枯否细胞分离的纳米粒子毒性测试
Summary
Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.
Abstract
绝大多数体外 nanotoxicological研究已经使用永生化细胞系为他们的实用性。然而，从纳米颗粒的毒性测试结果在永生化细胞系或原代细胞已经显示不符，强调需要延长使用的原代细胞的体外测定法。这个协议描述了小鼠肝巨噬细胞的分离，命名Kupffer细胞，以及它们的使用来研究纳米颗粒的毒性。 Kupffer细胞是最丰富的巨噬细胞群在体内和构成网状内皮系统（RES），负责循环纳米颗粒的捕获部分。这里报道的枯否细胞的分离方法是基于一种两步灌注法进行纯化上密度梯度。该方法的基础上，胶原酶消化和密度离心，适于从由Smedsrød 等人开发的原始协议。专为大鼠肝细胞的隔离和n和提供高产率（每鼠14×10 6个细胞）和枯否细胞的高纯度（> 95％）。这种隔离方法不需要复杂的或昂贵的设备，因此代表的复杂性和细胞产量之间的理想折衷。使用较重的小鼠（35-45克）提高了分离方法的产量，但也有利于显着的门静脉插管的过程。的官能化的碳纳米管˚F-CNTs毒性测定在该模型由改性的LDH测定。该方法通过测量缺乏的枯否细胞膜结构完整性孵育以f -CNTs后评估细胞生存力。毒性反应为f -CNTs可以使用一致此法来衡量，强调指出，孤立的枯否细胞对纳米颗粒的毒性测试非常有用。纳米毒理学的整体理解可以受益于这种模式，使得纳米颗粒的选择临床翻译更多艾菲cient。
Introduction
纳米毒理学研究领域的目的是表征纳米颗粒的生物效应。基于在体内研究毒理学研究仍然是最精确的方法。然而，它们的使用受到它们的成本，劳动和时间要求1限定。作为替代， 在体外测定法已被使用，因为它们的简单性和开发高通量体外测试平台2的可能性-如此延伸的测试条件的数量。最nanotoxicological研究使用体外测定与永生化的细胞系进行。然而，也有关于这些实验的结果外推到体内毒理学影响3担忧。事实上，永生化细胞系的性质可以是显著不同从组织它们衍生自， 例如 ，遗传转化4，关键形态特征恶化5，细胞极性6和功能的改变，如炎症介质7的调节的丧失。
Kupffer细胞是最丰富的巨噬细胞群中的主体，并直接与血液接触由衬肝血窦的壁上。作为网状内皮系统（RES）的一部分，这些巨噬细胞是负责循环纳米颗粒和因此的捕获，构成一个非常合适的模型来研究纳米颗粒的毒性。 体内 8 并在体外与暴露于纳米粒子枯否氏细胞相关的炎症反应9研究已在其他地方发表。枯否细胞也参与肝脏疾病的发病机制，如酒精性肝病10，肝纤维化11或病毒性肝炎12。据报道，孤立的枯否细胞提供了有益的启示来形容细胞机制INVolved肝破裂13,14。
几种方法已经报道了分离和纯化Kupffer细胞。细胞的分离可导致机械或酶解15。胶原酶消化节约表明枯否细胞的功能完整性，而导致高枯否细胞产量16的优势。许多实验方法，在复杂性和成本不同，已用于Kupffer细胞从其他肝细胞群中分离出来。例如，枯否细胞纯度可通过免疫亲和17，流电泳18，选择性粘附16或通过离心技术16，根据它们的大小和密度而选择的细胞来实现。这些方法的组合可以被选择为增加的人口16的纯度。没有关于对枯否细胞分离的理想方法共识的，因为它主要取决于应用和可用的equipmen吨。然而，在纳米颗粒的毒性测试的情况下，简单性和高收率与Kupffer细胞的功能保存相关的技术似乎是对本申请中最适合的。
这里报道的枯否细胞的分离方法是基于一种两步灌注法进行纯化上密度梯度。该方法是从由Smedsrød 等人开发的原始协议进行修改。16设计用于大鼠肝细胞的分离。大多数研究报告，并从大鼠肝脏描述Kupffer细胞的分离。在此，我们描述了一种方法，从小鼠肝脏分离Kupffer细胞，以高收率和纯度。使用小鼠降低了实验成本，并允许几个肝脏的处理，以获得纳米颗粒的毒性测试大量Kupffer细胞。
在下面的协议，枯否细胞与官能化的碳纳米管（六</EM> -CNTs）。碳纳米管的独特的物理化学性质- 即高长径比和表面积大，都取得了碳纳米管有趣候选作为用于治疗和诊断目的的载体。然而，忧虑已提出了关于碳纳米管19的毒性，以及新的体外测试的发展旨在提高CNT生物效应的理解。在枯否细胞毒性与缺乏细胞膜的结构完整性有关。这是通过胞质酶LDH的损失从细胞到上清液计量。这种方法的原理，因此，是除去任何释放LDH和测量什么留在电池20。这在要求完成测量所释放的LDH在上清液因为CNT的上清液中的存在干扰测定21。
我们建议使用这种简单和成本效益枯否细胞的分离实现方法具的d，来分离大量的功能枯否细胞。这允许范围的纳米颗粒的毒性的筛选，在一个相关的初级巨噬细胞的模型。
Protocol
Representative Results
Discussion
下面的步骤是关键的，以实现高产量和枯否氏细胞的高存活率。在无菌条件应用于限制细菌和真菌污染的风险。所有仪器使用前需要灭菌。试剂应在进行分离步骤之前新鲜制备。
Ⅳ型胶原酶的选择，具有低胰蛋白酶的活性，是至关重要的。不同批次来自同一供应商具有不同的酶活性和它们可能需要最初比较，以便选择最合适的批次库普弗细胞分离。这可以通过评估细胞产量?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by EU FP7-ITN Marie-Curie Network program RADDEL. MB and KAJ would like to acknowledge Joe Varguese and Rui Serra Maia for their help and suggestions along the optimization of the Kupffer cell isolation.
Materials
| Euthatal (pentobarbital sodium) | Merial | ||
| CD1 mice | Charles River | – | Mouse weight should vary from 35 to 45 g. We advise the use of male CD1 mice as their weight increase rapidly (e.g. a male CD1 mouse of 7 weeks old exceeds 35 g in weight). It is also advised to contact the animal supplier in advance to arrange for the delivery of older animals. Alternatively animals can be in-house to reach the desired weight. |
| HBSS (Ca2+ and Mg2+ free, with bicarbonate) | Life Technologies | 14175-053 | HBSS must be Ca2+ free. |
| Ethylene Glycol Tetraacetic Acid (EGTA) tetrasodium salt | Sigma-Aldrich | E8145 | EDTA can also be used but EGTA has the advantage of chelating Ca2+ selectively. |
| HEPES (1M) | Life Technologies | 15630-056 | 100 ml |
| Collagenase type IV | Worthington | CSL-4 | It is advised to test different batches of collagenase or at least mention to the supplier the product has to be suitable for liver cell isolation |
| Low glucose DMEM | Sigma-Aldrich | D5523 | 500 ml |
| RPMI (with sodium pyruvate and Glutamax®) | Life Technologies | 12633-012 | 500 ml |
| Penicillin/Steptomycin | Life Technologies | 15140-122 | 100 ml |
| Fetal Bovine Serum | First-Link | 60-00-850 | 500 ml |
| Trypan blue solution | Sigma-Aldrich | T8154 | 100 ml |
| Coated silica particle solution (Percoll®) | GE Healtcare | 17-0891-02 | Percoll® is very stable and can be kept for several years |
| 1x Phosphate Buffered Saline | Life Technologies | 10010-023 | 500 ml |
| 10x Phosphate Buffered Saline | Life Technologies | 70011-036 | 500 ml |
| Dimethyl sulfoxide | Fisher | D/4120/PB08 | 500 ml |
| LDH kit (CytoTox 96®) | Promega | G1781 | Keep protected from light |
| DMEM phenol red free | Life Technologies | 31053-028 | 500 ml |
| F4/80 antibody (Alexa 488) | AbD Serotec | MCA497A488 | Do not dilute, used neat for flow cytometry |
| Fluorescent beads | Sigma | L2778 | Latex beads, amine-modified polystyrene, fluorescent red. 1 ml |
| Name of the Material | Company | Catalog number | Comments/Description |
| Butterfly blood collection set (23G/305mm long tubing) | BD | 367288 | |
| Syringe Filters (0.22 μm Blue Rim) | Minisart | 16534-K | |
| Centrifuge Tubes (50 ml Blue Cap) | BD Biosciences, Falcon | 35 2070 | |
| Petri dish (90 x 15 mm) | Thermo Fisher Scientific | BSN 101VR20 | |
| 100 μm cell strainer | BD | 352360 | |
| Peristaltic pump | Watson Marlow | SciQ 300 | Rinse tubing before and after each usage with sterile PBS and 70% ethanol. |
| 24-well plates | Corning | 3526 | |
| 96-well plates | Corning | 3595 | |
| Centrifuge | Eppendorf | 5810R | |
| Plate reader | BMG Labtech | FLUOstar Omega | |
| Serrefine forceps | Hammacher GmbH | Art. Nr. HSE 004-35 / Cat. Nr. 221-0051 | The serrefine forceps allow to clamp the vessel cannulated with the 23G needle without the need of holding the forceps during the perfusion procedure. URL: (http://www.hammacher.de/Laboratory-Products/Clamps-forceps/Serrefines/HSE-004-35-Serrefine::25126.html) |
| Flow cytometry tubes | BD Biosciences, Falcon | 352052 | |
| Microcentrifuge tubes (1.5 ml) | Elkay | 000-MICR-150 | |
| Cell scraper | BD Biosciences, Falcon | 353086 | Cut blade extremities with a pair of scissors to scrape cells in 24-well plates. |
| Name of the Reagent | Company | Catalog number | Comments/Description |
| EGTA (Ethylene Glycol Tetraacetic Acid)/HBSS (Hank's Balanced Salt Solution) Solution | HBSS containing 0.5 mM EGTA and 25 mM HEPES. Adjust pH to 7.4. Prepare 50 ml for each liver to perfuse. | ||
| Collagenase Solution | DMEM low glucose containing collagenase type IV at 100 UI/ml, 15 mM HEPES and 1% Penicllin/Streptamycin (v/v). Adjust pH to 7.4. Prepare 100 ml for each liver to perfuse. After adding the collagenase, it is advised to warm up the solution for 30 min before use. This allows the collagenase activity to be optimum. | ||
| Kupffer Cell Isolation Medium | RPMI containing, 1% Non-Essential Amino-Acids (v/v),1% glutamax® (v/v) and 1% Penicllin/Streptomycin (v/v). Prepare at least 100 ml for 1-3 livers. | ||
| Kupffer Cell Culture Medium | RPMI containing 10% Fetal Bovine Serum, 1% Non-Essential Amino-Acids (v/v),1% Glutamax® (v/v) and 1% Penicllin/Streptamycin (v/v). Prepare at least 100 ml for 1-3 livers. | ||
| SIP (solution of isotonic coated silica particles) | Mix 1.7 ml of 10x Phosphate Buffered Saline with 15.3 ml of Percoll® to obtain 17mL of SIP. | ||
| 25% SIP solution | Mix 5 ml of SIP with 15 ml of 1x Phosphate Buffered Saline | ||
| 50% SIP solution | Mix 10 ml of SIP with 10 ml of 1x Phosphate Buffered Saline | ||
| Lysis Buffer | DMEM media with 0.9% Triton X-100 | ||
| PBS/BSA Solution | Prepare fresh Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin. | ||
Referencias
- Hoffmann, D., et al. Performance of novel kidney biomarkers in preclinical toxicity studies. Toxicol Sci. 116 (1), 8-22 (2010).
- Nel, A., et al. Nanomaterial Toxicity Testing in the 21st Century: Use of a Predictive Toxicological Approach and High-Throughput Screening. Accounts of Chemical Research. 46 (3), 607-621 (2013).
- Bregoli, L., et al. Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines. Toxicology. 262 (2), 121-129 (2009).
- Yamasaki, K., et al. Genomic Aberrations and Cellular Heterogeneity in SV40-Immortalized Human Corneal Epithelial Cells. Investigative Ophthalmology, & Visual Science. 50 (2), 604-613 (2009).
- Motoyoshi, Y., et al. Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria. Kidney International. 74 (10), 1262-1269 (1038).
- Prozialeck, W. C., Edwards, J. R., Lamar, P. C., Smith, C. S. Epithelial barrier characteristics and expression of cell adhesion molecules in proximal tubule-derived cell lines commonly used for in vitro toxicity studies. Toxicology in Vitro. 20 (6), 942-953 (2006).
- Chamberlain, L. M., Godek, M. L., Gonzalez-Juarrero, M., Grainger, D. W. Phenotypic non-equivalence of murine (monocyte-) macrophage cells in biomaterial and inflammatory models. J Biomed Mater Res A. 88 (4), 858-871 (2009).
- Kermanizadeh, A., et al. The role of Kupffer cells in the hepatic response to silver nanoparticles. Nanotoxicology. , (2013).
- Fischer, H. C., Hauck, T. S., Gomez-Aristizabal, A., Chan, W. C. W. Exploring Primary Liver Macrophages for Studying Quantum Dot Interactions with Biological Systems. Advanced Materials. 22 (23), 2520-2524 (2010).
- Wan, J. H., et al. M2 Kupffer Cells Promote M1 Kupffer Cell Apoptosis: A Protective Mechanism Against Alcoholic and Nonalcoholic Fatty Liver Disease. Hepatology. 59 (1), 130-142 (2014).
- Matsuoka, M., Tsukamoto, H. Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor β: Implication for a pathogenetic role in alcoholic liver fibrogenesis. Hepatology. 11 (4), 599-605 (1990).
- Polakos, N. K., et al. Kupffer Cell-Dependent Hepatitis Occurs during Influenza Infection. The American Journal of Pathology. 168 (4), 1169-1178 (2006).
- Tanner, A., Keyhani, A., Reiner, R., Holdstock, G., Wright, R. Proteolytic enzymes released by liver macrophages may promote hepatic injury in a rat model of hepatic damage. Gastroenterology. 80 (4), 647-654 (1981).
- Knittel, T., et al. Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-α and TGF-β1. Journal of Hepatology. 30 (1), 48-60 (1999).
- Branster, M. V., Morton, R. K. Isolation of Intact Liver Cells. Nature. 180 (4597), 1283-1284 (1038).
- Smedsrød, B., Pertoft, H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. Journal of Leukocyte Biology. 38 (2), 213-230 (1985).
- Morin, O., Patry, P., Lafleur, L. Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry. J Cell Physiol. 119 (3), 327-334 (1984).
- Miller, S. B., et al. Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis. Cancer Res. 43 (9), 4176-4179 (1983).
- Firme, C. P., Bandaru, P. R. Toxicity issues in the application of carbon nanotubes to biological systems. Nanomedicine. 6 (2), 245-256 (2010).
- Ali-Boucetta, H., Al-Jamal, K. T., Kostarelos, K. Cytotoxic assessment of carbon nanotube interaction with cell cultures. Methods in Molecular Biology. 726, 299-312 (2011).
- Wang, G., et al. Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay. Toxicology. 299 (2-3), 99-111 (2012).
- Wang, J. T. -. W., et al. Magnetically Decorated Multiwalled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents. Advanced Functional Materials. 24 (13), 1880-1894 (2014).
- Li, P. Z., Li, J. Z., Li, M., Gong, J. P., He, K. An efficient method to isolate and culture mouse Kupffer cells. Immunol Lett. 158 (1-2), 52-56 (2014).
- Froh, M., Konno, A., Thurman, R. G. Isolation of liver Kupffer cells. Curr Protoc Toxicol. Chapter 14, Unit14 14 (2003).
