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

We studied the interaction between bacteria and the host, and our internal and external factor modified this relationship to change health. Specifically, we're interested into understanding the role of bacteria in cancer development, progression, and therapeutics. We and other microbiome lab utilize ISO positive cage system to transfer fecal community from human subject into a recipient murine host to create stable and long-term microbial community.

This technology has enabled us to generate a new discovery on the role of relevant clinical bacteria in health and disease. Many prospective clinical trials have found associations between specific microbes and cancer development, progression, and treatment response, but correlation does not imply causation. Humanization of germ-free mice with human fecal specimens enables causative relationship to be validated in relevant cancer models.

Using the ISO positive cage system, we have demonstrated that specific bacteroides strains from human immunotherapy responder, non-small cell lung cancer patients confer a response to mice bearing orthotopic lung tumors. To begin, place 10 wipes into the chlorine dioxide sterilant dunk tank and ensure they're fully soaped. Then, using a graduated cylinder, fill a large plastic bag with at least 100 milliliters of chlorine dioxide sterilant.

Shake the bag to ensure all interior surfaces are soaked and place it aside. Remove a single ISO cage from the rack and place it into the chlorine dioxide sterilant dunk tank, ensuring full liquid contact. Use soaked wipes to scrub all cage surfaces.

After soaking the cage, have an assistant open the soaked plastic bag. Place the cage inside and immediately close the opening. Using a spray bottle, spray the bag opening with chlorine dioxide sterilant.

Once all cages and supplies are bagged, submerge chemical resistant gloves in chlorine dioxide sterilant. In the meantime, move the soaked wipes into the biosafety cabinet and soak all surfaces. For non-protected surfaces, squeeze the wipes over them without touching them.

After 20 minutes, using sterilized chemical resistant gloves, move each cage and bottle to the sterilized biosafety cabinet. To open the hermetically sealed ISO cage, lift the white tabs on the two clamps on the sides of the lid and pull out each clamp sideways to unlock the lid. Using the sterile forceps inside the cage on top of the wire rack, remove the empty water bottle and place it on the inside of the lid.

Remove the rubber seal and pour water into the bottle to fill it. Using sterile forceps, place the nozzle on the water bottle and press down firmly to seal. Then, with sterile forceps, lift the wire rack and slide it back several inches to create an opening to the cage bottom.

Rest the sterile forceps on the wire rack, ensuring that the handles do not come in contact with cage surfaces. Upon receiving the transfer disc, have the assistant hold and partially unwrap the plastic cover so that the soaked surface is exposed but not touched. Wearing chemical resistant gloves soaked in chlorine dioxide sterilant, remove the transfer disc from the plastic wrapping and place it on a flat surface inside the sterilized biosafety cabinet.

To open the transfer disc, peel off the tape sealing the disc circumference and then remove the lid. Next, using the sterile forceps previously rested on the wire rack, manipulate the compartment lid to align the opening with the mouse to be transferred. Employing sterile forceps, grasp the base of the mouse's tail through the opening in the transfer disc cover, lift and transfer the mouse into the ISO cage through the opening between the wire rack and the cage.

Once all mice are transferred, replace the wire rack with the sterile forceps. Lift the cage lid and place it back on top of the cage. Lift each clamp of the cage lid up and carefully lower it over the sides of the cage.

Push down the white tabs to securely seal the cage lid. Now, have the assistant spray each nozzle of the docking site on the cage rack with chlorine dioxide sterilant. Remove the cage from the biosafety cabinet and dock it on the cage rack.

Dawn a new sterile surgical gown and sterile surgical gloves in place of the chemical resistant gloves. To prepare the gavage needles, unwrap each sterilization pouch and use the interior of the pouch as a dry sterile resting surface. Connect the gavage needle to the syringe.

Open the fecal slurry tube and pull up 200 microliters of the slurry into each syringe. Using forceps, grasp the base of the mouse tail and place it on the wire rack. Gently restrain the mouse by scuffing the loose skin behind the neck.

While holding the mouse in an upright vertical position, insert the gavage needle and gently inject the fecal slurry. Immediately remove the needle after injection. Place the mouse directly into one of the sterilized cups for observation.

After all mice have received the gavage, use forceps to move each mouse back into the cage bed. The humanized mouse fecal community structure was significantly different between response phenotypes at all three time points. The responder feces colonized mice showed no difference in microbial community structure between weeks one and two, and weeks two and four.

While non-responder feces colonized mice showed a different microbial community structure between weeks one and two, but similar structure between weeks two and four. The human donor inoculums showed increased representation of Blautia, Eubacterium, ruminococcus, and streptococcus, while recipient mice had increased relative abundance of bacteroides, Lachnoclostridium, Marvinbryantia, and Parabecteroides.