By Corporate Marketing Dept. - Buguggiate, Italy -

Biocontainment applications – BSL3/4 lab Bioexclusion applications – Germfree, SPF, gnotobiotic

GermFree/SPF mice/compromised Immune System Bioexclusion Application in genetics, cancer, infective diseases, gastro……

Possible choices for mice housing and bioexclusion: Isolators IVCs

- High degree of protection for animals and users - Easy to install - Validation process: available - Very consistent negative/positive pressure achievable - Very low Cage density - Ergonomically poor - All animals within different cages are in “contact”. - Risk for canopy/gloves puncture Isolators advantages… … and disadvantages

Germ-Free and Isolators Germ-free are bred and stocked in Isolators In case one mouse is contaminated, ALL THE ISOLATOR IS “LOST”

Germ-Free and Isolators Germ-free are frequently differentiated to run experiment Moved in dedicated isolators

Improved Animal Environment Flexibility For (+)ve or (-)ve Operation Higher Density Separation Between Cages (no cross-contamination) Traditional IVC advantages… … and disadvantages No Cage Level HEPA Filtration Can’t Maintain (-)ve or (+) Pressure Off The Rack

Bioexclusion applications Fully Sealed Cage HEPA filter at cage level Pressure Driven Blowers Positive pressure Mode

Germ – Free and Iso Cage IsoCage P protects all the group in case one mouse is contaminated Isocage P means: - more experiments - less space - more flexibility - more research

Germ-Free and Research GF 1 GF 2GF 3 TG A TG B TG C 1A2A3A 1B2B 3B 1C 2C3C A huge possibility to combine germ-free strains with trangenic strains, and create infinite combinations of unique model to study 9 Isolators or 1 Isocage Rack

Isolator at cage level HEPA filter protection at cage level Safety closure clamps to prevent accidental opening, with locking system Hermetic cage with single piece silicon gasket between cage top and cage bottom High resistant polyphenylsulfone (PPSU) plastic guaranteed 8 years for cage bottom and top. Nylon plastic components for clamps, nozzles and HEPA filter grid

Isolator at cage level Self-closing nozzles with double gasket to prevent air exchange when the cage is moved from the rack Nozzles located at the top of the cage (lid) and not an the bottom, to avoid air drafts to the animals and prevent clogging of the valves due to bedding or dirt Positive pressure of 100Pa assured even when the cage is removed from the rack, for at least 15 minutes

14 X-Temp Internal bottle HEPA Filter on the supply Safe closure clamps Silicone gasket Double gasket, self closing nozzles Air nozzles on the top

Air Handling Unit Positive pressure (blower) Two HEPA filters H14, supply & exhaust Pre-filters G4 2 twin ECM DC-motors working simultaneously -45% of power consumption = GREEN POWER DOP certified and able to be tested at user site UPS system ensures safety for animals and operators Certified performance on 100% of AHUs Battery running time > 24 h = IMPROVED RELIABILITY

Air Handling Unit Alarms: pressure, Airflow, working time/life span for supply and exhaust HEPA filters, UPS battery low, UPS faulty User friendly touch screen Compatible with thimble for air exhaust out of the building Monitor cage with pressure indicator Double HEPA filtration (AHU and cage level) of supplied air Data Log and Error Log via USB key = PRECISION New 7’’ touch screen vertical type = IMPROVED ERGONOMICS

Available Test Reports Low Air speed at animal level = BELOW 0.2 m/s, COMPLIANT WITH GUIDELINES Smoke test proving intra-cage gas removal = COMPLETE SMOKE REMOVAL IN 3 MIN. Equal Pressure distribution across the rack = MAX DIFFERENCE ± 10 % Long-lasting battery = MUCH MORE THAN 24 h Silent = LESS THAN 50 dB Twin Blower for redundancy = ACHIEVED SET PRESSURE EVEN WITH JUST ONE BLOWER

Biological validation Aim: Validate the complete bioexclusion ensured by ISOcage P system. Method 100µl of cell suspension, with at least 10 6 viable cells (C. difficile for group 1 and E. coli for group 2) is placed on the nozzle of the rack (either inlet or outlet) randomly across the ISOCAGE P unit. A sterile cage containing germ free mice is placed onto the infected nozzles and left for 7 days untouched. The mice are then killed by schedule 1, organs (Ceacum) removed under sterile conditions and plated out on appropriate media. Colonies are counted following incubation overnight. Results Only in the positive controls (cages with microorganism directly exposed to the mice) the mouse was heavily colonized with the respective organism (positive control). No other mice were colonized with either of the 2 bacteria. The result was replicated in a further experiment which involved daily cage removal from the rack. Again, no mice were colonized except the positive controls.

A comparison between Isocage and 3 different Isolators was made to evaluate operational parameters

Cages/m²

Cage changes / operator

Additional Reasons for ISO CAGE Use Ergonomics: Flexibility: Hygiene:  Easy manoeuvrability of ISO Cage Systems  Cages and racks fully auto- clavable, H 2 O 2 decontamination of AHU; only chemical decontamination for Isolators (peracetic acid)  No need to work with Isolator Glove Box

Conclusions An effective barrier suitable for bioexclusion Complete Isolation at cage level Safe AHU Flexibility of use, ergonomics Increases density/capacity of the animal rooms Increases the operational efficiency and reduces the time per cage change Meets the advantages of IVC systems, ensuring improved protection for animals

Thank you