Name: Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice (Video) | JoVE Journal
Uploaded: 2025-02-28T14:45:00.000+00:00
Duration: 7 min 45 s
Description: Learn fecal transplantation into germ-free mice using ISO positive cage systems for microbiome and cancer research. This method enables stable human microbial communities in murine hosts for studying bacterial roles in health and disease.

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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 in&#8239;Fla. Stat. &#167; 381.915 and&#8239;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.

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The authors have no conflicts of interest.

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<s...

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.
<img alt="Rodent cage system diagram with XT HEPA filter, water bottle, and air intake setup." class="xfigimg" src="/files/ftp_upload/68029/68029fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/68029/68029fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bioexclusion system rack diagram, HEPA filter, monitoring screen, experimental cage setup." class="xfigimg" src="/files/ftp_upload/68029/68029fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/68029/68029fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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preparation and maintenance of bioexclusion isopositive cage experiment for human fecal transplantation into germ-free mice

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

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.

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 1 × 107 CFU of either responder or non-responder pooled human feces. The stool was then collected 1, ...

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 ...

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Research

JoVE Journal

Immunology and Infection

This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

This video demonstrates a procedure of fecal microbiota transplantation in an interleukin-10 deficient mouse with colon inflammation. Colonized fecal microbes in the gut release secondary metabolites, eliciting anti-inflammatory cytokine release and reducing colon inflammation.

Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Murine Fecal Isolation and Microbiota Transplantation

Here, we describe the structure and operating procedures, including microbial containment measures of a facility for "Wilding mice" using blood sampling for immunophenotyping as an example.

Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

This protocol describes the necessary steps for colonization of mice with pks+ Escherichia coli under non-inflammatory conditions, as well as non-invasive methods for validation of colonization and assessment of pks+ E. coli expansion in feces.

Colonization with Murine pks+ Escherichia coli under Non-Inflammatory Conditions

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks.  Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours.  We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Learn fecal transplantation into germ-free mice using ISO positive cage systems for microbiome and cancer research. This method enables stable human microbial communities in murine hosts for studying bacterial roles in health and disease.

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

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