Name: sampling strategies and processing of biobank tissue samples from porcine biomedical models
Uploaded: 2018-03-06T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models at JoVE.com

The authors thank Lisa Pichl for excellent technical assistance.

<ol>
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B., Kromann-Andersen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+between+the+effects+of+paraffin+and+plastic+embedding+of+the+normal+and+obstructed+minipig+detrusor+muscle+using+the+optical+disector.">A comparison between the effects of paraffin and plastic embedding of the normal and obstructed minipig detrusor muscle using the optical disector.</a> J Urol. 154, 2170-2173 (1995).</li><li>Schneider, J. P., Ochs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+mouse+lung+tissue+dimensions+during+processing+for+morphometry:+a+comparison+of+methods.">Alterations of mouse lung tissue dimensions during processing for morphometry: a comparison of methods.</a> Am J Physiol Lung Cell Mol Physiol. 306, L341-L350 (2014).</li><li>von Bartheld, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+particles+in+the+z-axis+of+tissue+sections:+Relevance+for+counting+methods.">Distribution of particles in the z-axis of tissue sections: Relevance for counting methods.</a> NeuroQuantology. 10, 66-75 (2012).</li><li>Baddeley, A. J., Gundersen, H. J., Cruz-Orive, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+surface+area+from+vertical+sections.">Estimation of surface area from vertical sections.</a> J microsc. 142, 259-276 (1986).</li><li>Blutke, A., Schneider, M. R., Wolf, E., Wanke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+hormone+(GH)-transgenic+insulin-like+growth+factor+1+(IGF1)-deficient+mice+allow+dissociation+of+excess+GH+and+IGF1+effects+on+glomerular+and+tubular+growth.">Growth hormone (GH)-transgenic insulin-like growth factor 1 (IGF1)-deficient mice allow dissociation of excess GH and IGF1 effects on glomerular and tubular growth.</a> Physiol Rep. 4, e12709 (2016).</li><li>Hermanns, W., Liebig, K., Schulz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembedding+immunohistochemical+demonstration+of+antigen+in+experimental+polyarthritis+using+plastic+embedded+whole+joints.">Postembedding immunohistochemical demonstration of antigen in experimental polyarthritis using plastic embedded whole joints.</a> Histochemistry. 73, 439-446 (1981).</li><li>Böck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Romeis Mikroskopische Technik. , 1-697 (1989).</li><li>Suvarna, K. S., Layton, C., Bancroft, J. D., Suvarna, K. S., Layton, C., Bancroft, J. 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The authors have nothing to disclose.

Generation of biobank sample collections from porcine animal models requires robust techniques and protocols for the determination of organ/tissue volumes, the reproducible generation of representative, redundant tissue samples suitable for a broad range of different analysis methods, and for randomization of the orientation of sample sections for quantitative stereological analyses. The methods described in the present article are adapted to the sizes of porcine organs and tissues, and have been developed to effectively meet these demands2,7. They are based on well recognized methodological principles, and have previously proven their practicability in different published studies2,7,12,21. The featured methods are important for various types of studies examining tissue/organ samples, since they provide a basis for generation of representative samples, and for acquisition of a variety of parameters which could otherwise not be determined. These methods can quickly be performed with little effort, and are compatible with virtually all kinds of downstream analyses. Therefore, they are considered suitable not only for porcine animal model biobank projects, but also are useful in studies involving quantitative histomorphological analyses of tissue samples of other large animal model species (e.g., sheep), as well as in veterinary studies. Taking the technical aspects of the different methods into account, a few critical steps and limitations have to be considered during implementation of the protocols of the respective techniques.
Submersion technique for determination of tissue/organ density
During determination of tissue densities using the submersion method (step 1.1, Figure 1, Figure 2), care must be taken not to touch the inner walls or the bottom of the beaker with the tissue sample or the sample holder while the tissue sample is submerged in saline. Otherwise, the scale will display the weight of the sample rather than the weight of the volume of saline displaced by the sample. For very small/light tissue samples (a few mg), the submersion method for determination of tissue density is limited, due to the surface tension of the saline and the adversely high quotient of sample volume to the volume of submersion liquid in the beaker, which is impeding the accuracy of the measurement. Here, the weight of the sample might be used for further calculations instead of the density-calculated volume. Determination of tissue densities by submersion is also not effective for fresh (unfixed) lung tissue, due to the inconsistent content of air in the tissue, and in some cases, where organs are experimentally perfused/instilled with fixatives.
Cavalieri method for determination of organ volumes
The method of Cavalieri volumetry of porcine organs and tissues is more cumbersome when compared to determination of the organ volume from the organ weight and density. However, it is suitable for organs that cannot appropriately be weighed due to their experimental processing (e.g., perfusion-fixed organs, or lungs instilled with fixation solution). Here, the Cavalieri volumetry method can ideally be combined with the volume-weighted systematic random sampling technique described in step 2 (Figure 5, Figure 6, Figure 12). During estimation of organ/tissue volumes from the areas of section profiles of parallel, equidistant slices of the organ/tissue (Cavalieri approach, step 1.2, Figure 3), maintenance of the orientation of the tissue slabs (upper and lower section plane), as well as the accurate determination of the area of all section profiles are of great importance. Particularly, if digital images or scans of organ slabs are analyzed planimetrically, overprojections of tissue (outside of the sectioned tissue plane) of the tissue slab (Figure 12G-H) must carefully be considered to provide reliable volume estimates.
Determination of three-dimensional embedding-related tissue shrinkage during processing of tissue samples for histology
The extent of embedding-related tissue shrinkage depends on the embedding medium and the type of tissues, and may also vary between differentially processed samples of the same tissue.
Whereas frozen sections are considered to display virtually no shrinkage in the X-Y plane, and plastic embedding generally causes little shrinkage of the tissue, embedding-related tissue shrinkage is particularly extensive in paraffin-embedded tissue samples, and will usually significantly impair quantitative morphological analyses of dimensional parameters in histological sections of these samples. In addition to the large extent of overall three-dimensional shrinkage, paraffin-embedding also causes a non-uniform, differential, anisotropic, and variable shrinkage of the sample and of different anatomical structures, tissue types and cell types within the tissue sample8,13. Moreover, particularly in thicker histological sections of paraffin-embedded tissue samples, the extent of tissue shrinkage may also significantly differ in the X-Y and the Z-direction of the section. This can be due to a differential shrinkage of the section area (in X-Y-direction) and the section height (i.e., the vertical section thickness) during stretching of the section in the warm tissue flotation water bath after the section is cut from the tissue block, and during the subsequent processing, staining, and coverslipping procedures of the section mounted on a glass slide (i.e., a homogenous collapse of the sections&#39; Z-axis)8. Additionally, in thick sections, there might be a comparably stronger compression of the section-plane-near tissue regions within the section while the section is cut from the tissue block (leading to a differential deformation of the sections&#39; Z-axis)19. These effects may then also distort the estimates of the number of tissue structures within the section by distinct quantitative stereological analysis methods, such as the optical disector. Therefore, prediction, monitoring, and correction of embedding-related tissue shrinkage is not feasible for paraffin-embedded tissue samples8.
In contrast, the extent of volume shrinkage of tissue samples embedded in plastic resins, such as GMA/MMA or epoxy, is significantly lower and, importantly, more uniform. Therefore, embedding in plastic resin with an assumed homogenous, isotropic, and global shrinkage of the tissue is advantageous for several analysis methods of different quantitative morphological parameters17,18. During estimation of tissue shrinkage related to embedding in plastic resin, the shape of the fixed tissue should not additionally be distorted during the embedding procedure, to allow for comparison of corresponding section profile areas of the fixed and embedded samples. This can be difficult in soft or easily compressible tissue samples such as adipose or lung tissue, or in tissue samples with a high fluid content. Here, embedding of the fixed sample in agar prior to sectioning of the tissue is helpful to stabilize the shape of the tissue sample during the subsequent plastic embedding procedure (protocol step 1.3., Figure 4). To achieve reliable data, repeated measurements of the embedding-related tissue shrinkage should be performed, using different samples of the same tissue. The extent of embedding-related tissue shrinkage is expressed as the linear tissue shrinkage factor fs (protocol step 1.3.9., Figure 4G). Examples for equations using fs for shrinkage correction of various length, surface area, and volume parameters of different tissue structures are provided in several quantitative stereological studies14,21,27.
Volume-weighted systematic random sampling by point counting and processing of tissue subsamples for different downstream analysis types
The presented volume-weighted sampling technique for porcine organs determines random sampling locations within the tissue once, and subsequently generates all necessary samples for further different analyses by subsampling of the tissue samples excised from these locations7. In volume-weighted systematic random sampling regimes, each possible sampling location within the total volume of the organ/tissue under examination has exactly the same random chance to be sampled, and generalizability is ensured by the collection of a sufficient number of specimens. Using volume-weighted systematic random sampling designs for generation of samples from parenchymal organs, therefore, effectively prevents analysis results possibly biased by (potentially unexpected and unrecognized) unequal distributions of distinct functional or morphological tissue properties within different locations of an organ, which have been demonstrated, e.g., for the mean volume and the numerical volume density of porcine hepatocytes in different regions of the liver parenchyma28. The featured &#34;tissue-slabbing and subsampling&#34; strategy for porcine organs is easily comprehensible, complies with the technical requirements of the downstream analyses methods, and can be quickly carried out and adapted to the demands of a specific study, thereby avoiding systematic sampling biases, reducing experimental variability, and increasing the precision of the overall experiment, while being more efficient than sampling strategies in which each single sampling location is determined randomly9,15. The number of specimens that have to be generated, obviously depend on the examined parameter and its variability within samples from different locations of the sampled organ/tissue. For generation of biobank sample collections designed to allow for examination of a maximum of different parameters (not specified at the time of sampling) by a variety of different analytical methods, such a forward-looking sampling strategy usually schedules generation of relatively high numbers of redundant samples per organ/tissue.
Generation of Isotropic Uniform Random (IUR) sections and Vertical Uniform Random (VUR) sections for quantitative stereological analyses
Some of the techniques used for generation of IUR and VUR sections for quantitative stereological analyses have somewhat complicated theoretical bases, and explanations are therefore often (unnecessarily) eschewed by many scientists, although their practical implementation is reasonably easy. VUR sections are particularly easy to generate, and are optimal for surface density/area estimations in combination with cycloid test systems8,9,20. They are often preferred due to the familiar orientation of the section plane. However, in contrast to IUR sections, VUR sections are not suitable for estimation of length-parameters8,9.
During preparation of IUR and VUR sections, the critical step is, basically, to maintain the IUR or VUR section surface of the fixed sample during embedding in plastic resin, and to ensure that the vertical axis of VUR sections can always be identified (step 3, Figure 9, Figure 10, Figure 11).
In the future, the broader application of the methods described above might substantially contribute to establish consistently high-quality standards for generation of comparable, multi-purpose biobank samples from porcine and other large animal models.

In translational medicine, pigs are increasingly common for use as large animal models1,2,3,4,5, due to several advantageous similarities between the porcine and human anatomy and physiology, and the availability of established molecular biological methods allowing for generation of tailored, genetically modified pig models for a wide range of disease conditions1,4.
However, compared to rodent models, the number of animals of a respective pig model that can be provided for experiments at any time is limited. This is due to the porcine generation interval of approximately one year, and the financial and time-intensive efforts required for the generation of porcine models and animal husbandry. Therefore, individual animals of a porcine model, as well as the samples that can be generated from these pigs, are very valuable, particularly if genetically modified porcine models and/or long-term experimental issues (e.g., late complications of chronic diseases) are examined in aged individuals2,6,7.
In the course of any study, performance of additional analyses which had not been scheduled in the initial experimental design of the study might later turn out to be relevant, e.g., to address distinct questions arising from previously discovered unexpected findings. If suitable samples for such additional experiments are not available, disproportionally high cost and time-intensive expenditures might be necessary to generate additional pigs and tissue samples. To be prepared for such eventualities, generation of biobank collections of conserved back-up samples of different organs, tissues, or bio-liquids, quantitatively and qualitatively suitable for a broad range of subsequent analyses, is considered an important approach2,6,7. Deriving optimal benefits from a porcine animal model, the availability of adequate biobank samples also offers the unique possibility to perform a broad spectrum of different analysis methods on identical sample materials on a multi-organ level in the very same individual animals, e.g., by distribution of samples to scientists of different working-groups organized in a research network2,6,7. Additionally, the &#39;&#39;forward-looking&#39;&#39; sampling strategy in biobanking also contributes to a reduction of the number of animals needed in a study. The advantages of porcine model biobanking have recently been demonstrated in a multi-organ, multiomics study, examining organ cross talk in a genetically modified porcine model of long-term diabetes mellitus, using specimens from the Munich MIDY Pig Biobank2.
There are some mandatory requirements biobank samples must generally comply with to establish the reliability and interpretability of the results of the subsequently performed analyses. The samples must be generated reproducibly, and they must be representative, i.e., adequately reflecting the interested morphological and molecular features of the tissue/organ the samples were taken from7. To be suitable for a wide range of downstream analysis types, the samples must be taken in sufficient quantities and processed according to the demands (including time and temperature conditions) of the different analytical methods, including descriptive histopathological analyses, such as cryohistology, paraffin and plastic histology, immunohistochemistry, in situ hybridization, ultrastructural electron microscopic analyses, and clinical laboratory diagnostic analyses, as well as molecular analyses of DNA, RNA, proteins, and metabolites.
To allow for the assessment of a wide range of distinct quantitative morphological parameters such as numbers, volumes, lengths, or surface areas of distinct tissue structures by quantitative stereological analyses, randomized section planes of the histological samples of the respective organs/tissues need to be prepared7,8,9,10,11. In quantitative morphological studies, the precise determination of the total volume of the tissue, organ, or organ compartment, the samples were taken from (i.e., the reference space) is crucially important7,9,12 to calculate the absolute quantities of the interested parameters within the respective organ, tissue, or organism. Eventually, the effect of embedding-related tissue shrinkage during preparation of histological sections has to be determined and taken into account13. Therefore, quantitative stereological analyses, especially of archived samples (fixed tissue samples, embedded tissue blocks, histological sections, etc.) from previous studies are sometimes severely limited or even impossible12, particularly if volumetry of the respective organs/tissues was not performed, if no adequate sampling designs were applied to warrant representative samples, if the numbers and amounts of available individual samples are insufficient, or if the processing of the samples is incompatible with estimation of the quantitative morphological parameter(s) of interest. Due to the manifold possible influencing factors, the suitability of archive-sample materials for analyses of distinct quantitative morphological parameters cannot unequivocally be answered, but depends on the careful assessment of each individual case.
Thus, as the location, size, number, processing, trimming direction, and orientation of samples will potentially affect the results of the subsequent analyses, these factors are of great importance and must be considered in the experimental design of any study. With regard to these aspects and the special features of the porcine anatomy, comprehensive, detailed, large-scale sampling guidelines adapted to porcine animal models have recently been established, providing a robust reference to standardized, reproducible, and efficient generation of redundant, adequately processed, high-quality samples from more than 50 different porcine organs and tissues6,7.
The methodological descriptions and the video tutorial shown in the present article provide detailed, illustrative, comprehensible, step-by step instructions for practical performance of a variety of techniques for volumetry, sampling of porcine tissues and organs, and processing of tissue samples for different downstream analysis methods. The featured techniques include methods for determination of organ/tissue volumes and densities based on the principles of Archimedes and Cavalieri9, including determination of the dimensions of three-dimensional shrinkage of tissue related to the embedding in different embedding media14 during processing for histological examination, application of practicable volume-weighted systematic random sampling approaches, processing of sampled tissue specimens for different subsequent analyses7,8,9,15, and generation of appropriately oriented and processed samples for potential quantitative stereological analyses7,8,9,10,11. Next to their application in porcine biobank projects, the demonstrated methods are generally appropriate for all studies examining quantitative histo-morphological properties of organs/tissues. Moreover, systematic random sampling designs are particularly beneficial for generation of representative samples in experiments using molecular analysis methods to detect abundance alterations of, e.g., RNAs, proteins, or metabolites in various organs and tissues.
The next paragraphs provide a brief introduction to these methods, while their practical performance is described in the protocol section.
Determination of organ/tissue volumes 
Determination of organ weights and volumes is important in several experimental settings, as these factors might indicate changes, potentially related to experimentally examined factors of interest. The total volume of an organ/tissue is also commonly required to calculate absolute quantitative parameters, (e.g., the total cell number), from stereologically estimated numerical volume densities (i.e., the number of cells per volume unit of tissue)7,12. Apart from techniques using complex technical equipment, such as computer tomography, there are basically three practical methods commonly used to determine the absolute volume of an organ or tissue. The volume of an organ can be determined by &#34;direct volumetric measurement&#34; according to the principle of Archimedes, i.e., measuring the volume of water or saline displaced by the structure when completely submerged. However, for comparably large porcine organs, these approaches are impractical and prone to imprecision, since they require very large volumetric/measuring flasks. More conveniently, the volume of an organ/tissue can be calculated from its weight and density7,12,16, which can efficiently be determined using the &#34;submersion method&#34;7,12,16 (protocol step 1.1.). Organ/tissue volumes can also be estimated using volumetry approaches based on the &#34;principle of Cavalieri&#34; (1598&#8211;1647). In simple terms, the Cavalieri principle states, that if two objects are sectioned in planes parallel to a ground plane, and the profiles of the sections cut through the two objects at corresponding distances from the ground plane have the same areas, the two objects have the same volume. Thus, the volume of arbitrarily shaped objects can be estimated as the product of their section profile areas in parallel, equally distant section planes and the distance between the section planes. This is comprehensible with the following analogy: consider two stacks consisting of the same number of identical coins are placed side by side, one stack with the coins orderly stacked on top of one another yielding a cylindrical shape of the coin stack, and the other stack of coins with off-center positioned coins (Figure 3A). Although the shapes of both coin stacks are different, their volumes are the same, since the areas of the coins at corresponding levels of both stacks (i.e., the areas of profiles of parallel sections cut through both coin stacks in equal distances from the ground) are identical. Estimation of the volumes of porcine organs and tissues using the Cavalieri principle7,12,15 is described in step 1.2.
Determination of the extent of tissue shrinkage related to histological embedding 
In analyses of several quantitative morphological parameters measured in histological tissue sections, the effect of embedding-related tissue shrinkage occurring during tissue processing for histology has to be determined and taken into account. The extent of embedding-related tissue shrinkage may be variable, and depends both on the tissue, its processing, and the embedding medium8,13,17,18,19. Generally, embedding-related changes of the volume of a tissue sample (i.e., mostly shrinkage) occur in all three dimensions of space, and, therefore, affects all dimensional parameters estimated by quantitative stereological analyses8. Basically, the extent of embedding-related tissue shrinkage, expressed as the linear tissue shrinkage factor (fS), can be estimated as shown in step 1.3. and used for correction of (shrinkage-sensitive) quantitative morphological parameters14.
Volume-weighted systematic random sampling of organs/tissues 
For establishment of a biobank collection of porcine organ/tissue samples, volume-weighted systematic random sampling approaches such as described in step 2 have proven to be practical, time-saving, and efficient techniques for generation of representative, multi-purpose tissue samples7,8,9,15.
Generation of Isotropic Uniform Random sections and Vertical Uniform Random sections for quantitative stereological analyses 
Biobank tissue samples need to be suitable for a wide range of different quantitative stereological analysis methods for estimation of a maximum of parameters that could not be determined without an adequately prepared specimen. Nearly all quantitative stereological parameters can be determined, using &#34;isotropic (independent) uniform random (IUR) sections&#34;8,9. In IUR sections, the three-dimensional orientation of the section plane of the tissue sample is randomized. This can be achieved by randomization of the position of the tissue sample relative to the position of the section plane, as applied in the &#34;Isector&#34; method11 (protocol step 3.1), or by randomization of the orientation of the section plane relative to the tissue sample, as in the &#34;Orientator&#34; method10 (protocol step 3.2). In tissue samples, such as skin- or mucosa specimen displaying a naturally present, or defined and properly identifiable vertical axis, preparation of &#34;vertical uniform random (VUR) sections&#34; (protocol step 3.3.) strictly sectioned within the plane of their vertical axis is advantageous8,20. For a complete discourse of the theoretical foundations of IUR/VUR sampling and a comprehensive discussion of potential downstream quantitative stereological analyses, the interested reader is referred to the textbooks of quantitative stereology in life sciences8,9.

<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 &amp;amp; Labortechnik, Ederm&amp;uuml;nde, Germany</td><td>Einbettsch&amp;auml;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 &lt;strong&gt;Albl et al.&lt;/strong&gt;&amp;nbsp; 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 &lt;strong&gt;Nyengaard &amp;amp; Gundersen&lt;/strong&gt;. Eur Respir Rev. 15, 107-114, doi: 10.1183/09059180.00010101 (2006) and in &lt;strong&gt;Gundersen et al.&lt;/strong&gt; Stereological Principles and Sampling Procedures for Toxicologic Pathologists. In: Haschek and Rousseaux&amp;acute;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&amp;uuml;romarkt B&amp;ouml;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&amp;reg; , 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 &amp;ndash; 100 ml volume will do.</td></tr><tr><td>Laboratory scale(s)</td><td>Mettler Toledo GmbH, Gie&amp;szlig;en, Germany</td><td>PM6000</td><td>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&amp;ouml;ttingen, Germany</td><td>BP61S</td><td></td></tr><tr><td>Microtome blades</td><td>Engelbrecht Medizin &amp;amp; Labortechnik, Ederm&amp;uuml;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>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><td></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&amp;nbsp; will do.</td></tr><tr><td>Plastic transparencies</td><td>Avery Zweckform GmbH, Oberlaindern, Germany</td><td>Laser Overhead-Folie DINA4 Cat.:&amp;nbsp; 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&amp;uuml;romarkt B&amp;ouml;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, &gt;99% Cat.: 0601.1</td><td>To prepare 0.9% saline, dissolve 9 g NaCl in 1000 ml of distilled water at 20&amp;deg;C.</td></tr><tr><td>Scalpel blades</td><td>Aesculap AG &amp;amp; Co KG, Tuttlingen, Germany</td><td>BRAUN Surgical blades N&amp;deg;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 &lt;strong&gt;Knust, et al.&lt;/strong&gt; Anatomical record. 292, 113-122, doi: 10.1002/ar.20747 (2009) and in the supplemental data file of&lt;strong&gt; Albl et al.&amp;nbsp; &lt;/strong&gt;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 &amp;amp; Hobby Schobes, Stra&amp;szlig;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 &amp;amp; Co KG, N&amp;uuml;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

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

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

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

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

sampling-strategies-processing-biobank-tissue-samples-from-porcine

Nanyang Technological University

Research

JoVE Journal

Medicine

15.6K Views.  LMU Munich. De praktische toepassing en de prestaties van methoden voor generatie van representatieve weefselmonsters van varkens diermodellen voor een breed spectrum van downstream analyses in biobank projecten worden gedemonstreerd, waaronder volumetrie, systematische steekproefsgewijze bemonstering, en differentiële verwerking van weefselmonsters voor kwalitatieve en kwantitatieve analyses van morfologische en moleculair typen.

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

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

Bioengineering

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

Cancer Research

Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and associated data for research, diagnosis, and personalized medicine. The Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology (IEO) is a landmark in this field. Biobanks collaborate with clinical divisions, internal and external research groups, and industry, supporting patients' treatment and scientific progress, including innovative diagnostics, biomarker discovery, and clinical trial design. Given the central role of biobanks in modern research, biobanking standard operating procedures (SOPs) should be extremely precise. SOPs and controls by certified specialists ensure the highest quality of samples for the implementation of science-based, diagnostic, prognostic, and therapeutic personalized strategies. However, despite numerous efforts to standardize and harmonize biobanks, these protocols, which follow a strict set of rules, quality controls, and guidelines based on ethical and legal principles, are not easily accessible. This paper presents the biobank standard operating procedures of a large cancer center.

Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks' standard operating procedures for the management of different types of human biological samples.

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and ...

Protocol for Prostatectomy Specimen Mapping and Dissection

Enhancing Prostate Tumor Biobanking Reliability with Improved Sampling Technique and Histological Characterization

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be particularly challenging in the context of prostate cancer (PC) due to the unique nature of this organ and the high heterogeneity associated with this tumor. On the other hand, histopathologically characterizing samples before their storage without causing significant tissue alterations is also an intriguing challenge. In this context, we present a new method for acquiring, mapping, characterizing, and micro-dissecting resected prostate tissue based on anatomopathological criteria.
Unlike previously published protocols, this method reduces the time required for histopathological analysis of the prostate specimen without compromising its structure, which is crucial for assessing surgical margins. Furthermore, it enables the delineation and micro-macro dissection of fresh prostate tissue samples, with a focus on histological tumor areas defined by pathological criteria such as Gleason score, precursor lesions (high-grade prostatic intraepithelial neoplasia - PIN), and inflammatory lesions (prostatitis). These samples are then stored in a Biobank for subsequent research analyses.

Author Spotlight: Advancing Prostate Cancer Research Through Improved Tissue Sampling and Biobanking

This protocol describes a method for facilitating the collection of samples from radical prostatectomy specimens. The goal is to map, characterize, and micro-macro dissect tissue samples from the specimens based on anatomopathological criteria before storing them in a Biobank.

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

Neuroscience

Intubation, Central Venous Catheter, and Arterial Line Placement in Swine for Translational Research in Abdominal Transplantation Surgery

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries does not solely rely on the research team's surgical skills; perioperative care and management procedures, like intubation, central venous line, and arterial line placement, are necessary and of the utmost importance for favorable experiment results. As it is uncommon for research teams to have anesthesiologists or any other staff other than the surgical team, the surgical team involved in translational research must acquire and/or develop the skills to perform the perioperative care. The purpose of this paper is to show the techniques of intubation, central venous catheter, and arterial line placement used and perfected at the Toronto Organ Preservation Laboratory over the last 10 years, to be used as a reference for future researchers joining either this team or any other lab performing translational research protocols in swine and/or abdominal transplantation.

To obtain the best possible results, surgeons performing translational research experiments should be proficient at intubation and vascular line placement. This paper describes the techniques used by the Toronto Organ Preservation Laboratory to perform these procedures.

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries ...

An Efficient High-Speed Pneumatic Drill Craniectomy to Access the Porcine Brain

Accessing the Porcine Brain via High-Speed Pneumatic Drill Craniectomy

The use of pigs as an experimental animal model is especially relevant in neuroscience research, as the porcine and human central nervous systems (CNS) share many important functional and architectural properties. Consequently, pigs are expected to have an increasingly important role in future research on various neurological diseases. Here, a method to perform an anterior craniectomy through the porcine frontal bone is described. After a midline incision and subsequent exposure of the porcine frontal bone, anatomical landmarks are used to ensure the optimal location of the craniectomy. By careful and gradual thinning of the frontal bone with a rounded drill, a rectangular opening to the dura mater and underlying cerebral hemispheres is achieved. The presented method requires certain surgical materials, including a pneumatic high-speed drill, and some degree of surgical experience. Potential complications include unintended lesions of the dura mater or dorsal sagittal sinus. However, the method is simple, time-efficient, and offers a high degree of reproducibility for researchers. If performed correctly, the technique exposes a large portion of the unaffected pig brain for various neuromonitoring or analyses.

Author Spotlight: A Reproducible and Efficient Method for Accessing Porcine Brain via Craniectomy

This protocol describes performing a craniectomy using a high-speed pneumatic drill on a 3-month-old Danish Landrace pig. The access is made through the frontal bone and reveals the ventral dura mater and underlying cerebral hemispheres. This procedure allows for access to a large portion of the pig brain.

The use of pigs as an experimental animal model is especially relevant in neuroscience research, as the porcine and human central nervous systems (CNS) ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

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

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