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

Summary

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.

Abstract

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 "Orientator" and "Isector" methods, and vertical uniform random (VUR) sections.

Introduction

In translational medicine, pigs are increasingly common for use as large animal models^1,2,3,4,5, 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 conditions^1,4.

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 individuals^2,6,7.

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 approach^2,6,7. 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 network^2,6,7. Additionally, the ''forward-looking'' 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 prepared^7,8,9,10,11. 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 important^7,9,12 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 tissues^6,7.

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 analyses^7,8,9,15, and generation of appropriately oriented and processed samples for potential quantitative stereological analyses^7,8,9,10,11. 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 "direct volumetric measurement" 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 density^7,12,16, which can efficiently be determined using the "submersion method"^7,12,16 (protocol step 1.1.). Organ/tissue volumes can also be estimated using volumetry approaches based on the "principle of Cavalieri" (1598–1647). 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 principle^7,12,15 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 medium^{8,13,17,18,19}. 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 (f_S), 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 samples^7,8,9,15.

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 "isotropic (independent) uniform random (IUR) sections"^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 "Isector" method¹¹ (protocol step 3.1), or by randomization of the orientation of the section plane relative to the tissue sample, as in the "Orientator" 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 "vertical uniform random (VUR) sections" (protocol step 3.3.) strictly sectioned within the plane of their vertical axis is advantageous^8,20. 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 sciences^8,9.

Protocol

All methods described here use tissue samples derived from dead animals and fully comply with the German legal regulations of animal welfare.

1. Volumetry

1. Submersion Technique for Determination of Tissue/Organ Densities (Figure 1 and Figure 2)^7,12,16
   1. Prepare materials: Scalpel blades, paper towels, fine forceps, standard laboratory scales, glass or plastic beakers, 0.9% saline, and self-constructed specimen holders (Figure 2A).
   2. Excise a piece of tissue (maximum size: 2 x 2 x 2 cm³) from the organ/tissue, specifically from the organ compartment of interest. Small organs, such as the pituitary, or the pineal gland, are measured in toto.
      CAUTION: Ensure that the size of the sample is notably smaller than the inner diameter of the beaker (step 1.1.5 et seq.), and its filling level to allow complete submersion of the sample without contacting the inner walls of the beaker in step 1.1.7.
   3. Carefully swab the sample with a paper towel to remove excess blood/tissue fluid.
   4. Weigh the sample on a precision scale and record the weight of the sample (m_S). Determine the weight of small tissue samples to the nearest mg (Figure 1A).
   5. Place a beaker filled with room-temperature 0.9% saline on the scale. Do not completely fill the beaker, to allow for a submersion of the tissue sample in the subsequent step without overflowing.
      CAUTION: Use a beaker size appropriate to the size and weight of the tissue sample(s) to be measured and the effective measurement range of the scale. For larger samples up to 2 x 2 x 2 cm³, a beaker size of 50–100 mL is appropriate in combination with a scale measuring from approximately 100 mg to 500 g, whereas for small samples, use beakers of 5–10 mL volume in combination with precision scales with measuring ranges between approximately 0.1 mg and 20 g.
   6. Submerge the sample holder (i.e., a sufficiently rigid loop of thin wire or something similar, Figure 2A) in the saline to a marked position (arrows in Figure 1B, Figure 2B, and Figure 2C). Then, reset (tare) the display of the scale to zero.
   7. Carefully attach the tissue sample to the sample holder and completely submerge the sample in the saline until the marked position on the sample holder is reached (arrows in Figure 1B, Figure 2B, and Figure 2C).
      CAUTION: The submerged sample and the sample holder must not have contact with the inner walls or the bottom of the beaker or the surface of the saline.
   8. While holding the sample holder and the submerged sample in that position, record the weight displayed on the scale (m_L), referring to the weight of saline displaced by the tissue sample (Figure 1C, Figure 2B, and Figure 2C).
   9. Calculate the volume of the sample (V_S) from m_L, and the density of saline at room temperature (20 °C) (ρ_saline = 1.0048 g/cm³) as V_S = m_L/ρ_saline [g/g/cm³] (Figure 1).
   10. Calculate the density of the tissue sample (ρ_sample) from the weight (i.e., mass) of the sample (m_S) and its volume (V_S): ρ_sample = m_S/V_S [g/cm³] (Figure 1).
   11. For organs measured in toto, repeat the measurement three times and calculate the average organ density from the single measurement values. For large organs/tissues, perform repeated measurements with different samples of the same organ/tissue/organ compartment, and calculate the average density from the single measurements, accordingly.
   12. Calculate the total volume of the organ/tissue/organ compartment from its weight and density (Figure 1).
2. Application of the Cavalieri-Method for Determination of Porcine Organ Volumes⁷
   1. Prepare materials: Ruler, caliper, knife, scissors, forceps, waterproof pen, plastic transparencies, scanner, photo camera, and cross grids printed on plastic transparencies.
   2. Place the entire organ/tissue on a plain surface (cutting base) and measure the length (l) of the organ along its longitudinal axis (Figure 3B, Figure 5A).
   3. Cut the complete organ/tissue into equidistant parallel slices orthogonal to the longitudinal organ axis (Figure 3C, Figure 5B). Choose a distance d between two sections (i.e., the sectioning interval/section thickness, usually approximately 1 cm) sufficiently small to receive a sufficient number of tissue/organ slabs. Randomly position the first section within a distance between 0 and the sectioning interval from the margin of the organ. While slicing, visually judge the position and orientation of each section plane, to obtain approximately parallel organ/tissue slabs of roughly uniform thickness.
      NOTE: The necessary number of tissue/organ slabs depends on the shape and the size of the examined organ/tissue. If small organs or samples have to be sectioned in thin slabs of ≤5 mm, embed the samples in agar prior to sectioning (see step 1.3.3.) and use a technical device for slicing of the agar-embedded sample. Empirically recommendable sectioning intervals for most porcine organs and tissues, as well as examples for slicing devices, are indicated in the Supplementary Material of "Tissue Sampling Guides for Porcine Biomedical Models"⁷.
   4. Place all organ/tissue slabs on the same surface facing down on the cutting base (i.e., consistently on either the right or the left section plane of each organ slab, Figure 3D, Figure 5C) and count the slabs (n).
   5. Obtain section profiles of the tissue slabs by one of the following approaches:
      1. Carefully place the tissue slabs on appropriately labeled plastic transparencies, while maintaining the orientation of their upper and lower section surfaces. Trace the outlines of the tissue slabs on the plastic transparencies using a waterproof pen (Figure 3E1-2).
      2. Take photographic images of the tissue slabs, holding the camera vertically above the section surfaces (Figure 3F). For calibration, place a size ruler next to the tissue slabs.
      3. Scan the tissue slabs on a flatbed scanner while maintaining the orientation of their upper and lower section surfaces (Figure 3G). For calibration, place a size ruler next to the tissue slabs.
   6. Measure the areas of the (traced, photographed, or scanned) section profiles of all tissue slabs by one of the following approaches:
      1. Overlay or superimpose the traced organ slab profiles with an appropriately sized, calibrated grid of equally spaced crosses printed on a plastic transparency and count all crosses hitting the profile area (Figure 3E3-4; compare to Figure 5D). Calculate the section profile area of each organ slab by multiplying the number of crosses hitting the profile area by the area corresponding to one cross.
         NOTE: To receive sufficiently precise volume estimates, choose a cross grid with a sufficiently small distance between adjacent crosses, so that an average of at least 100 crosses will hit the section surfaces of the slabs of one organ in each examined case of the study. Empirically recommendable cross grid sizes for most porcine organs and tissues are indicated in the Supplementary Material of "Tissue Sampling Guides for Porcine Biomedical Models"⁷.
      2. Measure the areas of the tissue slabs in the digital images of the photos/scans using appropriate commercially available or freeware morphometry soft- and hardware applications (Figure 3H), such as a commercial image analysis system²¹, or ImageJ²².
         CAUTION: Note that one tissue slab (either the first or the last) is placed on the scanner resting on its natural surface, respectively, faces the camera with its natural surface. Therefore, the scanned image, the photo image of this slab, will not show a section surface. Therefore, no section area profile is measured in the scanned image/photo image of this tissue slab (Figure 3I). Also note over-projection present in scanned images and photographs of organ/tissue slabs, i.e., only measure the areas of the actual section profiles, but not of the tissue in the image lying behind the slab section surface (Figure 12G-H).
   7. Calculate the estimated organ volume as the product of the sum of all corresponding section profile areas of all tissue slabs per case (i.e., consistently of either the right or the left, respectively, the upper or lower section surface of each organ slab and the mean thickness of the slabs (i.e., the quotient of the measured length of the vertical organ axis (l) and the number of slabs)¹⁵.
3. Determination of the Extent of Three-Dimensional Embedding-Related Tissue Shrinkage during Processing of Tissue Samples for Histology
   1. Prepare materials: Microtome blades, forceps, agar, metal casting molds, digital scanner, and size ruler (e.g., graph paper).
   2. Cut a fresh, plane section surface from a fixed tissue sample.
      NOTE: If using samples of easily deformable (soft) tissues (fat tissue, gelatinous tissues), embed the fixed tissue sample in agar prior to sectioning (Figure 4A).
   3. To embed sample in agar:
      1. Mix standard agar powder as used for microbiology culture medium with an appropriate volume of water (approximately 0.5–1 g agar/10 mL water) in a glass beaker. Stir the mixture and heat it in a microwave oven at 700 W until boiling for 3–5 s. Stir the mixture and bring to a boil again for 3–5 s.
      2. Optionally, to increase the contrast of the agar to the tissue sample, dye the liquid agar, e.g., with black ink (add 1 mL ink to 10 mL of hot liquid agar and stir vigorously).
      3. Pour the hot agar into a casting mold (e.g., a metal mold used for paraffin embedding, Figure 10A-D) and submerge the fixed tissue sample in the warm agar. Let the agar cool until solidification, remove the mold, and cut the agar block with the embedded tissue using a microtome or razor blade.
         CAUTION: While handling hot liquid agar, wear protective goggles and gloves. Process tissue samples fixed in formaldehyde solution under an exhaust hood and wear protective goggles and laboratory gloves.
   4. Place the sample with its section surface facing down on a flatbed scanner, together with a size ruler and scan the section surface (Figure 4A,B).
   5. Determine the area of the section surface of the fixed tissue sample (A_f) in the digital scan, using one of the techniques described in step 1.2.6 (Figure 4B).
   6. Embed the sample in plastic embedding medium, such as epoxy (e.g., Epon) or glycolmethacrylate/methylmethacrylate (GMA/MMA)²³, following standard protocols^23,24,25 (Figure 4C). Ensure that the section surface of the fixed sample scanned in the previous step (1.3.4) is maintained in the plastic-embedded sample.
      NOTE: To maintain the orientation of the section surface of the sample during processing of the sample, consistently place the sample with its intended section surface facing downwards into the embedding cassette or the casting mold, or mark the intended section surface (or the opposite side of the sample) with ink.
   7. Cut a histological section from the plastic block corresponding to the original section surface of the fixed tissue sample (step 1.3.2) using a microtome (Figure 4D), mount the section on a glass slide (Figure 4D) and stain it routinely (e.g., Hematoxylin and eosin stain, H&E)^24,25.
      CAUTION: To receive a histological section approximately in the same plane as the original section surface of the fixed tissue sample, carefully adjust the position of the plastic block in the mount of the microtome before sectioning.
   8. Place the slide with the stained section facing downwards on a flatbed scanner together with a size ruler and scan the section (Figure 4E).
   9. Determine the area of the section of the plastic-embedded tissue sample (A_e) in the digital scan, using one of the techniques described in step 1.2.6 (Figure 4F).
   10. Calculate the average embedding-related tissue shrinkage (for the respective tissue and embedding medium) from the measured areas of corresponding section profiles of tissue samples before and after embedding in plastic embedding medium. The linear shrinkage factor f_s is calculated as the square root of the quotient of the areas of the section profiles of n tissue samples after embedding in plastic embedding medium (A_e) and the areas of the corresponding section profiles of the same tissue samples before embedding in plastic embedding medium (A_f) (Figure 4G)¹⁴.

2. Volume-weighted Systematic Random Sampling by Point Counting and Processing of Tissue Subsamples for Different Downstream Analysis-types⁷

1. Prepare materials: Ruler, caliper, knife, scissors, forceps, waterproof pen, point/cross grids printed on plastic transparencies, and random number tables.
   NOTE: Copy templates for cross grids (5–60 mm) are provided in the Supplementary Material of "Tissue Sampling Guides for Porcine Biomedical Models"⁷.
2. Place the organ/tissue on a plain surface (cutting base) and measure the length (l) of the organ along its longitudinal axis (Figure 5A, Figure 6A).
3. Cut the complete organ/tissue into equidistant parallel slices orthogonal to its longitudinal axis (Figure 5B). Choose a distance d between two sections (i.e., the sectioning interval/section thickness, usually approximately 1 cm) small enough to obtain a sufficient number of tissue/organ slices. Randomly position the first section within a distance between 0 and the sectioning interval from the margin of the organ. While slicing, visually judge the position and orientation of each section plane to obtain approximately parallel organ/tissue slabs of roughly uniform thickness.
   NOTE: The necessary number of tissue/organ slabs depends on the size of the examined organ/tissue and the number of sampled tissue locations. If small organs or samples have to be sectioned in thin slabs of ≤5 mm, embed the samples in agar prior to sectioning (see step 1.3.3.) and use technical devices for slicing of the agar-embedded sample. Empirically recommendable sectioning intervals for most porcine organs and tissues, as well as examples for slicing devices are indicated in the Supplementary Material of "Tissue Sampling Guides for Porcine Biomedical Models"⁷.
4. Place all organ/tissue slabs on the same surface facing down on the cutting base (Figure 6B).
5. Overlay the tissue slabs with an appropriately sized cross grid printed on a plastic transparency by placing the outermost left upper cross of the grid over a random point out of the tissue (Figure 5C-D, Figure 6B).
   NOTE: Choose a cross grid with a sufficiently small distance between adjacent crosses, so that in each examined case of the study, at least twice as many crosses will hit the section surface(s) of the tissue compartment to be sampled, as the number of samples that have to be taken from that tissue compartment. Empirically recommendable cross grid sizes for most porcine organs and tissues are indicated in the Supplementary Material of "Tissue Sampling Guides for Porcine Biomedical Models"⁷.
6. Mark and count all crosses hitting the tissue (respectively, the tissue sub-compartment to be sampled). Consistently apply a uniform mode of counting and numbering of the crosses hitting the tissue compartment to be sampled in all tissue slabs, e.g., by consecutive numbering of the respective crosses in each line by line, from the left to the right and from top to bottom, or, e.g., by numbering the crosses in one tissue slab after another in clockwise direction, starting with the cross closest to the twelve o'clock position, as exemplarily demonstrated in Figure 5E.
7. Divide the number of crosses hitting the tissue/tissue compartment to be sampled (n) by the number of samples to be generated to obtain the systematic sampling interval (i).
8. Determine the first sampling position by choosing a random number x in the interval between 1 and i. For this, use a random-number table. Mark the first sampling position (x) and every next x + i, x + 2i, x + 3i, etc., cross hitting the tissue/tissue compartment to be sampled on the plastic transparency using a waterproof pen (Figure 5F).
   NOTE: Random number tables can be conveniently and quickly generated using an online random number generator.
9. Tag the tissue locations corresponding to the marked crosses by slightly raising the plastic transparency and placing a small piece of clean, blank confetti paper on the surface of the tissue slab using a pair of tweezers (Figure 5G, Figure 6E).
10. Excise specimen of tissue from the sampled locations (Figure 5H, Figure 6F, Figure 7A) and further subdivide them for different types of subsequent analyses (Figure 6G, Figure 7A-B), as specified in Table 1.
11. After sampling, clean the plastic transparencies with warm water and soap, dry, and reuse them.

3. Generation of Isotropic Uniform Random (IUR) Sections and Vertical Uniform Random (VUR) Sections for Quantitative Stereological Analyses

1. "Isector" Technique
   1. Prepare materials: Razor or microtome blades, agar, spherical casting molds (e.g., casting molds for pralines, which can be obtained from confectioner suppliers), foldback clamps, and forceps.
   2. Place an adequately sized piece (1 x 1 x 1 cm³) of fixed, systematically randomly sampled tissue in a spherical casting mold, hold together by foldback clamps, and fill the mold with warm liquid agar (Figure 8A-E).
   3. Remove the agar sphere (Figure 8F) from the casting mold after solidification of the agar.
   4. Roll the agar sphere with the embedded tissue sample across the table, stop, and section it at a random position.
      NOTE: The resulting section plane is an IUR section (Figure 8F-G).
   5. Proceed to embed the tissue sample in plastic resin such as GMA/MMA, maintaining the orientation of the IUR section plane (see 1.3.5).
2. "Orientator" Technique
   1. Prepare the materials: Razor or microtome blades, agar, forceps, random number table(s), prints of equiangular, and cosine-weighted circles.
      NOTE: Copy templates of circles are provided in previous publications^8,26.
   2. Place the sample of fixed tissue (or of agar-embedded fixed tissue) on a print of an equiangular circle with one edge parallel to the 0–180° direction (Figure 9A, Figure 10E).
   3. Determine a random angle by using the random number table. Find the corresponding marks at the scale of the equiangular circle, which the sample rests on. Using these marks, cut a section through the sample (or through the agar surrounding the embedded tissue sample), with the section plane being oriented parallel to the direction of the random angle indicated on the scale of the equiangular circle, and vertical to the resting surface of the sample (Figure 9B-C, Figure 10F).
   4. Place the tissue block with the section surface generated in the previous step facing downside on a cosine-weighted circle with the edge of the resting surface placed parallel to the 1-1 direction (Figure 9D, Figure 10H).
   5. Repeat step 3.2.3 and cut a new section through the sample at a random angle determined using the random number table (Figure 9E-F, Figure 10I-J).
      NOTE: The resulting section plane is an IUR section.
   6. If appropriate, determine the area of the IUR section profile of the fixed tissue sample for determination of the embedding-related tissue shrinkage (Figure 9G-J) as described in step 1.3, and proceed to embed the tissue sample in plastic resin such as GMA/MMA.
3. Generation of Vertical Uniform Random (VUR) Sections
   1. Prepare materials: Razor or microtome blades, agar, forceps, random number table(s), and prints of equiangular circles.
      NOTE: Copy templates of circles are provided in previous publications^8,26.
   2. Define a vertical axis within the fixed tissue sample that is always recognizable in the sample/sections during the subsequent steps.
      NOTE: Typically, the axis vertical to the natural surface of the tissue sample is chosen as the vertical axis.
   3. If appropriate, embed the sample in agar (Figure 11B).
      NOTE: Agar-embedding prior to VUR- or IUR-sectioning of the fixed sample is generally recommendable for small, thin, fragile, or soft samples. Also use agar-embedding of samples to facilitate the positioning of the VUR sectioned sample during the subsequent embedding of the sample in plastic resin medium.
   4. Place the sample on a print of an equiangular circle, with the vertical axis being orthogonally oriented to the plane of the table/paper table (Figure 11C).
   5. Cut the sample at a random angle (determined using a random number table) with the section plane orthogonal to the table and parallel to the vertical axis to receive a VUR section plane (Figure 11D).
   6. If appropriate, determine the area of the IUR section profile of the fixed tissue sample for determination of the embedding-related tissue shrinkage as described in step 1.3 (compare to Figure 9G-J) and proceed to embed the tissue sample in plastic resin such as GMA/MMA.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Agar	Carl Roth GmbH, Germany	Agar (powder), Cat.: 5210.3	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.
Caliper	Hornbach Baumarkt GmbH, Bornheim, Germany	Schieblehre Chrom/Vernickelt 120 mm Cat.: 3664902	Any kind of caliper (mechanical or electronic) will do as well.
Casting molds (metal)	Engelbrecht Medizin & Labortechnik, Edermünde, Germany	Einbettschälchen aus Edelstahl, 14 x 24 x 5 mm, Cat.: 14302b	Any other kind of metal casting mold used for paraffin-embedding will do as well.
Copy templates of cross grids (5mm - 6 cm)	n.a.	n.a.	Copy templates of cross grids (5mm - 6 cm) are provided in the supplemental data file of Albl et al.  Toxicol Pathol. 44, 414-420, doi: 10.1177/0192623316631023 (2016)
Copy templates of equiangular and cosine-weighted circles	n.a.	n.a.	Copy templates of equiangular and cosine-weighted circles are provided in Nyengaard & 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´s Handbook of Toxicologic Pathology. 3rd ed, 215-286, ISBN: 9780124157590 (2013).
Foldback clamps (YIHAI binder clips, 15 mm and 19 mm)	Ningbo Tianhong Stationery Co ltd., China	Y10006 and Y10005	Any other type of standard office foldback clamps will do as well.
Forceps (anatomical)	NeoLab Migge GmbH, Heidelberg, Germany	neoLab Standard -Pinzette 130 mm, anatomisch, rund, Cat.: 1-1811	Any type of anatomical forceps will do.
Formaldehyde-solution 4%	SAV-Liquid Produktion GmbH, Flintsbach, Germany	Formaldehyd 37/40 %, Cat.: 1000411525005	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.
Graph paper (for calibration)	Büromarkt Böttcher AG, Jena, Germany. www.bueromarkt-ag.de	Penig Millimeterpapier A4, Cat.: 2514	Any type of graph paper (scaled in millimeter) will do.
Laboratory beakers (5ml, 10 ml, 50 ml, 100 ml)	NeoLab Migge GmbH, Heidelberg, Germany	Becherglas SIMAX® , niedrige Form, Borosilikatglas 3.3 Cat.: E-1031, E-1032, E-1035, E-1036	Any kind of glass- or plastic beakers of 5 – 100 ml volume will do.
Laboratory scale(s)	Mettler Toledo GmbH, Gießen, Germany	PM6000	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
	Sartorius AG, Göttingen, Germany	BP61S
Microtome blades	Engelbrecht Medizin & Labortechnik, Edermünde, Germany	FEATHER Microtome blasdes S35, Cat.:14700	Any kind of single-use microtome blades will do.
Morphometry/planimetry software/system	National Institute of Health (NIH)	ImageJ	Download from https://www. imagej.nih.gov/ij/ (1997).
Zeiss-Kontron, Eching, Germany	VideoplanTM image analysis system	Out of stock
Photo camera	Nikon	D40	Any kind of digital photocamera that can be mounted to a tripod  will do.
Plastic transparencies	Avery Zweckform GmbH, Oberlaindern, Germany	Laser Overhead-Folie DINA4 Cat.:  3562	Any (laser)-printable plastic transparency will do.
Random number tables	n.a.	n.a.	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/
Razor blades	Plano GmbH, Wetzlar, Germany	T5016	Any kind of razor blades will do.
Ruler	Büromarkt Böttcher AG, Jena, Germany. www.bueromarkt-ag.de	Office-Point Lineal 30 cm, Kunststoff, transparent, Cat.: ln30	Any kind of cm-mm-scaled ruler will do as well.
Saline (0.9%)	Carl Roth GmbH, Germany	Natriumchlorid, >99% Cat.: 0601.1	To prepare 0.9% saline, dissolve 9 g NaCl in 1000 ml of distilled water at 20°C.
Scalpel blades	Aesculap AG & Co KG, Tuttlingen, Germany	BRAUN Surgical blades N°22	Any kind of scalpel blades will do.
Scanner	Hewlett-Packard	hp scanjet 7400c	Any type of standard office scanner capable of scanning with resolutions from 150-600 dpi will do.
Slicing devices	n.a.	n.a.	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.  Toxicol Pathol. 44, 414-420, doi: 10.1177/0192623316631023 (2016).
Spherical casting molds (e.g., in 25.5 mm diameter)	Pralinen-Zutaten.de, Windach, Germany	Pralinen-Hohlkugeln Vollmilch, 25.5 mm	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)
Thin wire	Basteln & Hobby Schobes, Straßfurth, Germany. www,bastel-welt.de	Messingdraht (0.3 mm) Cat.: 216464742	Any other kind of thin wire will also do.
Tissue paper	NeoLab Migge GmbH, Heidelberg, Germany	Declcate Task Wipes-White, Cat.: 1-5305	Any other kind of laboratory tissue paper will do as well.
Waterproof pen	Staedler Mars GmbH & Co KG, Nürnberg, Grmany	Lumocolor permanent 313, 0.4 mm, S, black, Cat.: 313-2	Any other kind of waterproof pen will do as well.