Summary

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

Published: December 27, 2014
doi:

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 artery1-3. Monocytes also mature to dendritic cells, which are frequently used for immunological studies4,5 and cancer research6,7.

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 interpretation10,11. 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

Este estudio se realizó con permiso del Estado de Sajonia y Sajonia-Anhalt, Regierungspraesidium Dresden / Halle, de acuerdo con el Artículo 8 de la Ley alemana de protección de los animales (24D-9168,11-1 / 2008-24). Aislamiento 1 celular 1.1 Preparación de fémur y la tibia Anestesiar al ratón usando una concentración de isoflurano 5% mediante la vaporización de isoflurano en un contenedor cerrado. Después de que el ratón ha dejado de moverse d…

Representative Results

La solución de células extraída de la médula ósea murina se compone de diversos tipos de células. Los tipos de células principales son los linfocitos, granulocitos y monocitos. Los tipos de células pueden ser estimados por el tamaño y la granularidad, que se muestra en la Figura 1 para ambas suspensiones nativas y células cosechadas después de 5 días de diferenciación. Tenga en cuenta la composición celular cambiando durante el cultivo. Sin embargo, la clasificación exacta de las poblacio…

Discussion

Se describe un método simple y rentable para aislar grandes cantidades de monocitos murinos de la médula ósea. En comparación con otros protocolos que utilizan la sangre periférica, que obtienen rendimientos de monocitos 5 de 1,4 x10 6, somos capaces de obtener mayores rendimientos de 11 x 10 6 ± 3 x 10 6 monocitos de un solo ratón donante.

Al considerar los desafíos con este método, es importante mencionar la posibilidad de contaminación…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center).

Thanks to Hans-Holger Gärtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.

Materials

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, 2mM 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/1000µL
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 1mL Omnifix® -F insuline syringe
Tubes with cap Greiner bio one GmbH, Frickenhausen, Germany 15mL/50mL Cellstar tubes
Warm water bath Julabo Labortechnik GmbH, Seelbach, Germany Julabo SW22

Referências

  1. Herold, J., et al. Transplantation of monocytes: a novel strategy for in vivo augmentation of collateral vessel growth. Hum Gene Ther. 15 (1), 1-12 (2004).
  2. Herold, J., Tillmanns, H., Xing, Z., Strasser, R. H., Braun-Dullaeus, R. C. Isolation and transduction of monocytes: promising vehicles for therapeutic arteriogenesis. Langenbecks Arch Surg. 391 (2), 72-82 (2006).
  3. Francke, A., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C., Herold, J. Transplantation of bone marrow derived monocytes: a novel approach for augmentation of arteriogenesis in a murine model of femoral artery ligation. American Journal Of Translational Research. 5 (2), 155-169 (2013).
  4. Leon, B., et al. Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8- and CD8+ splenic dendritic cells. Blood. 103 (7), 2668-2676 (2004).
  5. Timmerman, J. M., Levy, R. Dendritic cell vaccines for cancer immunotherapy. Annu Rev Med. 50, 507-529 (1999).
  6. Salem, M. L. The use of dendritic cells for Peptide-based vaccination in cancer immunotherapy. Methods Mol Biol. 1139, 479-503 (2014).
  7. Timmerman, J. M., et al. Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood. 99 (5), 1517-1526 (2002).
  8. Berthold, F. Isolation of human monocytes by Ficoll density gradient centrifugation. Blut. 43 (6), 367-371 (1981).
  9. Houthuys, E., Movahedi, K., De Baetselier, P., Van Ginderachter, J. A., Brouckaert, P. A method for the isolation and purification of mouse peripheral blood monocytes. Journal Of Immunological Methods. 359 (1-2), 1-10 (2010).
  10. Seeger, F. H., Tonn, T., Krzossok, N., Zeiher, A. M., Dimmeler, S. Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction. European Heart Journal. 28 (6), 766-772 (2007).
  11. Auffray, C., et al. CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation. The Journal Of Experimental Medicine. 206 (3), 595-606 (2009).
  12. Francke, A., Herold, J., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C. Generation of mature murine monocytes from heterogeneous bone marrow and description of their properties. J. Histochem. Cytochem. 59 (9), 813-825 (2011).
  13. Sneider, E. B., Nowicki, P. T., Messina, L. M. Regenerative medicine in the treatment of peripheral arterial disease. Journal Of Cellular Biochemistry. 108 (4), 753-761 (2009).
  14. Van Royen, N., Piek, J. J., Schaper, W., Fulton, W. F. A critical review of clinical arteriogenesis research. J Am Coll Cardiol. 55 (1), 17-25 (2009).
  15. Lawall, H., Bramlage, P., Amann, B. Treatment of peripheral arterial disease using stem and progenitor cell therapy. J Vasc Surg. 53 (2), 445-453 (2011).
  16. Herold, J., et al. Tetanus toxoid-pulsed monocyte vaccination for augmentation of collateral vessel growth. Journal of the American Heart Association. 3 (2), e000611 (2014).

Play Video

Citar este artigo
Wagner, M., Koester, H., Deffge, C., Weinert, S., Lauf, J., Francke, A., Lee, J., Braun- Dullaeus, R. C., Herold, J. Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes. J. Vis. Exp. (94), e52347, doi:10.3791/52347 (2014).

View Video