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

The authors thank Lisa Pichl for excellent technical assistance.

All methods described here use tissue samples derived from dead animals and fully comply with the German legal regulations of animal welfare.
1. Volumetry
<ol>
	<li>Submersion Technique for Determination of Tissue/Organ Densities (Figure 1 and Figure 2)7,12,16
	<ol>
		<li>Prepare materials:...

<ol>
	<li>Aigner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+pigs+as+models+for+translational+biomedical+research.">Transgenic pigs as models for translational biomedical research.</a> J Mol. Med. 88, 653-664 (2010).</li><li>Blutke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Munich+MIDY+Pig+Biobank:+A+unique+resource+for+studying+organ+crosstalk+in+diabetes.">The Munich MIDY Pig Biobank: A unique resource for studying organ crosstalk in diabetes.</a> Mol Metab. 6, 931-940 (2017).</li><li>Klymiuk, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin-deficient+pigs+provide+new+insights+into+the+hierarchy+of+physiological+derangements+of+dystrophic+muscle.">Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle.</a> Hum Mol Genet. 22, 4368-4382 (2013).</li><li>Klymiuk, N., Seeliger, F., Bohlooly, Y. M., Blutke, A., Rudmann, D. G., Wolf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+pig+models+for+preclinical+efficacy+and+safety+testing+of+targeted+therapies.">Tailored pig models for preclinical efficacy and safety testing of targeted therapies.</a> Toxicol Pathol. 44, 346-357 (2016).</li><li>Renner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+neonatal+diabetes+in+INSC94Y+transgenic+pigs.">Permanent neonatal diabetes in INSC94Y transgenic pigs.</a> Diabetes. 62, 1505-1511 (2013).</li><li>Abbott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inside+the+first+pig+biobank.">Inside the first pig biobank.</a> Nature. 519, 397-398 (2015).</li><li>Albl, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+sampling+guides+for+porcine+biomedical+models.">Tissue sampling guides for porcine biomedical models.</a> Toxicol Pathol. 44, 414-420 (2016).</li><li>Gundersen, H. J. G., Mirabile, R., Brown, D., Boyce, R. W., Haschek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+principles+and+sampling+procedures+for+toxicologic+pathologists.">Stereological principles and sampling procedures for toxicologic pathologists.</a> Haschek and Rousseaux's Handbook of Toxicologic Pathology. , 215-286 (2013).</li><li>Howard, C. V., Reed, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Unbiased Stereology: Three-Dimensional Measurement in Microscopy. , 1-277 (2005).</li><li>Mattfeldt, T., Mall, G., Gharehbaghi, H., Moller, P. <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+and+length+with+the+orientator.">Estimation of surface area and length with the orientator.</a> J Microsc. 159, 301-317 (1990).</li><li>Nyengaard, J. R., Gundersen, H. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isector:+A+simple+and+direct+method+for+generating+isotropic,+uniform+random+sections+from+small+specimens.">The isector: A simple and direct method for generating isotropic, uniform random sections from small specimens.</a> J Microsc. 165, 427-431 (1992).</li><li>Tschanz, S., Schneider, J. P., Knudsen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereology:+Planning,+volumetry+and+sampling+are+crucial+steps+for+a+successful+study.">Design-based stereology: Planning, volumetry and sampling are crucial steps for a successful study.</a> Ann Anat. 196, 3-11 (2014).</li><li>Dorph-Petersen, K. A., Nyengaard, J. R., Gundersen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+shrinkage+and+unbiased+stereological+estimation+of+particle+number+and+size.">Tissue shrinkage and unbiased stereological estimation of particle number and size.</a> J Microsc. 204, 232-246 (2001).</li><li>Mattfeldt, T., Doerr, W., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereologische+Methoden+in+der+Pathologie+[Stereologic+methods+in+pathology].">Stereologische Methoden in der Pathologie [Stereologic methods in pathology].</a> Normale und pathologische Anatomie. , (1990).</li><li>Gundersen, H. J., Jensen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+systematic+sampling+in+stereology+and+its+prediction.">The efficiency of systematic sampling in stereology and its prediction.</a> J Microsc. 147, 229-263 (1987).</li><li>Scherle, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+volumetry+of+organs+in+quantitative+stereology.">A simple method for volumetry of organs in quantitative stereology.</a> Mikroskopie. 26, 57-60 (1970).</li><li>Nielsen, K. K., Andersen, C. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bancroft's theory and practice of histological techniques. , 1-654 (2013).</li><li>Knust, J., Ochs, M., Gundersen, H. J., Nyengaard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+estimates+of+alveolar+number+and+size+and+capillary+length+and+surface+area+in+mice+lungs.">Stereological estimates of alveolar number and size and capillary length and surface area in mice lungs.</a> Anat Rec (Hoboken). 292, 113-122 (2009).</li><li>Nyengaard, J. R., Gundersen, H. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+for+stereology+in+lungs.">Sampling for stereology in lungs.</a> Eur Respir Rev. 15, 107-114 (2006).</li><li>Junatas, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+analysis+of+size+and+density+of+hepatocytes+in+the+porcine+liver.">Stereological analysis of size and density of hepatocytes in the porcine liver.</a> J Anat. 230, 575-588 (2017).</li></ol>

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...

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 &#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

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.

Submersion technique for determination of tissue/organ density
Figure 12A-B shows the representative determination of the density and volume of a porcine kidney using the submersion technique described in step 1.1 (Figure 1, Figure 2). More representative results of density measurements of additional porcine organs and tissues...

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

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 ...

This video demonstrates two key techniques for sampling and processing biobank porcine tissue specimens. The submersion technique reliably estimates total organ and tissue volume and the orientator technique generates random isotropic tissue sections for unbiased estimation of quantitative morphological parameters. The main advantage of the submersion technique for tissue density determination is that it can be applied to any kind of tissue and that it produces reliable results for small as well as for large samples.The advantages of the orientator technique are that it is highly efficient and also works with large tissue samples. To begin volumetry for a large organ excise an appropriately sized piece of tissue from the organ. Carefully swab the sample with a paper towel to remove excess blood and tissue fluid.Then weigh the sample on a precision scale and record the weight of the sample. Add 0.9%saline to a beaker of suitable size. Do not completely fill the beaker to prevent overflow during tissue emersion.Place the beaker on the scale then submerge a forceps or a self-constructed sample holder in the saline until the mark on the forceps or sample holder reaches the liquid. Next, reset the display of the scale to zero. Carefully attach the tissue sample to the forceps or sample holder and completely submerge the sample in the saline until the marked position is reached.To record reliable density measurements it is important to ensure that the sample is completely submerged and to avoid any contact of the submerged sample to the inner walls or the bottom of the beaker. While holding the sample in position, record the weight displayed on the scale. Use this measurement and the density of saline at room temperature to calculate the volume of the sample.Then calculate the density of the tissue sample from the weight of the sample and its volume. For large organs and tissues, such as this kidney, perform repeated measurements with multiple samples and calculate the average density from the single measurements. For small samples and small organs measured in total such as the pituitary gland, repeat the measurement three times before calculating the average organ density from the single measurement values.Finally, calculate the total volume of the organ, tissue or organ compartment from its weight and density. Place a fixed tissue specimen on a print of an equiangular circle with one edge parallel to the zero to 180 degree direction. Then use a random number table to determine a random angle.Find the corresponding marks on the scale of the equiangular circle. Using these marks, cut a section through the sample with the section plane oriented parallel to the direction of the random angle indicated on the scale of the equiangular circle and a vertical to the resting surface of the sample. Place the tissue block with the section surface facing down on a cosine weighted circle.With the edge of the resting surface placed parallel to the one-to-one direction. Now cut a new section through the sample at a random angle determined using the random number table. The resulting section plane is an isotropic uniform random section.This table shows the representative densities of several porcine tissues and organs determined using the submersion technique. Most porcine tissues display density values slightly higher than one referring to the density of water. Whereas tissues swimming in saline, such as adipose tissue for example, display densities lower than one.This figure shows four representative IUR sections of porcine left ventricular myocardium prepared with the orientator technique. Note the changed cutting angles in the different IUR sections. Once mastered, the submersion technique for tissue density determination can be done in a few minutes.If it is performed properly. Isotropic uniform random tissue sections generated with the orientator technique are suitable for estimation of any quantitative morphological parameters using appropriate stereological analysis methods. digital music.

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