M cells are involved in particulate uptake. Thus, to get a clear understanding of how NMs interact with the intestinal mucosa, in vitro models are necessary that integrate the main cell types. This protocol highlifghts the step necessary for the development of a a triple culture: Caco-2 cells, mucus-secreting goblet cells and Microfold (M) cells.
Standard operating protocol (SOP) for LEVITATT algal test system 5501 Toxicology: Eco This document describes how to perform an algal growth inhibition test to determine the ecotoxicity of nanomaterials using the LEVITATT (LED Vertical Illumination Table for Algal Toxicity Tests) test setup. This protocol is based on previous SOP work carried out in the NANoREG project and the standardized ISO-Guideline 8692:2012 and OECD-Guideline 201 “Algal growth inhibition test”. The method provides a versatile small-scale algal toxicity test complying with standards such as OECD 201 and ISO 8692. The method is optimized to increase reproducibility of standard algal tests by 1) utilizing LED light technology to ensure inform light conditions with minimal heat generation, 2) providing adequate sample volume for chemical/biological analysis while maintaining constant pH, CO2 levels, 3) enables the use of versatile test container material.
Biological testing of the DALI system 3205 Toxicology: In vitro  
Guidance Document for cell culture of lung epithelial cell-line (A549) 3101 Toxicology: In vitro A549 Cell Culture; This document provides a step-by-step description of how to successfully culture alveolar type-II like epithelial cells (A549) upon transwell-membrane inserts at the air-liquid interface.
SOP: Tissue characterisation, nanomaterial treatment and toxicological assessment in 3D primary human liver microtissues 4103 Toxicology: In vitro It is essential to establish more advanced, physiologically relevant in vitro assessment tools for improved prediction of the adverse effects caused by chronic nanomaterial (NM) exposure in humans. The utilisation of human primary hepatic cells is the closest representative in vitro model for the human liver. However, these cells are phenotypically unstable in 2D cultures and have an extremely limited life span (typically no longer than 7 days - with continued reduced viability, functional and metabolic activity). Moreover, in most traditional 2D hepatic models, non-parenchymal cell (NPC) populations are not included or considered. In an attempt to address this issue, the use of scaffold free 3D liver microtissue (MT) model composed of primary human hepatocytes and primary human liver-derived NPC could be beneficial.
Standard operating protocol: PATROLS 4.3 SOP Reverse Transcriptase PCR for Hepatocarcinogenicity Biomarkers in 3D HepG2 Liver Spheroids SU 4301 Toxicology: In vitro This document includes a description of the setup and standard operation procedure for the evaluation of transcriptional profiles changes associated with carcinogenesis in 3D HepG2 liver models following ENM exposure over both an acute or long-term, and repeated dose regime. This method allows for the identification of an advanced endpoint liver associated biomarker panel that can be used as an effective screening approach. This SOP uses predefined disease pathway PCR array plates can be costly and therefore it may not be feasible to run each sample in triplicate, however, it allows for the identification of a panel of key genes that can be explored further.
SOP for assembly and use of DALI 3203 Toxicology: In vitro See the introduction in the document
Vitrocell dry powder system 3206 Toxicology: In vitro This document describes the setting up, exposure, monitoring and cleaning of the Vitrocell dry powder exposure system. The SOP details assembly of microbalances for monitoring of deposited particles, exposure of cells cultured on tissue culture inserts, and the disassembly and cleaning steps required after exposure.
Standard operating protocol: Evaluation of NM-induced hepatotoxicity in a primary human multi-cellular MT model with emphasis on physiologically meaningful toxicological end-points 4203 Toxicology: In vitro It is essential to establish more advanced, physiologically relevant in vitro assessment tools for improved prediction of the adverse effects caused by chronic nanomaterial (NM) exposure in humans. The utilization of human primary hepatic cells is the closest representative in vitro model for the human liver. However, these cells are phenotypically unstable in 2D cultures and have an extremely limited life span (typically no longer than 7 days - with continued reduced viability, functional and metabolic activity). Moreover, in most traditional 2D hepatic models, non-parenchymal cell (NPC) populations are not included or considered. In an attempt to address this issue, the use of scaffold free 3D liver microtissue (MT) model composed of primary human hepatocytes and primary human liver-derived NPC could be beneficial.
Guidance document for assessing the direct effect of NMs on fibroblast proliferation and expression of pro-fibrotic biomarkers 3208 Toxicology: In vitro  
Standard operating protocol: 3D In Vitro HepG2, Kupffer cell co-Culture spheroid model 4201 Toxicology: In vitro This document includes a description of the standard operation procedure for the setup of an advanced, hepatic co-culture spheroid model suitable for both chemical and ENM exposure over both an acute or prolonged, and repeated dosing regime. This method utilises the HepG2 cell line and primary, liver resident macrophages, human kupffer cells. This model can be applied for in vitro chemical and ENM hazard assessment including cytotoxicity, liver-like functionality, inflammatory response and genotoxicity evaluation.
Guidance Document for cell culture of lung epithelial cell-line (Cl-hAELVi) 3102 Toxicology: In vitro  
Guidance Document for cell culture of lung epithelial cell-line (NCI-H441) 3103 Toxicology: In vitro NCI-H441 Cell Culture; This document provides a step-by-step description of how to successfully culture lung epithelial cells (NCI-H441) upon transwell-membrane inserts at the air-liquid interface.
Guidance Document for a quasi-air-liquid interface exposure of nanoparticles to cells grown at an air-liquid interface 3106 Toxicology: In vitro Quasi-ALI nanoparticle exposure; This document provides a step-by-step description of how to successfully expose a culture already at an air-liquid interface to nanomaterials using a quasi-ALI method.
Guidance Document for the isolation and differentiation of peripheral blood monocytes and further assembly into co-culture models with epithelial cells 3109 Toxicology: In vitro  
Guidance Document for co-culture of an lung epithelial cell-line (NCI-H441), and macrophages derived from a monocytoic cell line (dTHP-1) 3111 Toxicology: In vitro NCI-H441 and dTHP-1 Cell Culture; This document provides a step-by-step description of how to successfully culture NCI-H441 cells, differentiate THP-1 cells (dTHP-1) and then culture together upon transwell-membrane inserts at the air-liquid interface.
SOPs for advanced mechanism-based highthroughput in vitro screening 4401 Toxicology: In vitro This SOP describes the setup and approach taken for conducting high throughput 384-well-based 3D spheroid screens using HepG2 cells. The screens were carried out using 5 endpoints measurements (two cell viability measurements, ROS, cytotoxicity and apoptosis), 4 biological replicates and 6 concentrations over 24 and 120 hour timepoints. Parallel 2D monoculture screens were carried out for comparison purposes using the same assay setup. All nanomaterials made available though PATROLS were used in the screens. The results and the SOP are presented in deliverable 4.4 and the data is stored in the PATROLS database.
Reverse Transcriptase PCR for lung models 3303 General toxicology PCR analysis; This document provides a step-by-step description of how to successfully complete a bio-rad PCR analysis of selected genes on a lung model pre-exposed to various nanomaterial.
Daily oral exposure of rats to nanomaterial in snacks for improved animal welfare and control of adminstered dose 2401 Toxicology: In vitro This document describes a standard operating procedure for homogenous mixing of nanomaterial in a snack for rats and how to serve the snack daily with control of delivered dose.
Guidance Document for co-culture of an lung epithelial cell-line (A549), and macrophages derived from a monocytoic cell line (dTHP-1) 3110 Toxicology: In vitro A549 and dTHP-1 Cell Culture; This document provides a step-by-step description of how to successfully culture A549 cells, differentiate THP-1 cells (dTHP-1) and then culture together upon transwell-membrane inserts at the air-liquid interface.
SOP experimental setup for environmental dosimetry data acquisition 6401 Toxicology: Eco  
Guidance Document for cell exposure at the air-liquid interface using VITROCELL® automated exposure station 3107 Toxicology: In vitro  
SOPs for advanced mechanism-based highthroughput in vitro screening 4402 Toxicology: In vitro This dataset is derived from the setup and approach taken for conducting high throughput 384-well-based 3D spheroid screens using HepG2 cells. The screens were carried out using 5 endpoints measurements (two cell viability measurements, ROS, cytotoxicity and apoptosis), 4 biological replicates and 6 concentrations over 24 and 120 hour timepoints. Parallel 2D monoculture screens were carried out for comparison purposes using the same assay setup. All nanomaterials made available though PATROLS were used in the screens. The results and the SOP are presented in deliverable 4.4 and the data is stored in the PATROLS database.
Procedure for Examining the PEG Modification of the Surface of Gold Nanoparticles Using Time-of-Flight Secondary Ion Mass Spectroscopy 1102 Physics and chemistry This procedure guide describes the procedure for confirming the PEG modification of the surface of AuNPs. PEG is a biocompatible substance that inhibit the non-specific adsorption. To examine the PEG modification of the surface of AuNPs, free PEG ligands unadsorbed on the AuNPs surface were removed from the solution by centrifuging it for four times. Sampling was carried out by spotting 10 nL of the PEG-modified AuNPs solution on a silicon (Si) wafer. This procedure guide presents the ToF-SIMS measurement and data processing methods for examining the PEG surface modification of AuNPs.
SOP for pulmonary dissolution testing by Continuous Flow Cells based on ISO19057 1307 Physics and chemistry  
Method of dose-dependent EPR reactivity testing based on ISO18827:2017 1308 Physics and chemistry  
Method of robust oral dissolution & transformation testing by sequential incubation based on DIN19738:2017 1309 Physics and chemistry  
Atmosphere-Temperature-pH-controlled Stirred batch Reactor Dissolution and reactivity testing in water and biologically relevant media 1303 Physics and chemistry  
Electrochemical measurement of the redox potential of nanoparticles in biological media 1301 Physics and chemistry This document includes a description of the setup and standard operation procedure for conducting direct measurements of ENM redox potential, calculated from the reduction and oxidation peak that appear in a cyclic voltammogram. We deposited ENM on an inert electrode (carbon). We then scanned the potential of the loaded electrode in contact with a supporting electrolyte. The redox potential of the nanoparticles was calculated from the positive and negative peaks of current (halfway of the two redox potentials). We used this method to investigate the electrochemical behavior of nanoparticles in different biological relevant media: DQ water + KCl, DMEM, Gamble’s and PSF.
Sensor Dish Reader Method for solubility and pH and O2 reactivity testing under in in vitro test conditions 1302 Physics and chemistry  
Technical Description: Preparation and mechanical characterization of Bionate membranes 3204 Toxicology: In vitro  
Guidance Document for the induction of the inflamed alveolar epithelial model 3207 Toxicology: In vitro  
Experimental validation DG dosimetry model by assesing the concentration at half height (testing in column water) 1201 Toxicology: In vitro  
PATROLS Database: An instance of DB implemented in ENanoMapper database system 6101    
DosiGUI: a graphical user interface for dosimetry estimation using DG, ISD3 and ISDD models 6201 Toxicology: In vitro  
In situ dectection of particle size distribution and concentration of ions released in ecotox media 1502 Physics and chemistry This document includes a description of the setup and standard operation procedures for assessing profiles of colloidal stability and concentration of ENM in eco-tox water column experimental model, vs concentration and time of exposure. The sedimentation behavior of ENM was assessed by measuring concentration and turbidimetry intensity at half height of water column. For the quantitative measurement of the concentration we incubated nanoparticles in water media, collecting a fraction of sample from the center of the water-column. The collected sample was then acid digested and analyzed by ICP-OES. The overall colloidal stability of samples was assessed by measurements of DLS-Size, ELS-Zeta Pot and CLS-Hydrodynamic Diameter varying concentration and incubation time, supporting outcomes of the sedimentation study.
PBPK Model for nanomaterials 6301 Toxicology: In vitro This document describes the setup and standard operation procedure for applying and parametrizing a physiologically-based (pharmaco)kinetic (PBPK) model to simulate the kinetic distribution of nanomaterials in rats after inhalation exposure. The PBPK model consists of multiple organ compartments and parameters that describe the transfer of nanomaterials between the compartments. Optimization of these parameters was performed based on a Bayesian parameter estimation approach in combination with Markov Chain Monte Carlo sampling. All necessary software packages, PBPK model equations, and the Bayesian parameter estimation approach have been described in detail. Since models parametrized using this method are specific to one particular nanomaterial, application to different nanomaterials may require re-parametrization of the model.
Standard operating protocol: Investigating NM-induced toxicological effects in multiple cell primary liver MTs representative of mild and severe steatotic disease 4202 Toxicology: In vitro See the introduction in the document
Development of dose- and nano-specific QSAR models for environmentally relevant endpoints according to OECD recommendations 6402 Toxicology: Eco  
In situ dectection of ENM elemental distribution within different compartments of 2D epithelial tissue in-vitro model 1401 Toxicology: In vitro This document includes a description of the setup and standard operation procedure for measuring the elemental distribution within different compartments of 2D in-vitro lung model used for assessing the long-term fate of ENM. Lung cells (A549) were grown to a confluent monolayer on 12-well inserts and exposed to nanoparticles. Single and repeated exposure were evaluated, considering an acute and long-term repeated exposure concentration. Apical, wash, cells and basal fractions were collected and analyzed by ICP-OES. An acid digestion was performed to obtain the nominal concentration of nanoparticles in medium using ICP-OES technique.
IVD model for nanomaterials 6205 Toxicology: In vitro A kinetics model of the deposition of engineered nanomaterials (ENM) was developed and tested with data from T1.4. The model is a set of ordinary differential equations, the parameters of which describe the kinetics of deposition of the ENM. The model was calibrated with a single dose in vitro experiment with DQ12, BaSÕ, CèO and TiO2. It was also used for a repeated dosing experiment with the same ENM.
QSAR modelling of environmentally relevant fate and effect endpoints of nanomaterials 6403 Toxicology: Eco  
PBPK model for nanomaterials 6206 Toxicology: In vitro The dynamics of deposition of Engineered Nanomaterials (ENM) in a in vitro setting can be described mathematically. The mathematical construction of the time course of deposition of ENM, the in vitro dosimetry (IVD) model, is based on a system of ordinary differential equations. The kinetics parameters of the equations are estimated using a non-linear least squares methods. The model was used to describe the kinetics of deposition of several ENM (DQ12 Quartz, CeO2, BaSO4 and TiO2) in a single and a repeated dosing experiments.