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 the "standardization fallacy": assuming that minimizing variation in experimental conditions improves translational outcomes. However, in reality, it limits the biological relevance of findings^1,2. For example, research has shown that the absence of microbial and environmental diversity in SPF mice can result in an underdeveloped immune system, undermining the validity of immunological and preclinical studies³.

Several approaches have been proposed to address biological variation in mouse models, each with its own advantages and limitations, including co-housing with feral and pet shop mice^3,4,5,6,7,8, sequential exposure to commensals⁹, keeping the animals in outdoor enclosures¹⁰ or on bedding from large animals¹¹, and fecal transplants from wild mice¹². A promising new mouse model for preclinical and toxicological research is the "Wildling mice" model, which consists of standard laboratory mouse strains harboring a natural microbiome¹³. These "Wildling mice" are generated by transplanting embryos from laboratory mouse strains into wild-caught mice. During birth, the laboratory mouse strains acquire the natural microbiome of their surrogate mothers, mimicking the natural inoculation that occurs during human delivery¹³. "Wildling mice" can be bred like any other laboratory mouse strain, with their natural microbiome preserved across generations.

"Wildling mice" host a diverse microbiota - including bacteria, viruses, and parasites - that are typically excluded from SPF mouse facilities. Consequently, maintaining a natural microbiome in research facilities presents challenges, as these microbes must be contained without compromising the overall SPF hygiene standards.

At Charité - Universitätsmedizin Berlin, a dedicated facility for "Wildling mice" was established, separated from the SPF areas by strict biocontainment measures. The facility includes breeding and experimentation rooms, ensuring that the natural microbiome of "Wildling mice" is maintained while SPF areas are protected (Figure 1).

The founder pairs of Charité's colony were imported from the "Wilding mice" colony established at the Department of Microbiome Research, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Germany. They are supplied with a health certificate and monitored with an extended panel for zoonotic pathogens before the import of the colony founders. Sentinels will be used to monitor the microbiome over time. Both SPF and "Wildling mice" are housed under the same conditions. The mice are bred and kept preferentially in individually ventilated cages (IVC) type II long in groups of five mice. The temperature inside the facility is 22 °C, and the light/dark cycle is 12 h. The mice receive standard grain-based chow and tap water. Sterilization of bedding and enrichment items is unnecessary for "Wildling mice". However, autoclaving these items prevents mix-ups of materials in areas where SPF mice are housed.

In this protocol, the immunophenotyping procedures for both SPF and "Wildling mice" are demonstrated, highlighting the stringent microbial containment protocols at the "Wildling mouse" facility. These measures ensure the integrity of SPF environments while providing the benefits of working with mice carrying a natural microbiome.

Figure 1: Layout of the facility for Wildling mice. E1 = access to the facility. Arrows indicate the entry route into the facility. E2 = access to the laminar airflow cabin from outside the facility. PA = personnel airlock with air shower. AS = air shower. R1, R2 = rooms for Wildling mice breeding. R3 = room for keeping Wilding mice. R4 = room for keeping SPF mice. PR1 = procedure room for SPF mice. PR2 = procedure room for Wildling mice. SB = sterile working bench. EE = emergency exits. CR = changing room in front of LAF cabin. LAF = laminar airflow cabin for interventions under protective airflow. A = autoclave. ER = equipment room. Green arrows represent routes accessible when working with SPF animals, and yellow arrows indicate paths available for working with Wildling mice after air showering. Blue arrows indicate access for husbandry staff only. The red line marks the glass wall inside the LAF cabin, which divides the space into two sections that can either be accessed from E1 via PR2 or from E2 via CR. Please click here to view a larger version of this figure.

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 the online booking system.

2. Entering the facility for "Wildling mice"

1. Deposit the clothes in the changing room.
2. Change into area clothing: pants, kasak, and disposable shoe covers (Figure 3).

3. Sampling blood from SPF mice in the SPF area

1. Transfer the SPF mice (male and female, 8-20 weeks) from the room where they are kept (R4) to the procedure room for SPF animals (PR1) in a closed IVC cage.
2. Switch on the laminar flow bench and disinfect the workspace surface with 70% Vol. ethanol. Work inside the bench.
3. Inspect capillary tubes for broken or chipped sections to avoid increased risk of breakage or harm.
4. Under brief general anesthesia with isoflurane (induction at 5% isoflurane in oxygen-enriched medical air, followed by 1.5%-2.0% for maintenance), after loss of pedal withdrawal reflex (as indicated via toe pinch), compress the neck veins of the mice by gripping the scruff of the neck.
5. Using one hand, secure the animal's head using the thumb and forefinger. Insert a fresh capillary into the medial canthus of the eye under the nictitating membrane at a 45° angle. Ensure that the capillary is positioned between the eyeball and the bony orbit of the eye.
   NOTE: To avoid trauma, do not let the tip of the capillary tube touch the surface of the eye.
6. Insert the capillary through the sinus membrane with a gentle axial rotating motion. Continue rotating the tube at the back of the orbit until blood flows. Collect a minimum 15 µL of blood in a blood collection tube containing ethylenediaminetetraacetic acid (EDTA)(approximately 1 drop).
7. Before removing the capillary, loosen the grip on the scruff of the neck to keep bleeding into the tissue to a minimum. To ensure good hemostasis, use a clean gauze pad or swab to stop the bleeding.
8. Slightly swivel the tube to mix blood and EDTA. Do not shake the tube. Store the blood sample on ice.
9. Disinfect the working surface and switch off the laminar flow bench. Return SPF mice in a closed IVC to the SPF housing room (R4).

4. Entering the area for "Wildling mice"

1. Remove shoes and socks in the anteroom of the air shower (PA). Put on area socks and full-body personal protective equipment (PPE) consisting of Tyvek overall, hair net, if necessary, cotton gloves, nitrile gloves, and facemasks (Figure 3).
2. Enter the air shower in socks via the adhesive floor covering. While air showering, raise the arms and perform a 360° turn.
3. Exit the air shower and put on area shoes on the other side.

5. Sampling blood from "Wilding mice"

1. Transfer "Wildling mice" (male and female, 8 - 20 weeks) from the room where they are kept (R3) in a closed IVC cage to the procedure room (PR2).
2. Switch on the laminar flow bench and disinfect the workspace surface.
3. Follow the procedures described for sampling SPF mice in section 3, steps 2-8.
4. After completing blood sampling, change gloves. Disinfect all materials and surfaces. Switch off the laminar flow bench.

6. Exporting blood samples from the "Wildling mice" area via the laminar airflow (LAF) cabinet

NOTE: The procedure room (PR2) contains a LAF cabinet, which serves as a material lock and sterile intervention room. Samples are transferred out via the LAF cabin. The interior is accessible from both inside (via E1 and PR2) and outside (via E2 and CR) the Wildling area and is divided in the middle by a glass wall with a sliding door (Figure 1). Two people are required to export materials: Person 1 (inside of the Wildling area [via PR2]) will perform steps 6.1 and 6.2. Person 2 (outside of the Wildling area [via E2]) will perform steps 6.3-6.5.

1. Disinfect sample containers. Remove sample containers via the sliding door in the glass wall of the LAF cabin.
2. After the transfer of sample containers to person 2 is completed, receive protective clothing from person 2 (wears protective clothing in the smock lock of the cabin) on the other side of the LAF cabin and dispose of it. Disinfect all surfaces.
3. Put on protective clothing in the access LAF in front of the LAF cabin (CR, Figure 1).
4. Receive disinfected sample containers from the Wildling area without contact through the sliding door in the glass wall of the LAF cabin.
5. Remove protective clothing and pass it through the sliding door into the interior area of the LAF cabin for disposal.

7. Exiting the facility for "Wildling mice"

1. Leave disposable overalls and area shoes inside the area. Dispose of hair net, rubber gloves, and facemask.
   NOTE: Shoes and overalls can be reused.
2. Enter the air shower immediately in area socks. Raise arms and perform a 360° turn while air showering.
3. Leave the air shower and place area socks in the laundry collector. Put on the socks and shoes and disposable shoe covers. Deposit area clothing in the changing room in the laundry collector. Change into the clothes in the changing room

8. Processing and analyzing blood samples

1. Bring blood samples to the laboratory.
   1. Prepare fluorophore-conjugated antibody staining cocktail: For each sample, add 0.25 µL of Alexa Fluor 700 anti-mouse CD45, 0.25 µL of Brilliant Violet 605 anti-mouse TCR-β chain, 0.25 µL of APC anti-mouse CD4, 0.25 µL of Brilliant Violet 785 anti-mouse CD8, 0.25 µL of FITC anti-mouse CD44 and 0.25 µL of PerCP/Cyanine5.5 anti-mouse CD62L antibody to 50 µL of flow cytometry buffer (phosphate-buffered saline, 2 mM EDTA, 0.1% Bovine Serum Albumin, 4 °C). Keep in the dark on ice until step 8.8.
      NOTE: The fluorophores used can be adapted to the configuration of the flow cytometer that will be used for sample acquisition.
2. Disinfect blood collection tubes. Add 2 mL of ice-cold flow cytometry buffer to a 5 mL round-bottom polystyrene tube. Place the tube on ice.
3. Open blood collection tube. Add 10 µL blood to the tube containing the flow cytometry buffer and pulse vortex.
4. Spin down at 400 x g for 5 min at 4 °C.
5. Aspirate the supernatant. Add 1 mL of red blood cell (RBC) lysis buffer, pulse vortex, and incubate for 3 min at room temperature (RT).
6. Add 100 µL of 10x PBS. Spin down at 400 x g for 5 min at 4 °C.
7. Repeat steps 8.5-8.6.
8. Aspirate the supernatant. Add 50 µL of antibody staining cocktail, vortex, and incubate for 20 min on ice in the dark.
9. Add 1 mL of flow cytometry buffer. Spin down at 400 x g for 5 min at 4 °C.
10. Aspirate the supernatant. Add 180 µL of flow cytometry buffer.
11. Add 20 µL of SYTOX Blue Dead Cell Stain (final concentration 1:1000) for live/dead discrimination.
12. Acquire samples on a flow cytometer and analyze the data using the flow cytometer acquisition software or flow cytometry data analysis program.

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 (Figure 1). To realize this design, an existing mouse facility underwent extensive reconstruction, including the installation of an air shower and a laminar airflow cabin. The areas where "Wildling mice" and SPF mice are bred and kept are separated by the air shower and both areas have designated procedure rooms. Thermostable material leaves the area for "Wildling mice" only through the autoclave, while thermolabile materials, such as samples, can be exported via the LAF cabinet for further processing in laboratory areas (Figure 2). Strict adherence to all biocontainment measures is essential to prevent contamination of areas where SPF hygiene conditions are maintained (Figure 3). Collectively, the design and biocontainment measures of the facility for "Wilding mice" at Charité - Universitätsmedizin Berlin provide scientists with the opportunity to conduct research with mice harboring a natural microbiome while effectively preventing the spread of microorganisms to areas of the animal facility with higher hygiene levels.

Flow cytometry is the gold-standard method in cellular immunology and is used to interrogate immune cell populations in blood and tissues in response to altered environmental conditions. For example, adult humans have a substantial number of memory T cells circulating in the blood, which develop upon exposure to microorganisms from the environment³. Here, flow cytometry was used to probe for memory T cells in the blood of mice bred and kept under SPF conditions and in the blood of "Wildling mice" (Figure 4). "Wildling mice" colonized with a natural microbiome, present a distinct population of memory T cells. In contrast, mice bred and kept under SPF conditions have greatly reduced memory T cells in the blood circulation. Hence, flow cytometry can characterize the impact of different microbial environments on the immune compartment. Data granulation can be increased by including additional immune cell markers. Recently developed approaches in flow cytometry, such as Cytometry by Time-of-Flight (CyTOF) and spectral flow cytometry, allow for the acquisition of information on a multitude of parameters and, thereby, expanding the number of immune cell (sub-)populations that can be quantified in the same blood and tissue sample. Furthermore, cellular cytokine production can also be assessed by flow cytometry, providing insights into the function of cells in response to a natural microbiome.

Figure 2: Workflow in the Wildling facility. Blood sampling and sample processing are examples but can be replaced by other procedures, such as surgical interventions with tissue sampling and histology. For abbreviations, see Figure 1 legend. Please click here to view a larger version of this figure.

Figure 3: Comparison of personal protective equipment (PPE) requirements for SPF and Wildling areas. In the SPF area (left), standard PPE consists of a single layer of protective clothing. In contrast, the Wildling area (right) requires a three-step PPE process before entering the air shower: a kasac (protective undergarment = step 1) is worn beneath the overall (step 2). This additional layer helps maintain hygiene and prevent cross-contamination between areas. After the air shower, designated shoes for the Wildling area must be worn (step 3). Please click here to view a larger version of this figure.

Figure 4: Immunophenotype of "Wildling mice". Blood samples from male SPF mice (n = 5 mice and "Wilding mice" (n = 5 mice), age 14 weeks, were stained with a cocktail of fluorophore-conjugated antibodies against CD45, TCR-β chain, CD4, CD8, CD44, and CD62L. Numbers are the proportion of parental gate ± standard deviation. The gating strategy is shown for SPF mice, except for the last two blots from SPF and "Wildling mice". In "Wildling mice" central and effector memory T cells are increased compared to mice that are bred and maintained under SPF conditions. Please click here to view a larger version of this figure.

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^{3,7,8,9,10,11,12}. "Wildling mice" combine the benefits of a controlled laboratory environment with a natural microbiome. This approach maintains stability in experimental conditions while preserving the natural microbial diversity necessary for studying complex biological interactions. Consequently, biomedical therapies developed and tested in "Wildling mice" hold great potential for successful translation into clinical applications.

However, it still has to be determined in which preclinical disease models "Wildling mice", are superior to SPF mice. Furthermore, mice with a natural microbiome harbor microorganisms such as highly transmissible worms that are typically excluded from institutional facilities. Therefore, breeding and working with these mice present challenges to animal husbandry practices within research institutions.

The establishment of a facility for "Wildling mice" involved a collaborative effort of scientists, veterinarians, and institutional officials at Charité - Universitätsmedizin, the German Centre for Protection of Laboratory Animals (Bf3R), and the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU). After developing the facility concept, an existing mouse facility underwent extensive reconstruction to incorporate stringent biocontainment measures. The facility is certified for biosafety level 2 (BSL-2) operations regarding research with human pathogens.

Here, operating and biocontainment procedures in the facility for "Wildling mice" are described using blood sampling for immunophenotyping by flow cytometry as an example. Strict adherence to biocontainment measures while working in the facility for "Wildling mice" is mandatory to prevent the spread of microorganisms to other areas of the animal facility. This includes the restriction of access to the facility to as few people as possible per the research group. Additionally, a mandatory introductory session conducted by the institutional animal welfare officer is required before gaining initial access to the facility. General information and Standard Operating Procedures (SOP) for working within the Wildling facility are also available on an internal website ("Wildling wiki")^14,15.

While working inside the facility, PPE must be worn properly. For instance, it is essential to ensure that no hair is exposed outside the hair net, as parasites' eggs could potentially attach to it. If the experiment involves both "Wildling mice" and SPF mice, SPF mice must be handled first. The areas for "Wildling mice" and SPF mice within the facility are separated by an air shower, and each area is equipped with designated procedure rooms. These rooms can be booked through an online booking system, ensuring smooth working processes and a limited number of people inside the facility. A maximum of two people per area per procedure room (PR1, PR2, LAF) are allowed to work in each barrier at the same time. Exceptions apply to experiments requiring additional personnel to ensure ethical and proficient execution of the procedures. Mice are transferred between rooms within their respective areas in closed IVC cages, which can only be opened under a laminar flow bench to ensure microbial containment and work safety. The procedure room in the "Wilding mice" area also contains a laminar airflow (LAF) cabin. This LAF cabin must be used for the dissection and surgery of "Wildling mice", including embryo transfer. Embryo transfer is a critical procedure in the "Wilding mice" context, as it is used to generate new "Wildling mouse" models. For example, transferring embryos from genetically modified (GMO) mouse strains into wild-type "Wilding mice" is used to create Wildling GMO strains. The LAF cabin is also used for the export of samples, which requires coordination with a person outside the "Wildling mice" area. Further sample processing can be performed in adjacent laboratory space, allowing researchers to handle time-sensitive materials on-site while limiting the locations where samples from "Wildling mice" are processed.

All other materials must remain inside the Wildling facility or need to be autoclaved before leaving the facility. After work, all materials and surfaces are disinfected with disinfectants appropriate for BSL2 conditions. People who entered the facility for "Wildling mice" are prohibited from entering any other animal facility on the same day.

Research in the facility is conducted under the framework of the scientific consortium "The Wildling Mouse Model in Health and Disease (Wildling HeaD)" facilitating the communication among scientists working with "Wildling mice", as well as the exchange of methods and expertise. The consortium is supported by Responsible PrecliniX at the Berlin Institute of Health (BIH) QUEST Center for Responsible Research, which aids in animal study preregistration procedures, robust experimental design, and quality management (www.bihealth.org/en/quest/service/service/responsible-preclinix).

In summary, a state-of-the-art facility has been created, equipped with stringent biocontainment measures, to support safe and ethical preclinical research. This facility facilitates the study of "Wildling mice," which harbor a natural microbiome, effectively bridging the gap between controlled laboratory conditions and real world-like microbial environments. For constructing a facility like the one described, it is essential to prioritize team science and interdisciplinary collaboration, incorporating expertise from scientists, veterinarians, and institutional officials to ensure effective design and operation of high-biocontainment environments. Additionally, implementing stringent biocontainment measures, providing comprehensive training, and developing clear Standard Operating Procedures (SOPs) are crucial. Establishing an efficient communication structure, such as an internal wiki, and continuously monitoring and validating experimental conditions will further enhance the facility's efficacy and safety in managing complex models like "Wildling mice".

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