Name: sampling strategies and processing of biobank tissue samples from porcine biomedical models
Uploaded: 2018-03-06T14:00:00
Description: Watch this Scientific Journal Video about Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models at JoVE.com

作者感谢丽莎 Pichl 出色的技术援助。

这里描述的所有方法都使用来自死动物的组织样本, 完全符合德国动物福利法律规定。
1. 容量
<ol><li>用于测定组织/器官密度的淹没技术 (图 1 和图 2)7,12,16<ol>
<li>准备材料: 手术刀刀片, 纸巾, 细钳, 标准实验室秤, 玻璃或塑...

<ol>
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从猪动物模型中产生的生物库样本收集需要强健的技术和协议, 以确定器官/组织体积, 可再现的代表性, 多余的组织样本, 适合广泛的不同的分析方法, 并对抽样剖面的随机化进行定量视学分析。本条所述方法适用于猪器官和组织的大小, 并已开发出有效满足这些要求的2,7。它们基于公认的方法论原则, 并在不同的发布研究中证明了它们的实用性<sup class="xr...

在翻译医学中, 猪越来越普遍地使用作为大动物模型1,2,3,4,5, 由于猪的几个有利相似性和人体解剖和生理学, 以及现有的分子生物学方法, 允许为多种疾病条件生成量身定做的基因改良猪模型1,4。
然而, 与啮齿动物模型相比, 可以随时为实验提供的不同猪模型的动物数量是有限的。这是由于猪的世代间隔大约一年, 并且为猪模型和畜牧业的世代需要的财政和时间密集的努力。因此, 猪模型的个体动物, 以及可以从这些猪身上生成的样本, 都非常有价值, 特别是如果转基因猪模型和/或长期实验性问题 (例如, 晚期并发症的慢性病) 在老年个体检查2,6,7。
在任何研究过程中, 在研究的初步实验设计中尚未排定的额外分析的表现可能后来变成相关的,例如, 以解决由于先前发现的不同问题意想不到的发现。如果没有合适的样品进行这种额外的试验, 不成比例可能需要花费高昂的成本和耗费大量时间的开支来产生更多的猪和组织样本。为应对这种可能性, 生物库收集不同器官、组织或生物液体的保守备份样本, 在数量上和质量上都适合广泛的后续分析, 被认为是重要的方法2,6,7。从猪的动物模型中获得最佳的好处, 充分的生物库样本的可用性也提供了独特的可能性, 在同一样本材料上执行广泛的不同分析方法在一个多器官水平上, 在同一单个动物,例如, 通过在研究网络2、6、7中组织的不同工作组的科学家分发样本。此外, biobanking 的 "前瞻性" 抽样策略也有助于减少研究中需要的动物数量。猪模型 biobanking 的优势最近已在多器官, multiomics 研究, 检查器官交叉谈话的基因改良猪模型长期糖尿病, 使用标本从慕尼黑米迪猪生物库2。
有一些强制性要求生物库样品必须普遍遵守, 以建立可靠性和解读的结果, 随后进行分析。必须重现性生成样本, 并且它们必须具有代表性, 即, 充分反映了从7中抽取样本的组织/器官感兴趣的形态学和分子特征. 为了适合广泛的下游分析类型, 样品必须采取足够的数量和处理, 根据要求 (包括时间和温度条件) 的不同分析方法, 包括描述性病理学分析, 如 cryohistology, 石蜡和整形组织学, 免疫组化,原位杂交, 超微结构的电子显微分析, 临床实验室诊断分析, 以及分子分析 DNA, RNA, 蛋白质和代谢物。
通过定量视学分析, 允许对多种不同的定量形态学参数 (如数字、体积、长度或不同组织结构的表面积) 进行评估, 随机断面平面各器官/组织的组织学样本需要准备7,8,9,10,11。在定量形态学研究中, 对组织、器官或器官室总容积的精确测定, 样本取自 (即, 参考空间) 至关重要7,9,12计算相应器官、组织或有机体中相关参数的绝对数量。最终, 在组织学切片的制备过程中, 嵌入相关组织收缩的影响必须确定并考虑到13。因此, 以往研究中的定量视学分析, 特别是存档样本 (固定组织样本、嵌入组织块、组织学切片、等等) 有时会受到严重限制, 甚至是不可能的12,特别是如果没有执行有关的器官/组织的容量, 如果没有充分的抽样设计适用于保证代表性样品, 如果数量和数量的可利用的单独样品是不足的, 或者, 如果处理样本与感兴趣的定量形态学参数的估计不相容。由于各种可能的影响因素, 档案样本材料对不同数量形态参数分析的适用性不能明确地得到回答, 而是取决于对个别案件的仔细评估。
因此, 由于样品的位置、大小、数量、加工、修剪方向和方向等因素对后续分析的结果有潜在影响, 因此这些因子具有重要意义, 必须在任何研究的实验设计中加以考虑。关于这些方面和猪解剖学的特点, 最近建立了适合猪动物模型的全面、详细、大规模的抽样准则, 为标准化、可重现性提供了有力的参考。, 并高效生成冗余、经过充分处理的高质量样品, 来自50多个不同的猪器官和组织6,7。
本文所示的方法学描述和视频教程提供了详细的、说明性的、可理解的、循序渐进的指导, 以实现各种容量技术的实际性能、猪组织取样和器官, 并处理组织样品的不同下游分析方法。精选的技术包括根据阿基米德和卡瓦列里的原理确定器官/组织体积和密度的方法9, 包括确定与所涉组织的三维收缩的尺寸嵌入到不同的嵌入媒体14在组织学检查过程中, 应用实际体积加权系统随机抽样方法, 对不同后续的取样组织标本进行处理。分析7,8,9,15, 并为潜在的定量视学分析生成适当的面向和处理的样本7,8, 9,10,11。除了在猪生物库项目中的应用, 所展示的方法一般适用于检查器官/组织的定量历史形态特性的所有研究。此外, 系统随机抽样设计特别有利于在实验中产生代表性样品, 方法是利用分子分析法检测在各种器官和组织。
下段简要介绍了这些方法, 而它们的实际性能则在 "协议" 部分中进行了说明。
器官/组织体积的测定 在几个实验环境中, 器官重量和体积的测定是很重要的, 因为这些因素可能表明变化, 可能与实验性研究的兴趣因素有关。一个器官/组织的总体积也通常需要计算绝对数量参数, (例如, 总细胞数), 从 stereologically 估计的数字体积密度 (即, 每个体积单位的细胞数组织)7,12。除了使用复杂的技术设备 (如计算机断层扫描) 技术外, 基本上还有三种常用的方法来确定器官或组织的绝对体积。一个器官的容量可以由 "直接容积测量" 决定根据阿基米德的原则,即, 测量水的容量或盐水由结构完全地淹没时偏移。然而, 对于比较大的猪器官, 这些方法是不切实际的, 容易不精确, 因为它们需要非常大的容积/测量烧瓶。更方便的是, 一个器官/组织的体积可以计算从它的重量和密度7,12,16, 可以有效地确定使用 "淹没方法"7,12 ,16 (协议步骤 1.1)。器官/组织体积也可以根据 "卡瓦列里原则" (1598–1647) 使用容量方法估计。简单地说, 卡瓦列里原则规定, 如果两个物体在平行于地面平面的平面上切片, 而在与地面平面相对应的距离上切割两个物体的剖面轮廓有相同的区域, 则两个物体有相同的音量。因此, 任意形状物体的体积可以被估计为其截面剖面区域的乘积, 平行, 同样遥远的剖面平面和截面平面之间的距离。这是可理解的与以下类比: 考虑两个栈组成相同数量的相同的硬币是并排放置, 一个栈与硬币有序堆放在一起产生圆柱形的硬币栈, 和其他用离中心位置的硬币堆叠的硬币 (图 3A)。虽然两个硬币栈的形状是不同的, 它们的体积是相同的, 因为硬币的面积在两个栈的相应级别 (即, 平行截面轮廓的区域通过两个硬币栈在相同的距离切割地面) 是相同的。使用卡瓦列里原理估计猪器官和组织的体积7,12,15在步骤1.2 中描述。
组织学埋植相关组织收缩程度的测定 在对组织学组织切片测量的几种定量形态学参数进行分析时, 必须确定并考虑组织处理过程中嵌入相关组织收缩对组织学的影响。嵌入相关组织收缩的程度可能是可变的, 并且取决于组织、处理和嵌入介质8,13,17,18,19。通常, 组织样本体积的嵌入相关变化 (即, 主要是收缩) 发生在所有三维空间中, 因此影响由定量视学分析估计的所有维度参数8.基本上, 嵌入相关组织收缩的程度, 表示为线性组织收缩因子 (fS), 可以估计如步骤1.3 所示。用于校正 (收缩敏感) 定量形态学参数14。
器官/组织体积加权系统随机抽样 为建立生物库收集猪器官/组织样本, 体积加权系统随机抽样方法, 如步骤2所述, 证明是切实可行的, 节省时间, 和有效的技术, 以产生代表,多用途组织样本7,8,9,15。
定量视学分析的各向同性均匀随机剖面和垂直均匀随机剖面的生成 生物库组织样本需要适用于多种不同的定量视学分析方法, 用于估计在没有充分制备标本的情况下无法确定的最大参数。几乎所有定量视学参数都可以用 "各向同性 (独立) 均匀随机 (产学研)" 部分 "8,9" 来确定。在产学研切片中, 组织样品断面平面的三维方向是随机的。这可以通过随机化组织样本的位置相对于截面平面的位置, 如应用于 "Isector" 方法11 (协议步骤 3.1), 或通过随机化截面平面相对于组织样本, 如 "定位装置" 方法 10 (协议步骤 3.2).在组织样本中, 如皮肤或黏膜标本显示自然存在, 或定义和正确地可识别的垂直轴, 准备 "垂直均匀随机 (VUR) 部分" (协议步骤 3.3.) 严格地切片在他们的飞机垂直轴有利8,20。为了全面论述产学研/VUR 抽样的理论基础, 并对潜在的下游定量视学分析进行了综合讨论, 读者可以参考生活中定量视学教科书。科学8,9。

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Carl Roth GmbH, Germany</td>
			<td>Agar (powder), Cat.: 5210.3</td>
			<td>Dissolve approximately 1 g of agar in 10 ml cold water in a glass or plastic beaker, heat in microwave-oven at 700 W, boil the solution twice with rigorous stirring. Cast into mold while still warm and let solidify. Caution: While handling with hot liquid agar, wear protective goggles and gloves.</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Hornbach Baumarkt GmbH, Bornheim, Germany</td>
			<td>Schieblehre Chrom/Vernickelt 120 mm Cat.: 3664902</td>
			<td>Any kind of caliper (mechanical or electronic) will do as well.</td>
		</tr>
		<tr>
			<td>Casting molds (metal)</td>
			<td>Engelbrecht Medizin &#38; Labortechnik, Ederm&#252;nde, Germany</td>
			<td>Einbettsch&#228;lchen aus Edelstahl, 14 x 24 x 5 mm, Cat.: 14302b</td>
			<td>Any other kind of metal casting mold used for paraffin-embedding will do as well.</td>
		</tr>
		<tr>
			<td>Copy templates of cross grids (5mm - 6 cm)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Copy templates of cross grids (5mm - 6 cm) are provided in the supplemental data file of Albl et al.&#160; Toxicol Pathol. 44, 414-420, doi: 10.1177/0192623316631023 (2016)</td>
		</tr>
		<tr>
			<td>Copy templates of equiangular and cosine-weighted circles</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Copy templates of equiangular and cosine-weighted circles are provided in Nyengaard &#38; Gundersen. Eur Respir Rev. 15, 107-114, doi: 10.1183/09059180.00010101 (2006) and in Gundersen et al. Stereological Principles and Sampling Procedures for Toxicologic Pathologists. In: Haschek and Rousseaux&#180;s Handbook of Toxicologic Pathology. 3rd ed, 215-286, ISBN: 9780124157590 (2013).</td>
		</tr>
		<tr>
			<td>Foldback clamps (YIHAI binder clips, 15 mm and 19 mm)</td>
			<td>Ningbo Tianhong Stationery Co ltd., China</td>
			<td>Y10006 and Y10005</td>
			<td>Any other type of standard office foldback clamps will do as well.</td>
		</tr>
		<tr>
			<td>Forceps (anatomical)</td>
			<td>NeoLab Migge GmbH, Heidelberg, Germany</td>
			<td>neoLab Standard -Pinzette 130 mm, anatomisch, rund, Cat.: 1-1811</td>
			<td>Any type of anatomical forceps will do.</td>
		</tr>
		<tr>
			<td>Formaldehyde-solution 4%</td>
			<td>SAV-Liquid Produktion GmbH, Flintsbach, Germany</td>
			<td>Formaldehyd 37/40 %, Cat.: 1000411525005</td>
			<td>Dilute to 4% from concentrated solution. Buffer to neutral pH. Wear appropriate eye-, hand- and respiratory protection. Process tissue samples fixed in formaldehyde solution under an exhaust hood and wear protective goggles and laboratory gloves.</td>
		</tr>
		<tr>
			<td>Graph paper (for calibration)</td>
			<td>B&#252;romarkt B&#246;ttcher AG, Jena, Germany. www.bueromarkt-ag.de</td>
			<td>Penig Millimeterpapier A4, Cat.: 2514</td>
			<td>Any type of graph paper (scaled in millimeter) will do.</td>
		</tr>
		<tr>
			<td>Laboratory beakers (5ml, 10 ml, 50 ml, 100 ml)</td>
			<td>NeoLab Migge GmbH, Heidelberg, Germany</td>
			<td>Becherglas SIMAX&#174; , niedrige Form, Borosilikatglas 3.3 Cat.: E-1031, E-1032, E-1035, E-1036</td>
			<td>Any kind of glass- or plastic beakers of 5 &#8211; 100 ml volume will do.</td>
		</tr>
		<tr>
			<td>Laboratory scale(s)</td>
			<td>Mettler Toledo GmbH, Gie&#223;en, Germany</td>
			<td>PM6000</td>
			<td rowspan="2">Any standard laboratory scales with measuring ranges between 0.1 mg to approximately 20 g, respectively between 100 mg to approximately 500 g will do</td>
		</tr>
		<tr>
			<td></td>
			<td>Sartorius AG, G&#246;ttingen, Germany</td>
			<td>BP61S</td>
		</tr>
		<tr>
			<td>Microtome blades</td>
			<td>Engelbrecht Medizin &#38; Labortechnik, Ederm&#252;nde, Germany</td>
			<td>FEATHER Microtome blasdes S35, Cat.:14700</td>
			<td>Any kind of single-use microtome blades will do.</td>
		</tr>
		<tr>
			<td rowspan="2">Morphometry/planimetry software/system</td>
			<td>National Institute of Health (NIH)</td>
			<td>ImageJ</td>
			<td>Download from https://www. imagej.nih.gov/ij/ (1997).</td>
		</tr>
		<tr>
			<td>Zeiss-Kontron, Eching, Germany</td>
			<td>VideoplanTM image analysis system</td>
			<td>Out of stock</td>
		</tr>
		<tr>
			<td>Photo camera</td>
			<td>Nikon</td>
			<td>D40</td>
			<td>Any kind of digital photocamera that can be mounted to a tripod&#160; will do.</td>
		</tr>
		<tr>
			<td>Plastic transparencies</td>
			<td>Avery Zweckform GmbH, Oberlaindern, Germany</td>
			<td>Laser Overhead-Folie DINA4 Cat.:&#160; 3562</td>
			<td>Any (laser)-printable plastic transparency will do.</td>
		</tr>
		<tr>
			<td>Random number tables</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Random number tables can conveniently be generated (with defined numbers of random numbers and within defined intervals), using random number generators, such as: https://www.random.org/</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Plano GmbH, Wetzlar, Germany</td>
			<td>T5016</td>
			<td>Any kind of razor blades will do.</td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>B&#252;romarkt B&#246;ttcher AG, Jena, Germany. www.bueromarkt-ag.de</td>
			<td>Office-Point Lineal 30 cm, Kunststoff, transparent, Cat.: ln30</td>
			<td>Any kind of cm-mm-scaled ruler will do as well.</td>
		</tr>
		<tr>
			<td>Saline (0.9%)</td>
			<td>Carl Roth GmbH, Germany</td>
			<td>Natriumchlorid, &#62;99% Cat.: 0601.1</td>
			<td>To prepare 0.9% saline, dissolve 9 g NaCl in 1000 ml of distilled water at 20&#176;C.</td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Aesculap AG &#38; Co KG, Tuttlingen, Germany</td>
			<td>BRAUN Surgical blades N&#176;22</td>
			<td>Any kind of scalpel blades will do.</td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>Hewlett-Packard</td>
			<td>hp scanjet 7400c</td>
			<td>Any type of standard office scanner capable of scanning with resolutions from 150-600 dpi will do.</td>
		</tr>
		<tr>
			<td>Slicing devices</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Examples forself constructed slicing devices can be found in Knust, et al. Anatomical record. 292, 113-122, doi: 10.1002/ar.20747 (2009) and in the supplemental data file of Albl et al.&#160; Toxicol Pathol. 44, 414-420, doi: 10.1177/0192623316631023 (2016).</td>
		</tr>
		<tr>
			<td>Spherical casting molds (e.g., in 25.5 mm diameter)</td>
			<td>Pralinen-Zutaten.de, Windach, Germany</td>
			<td>Pralinen-Hohlkugeln Vollmilch, 25.5 mm</td>
			<td>Spherical casting molds can as well be be self-constructed, or obtained from other confectioner suppliers (for for pralines). The casting molds indicated here are actually the package/wrapping of hollow pralines bodies (first eat the pralines and then use the package for generation of i-sector sections)</td>
		</tr>
		<tr>
			<td>Thin wire</td>
			<td>Basteln &#38; Hobby Schobes, Stra&#223;furth, Germany. www,bastel-welt.de</td>
			<td>Messingdraht (0.3 mm) Cat.: 216464742</td>
			<td>Any other kind of thin wire will also do.</td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>NeoLab Migge GmbH, Heidelberg, Germany</td>
			<td>Declcate Task Wipes-White, Cat.: 1-5305</td>
			<td>Any other kind of laboratory tissue paper will do as well.</td>
		</tr>
		<tr>
			<td>Waterproof pen</td>
			<td>Staedler Mars GmbH &#38; Co KG, N&#252;rnberg, Grmany</td>
			<td>Lumocolor permanent 313, 0.4 mm, S, black, Cat.: 313-2</td>
			<td>Any other kind of waterproof pen will do as well.</td>
		</tr>
	</tbody>
</table>

sampling strategies and processing of biobank tissue samples from porcine biomedical models

在翻译医学研究中, 猪模型逐渐变得越来越受欢迎。考虑到个别动物的高价值, 特别是基因改良猪模型, 以及这些模型的可用动物数量有限, 建立了适当加工的组织样本 (生物库), 适合于广泛的后续分析方法, 包括没有在取样时间点指定的分析, 代表了充分利用模型的平移值的有意义的方法。关于猪解剖学的特殊性, 最近建立了综合指导方针, 以标准化生成代表性的, 高质量的样品从不同的猪器官和组织。这些指导原则是结果重现性的必要条件, 也是不同研究和调查人员之间的可比。基本数据的记录, 例如器官重量和体积、取样位置的确定和要生成的组织样本数量, 以及它们的方向、大小、加工和修剪方向, 都是相关因素。确定标本的概化和可用性的分子, 定性, 定量形态学分析。本文介绍了猪组织中具有代表性的、多用途的生物库标本最重要的生产技术。这里描述的方法包括测定器官/组织体积和密度, 用点计数法对实质器官进行容积加权系统随机抽样程序的应用, 确定组织收缩程度与样本的组织学嵌入有关, 并生成随机定向样本进行定量视学分析, 如由 "定位装置" 和 "Isector" 方法产生的各向同性均匀随机 (产学研) 剖面, 以及垂直均匀随机 (VUR) 部分。

浸没法测定组织/器官密度
图 12A-b显示使用步骤 1.1 (图 1,图 2) 中描述的淹没技术来确定猪肾脏密度和体积的代表性。其他猪器官和组织的密度测量的更有代表性的结果载于表 2。"猪生物医学模型的组织取样指南"7显示了...

Watch this Scientific Journal Video about Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models at JoVE.com

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

猪生物医学模型生物库组织样品的取样策略与处理

在生物库项目的下游分析中, 展示了猪动物模型代表性组织样本生成方法的实际应用和性能, 包括容量、系统随机抽样、组织样品的鉴别处理, 用于定性和定量的形态学和分子分析类型。

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Research

JoVE Journal

Medicine

Generation of Alginate Microspheres for Biomedical Applications

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

科研

医学

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

如何从MR图像测量皮质折叠：一个循序渐进的教程，以计算本地Gyrification指数

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

牛龙在生物医学研究：视觉引导支气管镜，支气管内接种和<em&gt;在体内</em&gt;抽样技术

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

临床样品RNA病毒的无偏深度测序

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

组织和采血程序标准化改善闭合性胸猪心肌梗死模型的研究

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

使用微型吸脂技术进行人体皮下脂肪组织取样

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...