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

Summary

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.

Abstract

The use of laboratory mice with a natural microbiome, such as "Wildling mice", offers a promising research tool for both basic and applied science due to their close resemblance to the human superorganism. However, the breeding and maintenance of these mice, which harbor a diverse microbiome including bacteria, viruses, and parasites, pose significant challenges for animal husbandry facilities at research institutions. To address these challenges, a specialized facility concept was developed for housing "Wildling mice" at Charité - Universitätsmedizin Berlin. This approach involved designing a facility with specific structural features and operational protocols to effectively contain the natural microbiome, thereby protecting areas with higher hygiene standards.

A methodology for blood sampling from both specified pathogen-free (SPF) and "Wildling mice" for immunophenotyping is demonstrated, highlighting the workflow and biocontainment measures implemented in the facility. Remarkable results reveal that "Wildling mice" exposed to a natural microbiome develop distinct immune cell populations, which are significantly reduced in mice bred and maintained under stringent hygiene conditions.

The significance of this study lies in its potential to provide researchers with access to mice that possess a natural microbiome and a mature immune system similar to that of human adults. This approach could enhance the translatability of preclinical findings into clinical practice, thereby advancing the field of biomedical research.

Introduction

Experimentation in mice is still indispensable in basic and applied science, such as preclinical and toxicological research. However, the standardization of hygiene in laboratory environments, aimed at reducing biological noise and minimizing variability in experimental results, has led to the exclusion of natural microbiota to a large extent. Thus, the conditions under which hygienically standardized, specified pathogen-free (SPF) laboratory mice are born and kept differ from the real-world conditions to which humans and animals are normally exposed. This mismatch between laboratory conditions and the natural environments in which human diseases occur gives rise to t....

Protocol

The facility for "Wilding mice" and procedures involving living animals were approved by the responsible state office for animal experimentation, "Landesamt für Gesundheit und Soziales Berlin" (LAGeSo). The most important steps of the protocol are summarized in Figure 2.

1. Getting access to the Wilding facility

1. Get a personal introduction to the operating and biocontainment procedures of the facility from the responsible animal welfare officer.
2. Get the transponder key to enter the facility for "Wildling mice" and book the procedure rooms via t....

Results

"Wilding mice" potentially harbor microorganisms typically excluded from SPF facilities, which poses a challenge to animal husbandry practices within research institutions that uphold stringent hygiene standards. Over the past 4 years, scientists and veterinarians at Charité - Universitätsmedizin Berlin and the German Centre for Protection of Laboratory Animals (Bf3R) have developed a facility dedicated to mice with a natural microbiome, incorporating stringent biocontainment measures through a collaborative endeavor (

Discussion

Mice with a natural microbiome are a promising research tool for basic and applied science because of their closer resemblance to the human superorganism^3,9,10,11,12. Attempts to incorporate biological complexity into mouse models have led to the development of various approaches, each with its own advantages and limitations^3,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 700 anti-mouse CD45 antibody	BioLegend	103127	Clone 30F-11
Animal Chow	Altromin	1324
APC anti-mouse CD4 antibody	BioLegend	100515	Clone RM4-5
Blood collection tube	Greiner	450475	MiniCollect K3E, K3EDTA
Bovine Serum Albumin	Sigma-Aldrich	A9647-100G
Brilliant Violet 605 anti-mouse TCR-beta chain antibody	BioLegend	109241	Clone H57-597
Brilliant Violet 785 anti-mouse CD8 antibody	BioLegend	100749	Clone 53-6.7
Capillary	Hirschmann	9000210	Hirschmann minicaps, Na-hep
EDTA	Corning	46-034-CI
FITC anti-mouse CD44 antibody	BioLegend	103021	Clone IM7
PerCP/Cyanine5.5 anti-mouse CD62L antibody	BioLegend	104431	Clone MEL-14
Phosphate-buffered Saline (10x)	Gibco	12579099
Phosphate-buffered Saline (1x)	Gibco	14190094
RBC lysing buffer	BioLegend	420302
Round Bottom Polystyrene Tube	Sarstedt	55.476.005
SYTOX Blue Dead Cell Stain	Invitrogen	S34852
Tyvek overall (DuPont)	Fisher Scientific	11371633