隔离和小鼠骨髓的静脉注射单核细胞衍生

摘要

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).

摘要

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.

引言

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.

研究方案

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

1. 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。
2. 消毒后肢用乙醇（96％）。去除皮肤和肌肉分开的骨头用无菌手术刀。
   1. 开始与腹股沟皮肤切口，并延伸朝向内踝切口并执行围绕下部小腿皮肤的圆形解剖。由近端脱皮彻底去除腿部的皮肤。
   2. 从分离用锋利的手术刀股骨股四头肌，同时删除前腓骨和腓肠肌肌肉群并通过定位和解剖韧带disarticulate腿在髋关节。
   3. 启动向前，继续关节周围小心地旋转腿和横断韧带，从而脱节的联合。
3. 洗股骨和胫骨用96％的乙醇最少90秒，以保证无菌细胞制剂。继续洗涤过程中使用无菌PBS冲洗掉剩余的乙醇。
   注意:所有的后续步骤必须是无菌的，以避免污染。

1.2收获骨髓

1. 切割每个骨的近端和远端具有一对细剪刀来访问的股骨和胫骨的轴。小心这一步，因为这些骨头断裂非常容易。
2. 冲洗骨头用温介质（M199 + 10％胎牛血清（FCS），+ 1％青霉素/链霉素）。使用无菌28-G针，1ml注射器，和10-15毫升培养基每间骨冲洗。
3. 握住骨用细镊子和，直到它变成半透明一个冲洗之一。小心施加压力的注射器柱塞的端部，以减少细胞应激。
4. 通过70微米尼龙网过滤骨髓并收集滤液。以提取骨髓的最大数量，从所述远端和近端反复冲洗。
5. 冲洗用PBS筛，并收集滤液。小心撞出的碎片和细胞集团轻轻搅拌和移液。

1.3栽培

1. 离心细胞悬浮液在250×g离心在室温10分钟。弃上清，悬浮细胞与大约25毫升介质。重复一次。
2. 第二清洗步骤之后重悬细胞在6ml培养基。混合50微升的细胞溶液与50μl的台盼蓝。计数细胞在计数室在光学显微镜下。计算C的数使用此公式中的溶液ELLS:
   
3. 种子上6孔超低附着板表面的细胞，以防止永久附着在板的底部。使用每毫升10 ⁶个细胞的浓度以每孔最多至6ml。
4. 补充悬架20毫微克/毫升的M-CSF，促进细胞分化。
5. 培养在37℃下将细胞5天，5％二氧化碳和每日观察。

细胞收获1.4

注意:这些步骤应在冰上进行。继续无论1.4.1或1.4.2相应。

1. 在这一点上，收获整个培养，包括分化的巨噬细胞，将粘附到培养板，即使它们是超低附着表面板。
   1. 通过温和移液收获整个培养和4℃的PB中的溶液分离S，0.5％牛血清白蛋白（BSA）和2mM EDTA中。重复，直到这些板块都清楚剩余的贴壁细胞 - 确认在显微镜下，如果不能确定。
2. 可替代地，通过收获与细胞在悬浮液中，其中主要含有单核细胞的上清液丢弃粘附的巨噬细胞。
   1. 收获非贴壁细胞用的​​无EDTA的PBS中的溶液和0.5％牛血清白蛋白。不适用为了避免撞出轻度粘连的巨噬细胞用力过猛。

1.5 FACS分析（可选）

注意:可以通过使用MACS的耗尽CD117 ⁺茎和祖细胞的细胞悬浮液。使用制造商协议，在此过程中。

1. 根据要么步骤1.4.1或1.4.2收获细胞。
2. 离心细胞，在250×g离心10分钟，在4℃，并再次重复该步骤。
3. 悬浮至少250,000个细胞在300微升FACS的Buffer每个探头。
4. 转移的细胞溶液到96孔板（30每孔微升）。
5. 离心细胞悬浮液在250×g离心5分钟，在4℃。
6. 除去上清液，并添加25微升抗体，每孔（使用制造商稀释度）。
7. 染色1.5.1-1.5.6将细胞与抗体（按照生产商协议后步骤的细胞表型。常用标记的单核细胞/巨噬细胞识别包括的CD11b，F4 / 80（ 图3），CD115（图2），和GR- 1。
   注意:对于小区的解决方案的进一步的描述中，使用象CD4，CD8A，的CD11c，CD25，CD45，CD45R，CD62L，CD80，CD86，CD145，CD117，MHCⅡ，的Ly6C，Ly6G和CD192标记的建议。蜂窝特性进行先前描述的^12。
8. 孵育探针20分钟，在4℃。
9. 离心机的探针，在250×g离心5分钟，在4℃下重悬用200μl的FACS缓冲液中。重复次是步两次。
10. 转移探讨FACS管并进行FACS分析^12。
11. 执行在第0天，第3和5 FACS分析¹²来分析细胞的分化。

2.尾静脉注射

2.1准备

1. 暖在加热板上的动物，在37℃进行约10分钟。观察动物，同时升温至识别过热的迹象。
2. 悬浮的单核细胞，分离的步骤1.1- 1.4，在150微升的NaCl，并加载细胞到1 ml胰岛素注射器用30G针。装载注射器，以确保所有的单核细胞被注射之前轻轻旋涡振荡溶液。总是处理冰上细胞，避免热介导的附着和激活单核细胞。

2.2抑制

1. 避免在选择鼠标静脉注射干扰其他老鼠在笼子里。
2. 将鼠标放到限制器。避免过多公关essure和小心的后肢。 （ 图4）
   注:另外，使用全身麻醉，以保证无压力的动物处理。
3. 确保鼠标有呼吸的空间关闭限制器之前。

2.3注塑

1. 消毒注射部位以消毒剂。喷在小鼠的尾部，静置至少1分钟。
2. 通过应用温和的压力，侧尾侧注射部位以上，为更好的可视性静脉停止尾部静脉血流。
3. 打开尾90度，注射前。 （ 图5）
4. 注入以45度角。慢慢的注入，也没有每克超过5微升，以免伤及动物。
5. 如果有一个泡罩，这是一个失败的注入的标志，立即停止和更近侧重复注射。
6. 通过应用柔和的P止血注射侧ressure约1分钟。
7. 在该过程结束时，打开该卷收器，并放置在鼠标在其笼中。
8. 观察小鼠20分钟，以确保动物没有被注射伤害和胸骨斜卧被保持。延长观察时间，直到鼠标重新获得足够的意识。返回鼠标给其他动物的公司只后它已完全恢复。

结果

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?...

讨论

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?...

材料

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