We thank Vincent Schram from the NICHD microscopy and imaging core for assisting in the confocal image acquisition, and Tsg-Hui Chang for invaluable help with colony management and mice care. This research was supported by the Intramural Research Program of the NINDS, NIH, Bethesda, MD, to K.J.S. A.B. was supported by the Intramural Research Program of the NINDS, NIH, and by a Robert Wenthold Postdoctoral Fellowship from the intramural research program of the NIDCD.

The animal care and experimental procedures were performed following the guidelines for the Care and Use of Laboratory Animals, which were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke (Animal protocol #1336 to KJS).
1. Mice
<ol>
	<li>Set a couple of breeding pairs of C57BL/6J wild-type to breed in the animal facility to control the date of birth of the litters and keep track of the age of the pups.</li>
</ol>
<p class="jove_titl...

This protocol describes how to perform uptake experiments in murine organ of Corti explants with 3 kDa dextran Texas Red. The goal of this method is to test whether molecules larger than others previously tested were also able to specifically label auditory hair cells and permeate through the MET channel. Similar experimental protocols have been previously used to evaluate the permeability of hair cells to other fluorescent dyes such as FM1-43 (0.56 kDa)12,19<sup...

The hair cells of the inner ear are the sensory cells that detect sound and covert the mechanically stimuli in electrical signals, which are ultimately interpreted by our brain. These cells have a staircase-shaped bundle of three rows of actin-based filaments, known as stereocilia, which protrude from their apical region1,2. The mechanical stimuli deflect the stereocilia filaments toward the longest row and trigger the opening of the mechanotransduction (MET) channels3. The opening of the MET channels leads to an influx of cations that depolarizes the cell and consequently signals the release of synapse vesicles at the basal region of the hair cell.
The biophysical properties of the MET channel essential for hearing have been extensively characterized. Among other properties, these channels are cationic selective and have a relatively large conductance (150&#8211;300 pS in low Ca2+)4,5,6,7,8,9,10. Remarkably, large fluorescent molecules such as FM1-43 and Texas Red-labeled aminoglycosides are permeant blockers of the MET channel, resulting in their accumulation in the hair cell body that can be visualized using fluorescence microscopy11,12,13,14. Conversely, the molecular identity and the structure of the MET channel and its permeation pathway have remained elusive. Increasing experimental evidence indicates that the transmembrane-like channel protein 1 (TMC1) is a component of the MET channel in mature hair cells15,16,17,18,19. Mutations in the transmembrane-like channel 1 (TMC1) alter the MET channel properties19,20,21,22 and cause deafness. In addition, TMC1 localizes to the site of the MET channel18,23 and interacts with the tip-link responsible for transmitting the mechanical force to the MET channel24,25. Furthermore, recent bioinformatics analysis has identified the TMC proteins as evolutionary related to the mechanosensitive channels TMEM63/OSCA proteins and the TMEM16 proteins, a family of calcium-activated chloride channels and lipid scramblases26,27,28. A structural model of TMC1 based on the relationship between these proteins revealed the presence of a large cavity at the protein-lipid interface27. This cavity harbors the two TMC1 mutations that cause autosomal dominant hearing loss (DFNA36)27,29,30,31,32, and selective modification of cysteine mutants for residues in the cavity alter MET channel properties28, indicating that it could function as the permeation pathway of the MET channel. The large size of this predicted cavity in TMC proteins could explain the ability of large molecules to permeate the MET channel. To test the prediction that the MET channel contains an unusually large permeation pathway and to push the limits of the size of the cavity observed in TMC1, we developed a protocol to perform uptake experiments in the organ of Corti explants with a larger molecule, 3 kDa dextran fluorescently labeled with Texas Red.
Dextran is a complex branched polysaccharide composed of many D-glucose molecules bound by alpha-1,6 glycosidic linkages. Its high solubility in water, low cell toxicity, and bioinertity make it a versatile tool to study several cellular processes. In addition, dextran is available in a wide range of sizes and fluorescently labeled with fluorophores of several colors. Fluorescently labeled dextrans are commonly used in cell and tissue permeability research33,34, to study endocytosis in multiple cellular systems35,36, and for neural tracing37,38. In the auditory field, dextran molecules have also been used to assess the disruption of the cell-cell junction and loss of the auditory sensory epithelium integrity after exposure to intense noise in the chinchilla organ of Corti39,40.
In this work, we exploited the properties of some of the smallest (3 and 10 kDa) fluorescent dextrans to perform uptake experiments in murine inner ear hair cells and explore the size of the permeation pathway of the inner ear hair cell MET channel. In addition, we used a laser-scanning confocal microscope (LSM) 880 equipped with an Airyscan detector to visualize and localize fluorescent dextran at the stereocilia and the cell body of auditory hair cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>#1.5 glass coverslips 18mm</td>
			<td>Warner Instruments</td>
			<td>64-0714</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>ThermoFisher</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Phalloidin</td>
			<td>ThermoFisher</td>
			<td>A12381</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective</td>
			<td>Carl Zeiss</td>
			<td>420780-9970-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Amiloride hydrochloride</td>
			<td>EMD MILLIPORE</td>
			<td>129876</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchwaver 3-dimensional Rocker</td>
			<td>Benchmarks scientific</td>
			<td>B3D5000</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild-type mice</td>
			<td>strain 000664</td>
			<td>The Jackson Laboratory</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell impermeant BAPTA tetrapotassium salt</td>
			<td>ThermoFisher</td>
			<td>B1204</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Fluorescein, 10,000 MW, Anionic, Lysine Fixable</td>
			<td>ThermoFisher</td>
			<td>D1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Texas Red, 10,000 MW, Lysine Fixable</td>
			<td>ThermoFisher</td>
			<td>D1863</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Texas Red, 3000 MW, Lysine Fixable</td>
			<td>ThermoFisher</td>
			<td>D3328</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Aqueous Solution EM Grade</td>
			<td>Electron microscopy science</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>ThermoFisher</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>ThermoFisher</td>
			<td>14170</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J or FIJI</td>
			<td>NIH</td>
			<td><a href="http://fiji.sc/" target="_blank">http://fiji.sc/</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol 518F oil immersion media</td>
			<td>Carl Zeiss</td>
			<td>444970-9000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium, GlutaMAX Supplement</td>
			<td>ThermoFisher</td>
			<td>31415029</td>
			<td></td>
		</tr>
		<tr>
			<td>neomycin trisulfate salt hydrate</td>
			<td>Sigma</td>
			<td>N6386</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4</td>
			<td>ThermoFisher</td>
			<td>70011069</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-CF405M</td>
			<td>Biotium</td>
			<td>00034</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond antifade mounting media</td>
			<td>ThermoFisher</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slide</td>
			<td>Fisherbrand</td>
			<td>22-037-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Black 2.3 SP1 software</td>
			<td>Carl Zeiss</td>
			<td><a href="https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html" target="_blank">https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

dextran labeling and uptake in live and functional murine cochlear hair cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

We observed robust and specific labeling of hair cells after 2h incubation of organ of Corti explants from wild-type postnatal-day-6 (P6) mice with 3 kDa dextran fluorescently labeled with Texas Red (dextran-TR) (Figure 2A-B). Dextran labeling was observed in both inner and outer hair cells (IHC and OHC) at the basal, middle, and apical regions of the organ of Corti (Figure 2B).
Fluorescently labeled ...

Watch this Scientific Journal Video about Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells at JoVE.com

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

The overall goal of this protocol is to study the uptake of regular dextran fluorescently labeled with Texas Red in live hair cells with functional mechanotransduction channels. The main advantage of this technique is that the three kilodalton Texas Red dextran can be used to assess mechanotransduction channel function in live hair cells. To harvest the cochlea, place the skull of a postnatal day six mouse into a 60 millimeter culture dish containing Leibovitz's L-15 medium and use surgical scissors to make a superficial cut from the anterior end to the posterior end of the skull and across the external auditory canals.Fold the skin toward the nose to expose the cranium and make an incision from the back to the front of the skull and across the eye line. Separate the skull into two halves and use a small spatula to remove the brain. When the temporal bones can be observed, use small scissors to cut around these bones to excise the tissue.Submerge the temporal bones in the 35 millimeter dish containing fresh L-15 medium and place the dish under a stereo microscope equipped with a wide field eyepiece and an external alternating current halogen light source. Use forceps to remove the surrounding cochlear tissue, semi-circular canals and vestibular organs. Once the cochlea has been dissected and placed in a new dish with fresh L-15 media, use surgical forceps to make one puncture wound on the round window and one puncture at the apical cochlear region.When all of the cochleae have been harvested, place the tissue into a well of a nine-well glass depression plate containing 200 microliters of fresh L-15 medium. For dextran labeling of the isolated tissues, replace the medium from each sample well with 200 microliters of fresh L-15 medium supplemented with two milligrams per milliliter of the fluorescence conjugated dextran of interest. Incubate the samples for two hours at room temperature and 25 rotations per minute in a three-dimensional shaker at a tilted angle of 25 degrees protected from light.At the end of the incubation, wash the tissues one time with medium and one time with HBSS for two minutes per wash. After the second wash, fix the specimens in 4%paraformaldehyde for 30 minutes at room temperature followed by two quick and gentle washes in HBSS. After the second wash, use fine tip forceps to remove the cochlear bone, the spiral ligament, and the tectorial membrane and wash the isolated organ of Corti in HBSS.Take extra care when removing the transparent tectorial membrane since the tissue on the hair cells can be easily damaged during this step. To label the F-actin, permeabilize the samples in 0.5%Triton X-100 in PBS supplemented with 100 to 200 dilution of fluorescently labeled phalloidin for 30 minutes at room temperature protected from light. At the end of the incubation, wash the tissues two to three times in fresh HBSS per wash as demonstrated followed by a single wash in PBS.Use an appropriate mounting medium to mount the organ of Corti tissues onto glass microscope slides hair stereocilia sides up and image the samples on a fluorescent confocal microscope using the 63X oil immersion objective. After a two-hour incubation with three kilodalton dextran fluorescently conjugated with Texas Red, organ of Corti explant from wild type postnatal day six mice demonstrate a robust and specific labeling of both inner and outer hair cells. Imaging of the hair cell stereocilia and cell bodies reveals that only those cells that have incorporated three kilodalton dextran Texas Red in their cell body also demonstrated fluorescent labeling of their stereocilia.Importantly, a uniform fluorescent signal is observed along the stereocilia with enrichment at the tips of the shorter stereocilia rows where the mechanotransduction channel is located. Vesicle-like structures in the cell body of the hair cells and the neighboring supporting cells are also observed suggesting that dextran Texas Red can also be taken up by endocytosis. Larger 10 kilodalton dextrans also produced a vesicle-like pattern in the cell body of hair cells and supporting cells.Notably, the presence of a calcium chelator or mechanotransduction channel blockers prevents stereocilia labeling and the uptake of three kilodalton dextran Texas Red in hair cells. The vesicle-like pattern is still observed in the presence of mechanotransduction channel blockade, however, indicating that this pattern of uptake is independent of functional MET channels. It is very important to dissect the organ of Corti tissue carefully and to confirm the proper orientation of the hair cells before mounting the tissue for imaging.After dextran labeling of live and functional hair cells, immunostaining of the fixed tissue can be performed to identify specific cell types of proteins of interest.

dextran-labeling-uptake-live-functional-murine-cochlear-hair

Research

JoVE Journal

Bioengineering

7.2K ビュー.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. このプロトコルの全体的な目標は、機能的メカノトランスダクションチャネルを有する生毛細胞において、テキサスレッドで蛍光標識された規則的なデキストランの取り込みについて研究することです。この技術の主な利点は、3キロダルトンテキサスレッドデキストランが生きた毛細胞のメカノトランスダクションチャネル機能を評価するために使用できることです。?...

ビデオ: Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of...

live fluorescence, inverse imaging of cell ruffling, and macropinocytosis

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.<br...

isolation and culture of primary cochlear hair cells from neonatal mice

Author Spotlight: Advancements in Cultivating Mouse Hair Cells for Auditory Research 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged...

stereocilia bundle imaging with nanoscale resolution in live mammalian auditory hair cells

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair...

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic...

labeling of extracellular vesicles for monitoring migration and uptake in cartilage explants

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian...

development and functional characterization of murine tolerogenic dendritic cells

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic...

neonatal murine cochlear explant technique as an in vitro screening tool in hearing research

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

culture of embryonic mouse cochlear explants and gene transfer by electroporation

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear.  We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

cochlear surface preparation in the adult mouse

Cochlear Surface Preparation in the Adult Mouse

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse...

labeling and imaging cells in the zebrafish hindbrain

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Efficient In Vitro Cultivation of Mouse Cochlear Hair Cells

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transsynaptic tracing from peripheral targets with pseudorabies virus followed by cholera toxin and biotinylated dextran amines double labeling

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of...

murine colitis modeling using dextran sulfate sodium (dss)

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of...

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

optical monitoring of living nerve terminal labeling in hair follicle lanceolate endings of the ex vivo mouse ear skin

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

cochlear implant surgery and electrically-evoked auditory brainstem response recordings in c57bl/6 mice

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem...

isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking...

isolation and culture of neural crest stem cells from human hair follicles

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...