隔离和小鼠骨髓的静脉注射单核细胞衍生

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

与萨克森州和萨克森 – 安哈尔特州，Regierungspraesidium德累斯顿/哈勒的国家批准进行这项研究，根据德国法律的保护动物（24D-9168.11-1 / 2008-24）第8。 1细胞分离 1.1准备股骨和胫骨的用5％异氟烷浓度通过在一个封闭的仓汽化异氟烷麻醉小鼠。后的小鼠停止移动，持续3秒，执行颈椎脱位。 消毒后肢用乙醇（96％）。去除皮肤和肌肉分开的骨头用无?…

Representative Results

来自鼠骨髓中提取的细胞溶液包含各种细胞类型。主要的细胞类型是淋巴细胞，粒细胞和单核细胞。细胞类型可以通过尺寸和粒度，其示于图1中为5天后分化的收获两个天然悬浮液和细胞来估计。注意培养过程中的换档细胞组成。然而，人口的准确分类必须依靠细胞标记鲜明的表达。 用于识别的MPS-系统的细胞中最重要的标记是CD115，其功能是作为对M-CSF受体和专?…

Discussion

我们描述了一个简单而具有成本效益的方法，以分离大量骨髓的小鼠单核细胞。相较于使用外周血其他协议，其中获得的单核细胞产率⁵ 1.4×10 ^6，我们能够从一个单一的供体小鼠获得的11×10 ^{6±3×10 6}单核细胞更高的产量。

当考虑使用这种方法的挑战，它在非无菌条件下工作时，提供潜在的污染是重要的。如果冲洗和不遵守以下洗涤步骤正确，污染的细?…

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