This work was funded by an NIH grant Ey10540 to PAT.

1. Iris Cell Culture
<ol>
	<li>Collect eyeballs from 7 newts (anesthetized in 0.1% ethyl 3-aminobenzoate methane sulfonic acid prepared in PBS) and place in calcium magnesium free Hanks solution (CMF).</li>
	<li>Change gloves and work in the tissue culture hood.</li>
	<li>Sterilize eye balls in Lugol's-EtOH for 3 seconds.</li>
	<li>Wash 2 times in CMF.</li>
	<li>Transfer eyes to Hanks and begin dissection.</li>
	<li>Dissect iris-corneal complex and remove neural retina.</li>
	<li>Transfer complexes into a new dish of Hanks.</li>
	<li>Using #15 scalpel, separate complexes into dorsal and ventral halves.</li>
	<li>Remove iris fragments (ventral first) and place in a dish containing 1 ml L-15.</li>
	<li>Add 15% (150 ul) volume of dispase (7.5 units/ml concentration).</li>
	<li>Incubate at 27&deg;C for 2 hours (wrap dish with parafilm).</li>
	<li>Isolate IPE cells from stroma (Place trypsin solution at 27&deg;C).</li>
	<li>Collect IPE cells into a 1.5 ml tube.</li>
	<li>Centrifuge at 1000 rpm at RT (room temperature) for 2 minutes, remove supernatant.</li>
	<li>Wash with 1 ml Hanks and centrifuge at 1000 rpm at RT for 2 minutes, remove supernatant.</li>
	<li>Add 1 ml trypsin solution; incubate for 2 hours at 27&deg;C.</li>
	<li>Centrifuge at 1000 rpm at RT for 2 minutes, remove trypsin.</li>
	<li>Add 1 ml L-15 to wash, centrifuge at 1000 rpm for 2 minutes.</li>
	<li>Add 800 ul L-15, pipet slowly up and down ~ 10 times (more than 12 times reduces adherence).</li>
	<li>Plate the IPE cells on a 24 well collagen coated plate and incubate at 27&deg;C for 2 weeks (Usually, 4 dorsal and 4 ventral wells are obtained from 7 newts).</li>
	<li>At this stage cells are suitable for gene transfection or treatment with factors to determine their role in inducing or interfering with lens regeneration.</li>
</ol>
2. Aggregation of Iris Cells
<ol>
	<li>To the plates containing iris cells add 20&mu;l of dispase solution to 400 &mu;l medium, swirl gently.</li>
	<li>Incubate plates at 27&deg;C for overnight.</li>
	<li>Pipet gently up and down to dislodge cells</li>
	<li>Place cells into Eppendorf tube and spin for 2 minutes at 1000 rpm at RT.</li>
	<li>Remove medium and wash by adding 1 ml complete L-15, spin 2 minutes at 1000 rpm at RT, remove medium.</li>
	<li>Use 2000-7000 cells per aggregate. Add 200 ul L-15 per aggregate into each tube.</li>
	<li>Split cells (200 &mu;l) into new eppendorf tubes.</li>
	<li>Spin at 1000 rpm for 2 min at RT.</li>
	<li>Incubate for 48 hrs at 27&deg;C.</li>
</ol>
3. Implantation of Aggregrated Cells
<ol>
	<li>Make a slit in the cornea of newt eye and remove the lens.</li>
	<li>After lens removal, place the IPE cell aggregate just below the corneal tissue on the ventral side of the eye.</li>
	<li>The newts are kept for a month to let them regenerate the lens.</li>
</ol>
4. Embedding of Newt Eye
<ol>
	<li>Remove the newt eyes implanted with the aggregate and place it in phosphate buffered saline (PBS).</li>
	<li>Fix in 4% paraformaldehyde at 4&deg;C for at least 4 hrs or overnight.</li>
	<li>Wash in PBS, 4&deg;C for 30 minutes.</li>
	<li>Treat it with 0.85% saline: 4&deg;C, 30 minutes.</li>
	<li>Wash in Saline/Ethanol (1:1): RT for 15 minutes.</li>
	<li>Wash in 70% Ethanol: RT, 15 minutes. Repeat it once.</li>
	<li>Wash in 85% Ethanol and 95% Ethanol at RT, 30 minutes each</li>
	<li>Wash in 100% Ethanol: RT, 30 minutes. Repeat it once.</li>
	<li>Store at 4&deg;C or continue.</li>
	<li>Wash in 100% xylene: RT, 30 minutes. Repeat it once.</li>
	<li>Treat with xylene/paraffin (1:1): 60&deg;C, 45 minutes.</li>
	<li>Treat with 100% Paraffin: 60&deg;C, 20 minutes. Repeat it two more times.</li>
	<li>Embed in embedding molds.</li>
</ol>
5. Sectioning
<ol>
	<li>Prepare 15 &mu;m sections of the embedded eye using a microtome.</li>
	<li>Place the eye sections on a gelatin coated slide and leave it on a slide warmer.</li>
</ol>
6. Staining
<ol>
	<li>Place slides in xylene for 10 minutes. Repeat it once more.</li>
	<li>Hydrate in: 100%, 95%, 80%, 70%, 30% ethanol for 1 minute each</li>
	<li>Rinse in DI water for a minute.</li>
	<li>Wash in PBS for 15 minutes.</li>
	<li>Wash in PBST (0.2% Triton-X 100 in PBS) for 15 minutes.</li>
	<li>Wash in PBS for 15 minutes.</li>
	<li>Place in blocking solution (10% goat secondary antibody) for an hour.</li>
	<li>Place in primary antibody for overnight.</li>
	<li>Wash primary antibody in PBS for 15 minutes.</li>
	<li>Wash in PBST for 15 minutes.</li>
	<li>Wash in PBS for 15 minutes.</li>
	<li>Place in secondary antibody for 2 hours in dark (1:100 dilution in PBS).</li>
	<li>Wash in PBS, PBST, and PBS for 15 minutes each and then mount.</li>
	<li>Observe the slides under a fluorescence microscope.</li>
</ol>
7. Representative Results:
This procedure of culturing newt iris cells has been utilized to study the regeneration potential of the dorsal and the ventral IPE cells. Moreover, it is also possible to study specific genes that contribute towards the lens regeneration mechanism in newt eye. When the cells have been cultured for 2 weeks (Figure 1) they can be transfected by genes to examine their function in lens regeneration. Of particular interest are genes that might induce the ventral iris. Since the ventral iris cells cannot transdifferentiate to lens (Figure 2) the inductive function of a candidate gene can be studied. In the past, using this technique we have shown that when six-3was over expressed in the presence of retinoic acid lens induction was observed from the ventral iris 6. In Figure 3we can see that the ventral iris aggregate gave rise to a fully grown and differentiated lens (arrowhead), not different than the host lens from the dorsal iris (arrow).
<img src="/files/ftp_upload/2713/2713fig1.jpg" alt="Figure 1" /> 
 Figure 1. a) Dorsal iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. b) Ventral iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. Note that pigmentation persists to this stage in both dorsal and ventral iris cells.
<img src="/files/ftp_upload/2713/2713fig2.jpg" alt="Figure 2" /> 
 Figure 2. Regeneration ability of cultured IPE cells. a) Lens induction from dorsal IPE cell aggregate (arrowhead). Host lens induction from dorsal iris (arrow), di: dorsal iris, vi: ventral iris, le: lens epithelium, lf: lens fibers. b) Absence of lens induction from ventral IPE cell aggregate (arrowhead). Host lens induction from dorsal iris (arrow).
<img src="/files/ftp_upload/2713/2713fig3.jpg" alt="Figure 3" /> 
 Figure 3. Lens induction from ventral aggregate transfected with six-3 and treated with retinoic acid. Host lens induction from dorsal iris (arrow). Lens induction from ventral aggregate (arrowhead).

<ol>
	<li>Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+vertebrates.">Regeneration in vertebrates.</a> Dev Biol. 221, 273-284 (2000).</li><li>Tsonis, P. A., Madhavan, M., Tancous, E. E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newt's+eye+view+of+lens+regeneration.">A newt's eye view of lens regeneration.</a> Int. J. Dev. Biol. 48, 975-980 (2004).</li><li>Del Rio-Tsonis, K., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+regeneration+at+the+molecular+age.">Eye regeneration at the molecular age.</a> Dev. Dyn. , 226-2211 (2003).</li><li>Okamoto, M., Ito, M., Owaribe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+between+dorsal+and+ventral+iris+in+lens+producing+potency+in+normal+lens+regeneration+is+maintained+after+dissociation+and+reaggregation+of+cells+from+the+adult+newt,+Cynops+pyrrhogaster.">Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.</a> Develop Growth Differ. 40, 11-18 (1998).</li><li>Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+system+for+functional+gene+analysis+in+adult+amphibian+lens+regeneration.">Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.</a> Develop Growth Differ. 43, 361-370 (2001).</li><li>Grogg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+inhibition-driven+regulation+of+six-3+underlies+induction+of+newt+lens+regeneration.">BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.</a> Nature. 438, 858-862 (2005).</li><li>Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+Newt+Lens+Regeneration:+Specific+Control+of+Proliferation+and+Evidence+for+miRNA+Networking.">miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking.</a> PLoS One. 5, e12058-e12058 (2010).</li><li>Madhavan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14848-14853 (2006).</li><li>Maki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-type+linker+histone+B4+is+required+for+transdifferentiation+of+somatic+cells+in+vivo.">Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo.</a> FASEB J. 24, 3462-3467 (2010).</li></ol>

No conflicts of interest declared.

This protocol has established an in vitro system to study lens regeneration mechanisms in newts. Since the aggregates (either dorsal or ventral faithfully follow their in vivo behavior during regeneration this technique can alleviate the tremendous effort required for transgenesis in newts and can be used for gain of function as well as loss of function experiments 7,8,9 . Also, the aggregates or the irises as a whole can easily be treated with growth factors and examine their effects as described in this protocol. For example the role of BMP pathway has been studied using this technique 6.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lugol's solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-6146</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-4034</td>
<td>&nbsp;</td>
<tr>
<td>Ethyl 3-aminobenzoate methanesulfonic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-10521</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 1:250</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>27250018</td>
<td>&nbsp;</td>
<tr>
<td>L-15 ( Leibovitz)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4386</td>
<td>&nbsp;</td>
<tr>
<td>DNase 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5025-150KU</td>
<td>&nbsp;</td>
<tr>
<td>Kanamycin Sulfate</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>100x, 15160</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone (amphotericin B solution)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2942</td>
<td>&nbsp;</td>
<tr>
<td>24 well collagen coated plate</td>
<td>&nbsp;</td>
<td>BD biosciences</td>
<td>354408</td>
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a system for culturing iris pigment epithelial cells to study lens regeneration in newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

Watch this Scientific Journal Video about A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt at JoVE.com

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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11.7K visualizações.  University of Dayton. Em newt, a lente regenera sempre a partir da íris dorsal pela transdiferenciação das células epiteliais pigmentadas da íris (EPIs). Aqui nós descrevemos um procedimento para células de cultura dorsal e ventral newt IPE e sua implantação para o olho newt. As células são então implantados estudado por corte de tecido e imuno-histoquímica.

Vídeo: A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...