This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

The following protocol complies with the Thomas Jefferson University Institutional Animal Care and Use Committee guidelines and with the ARVO Statement for the Use of Animals in Vision Research.
1. Setup and Preparation of Lenses for Ex&#160;Vivo Wound Culture
<ol>
	<li>Place three 100 mm petri dishes in a sterile, laminar flow hood. Fill two of the petri dishes halfway with Tris/Dextrose buffer (TD buffer; 140 mM NaCl, 5 mM KCl, 0.7 mM Na2PO4, 5 mM D-glucose, 8.25 mM Tris Base, pH to 7.4 with HCl) at RT, leaving the third empty. Pre-warm culture media (Media 199 supplemented with, 1% L-glutamine and 1% penicillin/streptomycin) to 37oC. 
	Note: The standard wound healing culture media is serum-free, as occurs in vivo; however, the wound-repair cultures can be grown successfully in defined media conditions that include serum or other factors.</li>
	<li>Remove fertile embryonic day 15 white leghorn chick egg from incubator (held at 37.7&#176; C with gentle rocking)</li>
	<li>Place selected egg in the laminar flow hood and clean outside of shell with 70% ethanol from a wash bottle. Conduct all procedures below under aseptic conditions in the laminar flow hood, using sterile solutions and instruments.</li>
	<li>Crack egg and place contents into the empty 100 mm petri dish. Decapitate embryo using standard forceps and fine scissors. Place the chick embryo head in a petri dish containing TD buffer and properly dispose of the remainder of the embryo. Optionally keep the chick embryo heads in TD buffer for a short period of time, no longer than 15 min.</li>
	<li>Place the chick embryo head on a petri dish lid. Using high precision forceps, remove the lens along with its attached vitreous humor from the eye in the following sequence. Pinch the back of the eye with the forceps to create a small opening in the back of the eye.</li>
	<li>Then, grasp the vitreous humor with forceps and gently tug on the vitreous with a rolling motion, the vitreous with the lens attached will be dislodged from the eye. Place lens/vitreous in the remaining petri dish containing TD buffer. Allow the lenses to remain in TD buffer for no longer than 30 min.</li>
	<li>Move the lens to a new petri dish lid under a dissecting microscope. From this point on perform all steps under a dissecting microscope. With high precision forceps carefully brush away any ciliary body (pigmented cells) that were dislodged with the lens using the edge of the forceps, taking caution not to damage the lens tissue. 
	Note: Removing the ciliary body ensures that cell types that are not endogenous to the lens are not included in the wound repair culture.</li>
	<li>Separate the lens from the vitreous humor with high precision forceps by pinching off the vitreous body from its association with the posterior lens capsule.</li>
	<li>Using high precision forceps transfer the lens into a small drop of TD buffer (about 200 &#181;l) in a 35mm tissue culture dish.</li>
</ol>
2. Performing Mock Cataract Surgery
<ol>
	<li>Orient the lens in the drop of TD buffer in the 35 mm dish with the anterior aspect of the lens facing up. 
	Note: The anterior of the lens is easily identified by the presence of a dense ring in the tissue that notes the border between the anterior and equatorial region of the lens epithelium. In contrast, there is an absence of markings on the posterior of the lens capsule to which the lens fiber cells are attached.</li>
	<li>Using two high precision forceps make a small incision (approximately 850&#181;m) in the center of the anterior lens capsule, the thick basement membrane that surrounds the lens tissue, and its associated anterior lens epithelium, by grasping the tissue with one forceps in each hand and gently tugging in opposing directions.</li>
	<li>Remove the fiber cell mass, which makes up the bulk of the lens tissue, dislodging it from its attachments to the lens epithelium and surrounding lens capsule by hydro-elution (an approach used in classic cataract surgery, modeled in Figure&#160;1A).</li>
	<li>Fill a 1 ml syringe with a 27.5 G needle tip with 300 &#181;l of TD buffer. Insert the needle tip into the incision made in the anterior lens capsule, and about halfway into the lens.</li>
	<li>Gently depress the syringe injecting the TD buffer into the lens fiber cell mass. Inject between 50 and 200 &#181;l TD, and never more than 300 &#181;l. Observe the fiber cell mass loosening itself from the epithelium and lens capsule.</li>
	<li>Using high precision forceps, remove the loosened fiber cell mass from the lens through the anterior incision site. 
	Note: This procedure leaves the posterior lens basement membrane capsule to which the fiber cells had been attached denuded of cells, and an injured lens epithelium just adjacent to this site.</li>
</ol>
3. Preparing the Wounded Lens for Ex Vivo Culture
<ol>
	<li>Flatten the lens capsular bag that results from the cataract surgery described above on the culture dish, cell side up, by making five cuts in the anterior aspect of the capsular bag.</li>
	<li>Cut perpendicular to the original incision site through to the equator of the lens. Flatten the resultant five &#8220;flaps&#8221; of lens capsule with attached epithelium on the culture dish capsule side down, cell side up. Note the ex vivo wounded lens now should take on a star or flowerlike shape (see Figure&#160;1B).</li>
	<li>To secure the capsule to the dish, press softly down with the forceps at each point of the star. This will make a small indentation at the five most outside tips of the explant and result in sustained attachment to the dish. 
	Note: It is possible to damage the capsule during this procedure and so it is important to secure the capsule to the dish as close to the tips of the flaps as possible, as well as to make the least amount of securing points as possible (generally two, maximum of three per flap).</li>
	<li>Remove the TD buffer from the 35mm dish and replace it with 1.5 ml&#160;of pre-warmed media. Cover the 35mm dish with its lid and place in the incubator (37&#176;C, 5% CO2).</li>
</ol>
4. Separation of the Central Migration Zone (CMZ), where Re-epithelialization of the Wounded Area of the Posterior Capsule Occurs, from the Original Attachment Zone (OAZ) of Lens Epithelial Cells, for Quantitative Analysis.
Note: The cells begin to move into the CMZ region immediately in response to injury. By day one in culture enough cells are migrating across the CMZ for molecular and biochemical analysis, following separation of the CMZ and the OAZ by micro-dissection23. This protocol involves removal of one flap (OAZ) at a time from the wounded area of the capsule.
<ol>
	<li>Observe a demarcation, clearly visible under the dissecting microscope, between the OAZ and CMZ (see Figure&#160;2A). Using two high precision forceps, grasp with both forceps at the edge of the OAZ/CMZ line, one just adjacent to the other, on either side of the line (see Figure&#160;2A, B, arrow).</li>
	<li>Using one hand/one forceps on the CMZ side continue to hold onto the wounded culture, while with the other hand/forceps, gently pull the OAZ along the OAZ/CMZ line. The CMZ easily separates from the OAZ along this line. Continue along this line around the entire culture until the two regions are completely separated.</li>
	<li>Study the separated OAZ and CMZ fractions for molecular analysis such as RNA-Seq 24 or biochemical analysis such as western blot or co-immunoprecipitation 23,25.</li>
</ol>

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The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

The clinically relevant, mock cataract surgery, ex vivo epithelial wound healing model described here was developed to provide a tool for investigating the mechanisms that regulate repair of epithelial tissues in response to an injury. Key features that were aimed for in creating this model included 1) providing conditions that closely replicated the in vivo response to wounding in a culture setting, 2) ease of modulating the regulatory elements of repair, and 3) ability to image the repair process, in its entirety, in real time. The challenge, therefore, was to create a culture model in which it was possible to study, and manipulate, epithelial wound repair in the cells&#8217; native microenvironment. The availability of this wound-repair model opens new possibilities for identifying the endogenous signaling cues from matrix proteins, cytokines and chemokines that regulate the repair process. In addition, the model is ideal for examining how an epithelium is able to move as a collective sheet to re-epithelialize the wound area 2,3, and for determining the lineage of mesenchymal leader cells at the wound edge that function in directing the collective migration of the injured epithelium 4. This model also provides a platform with which to identify therapeutics that could promote effective wound healing and prevent aberrant wound repair5.
There are already a number of available wound-repair models, both in culture and in vivo, which have provided most of what is known about the wound repair process today. In animal injury models, such as cornea 6-12 and skin13-17, there is the opportunity to study the response of the tissue to wounding in the context of all the repair mediators that could be involved in the process, including contributions from the vasculature and nervous system. However, there are limitations to manipulating the experimental conditions in vivo, and it is not yet possible to conduct imaging studies of the repair response in vivo, continuously over time. In contrast, most in vitro wound-repair culture models, such as the scratch wound, can be easily manipulated and followed over time but lack the environmental context of studying wound healing in the in vivo tissue. While ex vivo models offer the advantage of studying the injury repair process continuously over time in the context of the cells&#8217; microenvironment coupled with the ability to modulate the molecular regulators of repair at any time point in the process, there are few models that fit these parameters.
Here is described a procedure to generate highly reproducible ex vivo epithelial wound healing cultures that reproduce an epithelial tissue&#8217;s response to a physiological wounding. Using the chick embryo lens as a tissue source, an ex vivo mock cataract surgery is performed. The lens is an ideal tissue to use for these studies since it is self-contained within a thick basement membrane capsule, avascular, not innervated, and free of any associated stroma18,19. In the human disease, cataract surgery addresses vision loss due to opacification of the lens, and involves removal of the lens fiber cell mass, which comprises the bulk of the lens. Following cataract surgery vision is restored through the insertion of an artificial intraocular lens. The cataract surgery procedure, through removal of fiber cells, induces an injury response in the adjacent lens epithelium, which responds by re-epithelialization of the posterior area of the lens capsule that had been occupied by the fiber cells. In cataract surgery, as in most wound repair responses, there sometimes occurs an aberrant fibrotic outcome to the wound healing response, associated with the emergence of myofibroblasts, which in the lens is known as Posterior Capsule Opacification20-22. To generate the cataract surgery wound healing model, a cataract surgery procedure is mimicked in lenses removed from the chick embryo eye to produce a physiological injury. Microsurgical removal of lens fiber cells results in a very consistent circular wound area surrounded by the lens epithelial cells. This cell population remains firmly attached to the lens basement membrane capsule and is injured by the surgical procedure. The epithelial cells migrate onto the denuded area of the endogenous basement membrane to heal the wound, led by a population of vimentin-rich mesenchymal cells known in the repair process as leader cells1. With this model the response of an epithelium to injury can be easily visualized and followed with time in the context of the cells&#8217; microenvironment. The cells are readily accessible to modifications of the expression or activation of molecules expected to play a role in wound repair. A powerful feature of this model is the ability to isolate and study migration-specific changes in the framework of wound healing. The ability to prepare large numbers of aged matched ex vivo wound healing cultures for studies is another advantage of this model. Thus, this model system provides a unique opportunity to tease apart mechanisms of wound repair and test therapeutics for their effect on the wound healing process. The ex vivo mock cataract surgery model is expected to have wide applicability, providing a critical resource for studying mechanisms of injury repair.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Ex Vivo Model created to study the wound healing process in the cells&#8217; native microenvironment
To investigate mechanisms involved in regulating wound healing of an epithelium within the cells&#8217; native microenvironment, a clinically relevant ex vivo mock cataract surgery model was created. This model is created from lens tissue which offers many advantages due to its intrinsic properties: 1) the lens is a self-contained organ surrounded by a thick basement membrane ...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Research

JoVE Journal

Developmental Biology

6.5K visualizações.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

Vídeo: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...