Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Summary

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Abstract

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.

Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%± 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%± 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection.

This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Introduction

The isolation of monocytes is important and critical for many in vitro and in vivo studies. These cells are targets for diseases such as peripheral arterial disease, coronary heart disease, or other ischemic diseases, since collateral vessel growth is strongly driven by local inflammation. Inflammatory responses include endothelial activation and local recruitment of leukocytes, mainly monocytes, which then mature to macrophages and create a highly arteriogenic environment by secreting multiple growth factors to induce the remodeling of an arteriole into a functional collateral artery^1-3. Monocytes also mature to dendritic cells, which are frequently used for immunological studies^4,5 and cancer research^6,7.

Problematic in the approach for monocyte isolation from peripheral blood⁸ is the high number of donor animals needed to produce a sufficient amount of monocytes for most analyses. Former protocols describe methods such as density gradient centrifugation and cell depletion via MACS⁹ when isolating monocytes; however, these techniques can alter the characteristics and functionality of monocytes which can lead to difficulties in interpretation^10,11. Moreover, these methods are difficult and can reduce experimental reproducibility.

Our aim with this protocol is to provide a simple and cost effective method to generate large amounts of bone marrow-derived monocytes. Due to the high cell yield of 11 x 10⁶ ± 3 x 10⁶ cells obtained by this protocol, we can substantially reduce the number of mice required during the isolation of bone marrow-derived monocytes. The procedure can be completed within a minimal amount of time, and without using expensive and complicated techniques as referenced above. Here, we extract monocytes from native bone marrow suspension of donor mice, cultivate the suspension on ultralow attachment plates, and supplement the solution with 20 ng/ml M-CFS. On day 5 of incubation, cells are harvested and characterized to confirm functional and phenotypic properties.

For experiments in the field of arteriogenesis, intravenous transplantation of these bone marrow-derived monocytes into mice is an effective method of systemic drug delivery, which can be combined with femoral artery ligation in common peripheral arterial disease models.

Protocol

This study was performed with permission of the State of Saxony and Saxony-Anhalt, Regierungspraesidium Dresden/Halle, according to Section 8 of the German Law for animal protection (24D-9168.11-1/2008-24).

1 Cell Isolation

1.1 Preparation of Femur and Tibia

1. Anesthetize the mouse using a 5% isoflurane concentration by vaporizing isoflurane in a closed bin. After the mouse has stopped moving for 3 sec, perform the cervical dislocation.
2. Disinfect the hind limbs with ethanol (96%). Remove skin and muscles and separate the bones with a sterile scalpel.
   1. Start with an inguinal skin incision, and extend the incision toward the medial malleolus and perform a circular dissection of the skin around the lower calf. Remove the skin of the legs completely by peeling it proximally.
   2. Detach the quadriceps muscle from the femur with a sharp scalpel, remove both the anterior peroneus and the gastrocnemius muscle groups and disarticulate the leg at the hip joint by locating and dissecting the ligaments.
   3. Start anteriorly and proceed around the joint by carefully rotating the leg and transecting the ligaments, thereby disarticulating the joint.
3. Wash femur and tibia with 96% ethanol for a minimum of 90 sec to guarantee aseptic cell preparation. Continue the washing process with sterile PBS to rinse off remaining ethanol.
   Note: All subsequent steps must be sterile to avoid contamination.

1.2 Harvesting of Bone Marrow

1. Cut the proximal and distal end of each bone with a pair of fine scissors to gain access to the femoral and tibial shafts. Be careful with this step, since these bones break very easily.
2. Flush the bones with warm medium (M199 + 10% fetal calf serum (FCS), + 1% Penicillin/Streptomycin). Use a sterile 28-G needle, a 1 ml syringe, and between 10-15 ml medium per bone for rinsing.
3. Hold the bone with a fine forceps and rinse one by one until it turns semi-translucent. Carefully apply pressure to the end of the syringe plunger in order to minimize cell stress.
4. Filter the bone marrow through a 70 µm nylon web and gather the filtrate. To extract the maximum quantity of bone marrow, rinse repeatedly from the distal and proximal ends.
5. Rinse the sieve with PBS and gather the filtrate. Carefully dislodge debris and cellular conglomerates by gentle stirring and pipetting.

1.3 Cultivation

1. Centrifuge the cell suspension at 250 x g for 10 min at RT. Discard the supernatant and resuspend the cells with approximately 25 ml of medium. Repeat once.
2. Resuspend the cells in 6 ml of medium after the second washing step. Mix 50 µl of the cell solution with 50 µl of trypan blue. Count the cells in a counting chamber under a light microscope. Calculate the number of cells in the solution using this formula:
   
3. Seed the cells on 6-well ultra-low-attachment surface plates to prevent permanent adhesion to the bottom of the plate. Use a concentration of 10⁶ cells per ml with up to 6 ml per well.
4. Supplement the suspension with 20 ng/ml M-CSF to promote cell differentiation.
5. Culture the cells for 5 days at 37 °C and 5% carbon dioxide and observe daily.

1.4 Harvesting of Cells

Note: These steps should be performed on ice. Proceed with either 1.4.1 or 1.4.2 accordingly.

1. At this point, harvest the entire culture, including differentiated macrophages that will adhere to the culture-plates, even if they are ultra-low-attachment surface plates.
   1. Harvest the entire culture by gentle pipetting and detaching with a solution of 4 °C PBS, 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Repeat until the plates are clear of remaining adherent cells - confirm under a microscope if unsure.
2. Alternatively, discard adherent macrophages by harvesting the supernatant with the cells in suspension, which predominantly contain monocytes.
   1. Harvest the non-adherent cells with a solution of EDTA-free PBS and 0.5% BSA. Do not apply too much force in order to avoid dislodging mildly adherent macrophages.

1.5 FACS-Analysis (Optional)

Note: It is possible to deplete the cell suspension of CD117⁺ stem- and progenitor cells by the use of MACS. Use manufacturer protocols for this procedure.

1. Harvest the cells according to either steps 1.4.1 or 1.4.2.
2. Centrifuge the cells at 250 x g for 10 min at 4 °C and repeat this step again.
3. Resuspend at least 250,000 cells in 300 µl of FACS buffer per probe.
4. Transfer cell solution onto a 96-well-plate (30 µl per well).
5. Centrifuge the cell suspension at 250 x g for 5 min at 4 °C.
6. Remove the supernatant and add 25 µl of antibody to each well (use manufacturer dilution).
7. Stain the cells with antibodies (following manufacturer protocols for cell phenotyping after steps 1.5.1-1.5.6. Commonly used markers for monocyte / macrophage identification include CD11b, F4/80 (Figure 3), CD115 (Figure 2), and Gr-1.
   Note: For a further description of cell solutions, the use of markers like CD4, CD8a, CD11c, CD25, CD45, CD45R, CD62L, CD80, CD86, CD145, CD117, MHCⅡ, Ly6C, Ly6G, and CD192 is recommended. Cellular characteristics were previously described¹².
8. Incubate the probes for 20 min at 4 °C.
9. Centrifuge the probes at 250 x g for 5 min at 4 °C and resuspend with 200 µl of FACS buffer. Repeat this step twice.
10. Transfer the probes into FACS tubes and perform the FACS analysis¹².
11. Perform FACS analysis¹² on day 0, 3 and 5 to analyze cell differentiation.

2. Tail Vein Injection

2.1 Preparation

1. Warm the animals on a heating plate at 37 °C for about 10 min. Observe the animals while warming to recognize signs of overheating.
2. Resuspend the monocytes, isolated in steps 1.1- 1.4, in 150 µl of NaCl, and load the cells into a 1 ml insulin syringe with a 30G needle. Lightly vortex the solution before loading the syringe to ensure all monocytes are injected. Always handle cells on ice to avoid heat-mediated attachment and activation of monocytes.

2.2 Restraining

1. Avoid disturbing other mice in the cage when choosing a mouse for intravenous injection.
2. Place the mouse into the restrainer. Avoid too much pressure and be careful with the hind limbs. (Figure 4)
   Note: Alternatively, use general anesthesia to guarantee stress free handling of the animals.
3. Ensure that the mouse has space for breathing before closing the restrainer.

2.3 Injection

1. Disinfect the injection site with disinfection agent. Spray on the tail of the mouse and let stand for at least 1 min.
2. Stop venous blood flow of the tail by applying gentle pressure to the lateral tail side above the injection site, for better visibility of the veins.
3. Turn the tail 90 degrees, before injection. (Figure 5)
4. Inject at a 45 degree angle. Inject slowly and no more than 5 µl per g to avoid harming animals.
5. If there is a blister, which is a sign of a failed injection, discontinue immediately and repeat the injection more proximally.
6. Stop the bleeding at the injection side by applying gentle pressure for about 1 min.
7. At the end of the procedure, open the retractor and place the mouse in its cage.
8. Observe the mouse for 20 min to ensure that the animal was not harmed by the injection and sternal recumbency is maintained. Extend the time of observation until the mouse regains sufficient consciousness. Return the mouse to the company of other animals only after it has fully recovered.

Results

The cell solution extracted from the murine bone marrow consists of various cell types. The major cell types are lymphocytes, granulocytes and monocytes. Cell types can be estimated by size and granularity, which is shown in Figure 1 for both native suspensions and cells harvested after 5 days of differentiation. Note the shifting cellular composition during cultivation. However, accurate classification of populations must rely on distinctive expression of cellular markers.

Th...

Discussion

We describe a simple and cost-effective method to isolate large amounts of murine monocytes from bone marrow. In comparison to other protocols using peripheral blood, which obtain monocyte yields⁵ of 1.4 x10⁶, we are able to obtain higher yields of 11 x 10⁶ ± 3 x 10⁶ monocytes from a single donor mouse.

When considering challenges with this method, it is important to mention the potential for contamination when working under non-sterile conditio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well-ultra-low-attachment plate	Corning Incorporated, NY, USA		6-well-ultra-low-attachment plate, with cap, sterile
8- 12 week old, male, balb/c mice	Charles River, Sulzfeld, Germany
96-well-plate	Greiner bio one GmbH, Frickenhausen, Germany
Blue dead cell stain	Life technologies GmbH, Darmstadt, Germany
Bovine serum albumine	GE Healthcare, Freiburg, Germany		Fraction V, pH 7.0
Canules	B. Braun, Melsungen AG, Melsungen, Germany		28G, 30G
CD115	eBioscience, San Diego, USA	12-1152
CD11b	eBioscience, San Diego, USA	53-0112
Cell culture dish	Greiner Bio-One GmbH, Frickenhausen, Germany		With cap, steril
Centrifuge	Beckman Coulter GmbH, Krefeld, Germany		Allegra® X-15R centrifuge
Depilatory cream	Veet, Mannheim, Germany
Disinfection agent	Schülke&Mayr GmbH, Norderstedt, Germany		Kodan Tinktur forte
Disposable scalpel No.10	Feather safety razor Co.Ltd, Osaka, Japan
EDTA	Sigma Aldrich, Hamburg, Germany
Ethanol 96%	Otto Fischar GmbH und Co KG, Saarbrücken, Germany
Extraction unit Pipetus	Hirschmann Laborgeräte GmbH & Co.KG, Eberstadt, Germany
F4/80	AbD Serotec, Düsseldorf, Germany	MCA497APC
FACS buffer	Manufactured by our group with single components		PBS, 0.5% BSA, 0.1% NaN3
FACS device	Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA		BD FACS Canto II
FACS tubes	Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA
Falcon® pipette	Becton Dickenson Labware, NY, USA
Fetal calf serum	Sigma Aldrich, Hamburg, Germany
Fine forceps	Rubis, Stabio, Switzerland
Gloves	Rösner-Matby Meditrade GmbH, Kiefersfelden, Germany
Gr1	eBioscience, San Diego, USA	53-5931
Heating plate	Labotect GmbH, Göttingen, Germany		Hot Plate 062
Incubator	Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany		Incu safe
Isofluran	Baxter Deutschland GmbH, Unterschleißheim, Germany
Light microscope	Carl Zeiss SMT GmbH, Oberkochen, Germany		Axiovert 40 °C
Macrophage-Colony Stimulating Factor	Sigma Aldrich, Hamburg, Germany	SRP3110
Mechanical shaker	IKA, Staufen, Germany		ms2 minishaker
Medium 199	PAA Laboratories GmbH, Pasching, Austria		Warm in 37 °C water bath before use
Micro test tubes	Eppendorf AG, Hamburg, Germany
Microbiological work bench	Thermo Electron, LED GmbH, Langenselbold, Germany		Hera safe
Monocyte wash buffer	Manufactured by our group with single components		PBS, 0.5% BSA, 2 mM EDTA
Mouse restrainer	Various
NaCl	Berlin Chemie AG, Berlin, Germany
NaN3 (sodium acide)	Sigma Aldrich, Hamburg, Germany
Neubauer counting chamber	Paul Marienfeld GmbH und Co.KG, Lauda-Königshofen, Germany
Nylon cellsieve	Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA		Cell strainer, 70 µm mesh size
Penicillin/Streptomycin	Sigma Aldrich, Hamburg, Germany
Phosphate buffered saline	Life technologies GmbH, Darmstadt, Germany		pH 7.4, sterile
Pipettes	Eppendorf AG, Hamburg, Germany		10µl/100µl/200µl/1,000µl
Pipetting heads	Eppendorf AG, Hamburg, Germany
Serological pipette	Greiner Bio-One GmbH, Frickenhausen, Germany		Cellstar 5 ml, 10 ml
Suction unit	Integra bioscience, Fernwald, Germany		Vacusafe comfort
Surgical scissors	Word Precision Instruments, Inc., Sarasota, USA
Syringe	B. Braun, Melsungen AG, Melsungen, Germany		1 ml Omnifix® -F insuline syringe
Tubes with cap	Greiner bio one GmbH, Frickenhausen, Germany		15 ml/50 ml Cellstar tubes
Warm water bath	Julabo Labortechnik GmbH, Seelbach, Germany		Julabo SW22