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This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.
Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations.
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.
Germ-free mice are an essential tool in the repertoire of microbiome researchers, allowing one to dissect the contribution of the microbiota in host health and disease states. Germ-free mice are born completely sterile and remain axenic for their entire lives1. Colonization of germ-free mice with specific bacterial strains enables causative studies between those taxa and metabolic, immune, or other host functions2,3,4,5. Particularly advantageous is the ability to "humanize" germ-free mice at the level of the microbiota by transplanting feces obtained from human donors and, when housed in barrier conditions, prevent contamination from murine-derived microorganisms1. This approach has enabled many important discoveries in the field of microbiome, for instance, the effect of the human gut microbiome on cancer immunotherapy response6,7,8.
However, while humanized germ-free mice are invaluable to research efforts in the microbiome field, there are many limitations that have inhibited the wider adaptation of this approach. Germ-free mice are bred and maintained in semi-rigid or flexible-film large isolators, but functional experiments require separate mini-isolators to be set up, with one mini-isolator housing several cages but only under one experimental condition. This mini-isolator approach increases the space footprint and cost while severely limiting the number of experimental conditions that can be investigated in an experiment and the number of experiments that can be run in parallel. A promising solution is using an individual, modular caging system called the ISOcage P Bioexclusion System (here referred to as isocage system)9,10. The isocage system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages, enabling separate experimental conditions between each cage rather than between each mini-isolator. With the proper aseptic technique, animals can be housed in isocages for up to 12 weeks under germ-free conditions or humanized by human fecal transplant for use in any compatible experimental approach (i.e., can be performed under aseptic conditions). Multiple independent experiments can be run in parallel using the isocage system, and the space footprint and cost are dramatically less than running multiple experiments across mini-isolators.
The purpose of breeding germ-free mice in flexible film breeding isolators is to carefully preserve axenic status11. Techniques used to monitor germ-free status include routine swabs of mouse body surfaces and oral cavities, as well as the aseptic collection of fecal samples, which are both cultured and tested by PCR-based commercial assays. Bacterial, serological, and fungal testing of these samples are all required to determine germ-free status11. When germ-free mice are transferred from breeding isolators to isocages for experimental usage, the mice are swabbed and tested to validate their germ-free status upon transfer. Isocage sterility checks are performed through aseptic collection of fecal samples, which are then cultured for detection of bacterial, viral, and fungal contaminants. Carefully collecting and recording the results of these sterility checks from birth to the end of an experimental protocol is necessary to validate the germ-free status of these mice.
The isocage system is composed of individual cages (Figure 1), transfer disks for transport out of breeding isolators (Figure 1), and the isocage rack, which houses the cages (Figure 2). Each isocage contains a cage-level high-efficiency particulate air (HEPA) filter installed on the supply air intake and a silicone gasket which makes an airtight seal when closed, ensuring no contaminants can enter the cage through the air (Figure 1A). This cage lid can be used as a sterile working surface when placed upside down within a sterilized biosafety cabinet (Figure 1A). A wire rack within the cage holds the food and water bottle (Figure 1B). Forceps autoclaved within the cage are used for all manipulations that require contact with interior cage surfaces. The cage itself has notches for a removable cage card holder to identify animals on the outside and air intake and export nozzles that dock into the isocage rack (Figure 1C-E). Safe closure clamps and a tab lock on the lid seal the cage when it is ready to be redocked on the rack system (Figure 1F). The suggested bedding is Alpha-dri, and an autoclavable enrichment hut is also recommended (Figure 1F). Transfer disks are used to move germ-free mice from breeding isolators to the isocages and contain a rotatable compartment lid with a triangular opening to allow for manipulation of animals (Figure 1G-H). Disks come in sizes small (21.6 cm diameter) and large (28 cm diameter), both of which have a capacity of eight mice. Autoclaved tape is used to create airtight seals on the circumference and air holes of the disk, which is performed prior to soaking with sterilant and transport in a sterilant-soaked bag (Figure 1I). The rack system itself has a screen to monitor the air blowers, rack-level HEPA filter status, and emergency battery power for the rack, which are all included features of the system (Figure 2A). An enclosed Magnehelic gauge displays the positive pressure maintained by the cage system, and an automatic visual docking indicator shows the docking status of the cages (yellow tab out means no cage is docked, or the dock was unsuccessful) (Figure 2B-D). Also necessary for the manipulation of isocages is a standard certified biosafety cabinet.
The protocol presented here describes the proper methods for the successful transfer of germ-free mice from breeding isolators under aseptic conditions to the isocages while maintaining germ-free status, the humanization of germ-free mice with human donor fecal slurry, and the collection of feces from mice housed in the isocage for either confirmation of germ-free status or viability preservation for further functional studies. In this example, germ-free mice are humanized with pooled fecal specimens from human subjects treated with immunotherapy for lung cancer and dichotomized as responders or non-responders to therapy. In this instance, the response phenotype to immunotherapy response was transferred by the gut microbiota humanization to the recipient mice, who could then be further inoculated with tumor cells and treated with immunotherapy. The human fecal slurry protocol can be readily adapted to any human donor feces or any disease preclinical model that the investigator wishes. Using this protocol, it is possible to transfer any human fecal donor microbiota into the germ-free host, enabling further investigation into the role of microbiota in health and disease.
Figure 1: Schematic diagram of isocage and transfer disks. (A) Top-down view of the underside of the cage lid, with labels indicating the location of the internal cage-level HEPA filter and the silicone gasket seal. (B) Top-down view of the interior of the cage, with labels indicating the wire bar lid, the internal water bottle, and spout, and the location in the wire rack to hold autoclavable chow. (C) Front view of cage showing notches for the cage card holder. (D) Top-down view of a full cage with the lid on top, showing how the HEPA filter is installed on the air intake nozzle. (E). Rear view of cage showing air intake and export nozzles which dock to the isocage rack system. (F) Lateral view of a full cage with the lid on top, with labels indicating the safe closure clamps in the open position, with white tabs on each clamp that lock them in place. The interior of the cage shows Alpha-dri bedding layered at the bottom and suggested enrichment hut placed in bedding. (G) Top-down view of transfer disks with lid on top. (H) Top down view of the interior of the transfer disk, showing the rotatable compartment lid with a triangular opening to allow for manipulation of animals. (I) Lateral view of fully assembled transfer disk showing placement of autoclaved tape, which creates an airtight seal during transfer from breeding isolator to isocage. Please click here to view a larger version of this figure.
Figure 2: Schematic diagram of isocage rack system. (A) Complete isocage rack with cages docked and a label indicating the monitoring screen for air blower status, HEPA filter status, and emergency battery. On the bottom left side of the rack is the slot for the rack-level HEPA filter. (B) Enclosed Magnehelic gauge showing the positive pressure maintained by the rack. (C) A docked isocage with no visible yellow docking indicator, demonstrating a successful connection between the rack and the air nozzles. (D) An empty slot in the rack, with a visible automatic visual docking indicator indicating that no rack is in place and there is no connection of the air nozzles with an isocage. Please click here to view a larger version of this figure.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida (UF) and performed at UF Animal Care Facilities (IACUC Protocol #IACUC202300000005). Colonies of germ-free wild-type (GF WT; C57BL/6) mice were bred and maintained in isolators by UF Animal Care Services Germ-free Division. Mixed-gender GF WT mice were transferred from breeding isolators and placed into the ISOcage P Bioexclusion system to allow for microbial manipulation.
Human fecal samples were obtained from a prospective observational study that collected longitudinal stool samples from patients who received immune checkpoint inhibitor (ICI) treatment12. Informed consent was obtained from patients after study approval by Advarra IRB (MCC#18611, Pro00017235). Subjects received and completed a liquid dental transport medium (LDTM) stool collection kit meant to preserve bacterial viability for functional studies. Response assessment characterized n=4 samples as responders (R) and n=6 as non-responders (NR). The homogenized LDTM-preserved patient samples were individually thawed, each placed into an anaerobic chamber for no more than 90 s and pooled by response phenotype (R: n = 4, NR: n = 6). The pooled samples were then aliquoted and frozen at -80 Β°C for use in this protocol. To determine the anaerobic colony forming units (CFU) counts of the donor feces, the feces of each subject was serially diluted to 1Β Γ 10-5, and 10 Β΅L of each dilution was plated in duplicate on anaerobic brain heart infusion (BHI) and Luria Bertani (LB) agar plates and CFU counts per gram stool estimated. Equal CFU from each subject was pooled into fecal inoculum samples for gavage into mice.
1. Preparation of cages and autoclaving
2. Chlorine-dioxide sterilant preparation
CAUTION: Chlorine-dioxide sterilant is extremely corrosive once activated. Activated chlorine-dioxide sterilant expires 24 h from the mixing of the activator with the base. Chlorine-dioxide sterilant produces fumes, which can be irritating to mucosal surfaces and will cause irritation in contact with the skin. Ensure the room for sterilant preparation has access to a sink and proper ventilation. Don safety goggles, respirator, and chemical-resistant gloves when working with chlorine-dioxide sterilant in addition to the required personal protective equipment (PPE) for the animal housing facility.
3. Sterilization
4. Germ-free mouse transfer
5. Oral gavage of human fecal slurry into germ-free mice
6. Stool collection from humanized mice for viability preservation
Human fecal samples, pooled by ICI responder and non-responder phenotype (previously described in the protocol), were gavaged into mixed gender GF-WT mice housed in 3 isocages per group (n = 1-2 mice/cage, n =6 for responder and n = 5 for non-responder). Mice were allowed to acclimate for 1 week post-transfer. Fecal samples were then collected from these mice (germ-free conditions). Mice were then gavaged with 1Β Γ 107 CFU of either responder or non-responder pooled human feces. The stool was then col...
The protocol described here provides a reproducible, highly detailed method for the humanization of germ-free mice housed in experimental isocages. The ability to exclusively transplant fecal communities from human subjects into murine hosts is invaluable to microbiome research. Without contamination from mouse-specific commensal microbiota, one can study the impact of human-resident bacteria on a variety of health and disease states or the impact of interventions such as diet or drug administration on human microbiota
The authors have no conflicts of interest.
The authors are grateful to the Germ-Free Services Division of UF Animal Care Services for the assistance with gnotobiotic husbandry, to Dr. Brooke Bloomberg and Dr. Laura Eurell for veterinary and IACUC assistance, and Josee Gauthier for the assistance with 16S rRNA gene sequencing. This research was supported, in part, by the UF Health Cancer Center Funds (C.J.) and the UF Department of Medicine Gatorade Fund (C.J.). R.Z.G. was supported by UF Health Cancer Center funds. R.C.N. was supported by the National Institutes of Health TL1 Training Grant at the University of Florida (TL1TR001428, UL1TR001427), the National Cancer Institute of the National Institutes of Health Team-Based Interdisciplinary Cancer Research Training Program award T32CA257923 and the UF Health Cancer Center. Research reported in this publication was supported by the UF Health Cancer Center, supported in part by state appropriations provided inβ―Fla. Stat. Β§ 381.915 andβ―the National Cancer Institute of the National Institutes of Health under Award Number P30CA247796. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the State of Florida. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Name | Company | Catalog Number | Comments |
1 mL BD Slip Tip Syringe sterile, single use | Fisher Scientific | 309659 | |
2.0 mL Screw Cap Tube, NonKnurl,Skirted,Natural, E-Beam Sterile tube w/ attached cap | Fisher Scientific | 14-755-228 | |
36 x 32 x 48" 3 Mil Gusseted Poly Bags | Uline | S-13455 | |
5 gallon tank of Exspor chlorine-dioxide sterilant activatorΒ | Ecolab | 6301680 | |
5 gallon tank of Exspor chlorine-dioxide sterilant baseΒ | Ecolab | 6301194 | |
600 mL polypropylene beakers | Fisher Scientific | S01914 | |
ALPHA-dri bedding | Shepherd Specialty Papers | ||
Anaerobic chamber | Coy Lab Products | Type B | |
Biosafety cabinet class 2 | Nuaire | ||
Certified IsoCage autoclavable HEPA filter XT Extreme Temperature | Tecniplast | 1245ISOFHXT | |
Clear Lens LPX IQuity Safety GogglesΒ | Fastenal | 922205455 | |
DuPontΒ TyvekΒ Sleeve - 18" | Uline | S-13893E | |
DWK Life Sciences DURAN 45 mm Push-on Natural Rubber Cap | Fisher Scientific | 01-258-107 | Rubber cap for 1 L autclave bottles |
Dynalon Quick Mist HDPE Sprayer Bottles | Fisher Scientific | 03-438-12B | |
Fisherbran Polypropylene Graduated Cylinders | Fisher Scientific | 03-007-44 | |
FisherbranΒ Dissecting Blunt-Pointed Forceps | Fisher Scientific | 08-887 | |
Fisherbrand Instant Sealing Sterilization Pouches | Fisher Scientific | 01-812-51 | |
Fisherbrand Straight Broad Strong Tip General Application ForcepsΒ | Fisher Scientific | 16-100-107 | |
FisherbrandΒ lead Free Autoclave Tape | Fisher Scientific | 15-901-110 | |
Gavage needle, reusable stainless steel. Straight. 22 gauge needle, tip diameter 1.25 mm, length 38 mm or 1.5 inches(doz) | Braintree Scientific | N-PK 020 | |
H-B Instrument Durac Timer | Fisher Scientific | 13-202-015 | |
IsoPositive Cages and Rack (i.e. isocages) | TecniplastΒ Β | ISO30P | 30 cages (6 w x 5 h), single sided |
Nitrile Chemical Resistant Gloves Size S (7), M (8) or L (9) 18β long, 22 mil, Ansell | Grainger | 4T426 | |
Nitrile Exam Gloves, Medium, Non-Sterile, Powder-Free | MedSupply Partners | KG-1101M | |
Olive / Magenta Bayonet Gas & Vapor Cartridges / Particulate Filter 2CtΒ | Β 3M/Fastenal | 50051138541878 | |
Polycarbonate RadDisk Mini for Mice 8-75 x 4 | Braintree Scientific | IRD-P M | |
Polypropylene Bouffant Caps - 24", Blue | Uline | S-10480BLU | |
Puritan Cary-Blair Medium, 5 mL | Fisher Scientific | 22-029-646 | |
S, M and L Blue Silicone Dual-Mode Head Harness Half Mask RespiratorΒ | Β 3M/Fastenal | 50051131370826 | |
Sgpf Series Sterile Powder Free Latex Gloves, CT International, Thickness = 6.5 mm, Length = 30.5 cm (12), Glove Size = 8.5, Glove Color = White | Fisher Scientific | 18-999-102F | |
Skid Resistant Shoe Cover | Uline | Β S-25639 | |
Surgical Gown, Towel, Sterile, Large, 32/cs | Thomas Scientific | KIM 95111 | |
Teklad Global 18% protein extruded rodent diet (sterilizable)Β | Inotiv | 2018SX | |
Thermo Scientific Nalgene Heavy-Duty Rectangular LLDPE Tank with Cover (20 L volume) | Thermo Scientific | 14-831-330J | |
VERIFY Dual Species Self Contained Biological Indicators | Steris Healthcare | S3061 | |
WypAll L40 1β4 Fold Wipers | Uline | S-8490 |
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