VITROCELL Systems

Advanced in vitro exposure systems Engineering meets cell biology Catalog 03/2022

Exposure Systems for Inhalation Toxicology Introduction 4 Modules/Continuous Aerosol Flow for 6-well insert sizes System Options 10 6/3, 6/4 and 6/6 CF Stainless Steel 14 6/6 18 6/48 Climatic Chamber 20 for 12-well insert sizes System Options 24 12/3 and 12/4 CF Stainless Steel 26 12/6 Stainless Steel 30 12/6 CF Stainless Steel 32 12/12 36 12/12 Isokinetic Sampling System 40 for 24-well insert sizes 24/24 42 24/48 44 for 35 mm Petri dishes AMES 3 48 AMES 4 50 AMES 48 52 for suspension cells SC 3 56 SC 4 58 Modules/Aerosol Droplet Sedimentation for 6, 12 and 24-well insert sizes Cloud Alpha 6 60 Cloud Alpha 12 62 Cloud SEQ 24 64 Cloud Alpha 96 66 Cloud Alpha PowerVent 68 Cloud Alpha MAX 70 Cloud Advanced Nebulizer 72 Cloud Quartz Crystal Microbalance Sensor 73 Cloud ALI Starter Kit 74 Modules/Powder Sedimentation Powder Chamber 76 Automated Exposure Station Standard Version 78 Extended Version 80 Application Examples 82 Available Configurations 87 Dose Supply Gas supply systems Cylinder/Bag/ Mixing System 89 Spiking System 90 Aerosol Generators 4-Jet BioAerosol Generator 93 VAGF Nebulizing Generator 94 VAGK Nebulizing Generator 95 VRBG Generator 97 Abrasive Dust Generator 98 Smoking Machines VC 1 100 VITROCELL® Shisha Testing Device 106 VC 1 S-TYPE 108 VC 1/7 114 VC 10® 118 VC 10® S-TYPE 124 CHEMCONTROL Feature 136 Human Puff Profiles 138 Vapestarter 139 Comparison VITROCELL® Smoking Machines 140 Index © VITROCELL: Edition 03/2022 2

Dilution / Distribution / Humidification Dilution Systems 142 Distribution Systems for Gases 143 Isokinetic Distribution System 144 HD Distribution Systems 146 Sidestream Chamber 148 Humidification Station 150 Washing Bottle / Inline Humidifers 151 Racks & Carts 2 Modules / 5 Modules / 7 Modules 152 Cart Systems 155 Dose Monitoring Overview 156 Filter Housings 157 Photometer / Software 158 Microbalance Sensors / Software 160 VITROCELL® TEM Grid Holder 166 VITROCELL® CO Monitor / Software 167 VITROCELL® FTIR 168 VITROCELL® Photonion 170 Skin Exposure VITROCELL® Skin 172 VITROCELL® Skin Max 174 VITROCELL® Skin Aerosol Max 176 VITROCELL® Cloud Skin 178 VITROCELL® Skin Autosampler 180 VITROCELL® Skin Punching Tool 182 Auxiliary Equipment VITROCELL® Flow Control 184 VITROCELL® Flow Control Equipment 185 VITROCELL® RH/T-Controller 186 VITROCELL® Clean Air Purification Station 188 VITROCELL® Water Bath 189 VITROCELL® Vacuum Pump 190 VITROCELL® Precision Pump System 192 VITROCELL® EPITHELIX Nanopress 194 VITROCELL® Aerosolist 196 VITROCELL® Leak Tester 197 About VITROCELL® 200 3

VITROCELL® Exposure Systems for Inhalation Toxicology In vitro characterization of the effects of airborne substances When you require a specific solution for your in vitro studies in the field of inhalation toxicology, talk to us with confidence. We have over a decade of experience in designing, producing and installing customized exposure systems specifically for your research needs. VITROCELL® equipment offers you flexible solutions using biological systems for the accurate analysis of these airborne substances: ○ Gases ○ Complex mixtures ○ Nanoparticles ○ Fibers ○ Combustion exhaust ○ Cosmetics ○ Household chemicals ○ Indoor / outdoor air analysis ○ Viruses ○ Industrial chemicals ○ Pesticides ○ Pharmaceuticals ○ Tobacco smoke (whole smoke and gas phase) ○ E-cigarette aerosols Application Areas ○ Direct exposure to monolayer cell cultures, tissue constructs, suspension cells or bacteria ○ Dose-dependent results ○ Highest levels of design and manufacturing quality ○ Tailor-made solutions ○ Installation and application support Features Direct Exposure Technology at Air/Liquid Interface Primary cultures and cell lines, e.g. from the respiratory tract , can be observed in modules specifically designed for direct contact between cells and components of the test atmosphere at the air/liquid interface. 4

Exhaust Dilution / Distribution System Dilution Inlet Vacuum Calibration Valves Medium Supply Temperature Control Vitrocell® Module Exhaust Vacuum Pump In vitro Inhalation Toxicology A typical system flow chart Simulation of the Human Lung System Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems 5

In response to the scientific need to expose in physiologically relevant conditions, VITROCELL® exposure modules have been specifically designed and engineered to enable direct exposure of mammalian cells or tissue at the air/liquid interface where the cell systems are not covered with culture media. Researchers can thus use all cell types cultivated on microporous membranes. This approach allows for more credible and authentic results than by submerged exposure due to a closer replication of the human physiology. ○ No losses ○ No dissolution ○ No reaction of constituents with culture media ○ High sensitivity Optimized flow dynamics The advantages: VITROCELL® Exposure Systems for Inhalation Toxicology Direct Exposure Technology at Air/Liquid Interface 6

A Phases Gaseous ticle Semi-volatile Par The exposure of mammalian cells or tissues to airborne substances is frequently performed under submerged conditions. In doing so, the test substances are dosed into the culture media. This procedure results in an undesired interaction of the formerly airborne substances with the media, causing limitations for authentic analysis. Therefore VITROCELL® recommends the air/liquid interface exposure technology. Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Submerged Cultivation and Exposure in Incubator A Media above cells B Cells on membrane C Media below cells Suspension Cultivation and Exposure in Incubator A Cells in media Air / Liquid Cultivation and Exposure in Exposure Module A Direct and controlled exposure of test atmosphere to cells B Cells on membrane C Media below cells No losses No reaction of principle components with culture media High sensitivity of system Interaction of test components with culture media Low sensitivity Interaction of test components with culture media Low sensitivity Phases Gaseous ticle Semi-volatile Par A B C Phases A B C Gaseous ticle Semi-volatile Par VITROCELL® EXPOSURE AT THE AIR/LIQUID INTERFACE 7

Culture Media Supply Options For VITROCELL® modules The culture media supply may be static (gravimetric/syringe method) or intermittent/continuous („CF“ type modules). VITROCELL® 12 Static culture media supply VITROCELL® 12 CF Static, intermittent or continuous culture media supply VITROCELL® 6 CF Static, intermittent or continuous culture media supply VITROCELL® 6 Static culture media supply 8

In vitro ASSAY GUIDE Examples Category Module type VITROCELL 6 CF VITROCELL 6 Cloud 6 / MAX VITROCELL 12 CF VITROCELL 12/6 VITROCELL 12/6 CF VITROCELL 12/12 Cloud 12 VITROCELL 24 Cloud 24 VITROCELL AMES VITROCELL SC Number of inserts 3, 4 or 6 6 or 48 3 or 4 6 6 12 24 or 48 3, 4 or 48 3 or 4 Insert type (size) 6-well 6-well 12-well 12-well 12-well 12-well 24-well 35 mm Petri Dish 35 mm Petri Dish Culture Media Supply Continuous Static Continuous Static Continuous Static Static -- -- Cytotoxicity Number of viable cells x x xx xx xx xx xx – – LDH release xx xx xx xx xx xx x – – NRU uptake x x xx xx xx xx xx – – MTT x x xx xx xx xx xx – – XTT x x xx xx xx xx xx – – MTS x x xx xx xx xx xx – – Proliferation WST-1 x x xx xx xx xx xx – – Protein level x x xx xx xx xx xx – – Cellular Stress ATP xx xx x x x x x – – Cellular ATP/ADP ratio xx xx x x x x x – – GSH xx xx x x x x x – – GSSG xx xx x x x x x – – GSSG/GSH xx xx x x x x x – – Oxidative Stress Lipid peroxidation (MDA test) xx xx x x x x x – – Inflammation Cytokines like IL8, IL6, IL12 x x xx xx xx xx x – – Genotoxicity Comet assay x x xx xx xx xx – – – AMES/number of revertants – – – – – – – xx – "omic“ technologies ARN Sample for microarray xx xx – – – – – – – ARN Sample for QRT PCR xx xx – – – – – – – Molecular Biology Protein Sample for Western Blot xx xx – – – – – – – Assays for Suspension Cells – – – – – – – – xx Legend: x = well suited xx = particularly well suited 9

VITROCELL® 6/3 CF VITROCELL® 6/4 CF and 6/3 CF B VITROCELL® 6/6 System Options for 6-well insert sizes VITROCELL® 6 system setup with a number of different module options In this configuration 2 VITROCELL® 6/3 CF modules are used: One module houses 3 compartments for exposure, a secound VITROCELL® 6/3 module takes care with it ’s 3 compartments for clean air control. In this configuration there is the option to asses 3 different doses @ 3 replicates plus one row of compartments @ 3 replicates for clean air control. One compartment for each row for exposure may be equipped with a Quartz Crystal Microbalance sensors for validation or monitoring purposes. In one module 4 compartments are used for exposure to the substances and in another module 3 compartments for clean air control. 1–4 compartments of the exposure module may be equipped with Quartz Crystal Microbalance sensors for validation or monitoring purposes. C A A 10

Delivery Content Vacuum Pump 1 The vacuum diaphragm pumps supplied by VITROCELL® have been developed and manufactured to be chemically resistant . Thus, they are suitable for aggressive or corrosive gases and vapors alike. The dry-running devices are specifically developed for laboratory applications and are maintenancefree. The pump exhaust line must be guided to a laboratory hood for proper extraction. Flow Meter Mass flow meters are required to adjust the vacuum flow by means of vacuum calibration valves and to accurately set dilution and aerosol generator air flows. They are available in various ranges. 3 4 Flow Controller Mass flow controllers have been specifically designed to regulate dilution and aerosol generator airflow precisely. They are available various l/min. ranges. Vacuum Calibration Valve VITROCELL® flow calibration valves regulate the flow of the aerosol through the modules in the ml range. The valves are located between the vacuum pump and the aerosol exposure top. Mass flow meters enable accurate valve read-outs. The consistent and adjustable flow rate ranges from 0…20 ml/min. Alternative flow rate ranges are available upon request . The valves enable fast control of the flow rates prior to the experiment . They are manufactured from Teflon/stainless steel and can be easily cleaned. If the aerosol contains particles, we recommend installing disposable microfiber filters in front of the valves. 2 Water Bath VITROCELL® uses water baths to efficiently heat and pump water through the circuit of the exposure modules, ensuring accuracy in temperature control with a modern state-of-the-art design. These units provide best-inclass economical performance while remaining user-friendly by reducing unnecessary functions and focusing on reliability. Engineered from highestquality components and materials, such as stainless steel for the baths, durable heating thermostats with a long operation life are guaranteed. 5 Option: Quartz Crystal Microbalance Sensor System VITROCELL® Microbalance Sensor – optimal for dose-response measurement . With the advanced VITROCELL® software, deposition data in ng/cm2 is attainable online. 6 Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems 11

○ Suitable for COSTAR®, FALCON® and ThinCert® 6-well sized cell culture inserts ○ Adaptors for 12 and 24-well sized inserts ○ Individual media inlets and outlets for each cell culture insert compartment ○ Special inlet geometry of the aerosol exposure top for optimal particle deposition ○ Inlets made of stainless steel with a unique VITROGLIDE surface treatment ○ Base module made of electropolished stainless steel ○ Base module and aerosol exposure top with integrated water heating circuit ○ Control window for monitoring culture media levels ○ Autoclavability of all components ○ Suitable for VITROCELL® Quartz Crystal Microbalance Features 7 Option: High voltage charging for enhanced particle deposition The VITROCELL® 6/3, 6/4 and 6/6 CF modules can be equipped with electrodes for high voltage charging to increase particle deposition efficiency significantly. Increased particle deposition efficiency by electrical field 12

VITROCELL® 6/3, 6/4 and 6/6 CF Stainless Steel Exposure Module Universal test platform for 3, 4 or 6 cell culture inserts (6-well size) Normally two modules are required: one module for exposure to the substances and a second module for clean air control. The capacity can be easily increased by adding more modules. Each module can receive a different dose concentration, so that a complete dose/ response profile is obtained in one experiment . The cells are exposed at the air/liquid interface on cell culture inserts using low flow rates of the aerosol. After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). The VITROCELL® 6 CF module system has been specifically designed and engineered to facilitate the research of mammalian cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Culture media supply The media is supplied to the module by gravimetric method, syringe or using a media pump for intermittent /continuous media exchange. 14

Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped inlets. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nanoparticles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit , ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum. Aerosol exposure top Universal Module with optional adaptor sets for: ○ 12-well sized inserts ○ Snapwell® inserts ○ 24-well sized inserts ○ 35 mm Petri dishes (AMES test) Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Base module The base module is made of electropolished stainless steel for maximum durability and is fully autoclavable for 20 min. at 120° C and higher temperatures. A constant temperature within the unit (37° C / 99° F) is assured via water circuit heating. Therefore, an additional incubator is not required during exposure. The media are supplied individually to each well compartment . Optionally, media exchange can be carried out on a continuous basis using a precision media pump. This module is renowned in scientific circles for its reliability and durability. Cell culture insert (6-well size) Optional media inserts for media volume reduction units (10.8 ml) (static supply only for easier maintenance) Optional microbalance sensor for dose monitoring 15

Option: High voltage charging for enhanced deposition ○ Suitable for COSTAR®, FALCON® and ThinCert® 6-well sized cell culture inserts ○ Adaptors for 12 and 24-well sized inserts ○ Individual media inlets and outlets for each cell culture insert compartment ○ Special inlet geometry of the aerosol exposure top for optimal particle deposition ○ Inlets made of stainless steel with a unique VITROGLIDE surface treatment ○ Base module made of electropolished stainless steel ○ Base module and aerosol exposure top with integrated water heating circuit ○ Control window for monitoring culture media levels ○ Autoclavability of all components ○ Suitable for VITROCELL® Quartz Crystal Microbalance Features The VITROCELL® 6/3, 6/4 and 6/6 CF modules can be equipped with electrodes for high voltage charging to increase deposition efficiency significantly. VITROCELL® 6/3 CF VITROCELL® 6/4 CF VITROCELL® 6/6 CF Options for 3, 4 and 6 cell culture compartments 16

The VITROCELL® 6 modules can be equipped with microbalance sensors.

VITROCELL® 6/6 Exposure Module For 6 cell culture inserts (6-well size) The VITROCELL® 6/6 module system has been specifically designed and engineered to facilitate the research of mammalian cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Normally only one system is required: 1 dilution with 3 inserts is used for for exposure to the substances and 3 inserts in the same system for clean air control. The cells are exposed at the air/ liquid interface on 6 cell culture inserts using low flow rates of the aerosol. After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). 1 dose @ 3 replicates 1 control @ 3 replicates Compact Design Culture media supply The media is supplied to the module by gravimetric method, syringe or using a media pump for intermittent / continuous media exchange. 18

Base module Cell culture insert (6-well size) Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped inlets. They are available in stainless steel or stainless steel with unique VITROGLIDE surface Aerosol exposure top treatment for lowest adhesion results. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nanoparticles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit , ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum. Optional media volume reduction units (3 ml) (static supply only) Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Optional microbalance sensor for dose monitoring The base module has been designed for 6 cell culture inserts. The media are supplied individually to each well compartment . Optionally, media exchange can be carried out on a continuous Available adaptor set for: ○ 12-well sized inserts ○ Snapwell® inserts ○ 24-well sized inserts basis using a precision media pump. Temperature is electronically controlled at 37° C (99° F) by means of a heated base plate. Therefore, an additional incubator is not required while using this module. The base module is made of electropolished stainless steel. It is autoclavable at 121° C (250° F) for 20 min. This module is renowned in scientific circles for its reliability and durability. Features ○ Suitable for COSTAR®, FALCON® and ThinCert® 6-well sized cell culture inserts ○ Individual media inlets for each row of 3 insert compartments ○ Base module made of stainless steel for maximum durability ○ Control window for monitoring culture media levels ○ Hyperboloid geometry of inlets for optimized particle deposition and distribution ○ Available inlet materials: Stainless steel, stainless steel with unique VITROGLIDE surface treatment ○ Heating base plate with electronic control ○ An additional incubator is not required while using this module ○ Autoclavable at 121° C (250° F) 19

7 doses @ 6 replicates 1 control @ 6 replicates Higher Throughput VITROCELL® 6/48 Climatic Chamber Exposure Module For 48 cell culture inserts (6-well size) The VITROCELL® 6/48 module system has been specifically designed and engineered to facilitate the research of mammalian cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Only one system is required: 7 dilutions with 6 inserts are used for exposure to the substances and 6 inserts in the same system for clean air control. The cells are exposed at the air/ liquid interface on 48 cell culture inserts using low flow rates of the aerosol. A complete dose / response profile can be obtained in one experiment . After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). Easy access to all components Heating Systems for base module and climatic chamber to avoid effects of aerosol condensation. 20

Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Features Base module Aerosol exposure top inuscimporro Base module The base module has been designed for 48 cell culture inserts. The media is supplied individually to each well compartment . Temperature is electronically controlled at 37° C (99° F) by means of a heated base plate. Therefore, an additional incubator is not required while using this module. The base module is made of aluminum with media compartments of stainless steel. It is autoclavable at 121° C (250° F) for 20 min. This module is renowned in scientific circles for its reliability and durability. The docking station with special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped inlets. They are available in stainless steel or stainless steel with unique VITROGLIDE surface treatment for lowest adhesion results. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nanoparticles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit , ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum and titanium. ○ Suitable for COSTAR®, FALCON® and ThinCert® 6-well sized cell culture inserts ○ Base module compartments made of stainless steel for maximum durability ○ Hyperboloid geometry of inlets for optimized particle deposition and distribution ○ Available inlet materials: stainless steel, stainless steel with unique VITROGLIDE surface treatment ○ Heating base plate with electronic control ○ An additional incubator is not required while using this module ○ Option: VITROCELL® AMES 6/48 Plus with 7th row for dosimetry (for liquid trapping or microbalance) ○ Autoclavable at 121° C (250° F) Docking station Drawer and locking mechanism Cell culture insert (6-well size) Available adaptor set for: ○ 12-well sized inserts ○ Snapwell® inserts ○ 24-well sized inserts 21

Exposure module with 7th row on drawer Optional microbalance sensor in 7th position VITROCELL® 48 PLUS with 7th row for liquid trapping or microbalance Refined detail solutions Vacuum quick connector strips for fast exchange of exposure tops with alternative inlet sizes Heavy duty column guided lifting-closing mechanism for precise dosing Dimensions: 1 280 x 750 x 800 mm (L x W x H), Height opened: 1 115 mm Weight : 200 kg Voltage: 110–240 V / 50 Hz Rating: 1 200 W (max) Fuse: 2 x 6.3 A Technical Data 7 doses @ 7 replicates 1 control @ 7 replicates For Dosimetry 22

VITROCELL® 12/4 CF and 12/3 CF VITROCELL® 12/6 CF 2 VITROCELL® 12/12 System Options for 12-well insert sizes VITROCELL® 12 system setup with a number of different module options In this configuration 2 seperate module are used: The VITROCELL® 12/4 CF module houses 3 compartments for exposure plus one for the Quartz Crystal Microbalance sensor. A secound VITROCELL® 12/3 module takes care with it ’s 3 compartments for clean air control. In this configuration there is the option to asses 3 different doses @ 3 replicates plus one row of compartments @ 3 replicates for clean air control. One compartment for each row for exposure may be equipped with a Quartz Crystal Microbalance sensors for validation or monitoring purposes. In this module 3 compartments are used for exposure to the substances and 3 compartments for clean air control. 1-3 compartments for exposure may be equipped with Quartz Crystal Microbalance sensors for validation or monitoring purposes. 3 1 1 24

Delivery Content Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Vacuum Pump 1 The vacuum diaphragm pumps supplied by VITROCELL® have been developed and manufactured to be chemically resistant . Thus, they are suitable for aggressive or corrosive gases and vapors alike. The dry-running devices are specifically developed for laboratory applications and are maintenancefree. The pump exhaust line must be guided to a laboratory hood for proper extraction. Flow Meter Mass flow meters are required to adjust the vacuum flow by means of vacuum calibration valves and to accurately set dilution and aerosol generator air flows. They are available in various ranges. 3 4 Flow Controller Mass flow controllers have been specifically designed to regulate dilution and aerosol generator airflow precisely. They are available various l/min. ranges. Vacuum Calibration Valve VITROCELL® flow calibration valves regulate the flow of the aerosol through the modules in the ml range. The valves are located between the vacuum pump and the aerosol exposure top. Mass flow meters enable accurate valve read-outs. The consistent and adjustable flow rate ranges from 0…20 ml/min. Alternative flow rate ranges are available upon request . The valves enable fast control of the flow rates prior to the experiment . They are manufactured from Teflon/stainless steel and can be easily cleaned. If the aerosol contains particles, we recommend installing disposable microfiber filters in front of the valves. 2 Water Bath VITROCELL® uses water baths to efficiently heat and pump water through the circuit of the exposure modules, ensuring accuracy in temperature control with a modern state-of-the-art design. These units provide best-in-class economical performance while remaining user-friendly by reducing unnecessary functions and focusing on reliability. Engineered from highestquality components and materials, such as stainless steel for the baths, durable heating thermostats with a long operation life are guaranteed. 5 Option: Quartz Crystal Microbalance Sensor System VITROCELL® Microbalance Sensor – optimal for dose-response measurement . With the advanced VITROCELL® software, deposition data in ng/cm2 is attainable online. 6 25

VITROCELL® 12/3 and 12/4 CF Stainless Steel Exposure Module For 3 or 4 cell culture inserts (12-well size) Normally two modules are required: one module for exposure to the substances and a second module for clean air control. The capacity can be easily increased by adding more modules. Each module can receive a different dose concentration, so that a The VITROCELL® 12 CF module system has been specifically designed and engineered to facilitate the research of human cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. complete dose/ response profile is obtained in one experiment . The cells are exposed at the air / liquid interface on 3 cell culture inserts using low flow rates of the aerosol. After exposure, the cells are further processed to Culture media supply The media is supplied to the module by gravimetric method, syringe or using a media pump for intermittent /continuous media exchange. measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). 26

This modules’ casing is made of electropolished stainless steel. It is designed with three or four compartments for 12-well cell culture inserts and is fully autoclavable at 121° C (250° F) for 20 min. Through the employment of a special adapter set , this module can also be flexibly combined for use with 24-well cell culture inserts. A constant unit temperature is guaranteed using a regulated flow of temperature-controlled water through the module. Aerosol exposure top Base module Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped Cell culture insert (12-well size) inlets. They are available in stainless steel or stainless steel with unique VITROGLIDE surface treatment for lowest adhesion results. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nanoparticles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit, ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum. The media can be supplied individually to each well compartment . Flexible media exchange can be static, intermittent or continuous basis. This flexibility is afforded by use of a precision media pump. This module is renowned in scientific circles for its reliability and durability. Available adaptor set for: ○ 24-well sized inserts Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems 27

○ Suitable for COSTAR®, FALCON® and ThinCert® 12-well sized cell culture inserts ○ Autoclavability of all components ○ Base module casing made of electropolished stainless steel for extreme durability ○ Base module and aerosol exposure top with integrated heating circuits ○ Hyperboloid geometry of inlets for optimized particle deposition and distribution ○ Longer exposure cycles possible due to permanent media supply option ○ Control window for monitoring culture media levels ○ Option: Quartz Crystal Microbalance Features Option for 4 cell culture compartments Option for microbalance sensor VITROCELL® 12/4 CF VITROCELL® 12/4 CF base module The VITROCELL® 12/3 and 12/4 CF modules can be equipped with microbalance sensors for dose monitoring. Media insert for VITROCELL® 12 series Optional media inserts for easier cleaning. Media volume 2.5 ml 28

VITROCELL® 12/6 Stainless Steel Exposure Module for Static Culture Media Supply For 6 cell culture inserts (12-well size) The VITROCELL® 12/6 module system has been specifically designed and engineered to facilitate the research of human cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Culture media supply The media is supplied to the module by gravimetric method or syringe. VITROCELL® 12/6 with dilution system Normally two modules are required: one module for exposure to the substances and a second module for clean air control. The capacity can be easily increased by adding more modules. Each module can receive a different dose concentration, so that a complete dose/ response profile is obtained in one experiment . The cells are exposed at the air / liquid interface on 6 cell culture inserts using low flow rates of the aerosol. After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). 30

○ Suitable for COSTAR®, FALCON® and ThinCert® 12-well sized cell culture inserts ○ For higher troughput ○ Autoclavability of all components ○ Base modules’ casing made of electropolished stainless steel for extreme durability ○ Base module with water heated base plate ○ Integration of the hyperboloid geometry of inlets into the aerosol exposure top ○ Dynamic dilution system part of delivery ○ Option: Control window for monitoring culture media levels Features The VITROCELL® 12/6 modules are made of electropolished stainless steel. They are designed with six compartments for 12-well cell culture inserts and are fully autoclavable at 121° C (250° F) for 20 min. A transparent control window facilitates the external monitoring of media levels. Through the employment of a special adapter set , this module can also be flexibly Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped inlets. They are available in stainless steel or stainless steel with unique VITROGLIDE surface treatment for lowest adhesion results. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nanoparticles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit , ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum. combined for use with 24-well cell culture inserts. A constant unit temperature is guaranteed using a regulated flow of temperature-­ controlled water through the module. The media can be supplied gravimetrically using a bottle or syringe. This module is renowned in scientific circles for its reliability and durability. Base module Aerosol exposure top with central aerosol supply Available adaptor set for: ○ 24-well sized inserts Cell culture insert (12-well size) Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems 31

Culture media supply The media is supplied to the module by gravimetric method, syringe or using a media pump for intermittent /continuous media exchange. VITROCELL® 12/6 CF Stainless Steel Exposure Module For 6 cell culture inserts (12-well size) The VITROCELL® 12/6 CF module system has been specifically designed and engineered to facilitate the research of human cell cultures in direct exposure to airborne substan- ces such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Normally 3 compartments are used for exposure to the substances and 3 compartments for clean air control. The capacity can be easily increased by adding more modules. Each module can receive a different dose concentration, so that a complete dose/ response profile is obtained in one experiment . The cells are exposed at the air/liquid interface on 6 cell culture inserts using low flow rates of the aerosol. After expo- sure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). VITROCELL® 12/6 CF Dynamic Dilution System and rack 32

Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped inlets. They are available in stainless steel or stainless steel with unique VITROGLIDE surface treatment for lowest adhesion results. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nano-particles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit , ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum. Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Aerosol exposure top with individual aerosol inlets Base module The VITROCELL® 12/6 CF modules casing is made of electropolished stainless steel. It is designed with six compartments for 12-well cell culture inserts and is fully autoclavable at 121° C (250° F) for 20 min. Through the employment of a special adapter set , this module can also be flexibly combined for use with 24-well cell culture inserts. A constant unit temperature is guaranteed using a regulated flow of temperature-controlled water through the module. The media are supplied individually to each well compartment . Optionally, media exchange can be carried out on a continuous basis using a precision media pump. This module is renowned in scientific circles for its reliability and durability. Suitable for six cell culture insert (12-well size) – adaptor set for 24-well sized inserts available. The module can easily be locked hermetically with the two levers attached to the base. 33

The QCM sensor can be integrated in the VITROCELL® 12/6 CF exposure module. It is capable of measuring the deposited mass at a resolution of 10 nanogram/cm2 per second. Results are reported online by the VITROCELL® Monitor software. Data is presented in graphs and stored in MS Excel ®. The system can be combined with a separate single module for dosimetry. The dosimetry module can accomodate a stainless steel insert for trapping constituents of the aerosol in liquids. Alternatively it can be equipped with a microbalance sensor. A photometer can be added either directly in the aerosol flow of the Exposure Module or at a seperate port of the main aerosol flow. Option for microbalance sensor System available in different setups for dosimetry ○ Suitable for COSTAR®, FALCON® and ThinCert® 12-well sized cell culture inserts ○ For higher troughput ○ Autoclavability of all components ○ Base module made of electropolished stainless steel for extreme durability ○ Base module with water heating base plate ○ Hyperboloid geometry of inlets for optimized particle deposition and distribution ○ Option: Quartz Crystal Microbalance Features Rack with Dosimetry Module and Photometer connected to Exposure Module. Rack with Dosimetry Module and Photometer connected to the main aerosol flow. 34

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VITROCELL® 12/12 Exposure Module For 12 cell culture inserts (12-well size) The VITROCELL® 12/12 module system has been specifically designed and engineered to facilitate the research of human cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Normally only one system is required: 3 dilutions with 3 inserts each are used for for exposure to the substances and 3 inserts in the same system for clean air control. Each dilution represents a different dose concentration, so that a complete dose/ response profile can be obtained in one experiment . The cells are exposed at the air / liquid interface on 12 cell culture inserts using low flow rates of the aerosol. After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). Culture media supply The media is supplied to the module by gravimetric method or syringe. 3 doses @ 3 replicates 1 control @ 3 replicates Compact Design 36

○ Suitable for COSTAR®, FALCON® and ThinCert® 12-well sized cell culture inserts ○ Autoclavability of all components ○ Base module made of electropolished stainless steel for extreme durability ○ Electronic heating system ○ Base module and aerosol exposure top with integrated heating circuit ○ Hyperboloid geometry of inlets for optimized particle deposition and distribution ○ Control window for monitoring culture media levels Features Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Aerosol exposure top Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlet-stream flows through specifically shaped inlets. They are available in stainless steel or stainless steel with unique VITROGLIDE surface treatment for lowest adhesion results. The stainless steel / VITROGLIDE inlets are specifically designed for aerosols containing nanoparticles. The aerosol inlets are connected to the distribution or dilution systems. Extraction from the module takes place via small holes using a controlled vacuum flow. VITROCELL® calibration valves, which are connected to a vacuum pump, control flow rates effectively. The aerosol exposure top features an in/outlet for heating the water circuit , ensuring corresponding temperatures with the base module. It is made of high-quality anodized aluminum. Base module The VITROCELL® 12/12 modules’ casing is made of electropolished stainless steel. It is designed with 12 (4x3) compartments for 12-well cell culture inserts and is fully autoclavable at 121° C (250° F) for 20 min. The capability to expose with up to 4 dilutions or 3 dilutions plus 1 clean-air control in a compact module is its’ outstanding feature. A transparent control window facilitates the external monitoring of media levels. Through the employment of a special adapter set , this module can also be flexibly combined for use with 24-well cell culture inserts. A constant unit temperature is guaranteed using an electronic temperature control. The media can be supplied individually for each row of 3 compartments. This module is renowned in scientific circles for its reliability and durability. Cell culture insert (12-well size) Available adaptor set for: ○ 24-well sized inserts Optional microbalance sensor for dose monitoring 37

VITROCELL® 12/12 Docking Station Easy connection to distribution and dilution systems VITROCELL® 12/12 quick connect system replaces the mounting of tubing material. Vitrocell 12/12 Base Module VITROCELL® 12/12 incl. rack, docking station, quick-connect system, distribution/dilution systems and vacuum valves. ○ Complete rack system for fixation of components ○ Docking station with sliding base for fast and safe connections ○ Quick-connect system for distribution/ distribution system Features 38

Easy access to all components. Components can be separated with ease. Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems VITROCELL® 12/12 Climatic Chamber The professional solution to avoid condensation during the exposure to liquid aerosols System includes rack, docking station, quick-connect system, dilution systems and optional flow controllers for dilution air. ○ Dimensions (L x W x H): 1 280 x 900 x 870 (open 1 350) mm ○ Supplied with dynamic double-jet dilution system for 4 dilutions with 3 replicates ○ Integrated aerosol distribution system made of stainless steel /VITROGLIDE surface ○ Climatic Chamber with temperature monitoring ○ Unique quick-connect system for easier handling Features 39

VITROCELL® 12/12 Isokinetic Sampling System For uniform particle distribution – optional dilution systems 3 doses @ 3 replicates 1 control @ 3 replicates Compact Design Rack with Isokinetic Sampling and Dilution System and optional Microbalance Modules The aerosol is guided from the central duct to the modules via isokinetic sampling probes enabling high reproducibility and uniformity of results. The inlets aspirate directly from the constant flow of test substance.The isokinetic distribution system is made of stainless steel. The optional dilution systems are equipped with air jets for dynamic dilution to obtain different dose / response results per row @ 3 replicates. 40

○ Distribution from the source of the test substances to the module inlets ○ High reproducibility ○ Optional dynamic dilution system Features Integration of the controllers into the VITROCELL® Monitor Software for easy setting of target flows and reporting of actual flows. VITROCELL® Flow Monitor Software Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems Small flange connections for easy disassembly and cleaning. Isokinetic Sampling probes made of stainless steel. Dynamic dilution system with air jets featuring a constant flow rate over the entire system. The optional microbalance sensor is capable of measuring the deposited mass in seperate VITROCELL® 12/1 Modules at a resolution of 10 nanogram/cm2 per second. Back view – Isokinetic Sampling and Dilution System and optional Microbalance Modules 41

VITROCELL® 24/24 Exposure Module For 24 cell culture inserts (24-well size) The VITROCELL® 24/24 module system has been specifically designed and engineered to facilitate the research of human cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. 6 dilutions and controlled guidance of the aerosol to 4 wells per dilution make the VITROCELL® an efficient and extremely powerful analysis tool. Normally only one system is required: 5 dilutions with 4 inserts each are used for for exposure to the substances and 4 inserts in the same system for clean air control. Each dilution represents a different dose concentration, so that a complete dose/ response profile can be obtained in one experiment . The cells are exposed at the air / liquid interface on 24 cell culture inserts using low flow rates of the aerosol. After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). Culture media supply The media is supplied to the module by gravimetric method or syringe. 5 doses @ 4 replicates 1 control @ 4 replicates Compact Design 42

○ Supplied with dynamic double-jet dilution system for 6 dilutions with 4 replicates ○ 24 media compartments for COSTAR®, FALCON® and ThinCert® inserts in 24-well size ○ Base module made of electropolished stainless steel for extreme durability ○ Individual aerosol supply to each cell culture membrane ○ Base module and aerosol exposure top are autoclavable ○ Integrated, sensor-controlled heating plate ○ Option: Control window for monitoring culture media levels Features Base module The base module is made of electropolished stainless steel. An optional transparent control window facilitates the external monitoring of media levels and may be easily removed from the integrated heating base. Temperature can be programmed individually. This module is renowned in scientific circles for its reliability and durability. Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlets are already connected to the integrated distribution system. Evacuation from the module takes place via small holes using an individually controlled vacuum flow for each insert . The base module is made of electropolished stainless steel. It is autoclavable at 20 min. /121° C (250° F). Cell culture inserts (24-well size) Aerosol exposure top Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems 43

VITROCELL® 24/48 Exposure Module For 48 cell culture inserts (24-well size) The VITROCELL® 24/48 module system has been specifically designed and engineered to facilitate the research of human cell cultures in direct exposure to airborne substances such as gases, complex mixtures, nanoparticles and fibers. The system authentically simulates the conditions of human physiological exposure. Normally only one system is required: 7 dilutions with 6 inserts each are used for for exposure to the substances and 6 inserts in the same system for clean air control. Each dilution represents a different dose concentration, so that a complete dose/ response profile can be obtained in one experiment . The cells are exposed at the air / liquid interface on 48 cell culture inserts or on a HTS plate using low flow rates of the aerosol. After exposure, the cells are further processed to measure a wide range of endpoints, e. g. cytotoxicity, genotoxicity, proliferation, cellular and oxidative stress as well as inflammation (see also VITROCELL® assay guide). Culture media supply The media is supplied to the module by gravimetric method or syringe. VITROCELL® 24/48 incl. rack , locking device and dilution systems 7 doses @ 6 replicates 1 control @ 6 replicates Higher Throughput 44

Base module with unique locking device The base module is made of electropolished stainless steel. Temperature can be programmed individually. Special sealing and connection mechanisms guarantee a hermetic connection of the base module with the aerosol exposure top. The aerosol inlets are already connected to the integrated distribution system. Evacuation from the module takes place via small holes using an individually controlled vacuum flow for each insert . The flow rate is controlled by the VITROCELL® calibration valves which are connected to a vacuum pump. The aerosol exposure top with the integrated aerosol distribution system is made The base module is made of electropolished stainless steel and autoclavable at 20 min. /121° C (250° F). This module is renowned in scientific circles for its reliability and durability. Cell culture inserts (24-well size) Aerosol exposure top of stainless steel with VITROGLIDE surface treatment . The dynamic dilution system for up to 8 dilutions is part of the delivery. Skin Exposure Auxiliary Equipment Dose Monitoring Racks & Carts Dilution Dose Supply Exposure Systems 45

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