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

Summary

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.

Abstract

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.

Introduction

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 functions^2,3,4,5. Particularly advantageous is the ability to "humanize" germ-free mice at the level of the ....

Protocol

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 immun....

Discussion

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

Materials

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 activator	Ecolab	6301680
5 gallon tank of Exspor chlorine-dioxide sterilant base	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 Goggles	Fastenal	922205455
DuPont Tyvek 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
Fisherbran 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 Forceps	Fisher Scientific	16-100-107
Fisherbrand 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)	Tecniplast	ISO30P	30 cages (6 w x 5 h), single sided
Nitrile Chemical Resistant Gloves Size S (7), M (8) or L (9) 18” 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 2Ct	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 Respirator	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	S-25639
Surgical Gown, Towel, Sterile, Large, 32/cs	Thomas Scientific	KIM 95111
Teklad Global 18% protein extruded rodent diet (sterilizable)	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 1⁄4 Fold Wipers	Uline	S-8490