Name: an ex vivo tissue culture model for fibrovascular complications in proliferative diabetic retinopathy
Uploaded: 2019-01-25T15:00:00
Description: Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy at JoVE.com

作者非常感谢赫尔辛基大学医院眼科糖尿病科和玻璃体视网膜外科的医疗和外科医生、护士和全体工作人员积极参与招聘工作。的病人。我们感谢生物素分子成像设备的成像设施。我们感谢阿纳斯塔西亚·切尔年科提供的出色技术援助。这项研究得到了芬兰科学院 (kl) 赠款的支持, 赫尔辛基大学 (kl)、sigrid juselius 基金会 (kl)、k. albin johansson 基金会 (kl)、芬兰癌症研究所 (kl)、karolinska 学院 (kl)、芬兰眼科基金会 (sl)、眼睛和组织银行基金会 (sl)、mary 和 georg c. ehrnrooth 基金会 (sl) 和 huch 临床研究赠款 (tyh2018127 (tyh2018127), TYH2018127, sl), 糖尿病研究基金会 (sl, kl, ak, eg) 以及生物医学博士方案 (eg)。

这项研究得到了赫尔辛基大学医院机构审查委员会和伦理委员会的批准。每个病人都得到了签字的知情同意。
1. 解决方案、介质和设备的准备
<ol><li>在收集纤维血管组织 (ft) 之前准备以下设备, 以确保快速处理。<ol>
<li>灭菌高压灭菌器两个微解剖推子。</li>
		<li>在900毫升去离子水中溶解1个预称的 pbs 片 (0.14 m ncl, 0.0027 m kcl, 0.010 m po 4-3), 制备 1x磷酸盐缓冲盐水...

<ol>
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考虑到相关组织微环境对可靠的功能细胞和分子力学结果的重要性, 有必要找到适当的实验模型, 提供这种组织环境。本文介绍了纤维蛋白嵌入 ft 的体外pdr 培养模型, 可以研究 pdr 病理生理学在 pdr 临床样本的本机、复杂和多细胞环境中的机制。
协议中的关键步骤是适当的纤维蛋白凝胶形成、ft 的定位和染色过程中的适当清洗。由于纤维蛋白凝胶的形成主要取决于凝血?...

dr 是糖尿病的一种严重的眼部并发症, 在过去30年中, 这种疾病已达到巨大的比例1。诊断20年后, 几乎每个1型糖尿病患者和60% 的2型糖尿病患者都有视网膜病变的迹象, 使糖尿病本身成为正常工作年龄失明的主要原因之一.根据微血管变性和缺血损伤的程度, dr 分为非增殖 dr (非 pdr) 和增殖 dr (pdr)。终末期疾病 pdr 的特点是缺血和炎症引起的新生血管和纤维化反应在玻璃体视网膜界面。在未经治疗的情况下, 这些过程将导致失明, 由于玻璃体出血, 视网膜纤维化, 视网膜三度脱离, 和新生血管性青光眼3,4。尽管最近的进展, 目前的治疗方案只针对 dr 阶段, 包括糖尿病黄斑水肿和 pdr, 当视网膜损伤已经接踵而至。此外, 很大一部分 dr 患者没有从目前的治疗水族馆中受益, 这表明迫切需要改进治疗 4、5、6.
到目前为止, 已经开发出多个其他体内疾病发展模型和糖尿病动物模型, 但没有一个模型可以概括人类 pdr7,8中观察到的全部病理特征。此外, 越来越多的证据表明, 治疗反应与 ecm 组合以及细胞和无细胞微环境之间的空间排列和相互作用密切相关。因此, 我们开始开发一个临床相关的模型的人 pdr 利用 ft 病理材料, 通常是从眼睛接受玻璃体切除术作为 pdr10的手术管理的一部分。
这份手稿描述了3d 体外培养和定性的手术切除, pdr 患者衍生病理 ft 的协议。这里描述的方法已被用于最近的一份出版物, 证明了成功解构本地 3d pdr 组织景观, 并重述了 pdr 病理生理学的特征, 包括血管生成和纤维化反应的异常血管结构11。该模型还揭示了从薄薄的组织学切片中不易被欣赏的新特征, 如空间限制的凋亡和增殖以及血管胰岛形成11。玻璃体液已被其他人成功地应用于三维内皮球体培养物, 以评估其血管生成潜力和血管静止分子12的有效性。当与体外三维淋巴细胞内皮细胞 (lec) 球体萌发试验结合使用 pdr 玻璃体作为刺激物时, 我们的模型揭示了可溶性玻璃体因子以及新生血管组织中的局部线索对作为刺激物的贡献。但对 lec 参与 pdr 病理生理学的情况了解甚少,3, 11。在 pdr 的管理中, 玻璃体视网膜手术是一个例行执行但具有挑战性的程序。由于外科器械和技术正在看到不断的进步和成熟, 及时和保守地去除纤维血管增生标本不仅提高视力, 而且还提供了宝贵的组织材料研究人类组织微环境复杂翻译方面的 pdr 病理生理学和治疗反应。

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microforceps</td>
			<td>Medicon</td>
			<td>07.60.03</td>
			<td>Used for handling the FTs</td>
		</tr>
		<tr>
			<td>Disposable Scalpels - Sterile</td>
			<td>Swann-Morton</td>
			<td>0513</td>
			<td>Used for FT dissection</td>
		</tr>
		<tr>
			<td>Culture dish, vented, 28 ml (60mm)</td>
			<td>Greiner Bio-One</td>
			<td>391-3210</td>
			<td>Used for dissection and for testing fibrin gel formation</td>
		</tr>
		<tr>
			<td>Cell culture plates, 12-well</td>
			<td>Greiner Bio-One</td>
			<td>392-0049</td>
			<td>Used for FT dissection and whole-mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Reagent/centrifuge tube with screw cap, 15 mL</td>
			<td>Greiner Bio-One</td>
			<td>391-3477</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent/centrifuge tube with screw cap, 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>525-0384</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit, 0.22&#160;&#181;m, PVDF</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td>Used to sterile-filter the fibrinogen solution</td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>Used to sterile-filter the fibrinogen solution</td>
		</tr>
		<tr>
			<td>Polypropylene Microcentrifuge Tubes, 1.5 mL</td>
			<td>Fisher</td>
			<td>FB74031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Culture Treated Multidishes, 24-well</td>
			<td>Nunc</td>
			<td>142475</td>
			<td>Used for casting the FT/fibrin gels for native FT characterization and ex vivo culture</td>
		</tr>
		<tr>
			<td>Cell culture plates, 96-well, U-bottom</td>
			<td>Greiner Bio-One</td>
			<td>392-0019</td>
			<td>Used for whole-mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Round/Flat Spatulas, Stainless Steel</td>
			<td>VWR</td>
			<td>82027-528</td>
			<td>Used for whole-mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Coverslips 22x22mm #1</td>
			<td>Menzel/Fisher</td>
			<td>15727582</td>
			<td>Used for mounting</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>Kindler K102</td>
			<td>Used for mounting</td>
		</tr>
		<tr>
			<td>Absorbent paper</td>
			<td>VWR</td>
			<td>115-0202</td>
			<td>Used for mounting</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Medicago</td>
			<td>09-9400-100</td>
			<td>Used for preparing 1x PBS</td>
		</tr>
		<tr>
			<td>Fibrinogen, Plasminogen-Depleted, Human Plasma</td>
			<td>Calbiochem</td>
			<td>341578</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9394-500ML</td>
			<td>Used for preparing the fibrinogen and TA solution</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Used for preparing the blocking solution</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma-Aldrich</td>
			<td>H4522</td>
			<td>Aliquoted in -20 &#176;C, thaw before preparing the ex vivo culture media</td>
		</tr>
		<tr>
			<td>Gentamicin Sulfate 10mg/ml</td>
			<td>Biowest</td>
			<td>L0011-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell media MV Kit</td>
			<td>Promocell</td>
			<td>C-22120</td>
			<td>Contains 500 ml of Endothelial Cell Growth Medium MV, 25 mL of fetal calf serum, 2 mL of endothelial cell growth supplement,&#160; 500 &#956;L of recombinant human epidermal growth factor (10 &#956;g/ mL) and 500 &#956;L of hydrocortisone (1 g/ mL)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Used for storage of the native and ex vivo cultured FTs. TOXIC: wear protective gloves and/or clothing, and eye and/or face protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>32201-2.5L-M</td>
			<td>Used to prepare the post-fixation solution. HARMFUL: wear protective gloves and/or clothing. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>32213</td>
			<td>Used to prepare the post-fixation solution. TOXIC: wear protective gloves and/or clothing. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Triton X-100 (octyl phenol ethoxylate)</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>Used for whole-mount immunofluorescence. HARMFUL: wear protective gloves and/or clothing.</td>
		</tr>
		<tr>
			<td>Hoechst 33342, 20mM</td>
			<td>Life Technologies</td>
			<td>62249</td>
			<td>For nuclei counterstaining. HARMFUL: wear protective gloves and/or clothing, and eye and/or face protection.</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Wear protective gloves and/or clothing, and eye protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Mounting medium with nuclei counterstaining. Wear protective gloves and/or clothing, and eye protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Eukitt Quick-hardening mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>03989-100ml</td>
			<td>TOXIC: Wear protective gloves and/or clothing, and eye protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma, lyophilized powder</td>
			<td>Sigma-Aldrich</td>
			<td>T9549-500UN&#160;</td>
			<td>Dissolve at 100 units/ mL, aliquote and store at -20 &#176;C, avoid repeated freeze/ thaw</td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung, lyophilized powder</td>
			<td>Sigma</td>
			<td>A3428</td>
			<td>Dissolve at 50 mg/ mL, aliquote and store at -20 &#176;C, avoid repeated freeze/ thaw</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Growth factors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGFA</td>
			<td>R&#38;D Systems</td>
			<td>293-VE-010</td>
			<td>50 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Recombinant human VEGFC</td>
			<td>R&#38;D Systems</td>
			<td>752-VC-025</td>
			<td>200 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Recombinant human TGF&#946;</td>
			<td>Millipore</td>
			<td>GF346</td>
			<td>1 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Recombinant human bFGF</td>
			<td>Millipore</td>
			<td>01-106</td>
			<td>50 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 (JC70A)</td>
			<td>Dako</td>
			<td>M0823</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>CD34 (QBEND10)</td>
			<td>Dako</td>
			<td>M716501-2</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>CD45 (2B11+PD7/26)</td>
			<td>Dako</td>
			<td>M070129-2</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>CD68</td>
			<td>ImmunoWay</td>
			<td>RLM3161</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>Cleaved caspase-3 (5A1E)</td>
			<td>Cell Signalling</td>
			<td>9664</td>
			<td>Used at 1:200 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>ERG (EP111)</td>
			<td>Dako</td>
			<td>M731429-2</td>
			<td>Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td>Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Leica Microsystems</td>
			<td>NCL-Ki67p</td>
			<td>Used at 1:1500 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>Lyve1</td>
			<td>R&#38;D Systems</td>
			<td>AF2089</td>
			<td>Used at 1:100 dilution, Donkey anti Goat Alexa 568 Secondary Ab</td>
		</tr>
		<tr>
			<td>NG2</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td>Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>Prox1</td>
			<td>ReliaTech</td>
			<td>102-PA32</td>
			<td>Used at 1:200 dilution, Goat anti Rabbit Alexa 568 Secondary Ab</td>
		</tr>
		<tr>
			<td>Prox1</td>
			<td>R&#38;D Systems</td>
			<td>AF2727</td>
			<td>Used at 1:40 dilution, Chicken anti Goat Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>VEGFR3 (9D9F9)</td>
			<td>Millipore</td>
			<td>MAB3757</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>&#945;-SMA (1A4)</td>
			<td>Sigma</td>
			<td>C6198</td>
			<td>Used at 1:400 dilution, Cy3 conjugated</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor488 Donkey Anti-Mouse IgG</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor594 Goat Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-11012</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor568 Donkey anti-Goat IgG</td>
			<td>Thermo Scientific</td>
			<td>A-11057</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor568 Goat anti-Rabbit IgG</td>
			<td>Thermo Scientific</td>
			<td>A-11036</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor594 Chicken Anti-Goat IgG</td>
			<td>Molecular Probes</td>
			<td>A-21468</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 200 inverted epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>For imaging of the fresh and fibrin-embedded FT</td>
		</tr>
		<tr>
			<td>SZX9 upright dissection stereomicroscope</td>
			<td>Olympus</td>
			<td></td>
			<td>For FT dissection</td>
		</tr>
		<tr>
			<td>LSM 780 confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>For imaging of whole-mount immunostained FT</td>
		</tr>
		<tr>
			<td>AxioImager.Z1 upright epifluorescence microscope with Apotome</td>
			<td>Zeiss</td>
			<td></td>
			<td>For imaging of whole-mount immunostained FT</td>
		</tr>
	</tbody>
</table>

an ex vivo tissue culture model for fibrovascular complications in proliferative diabetic retinopathy

糖尿病视网膜病变 (dr) 是糖尿病最常见的微血管并发症, 也是工作年龄成年人失明的主要原因之一。目前没有糖尿病和氧引起的视网膜病变的动物模型会出现人类增殖性糖尿病视网膜病变 (pdr) 所表现出的全系列渐进性变化。因此, 对疾病发病机制和病理生理学的了解在很大程度上依赖于使用组织学切片和玻璃体样本的方法, 这些方法只能提供有关致病因素的稳态信息。越来越多的证据表明, 在三维 (3d) 微环境中的动态细胞和细胞外基质 (ecm) 相互作用对于新的治疗策略。因此, 我们假设用 pdr 手术切除眼睛的病理性纤维血管组织可以可靠地揭示这种毁灭性疾病的细胞和分子机制, 并测试新的临床潜力干预。为此, 我们开发了一种新的方法, 用于手术切除患者衍生的纤维血管组织 (ft) 的三维体外培养, 该方法将作为人类 pdr 病理生理学的相关模型。将 ft 分解成外植体, 并嵌入纤维蛋白基质中, 用于体外培养和三维表征。通过对原生 fts 和终点培养物的全安装免疫荧光进行彻底的研究, 从而可以对组织组成和多细胞过程进行彻底的研究, 突出了3d 组织级表征对发现相关特征的重要性。pdr 病理生理学。该模型将允许在 pdr 组织结构和微环境中动态生化和物理相互作用的复杂背景下同时评估分子机制、纤维素组织过程和治疗反应。由于该模型重述了 pdr 病理生理学, 因此也可进行测试或开发新的治疗方法。

对 pdr 纤维血管组织特性和蛋白表达的深入了解主要依靠玻璃体样品和薄组织学 ft部分 3,15,16,17。为了建立一种方法, 彻底研究三维组织组织和多细胞生理病理过程的 pdr, 我们提出了利用手术切除, 患者衍生的病理 ft 进行三维表征和体外培养。新鲜的 ft 被转移到研究实验室并进...

Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy at JoVE.com

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

在这里, 我们提出了一个方案, 研究增殖性糖尿病视网膜病变的病理生理学使用患者衍生的, 手术切除, 纤维血管组织的三维本地组织表征和体外培养。这种体外培养模型也适合测试或开发新的治疗方法。

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

糖尿病视网膜病变是糖尿病最常见的微血管并发症。该方法有助于回答期末疾病增殖性糖尿病视网膜病变病理生理学的关键问题。该技术可解构原生三维组织景观，并研究具有直接临床意义的活体患者材料中的组织病理生理学。有关手术和解剖中使用仪器的详细信息，请参阅文本协议。首先，将从接受转结微切口玻璃化手术的人体受试者中切除的纤维血管组织放在含有无菌PBS的细胞培养皿中。使用直立解剖立体显微镜，将纤维血管组织切成大约一平方毫米的碎片。将组织淹没在PBS中，用微解剖钳将组织放在位上。在组织中进行清晰的切割，用无菌的手术刀小心，以避免撕裂纤维血管组织。将每一块单独的组织放入含有一毫升无菌 PBS 的 12 井细胞培养板的井中。首先，在室温下准备25微升的纤维蛋白原溶液等分。将一块纤维血管组织放在24井板的井的中心，并去除任何多余的PBS。接下来，将 25 微升 TA 溶液添加到其中一个准备好的纤维蛋白原等分中。并使用另一个设置为 50 微升的移液器通过移液混合。将混合物分配到纤维血管组织和移液器上，以确保组织片包含在液滴中。纤维蛋白原形成的时间对外活培养的三维性至关重要，在协议中描述的空滴前组织嵌入中进行测试。在细胞培养孵化器中孵育板。30 至 60 分钟后倾斜板，检查纤维蛋白凝胶是否完全形成。接下来，用外活培养培养介质与阿普罗汀补充覆盖纤维血管组织纤维蛋白凝胶。在此之后，将培养板返回到所需时间段的细胞培养箱。首先，将外体培养基替换为每井一毫升的甲状甲醛溶液，以修复培养物。一小时后，用三个五分钟的PBS洗涤冲洗凝胶。接下来，用每井两毫升的阿齐德钠溶液代替PBS洗涤液。将固定凝胶存放在四摄氏度下。使用不锈钢方形边缘铲，小心地将液滴从板中提起，从边缘开始，然后提起液滴的中心。使用圆边铲将液滴转移到含有一毫升 PBS 的 12 孔板的单个孔中。在此之后，将板倾斜在支撑上，用一毫升冰冷的丙酮甲醇溶液将液滴固定一分钟。然后，用三到五两毫升的PBS洗涤冲洗。离心堵塞溶液15分钟，以清除任何碎屑。然后，将板倾斜在支撑上，在室温下将液滴孵育为 500 微升的阻消溶液中，两小时。根据文本协议准备原抗体混合物。然后使用圆边铲将液滴转移到圆底 96 井板。在4摄氏度下，在30微升原抗体混合物中孵育液滴。第二天，将液滴转移到一个12井板，每井含有两毫升的洗涤液。用三个五分钟的洗涤冲洗液滴。然后，用9个30分钟的洗涤液滴清洗。第二天，用PBS冲洗液滴三次。根据文本协议准备适当的氟磷结合二级抗体混合物。如先前证明，将液滴转移到圆底96井板中，并在室温下用30微升二次抗体混合物孵育板4小时，防止光线。四小时后，将液滴转移到一个12井板，每井含有两毫升的洗涤液。首先，用三个五分钟的洗涤冲洗液滴。然后，用四个30分钟的洗涤液滴清洗液滴。第二天，用洗涤溶液冲洗液滴5次，用30分钟清洗30分钟。第二天，将液滴转移到圆底96井板，如前所述。在室温下用霍赫斯特核污渍对液滴进行30分钟的反色。将液滴转移到每井含有两毫升 PBS 的 12 井板中，然后用 PBS 冲洗三次。接下来，沿方形盖玻璃的边缘应用一层狭窄的快速硬化安装介质，让其干燥一分钟。然后，将液滴浸入含有去压水的12井板的井中冲洗液滴。将液滴转移到显微镜幻灯片上。接下来，在液滴上分配 15 微升非硬化防褪色安装介质。将盖玻璃轻轻放在液滴上，安装介质朝向滑轨，让其稳定下来。让滑梯在室温下干燥两小时，并在夜间存放在四摄氏度。最后，使用直立的荧光显微镜在20倍或40倍的镜面上使用配备光学剖面功能的幻灯片进行成像。在该协议纤维血管组织被准备和成像。代表性数据表明，PDR外体培养物会诱发CD-31阳性内皮和血管保存的萌芽，以回应维格法。相反，TGF

Research

JoVE Journal

Medicine

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

患者组织及基因传递考试前的体外培养

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

科研

医学

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

人掌腱膜挛缩的<em&gt;离体</em&gt;文化为肌成纤维细胞的研究和细胞外基质相互作用

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

大动物模型肝组织血浆凝固的评价

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

糖尿病家兔欣德肢体缺血的临床前模型

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...