We thank Centro de Ecolog&#237;a y Biodiversidad CEBIO, Erika Paliza, Miluska S&#225;nchez, Jorge Carrera, Edgar Rengifo V&#225;squez, Harold Porocarrero Zarria, Jorge Ru&#237;z Leveau, Jaime Pecheco Castillo, Carlos Tello, Fanny Cornejo, and Fanny Fern&#225;ndez Melo for making tissue collection in Peru possible. In Colombia, we thank Colciencias and Instituto de Investigaci&#243;n de Recursos Biol&#243;gicos Alexander von Humboldt, as well as Alexandra Buitrago, Mailyn Gonz&#225;lez, Andr&#233;s Juli&#225;n Lozano Florez, Darwin M. Morales Martinez, Ana Maria Ospina, Adrian Pinzon, Paola Pulido-Santacruz, and Danny Rojas for making logistics, travel, capture, and filming possible. We also thank all members of Grupo Jaragua and Yolanda Leo&#769;n for making tissue collection in the Dominican Republic possible, as well as to Bernal Rodriguez Hernandez, Bernal Matarrita, and everyone at La Selva Biological Research Station in Costa Rica, for making sampling possible, and Kasia Sawicka for helping with procedures and reagents before the trip. We thank Ella Lattenkamp &#38; Lutz Wiegrebe for access and sample collection of wing punches from Phyllostomus discolor bats for cell culture generation. Phyllostomus discolor bats originated from a breeding colony in the Department Biology II of the Ludwig-Maximilians-University in Munich. Approval to keep and breed the bats was issued by the Munich district veterinary office. LMD, SJR, KTJD, and LRY were funded by NSF-DEB 1442142. LMD was funded by NSF-DEB 1838273. LRY was funded by the NSF-PRFB 1812035. SCV and PD were funded by a Max Planck Research Group Award, and a Human Frontiers Science Program (HFSP) Research grant (RGP0058/2016). SJR, JHTP, and KTJD were funded by the European Research Council (ERC Starting grant 310482 [EVOGENO]) awarded to SJR, ECT was funded by European Research Council grant (ERC-2012-StG311000).

The authors have nothing to disclose.

The protocol discussed in this manuscript describe the best sampling practices for various high-throughput molecular analyses of bats. All successful -omics studies require high quality tissue, but sampling bat tissue, as well as other non-model organisms, often occurs in field conditions that cannot be set to the same standards as those of a controlled laboratory setting. Sampling often occurs in remote locations, with minimal resources, including limited access to electricity and freezers. It is difficult, and often im...

High-throughput sequencing (HTS) methods have rapidly advanced in efficiency and decreased in cost, and it is now possible to scale these approaches to hundreds or thousands of samples. Insights obtained by the application of these technologies have enormous impacts across multiple scientific disciplines, from biomedicine to evolution and ecology1,2,3. Yet, many HTS applications rely critically on high quality nucleic acids from a living source. This limitation is likely to become increasingly problematic with the development of third generation sequencing based on long-read molecules4. For these reasons, there is a need to focus efforts on establishing best practices for collecting fresh tissue samples from wild organisms outside of the laboratory, which maximizes the utility of material and reduces the number of individuals that need to be collected.
Bat1K is an international consortium of scientists with an ongoing initiative to sequence the genome of every species of bat to chromosome-level assembly5. Bats represent 20% of mammalian diversity and have exceptional adaptations that have implications for understanding aging, disease ecology, sensory biology, and metabolism5,6. Many bats are also threatened or endangered due to human exploitation7 or are rapidly declining due to pathogens8,9, and genome-level sequencing is of great importance for conservation of these species. Although Bat1K currently aims to sequence the genomes of all bat species, the standardization of collection of tissue samples for high-quality genomic sequencing remains a key challenge across the community of organismal biologists. In addition to genomic data, functional understanding of the diversity of bat adaptations requires tissue-specific transcriptome and protein analyses, often requiring separate collection protocols. Moreover, as with all taxonomic groups, while optimal tissue collection and preservation is essential for obtaining the highest quality data for -omics analyses, communicating best practices is often difficult because of rapidly changing technologies and multiple research teams working independently.
The need to adopt best practices for bat -omics research is especially urgent, given that many bat species are rare or threatened. Unlike other small mammals such as rodents and shrews, bats are long-lived, attributable to exceptional DNA repair mechanisms10 and slow reproduction11, with most species giving birth to just one or (in a few cases) two young per year. For these reasons, bat populations can be slow to recover from disturbance, and collecting many individuals from the wild is neither advisable nor feasible. In other words, protocols must be optimized to obtain the maximum amount of data for a single specimen, thereby reducing the need to unnecessarily replicate sampling efforts.
Here, this protocol focuses specifically on standardized methods for the collection and sampling of bat tissue for genomic and transcriptomic sequencing and protein analyses. Its top priority is to ensure that bat tissues are collected ethically and responsibly, ranging from the permitting process for the collection, exportation of tissues to the minimization of stress to the animal, and long-term storage conditions. Elaborate dissections have been developed with the aim of future-proofing the usefulness of different materials collected. This manuscript provides a step-by-step guide to collect bats in a humane way that is intended to minimize impact on populations and maximize scientific value. While the focus of this protocol is specifically for use in bats, many of the steps are relevant to other vertebrate taxa, especially mammals.
Tissue collection overview 
The procedure for tissue collection, including the temperature for storage and the choice of preserving agent, will be determined by the nature of any downstream analyses planned. However, it is strongly recommended that, when possible, tissue is collected under a range of methods to maximize its future utility even if no specific analysis is planned. In general, tissue is collected and preserved for subsequent analyses of either nucleic acids (DNA and RNA), or protein. For each of these applications, tissue can be optimally preserved by directly flash freezing in liquid nitrogen (LN2). However, immediate immersion in LN2 is not always possible in the field. As technology advances, resources such as specialized vials to store DNA and RNA at ambient temperatures are becoming more readily available. While we have not validated all such materials in this protocol, we encourage other researchers to comparatively analyze the performance of new materials relative to what we present here. We do provide methods to ideally preserve tissue for different applications in situations where LN2 cannot be accessed, e.g., when LN2 transport is not possible due to site access via small plane in the Amazon. In addition, we provide a method for collecting tissue from which live cells can be grown and propagated. Below we outline key considerations for collecting material for each of these respective purposes and an overview of collection methods is given in Table 1.
Tissue for DNA 
For all harvested tissue collected, the storage media will determine if it can be used for either standard or high molecular weight (HMW) DNA extraction. HMW is required for long-read sequencing and currently required to generate chromosome level genome assemblies, or a &#8220;platinum standard&#8221; genome. Low molecular weight (LMW) DNA can be extracted from flash frozen, AllProtect (henceforth referred to as &#8220;tissue stabilizing solution&#8221;), or even RNAlater (henceforth &#8220;RNA stabilizing solution&#8221;)-preserved samples (although flash frozen samples remain optimal). DNA isolated by standard laboratory methods (e.g., silica gel membrane spin columns, phenol-chloroform), may still yield DNA fragments of up to ~20 kilobases (kb). Therefore, provided there is sufficient yield, this form of isolated DNA may be used for single insert size library preparation, in which the insert size is often ~500 base-pairs (bp), and short sequence reads of ~100 bp are generated12. This DNA is particularly useful for &#8220;resequencing&#8221; projects or studies in which full-length chromosomal data is not required. HMW DNA (10-150 kb) is more challenging and can only be reliably obtained using tissue that has been rapidly flash frozen in LN2 following harvest and maintained at a maximum of -80 &#176;C until extraction.
Low molecular weight or fragmented DNA is often sufficient for targeted approaches, including gene amplification via PCR and short-read sequencing13. PCR-based investigations using LMW DNA that target only one or a few genes have been highly informative in understanding adaptation and the molecular evolution of bat sensory biology6,14, physiology15, phylogenetics5,16, and conservation17,18. Successful targeted sequence recapture of low molecular weight and fragmented DNA has also been demonstrated for numerous vertebrate groups, including bats19. These methods are often cost-effective and minimally invasive to the bat, as fecal samples and non-lethal tissue sampling via buccal swabs or wing biopsy punches are also common ways to obtain DNA for low molecular weight analyses20,21.
However, the quality depends heavily on the type of media in which the sample is stored22. After systematic and quantitative comparisons of buccal swabs and biopsy punches, wing biopsy punches have been shown to yield consistently higher levels of DNA and were less stressful to the bat during collection22. These comparisons also showed that the best results were obtained when the wing punch was preserved in indicator silica (i.e., a type of desiccant made of silica gel beads that changes color when moisture is observed) rather than in other popular storage media such as ethanol or DMSO22; although, other storage media including tissue stabilization solution were not examined. Wing punches can also be used to grow fibroblast cells in culture, such as in Kacprzyk et al.23 and as described below (see section 6). For these methods, the wing or uropatagium should be extended gently, and a clean biopsy punch, typically 3 mm in diameter, should be used to obtain the sample. This approach appears to cause no lasting damage, with scars healing over within weeks in most cases24.
HMW DNA (10-150 kb) is more challenging and is currently only reliably obtained using tissue that has been rapidly flash frozen in LN2 following harvest and maintained at a maximum of -80 &#176;C until extraction. HMW DNA (10-150 kb) is crucial for long-read DNA sequencing and therefore for de novo genome assembly. Indeed, while most commercial kits can be used to isolate some standard HMW DNA, the resulting molecule sizes often do not meet the requirements of third generation sequencing technologies [e.g., those launched by companies such as Pacific Biosciences (PacBio), Oxford Nanopore Technologies, and 10x Genomics, or through assembly methods offered by Bionano Genomics or Dovetail Genomics]. As such, there is a new demand for &#8220;ultra HMW&#8221; DNA (&#62;150 kb). When obtaining ultra HMW DNA from bats, fresh samples of liver, brain, or muscle are all suitable, but these must be immediately flash frozen in LN2 without any storage buffer or cryoprotectant. A full description of these steps is beyond the scope of this paper but are available elsewhere25.
Tissue for RNA 
RNA is a single-stranded molecule that is less stable than DNA. Although there are many forms of RNA, -omics analyses tend to focus on mRNA (messenger RNA) and small RNAs (e.g., microRNAs). Following transcription, the mRNA is spliced to form a mature transcript that contains no introns and represents the coding portion of genes/genomes. Coding genes account for a tiny fraction of the genome size (1%-2%), making targeting mRNA a cost-effective means of obtaining sequence data for genes. MicroRNAs are a class of RNAs that regulate the process of translation of mRNA into proteins and are thus important regulatory effectors. RNA transcripts can be sequenced individually, or more commonly for -omics analyses26,27,28,29,30, as part of a transcriptome; that is, the total of all RNA transcripts present in a given sample.
Sequencing can be performed following several methods (i.e., via short-read RNA-seq or long-read whole isoform Seq), allowing analysis of both RNA abundance and isoform usage. As the quantity and diversity of mRNA transcripts varies among cells and tissues, RNA sequencing makes it possible to study and compare gene expression and regulation across samples. Interest in sequencing small RNAs and whole isoform sequencing is growing, as these methods are becoming increasingly more biologically informative. Preparation of tissue samples to sequence different classes of RNA can be performed in the same way as presented in this manuscript, with only the subsequent extraction methods differing31,32. Finally, because transcriptomes offer a high coverage subset of the protein-coding genome, the assembled dataset may be useful in genome assembly and annotation, making collection of RNA-seq data across a range of different tissues an important component of the Bat1K initiative.
In contrast to DNA, RNA is chemically unstable and also targeted by RNase enzymes, which are ubiquitously present in tissue lysates as a defensive strategy against RNA-based viruses. For these reasons, the RNA fraction in cells and tissues begins to degrade shortly after the point of sampling and/or euthanasia. Preserving the RNA therefore requires steps to prevent its degradation. This typically involves preserving freshly collected tissue at 4 &#176;C in a stabilizing agent such as RNA stabilizing solution to inactivate the RNases naturally present in tissues, followed by freezing for longer term storage. As a preferred alternative, tissue can be flash frozen in LN2; although as noted above, transporting LN2 into the field and maintaining levels to prevent the tissue thawing can be logistically challenging.
Tissue for protein 
Protein composition and relative abundance vary among cells and tissues in a similar way to what was discussed for RNA; however, proteins are on average more stable than RNA. Protein identification using proteomics typically matches a fraction of, and not the whole, protein sequence, but it can supply information on expression across tissues and characterize pathogens present. As many protein sequences are conserved across mammals, bat samples for proteomics can easily be contaminated with conserved human proteins, requiring sterile protocols (e.g., gloves, forceps) during collection. While flash-freezing in LN2 is the best way to prevent the degradation of proteins, use of dry ice, -20 &#730;C freezers, and even ice are suitable if there are no other means. As temperatures increase, the risk of differential protein breakdown also rises. Stabilizing agents such as tissue stabilization solution are effective in preserving the protein fraction of tissues at room temperature and are suitable for short-term preservation (up to one week) when flash-freezing is not viable.
The enzymatic profile of a given tissue directly influences the preservation of protein therein. Tissues with low enzymatic activity such as muscle can preserve protein profiles even at the higher temperatures in a household freezer. By contrast, liver tissue is enzymatically reactive, and its proteins have higher probabilities of degrading during preparation. The growing number of protocols for obtaining human proteomic profiles from formalin-fixed paraffin-embedded (FFPE) samples suggests that paraformaldehyde fixing of tissues holds promise for low-cost protein preservation when freezing upon collection is not feasible33,34. Although highly dependent on preservation time and condition, proteins have been identified via immunohistochemistry from formalin-fixed, ethanol-preserved bat specimens35. This approach is not scalable to proteomic-level sampling but highlights the potential for formalin-fixed bat tissues to yield protein profiles when flash-freezing is unavailable and other stabilizing agents are too costly.
Tissue for cell culture 
Sampling tissue and flash-freezing offers a finite amount of material to be used, and once the material is used, it is no longer available. Alternatively, cell cultures provide live cells that can be immediately used or preserved for future studies. Cultures also facilitate expansion of cells to increase yield when tissue samples are small. It is particularly useful in cases where tissue collection is limited, such as experiments with rare species in which non-lethal sampling is essential and therefore has wide implications for conservation. Described is a protocol in which cell culture is possible via non-lethal sampling of wing membrane tissue, but culturing is possible with multiple tissue types36,37. The protocol provided here selects for adherent cells. The combination of source tissue and growth media used makes this protocol suitable to select and grow fibroblasts, but if desired, alternate protocols can be used to select for other cell types. In the context of the Bat1K project, it is predicted that for rare and threatened species, non-lethal sampling of wing membranes and expansion of samples via culturing is essential to generate the volume of DNA needed for the multiple technologies employed5.
Bat capture 
All people handling bats should be trained by a bat-competent researcher and vaccinated against rabies with a series of pre-exposure injections. If bitten, a further series of post-exposure injections is still necessary. Standard methods for capturing bats include mist nets (Figure 1) and harp traps (Figure 2). Mist nets are most commonly used and ideal for areas with low to moderate activity, as they require the most care for minimizing bat distress. Small bats are particularly vulnerable and can die from stress if not tended to quickly. Frequent net inspections minimize bat injury and mortality as well as damage to the mist net. This detail is important because proper tissue collection requires the tissue to be fresh, and improper attention to the mist nets in bats can lead to unnecessary mortality or premature mortality before the researcher can properly process samples. Because several bats can rest in a harp trap with minimal distress, this approach is ideal for areas with high bat activity, such as near a cave or large roost. Detailed instructions for proper bat capture and data processing for collection of morphological and demographic information are available in the supplemental methods.

<table>
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf Safelock tubes</td>
			<td>Fischer Scientific</td>
			<td>10509691</td>
			<td>1.5 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>62,554,002</td>
			<td>15 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>2 mL cryovials</td>
			<td>Thomas Scientific</td>
			<td>1154P75</td>
			<td>cryogenic vials for tissues to be stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>3-mm biopsy punch</td>
			<td>Medline</td>
			<td>MIL3332</td>
			<td>wing biopsy punch for cell culture</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Greiner</td>
			<td>83,392</td>
			<td>Culture vessle</td>
		</tr>
		<tr>
			<td>Allprotect Tissue Reagent</td>
			<td>Qiagen</td>
			<td>76405</td>
			<td>for fecal samples; tissue stabilizing solution</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC10/TC20 cell counter, dual chamber</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
			<td>Chambers for the count cells using the automated cell counter TC20 by Bio-Rad</td>
		</tr>
		<tr>
			<td>collagenase IV</td>
			<td>Stemcell Technologies</td>
			<td>7909</td>
			<td>For dissociation of primary cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
			<td>Prevents crystalization of water during freezing of the cells.</td>
		</tr>
		<tr>
			<td>DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher</td>
			<td>12491-015</td>
			<td>culture media</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td>Balanced salt solution used for washing cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Fischer Scientific</td>
			<td>10270106</td>
			<td>Serum-supplement for in vitro cell culture of eukaryotic cells</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate salt</td>
			<td>Sigma Aldrich</td>
			<td>G1264-250MG</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0050</td>
			<td>Freezing container which provides 1&#176;C/min cooling rate</td>
		</tr>
		<tr>
			<td>PARAFILM</td>
			<td>Sigma</td>
			<td>P7793</td>
			<td>Wrapping tubes etc for sealing</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (pen-strep), 100x</td>
			<td>Invitrogen</td>
			<td>15140130</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) 10x, pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td>salt buffer used for washes and storage of bone tissue; dilute to 10X using de-ionized water</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermo Fisher</td>
			<td>AM7021</td>
			<td>RNA stabilizing solution</td>
		</tr>
		<tr>
			<td>RNAse away</td>
			<td>Genetech</td>
			<td>83931-250mL</td>
			<td>breaks down enzymes that lead to RNA degradation</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Fisher Scientific</td>
			<td>7631-86-9</td>
			<td>dessicant agent</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td>Cell stain used to assess cell viability</td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4049</td>
			<td>For dissociation of cells during splitting</td>
		</tr>
	</tbody>
</table>

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.

DNA 
For standard low molecular weight (LMW) analyses, DNA was extracted from three neotropical bat species. Tissue samples were collected in the field from the Dominican Republic and Costa Rica, following the protocols described in this paper. Following dissection, small pieces (&#60;0.5 cm at the thinnest section) of mixed tissues (brain, liver, and intestine) were placed in RNA stabilizing solution, flash frozen, then stored at -80 &#176;C. Extractions were carrie...

Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

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

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 ...

tissue-collection-of-bats-for-omics-analyses-and-primary-cell-culture

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.1K Views.  Yale University. 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.

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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...