鼠心肺复合物的脱细胞化
Summary
该协议旨在使小鼠的心脏和肺部脱细胞。可以对所得的细胞外基质（ECM）支架进行免疫染色和成像，以绘制其组分的位置和拓扑结构。
Abstract
我们在这里提出了一种小鼠心脏和肺部的去细胞化方案。它产生可用于分析ECM拓扑和组成的结构ECM支架。它基于一种微外科手术，旨在对安乐死小鼠的气管和主动脉进行导管插入，以用去细胞化剂灌注心脏和肺部。随后可以对去细胞化的心肺复合物进行免疫染色，以揭示结构性ECM蛋白的位置。整个过程可以在4天内完成。
由该协议产生的ECM支架没有尺寸失真。由于没有细胞，可以对ECM结构进行结构检查，直至亚微米分辨率的3D。该协议可应用于来自4周龄小鼠的健康和患病组织，包括纤维化和癌症的小鼠模型，为确定与心肺疾病相关的ECM重塑开辟了道路。
Introduction
ECM是由蛋白质和聚糖组成的三维网络，可容纳多细胞生物体中的所有细胞，使器官具有形状并在整个生命中调节细胞行为1。从卵子受精开始，细胞建立和重塑ECM，并反过来受到它的严格控制。该协议的目的是开辟一种分析和绘制小鼠ECM的方法，因为小鼠是哺乳动物病理生理学中最常用的模式生物。
该方法的发展是由表征和分离转移相关天然ECM2的需求所驱动的。由于肿瘤缺乏适当的解剖血管形成，并且小鼠是相对较小的生物体，因此显微外科手术被设计为逆行导管主动脉，同时分离导致肿瘤的主要血管（例如，肺静脉）的循环，从而聚焦试剂流并允许肿瘤脱细胞。该方法产生具有保守结构2 的ECM支架，可以进行免疫染色和成像，从而允许亚微米级细节的ECM结构映射。为了执行该协议，有必要获得手术和显微外科技术（解剖，显微缝合和导管插入术），这可能代表其使用的潜在限制。据我们所知，这种方法代表了本机ECM结构成像分析的最新技术2，3。
Protocol
这里包含的所有程序都经过哥本哈根大学管理实验医学伦理委员会的审查和批准，并同意丹麦和欧洲的立法。为了证明这一方案，我们使用了8-12周龄的雌性BALB / cJ小鼠和11周龄的MMTV-PyMT雌性小鼠。 注意：避免去细胞化的ECM支架的细菌污染可获得最佳的成像结果，并允许长期样品储存。因此，保持所有步骤无菌非常重要。因此，所有器械和手术材料，包括缝合线、微缝合线、?…
Representative Results
心肺脱细胞成功完成实验方案后，心脏和肺部以及主动脉弧等附属组织将没有细胞。去细胞化可以通过ECM支架的血氧锡 – 曙红染色（图1）来验证，与天然组织相比，显示细胞核的去除。这些支架保留了新鲜器官的尺寸，其不溶性ECM结构完好无损2。 图2 显示了成功灌注小鼠心肺复合体所需的关?…
Discussion
基于组织搅拌的去细胞化技术改变了ECM结构，使其不适合ECM结构分析4。灌注去细胞化，使用解剖途径，如气管的主动脉，允许到达毛细血管床或肺泡末端，并促进脱细胞剂在整个器官的递送。据报道，使用两性离子，阴离子和非离子洗涤剂使组织去细胞化4，5，6，然而，十二烷基硫酸钠（SDS，阴离子）线性?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Ivana Novak教授和Nynne Meyn Christensen博士（哥本哈根大学高级生物成像中心（CAB））提供显微镜访问。这项工作得到了欧洲研究理事会（ERC-2015-CoG-682881-MATRICAN;AEM-G，OW，RR和JTE）;伦德贝克基金会的博士奖学金（R286-2018-621;先生）;瑞典研究委员会 （2017-03389;清洁发展机制）;瑞典癌症协会，Cancerfonden（CAN 2016/783，19 0632 Pj和190007;清洁发展机制）;德国癌症援助组织（Deutsche Krebshilfe;RR）;和丹麦癌症协会（R204-A12454;RR）。
Materials
| MICROSURGERY | |||
| 6-0 suture, triangular section needle (Vicryl) | Ethicon | 6301124 | |
| 9-0 micro-suture (Safil) | B Braun | G1048611 | |
| Adson forceps | Fine Science Tools | 11006-12 | |
| Adson forceps with teeth | Fine Science Tools | 11027-12 | |
| Castroviejo microneedle holder | Fine Science Tools | no. 12061-01 | |
| CO2 ventilation chamber for mouse euthanasia | |||
| Deionized water (Milli-Q IQ 7000, Ultrapure lab water system) | Merck | ZIQ7000T0 | |
| Disposable polystyrene tray (~30 × 50 cm) | |||
| Dissection microscope (Greenough, with two-armed gooseneck) | Leica | S6 D | |
| Double-ended microspatula | Fine Science Tools | 10091-12 | |
| Dumont microforceps (two) | Fine Science Tools | 11252-20 | |
| Dumont microforceps with 45° tips (two) | Fine Science Tools | 11251-35 | |
| Hair clippers | Oster | 76998-320-051 | |
| Halsey needle holder (with tungsten carbide jaws) | Fine Science Tools | 12500-12 | |
| Intravenous 24-gauge catheter (Insyte) | BD | 381512 | |
| Intravenous 26-gauge catheter (Terumo) | Surflo-W | SR+DM2619WX | |
| Mayo scissors (tough cut, straight) | Fine Science Tools | 14110-15 | |
| Microforceps with ringed tips (two) | Aesculap | FM571R | |
| Micro-spring scissors (Vannas, curved) | Fine Science Tools | 15001-08 | |
| Minicutter | KLS Martin | 80-008-03-04 | |
| Molt Periostotome | Aesculap | D0543R | |
| Needles (27 gauge; Microlance) | BD | 21018 | |
| Paper towel (sterile) or surgical napkin | |||
| Serrated scissors (CeramaCut, straight) | Fine Science Tools | 14958-09 | |
| Spatula (Freer-Yasargil) | Aesculap | OL166R | |
| Syringes (1 mL; Plastipak) | BD | 3021001 | |
| Syringes (10 mL; Plastipak) | BD | 3021110 | |
| Tendon scissors (Walton) | Fine Science Tools | 14077-09 | |
| IMMUNOSTAINING | |||
| Alexa Fluor 488 donkey anti-guinea pig IgG | Thermo Fisher Scientific | A-11055 | |
| Alexa Fluor 594 donkey anti-rabbit IgG | Life Technologies | A11037 | |
| BSA(albumin bovine fraction V, standard grade, lyophilized) | Serva | 11930.03 | |
| Collagen IV polyclonal antibody (RRID: AB_2276457) | Millipore | AB756P | Host: rabbit |
| PBS (pH 7.4, 10×, Gibco) | Thermo Fisher Scientific | 70011044 | Host: goat |
| Periostin polyclonal antibody (a kind gift from Manuel Koch. RRID:AB2801621) | Host: guinea pig | ||
| Scalpel disposable with blade no.11 (pcs. 10) | VWR | 233-5364) | |
| Serum (normal donkey serum) | Jackson ImmunoResearch | 017-000-121 | |
| Tween 20 | Sigma-Aldrich | P9416-50ML | |
| IMAGING | |||
| Detectors (hybrid detector (Leica, HyD S model) and photomultiplier tubes (PMTs; ) | Leica | ||
| Fluorescence light source | Leica | EL6000 | |
| Microscope (inverted multiphoton microscope) | Leica | SP5-X MP | |
| Objective (lambda blue, 20×, 0.70 numerical aperture (NA) IMM UV) | Leica | HCX PL APO | |
| Two-photon Ti–sapphire laser (Spectra-physics, Mai Tai DeepSee model) | |||
| White-light laser (WLL) | Leica | ||
| DECELLULARIZATION | |||
| 70% Ethanol (absolute alcohol 99.9%); absolute alcohol must be adjusted to 70% (vol/vol) using deionized water | Plum | 1680766 | |
| Deionized water (Milli-Q IQ 7000, Ultrapure lab water system) | Merck | ZIQ7000T0 | |
| Luer-to-tubing male fittings (1/8 inch) | World Precision Instruments | 13158-100 | |
| PBS (pH 7.4, 10×, Gibco) | Thermo Fisher Scientific | 70011044 | |
| Penicillin-streptomycin | Gibco | 15140122 | |
| Peristaltic pump (with 12 channels) | Ole Dich | 110AC(R)20G75 | |
| Silicone tubing (with 2-mm i.d. and 4 mm o.d.) | Ole Dich | 31399 | |
| Sodium Azide | Sigma-Aldrich | 08591-1ML-F | |
| Sodium deoxycholate (DOC) | Sigma-Aldrich | D6750-100G | |
| Sodium Dodecyl Sulphate | Sigma-Aldrich | L3771-500G | |
| H&E STAINING | |||
| 4% PFA | Fisher Scientific | 15434389 | |
| 96% Ethanol | Plum | 201446-5L | |
| Absolute ethanol | Plum | 201152-1L | |
| Coverslips (24x50mm; 1000 pcs) | Hounisen | 422.245 | |
| Cryomolds Intermediate (15 x 15 x 5 mm; 100 pcs) | Tissue-Tek | 4566 | |
| Cryostat | Leica | CM3050S | |
| DPX mounting medium | Hounisen | 1001.0025 | |
| Eosin Y solution alcoholic 0.5% | Sigma | 1024390500 | |
| Feather microtome blade stainless steel,C35 (50 pcs) | Pfm medical | 207500003 | |
Fisherbrand Superfrost Plus slides (25 x 75 mm; 144 pcs) |
Thermofisher | 6319483 | |
| Mayers hematoxylin | Sigma | MHS32-1L | |
| OCT compound | VWR | 361603E | |
| Slide scanner (Nanozoomer) | Hamamatsu Photonics | ||
| Xylene | Sigma | 534056-4L |
References
- Hynes, R. O. Extracellular matrix: not just pretty fibrils. Science. 326, 1216-1219 (2009).
- Mayorca-Guiliani, A. E., et al. ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix. Nature Medicine. 23, 890-898 (2017).
- Mayorca-Guiliani, A. E., et al. Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D. Nature Protocols. 14, 3395-3425 (2019).
- White, L. J., et al. The impact of detergents on the tissue decellularization process: A TOF-sims study. Acta Biomaterialia. 50, 207-219 (2017).
- Ott, H. C., et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nature Medicine. 14, 213-221 (2008).
- Uygun, B. E., et al. Organ re-engineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nature Medicine. 16, 814-820 (2010).
- Susaki, E. A., et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nature Protocols. 10, 1709-1727 (2015).
- Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nature Protocols. 9, 1682-1697 (2014).
- Erturk, A., et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature Protocols. 7, 1983-1995 (2012).
- Wershof, E., et al. A FIJI Macro for quantifying pattern in extracellular matrix. BioRxiv. , (2019).
Cite This Article
Mayorca-Guiliani, A. E., Rafaeva, M., Willacy, O., Madsen, C. D., Reuten, R., Erler, J. T. Decellularization of the Murine Cardiopulmonary Complex. J. Vis. Exp. (171), e61854, doi:10.3791/61854 (2021).