Este trabajo fue financiado por una subvención del NIH Ey10540 a PAT.

1. Iris cultivo celular <ol><li> Recoger los globos oculares de 7 tritones (anestesiados en el 0,1% el 3-aminobenzoato de ácido metano sulfónico preparado en PBS) y el lugar en el calcio, magnesio libre Hanks solución (CMF). </li><li> Cambie los guantes y trabajar en la campana de cultivo de tejidos. </li><li> Esterilizar globos oculares en Lugol-EtOH durante 3 segundos. </li><li> Lavar dos veces en el CMF. </li><li> La transferencia de los ojos de Hanks y comenzar la disección. </li><li> Diseccionar iris-corneal complejo y eliminar la retina neural. </li><li> La transferencia de los complejos en un nuevo plato de Hanks. </li><li> # 15 complejos utilizando bisturí, separada en dos mitades dorsal y ventral. </li><li> Extraer los fragmentos del iris (ventral primero) y colóquelo en un recipiente que contenga 1 ml L-15. </li><li> Añadir un 15% (150 ul) de volumen de dispasa (7,5 unidades / ml de concentración). </li><li> Se incuba a 27 ° C durante 2 horas (plato envolver con parafilm). </li><li> Aislar las células del estroma de IPE (solución Colocar tripsina a 27 ° C). </li><li> Recoger las células IPE en un tubo de 1,5 ml. </li><li> Centrifugar a 1000 rpm a temperatura ambiente (temperatura ambiente) durante 2 minutos, retire el sobrenadante. </li><li> Lavar con 1 ml Hanks y centrifugar a 1000 rpm a temperatura ambiente durante 2 minutos, retire el sobrenadante. </li><li> Añadir 1 ml de solución de tripsina, se incuba durante 2 horas a 27 ° C. </li><li> Centrifugar a 1000 rpm a temperatura ambiente durante 2 minutos, retire la tripsina. </li><li> Añadir 1 ml L-15 para lavar, centrifugar a 1000 rpm durante 2 minutos. </li><li> Añadir 800 ul L-15, pipeta lentamente hacia arriba y hacia abajo ~ 10 veces (más de 12 veces se reduce la adherencia). </li><li> Placa de las células IPE en un plato de colágeno 24 pozos cubierto y se incuba a 27 ° C durante 2 semanas (por lo general, cuatro dorsal y ventral 4 pozos se obtienen a partir de 7 tritones). </li><li> En esta etapa las células son adecuados para la transfección de genes o el tratamiento con los factores para determinar su papel en la inducción o interferir con la regeneración de la lente. </li></ol> 2. La agregación de células Iris <ol><li> A las placas que contienen las células del iris añadir 20μl de solución dispasa a 400 l medio, revuelva suavemente. </li><li> Incubar las placas a 27 ° C durante la noche. </li><li> Pipeta suavemente hacia arriba y hacia abajo para separar las células </li><li> Células de lugar en el tubo Eppendorf y girar durante 2 minutos a 1000 rpm a temperatura ambiente. </li><li> Quitar y lavar medio mediante la adición de 1 ml L-15 completa, spin 2 minutos a 1000 rpm a temperatura ambiente, quite medio. </li><li> Use 2000-7000 células por agregada. Añadir 200 ul L-15 por el agregado en cada tubo. </li><li> Las células se dividen (200 l) en los nuevos tubos eppendorf. </li><li> Giran a 1000 rpm durante 2 min a temperatura ambiente. </li><li> Incubar durante 48 horas a 27 ° C. </li></ol> 3. La implantación de células Aggregrated <ol><li> Haga un corte en la córnea del ojo de tritón y quitar la lente. </li><li> Después de extracción del cristalino, coloque el agregado de células IPE justo por debajo del tejido de la córnea en la parte ventral del ojo. </li><li> Los tritones se mantienen durante un mes para dejar que la regeneración del lente. </li></ol> 4. Incorporación de los ojos Newt <ol><li> Quitar los ojos de tritón implantado con el agregado y lo coloca en tampón fosfato salino (PBS). </li><li> Fijar en paraformaldehído al 4% a 4 ° C durante al menos 4 horas o toda la noche. </li><li> Se lavan con PBS, 4 ° C durante 30 minutos. </li><li> Tratar con 0,85% de solución salina: 4 ° C, 30 minutos. </li><li> De lavado en solución salina / etanol (1:1): RT durante 15 minutos. </li><li> De lavado en etanol al 70%: RT, de 15 minutos. Repetir una vez. </li><li> Lave en el 85% de etanol y 95% de etanol a temperatura ambiente, 30 minutos cada uno </li><li> De lavado en etanol al 100%: RT, de 30 minutos. Repetir una vez. </li><li> Almacenar a 4 ° C o continuar. </li><li> Lave en xileno al 100%: RT, de 30 minutos. Repetir una vez. </li><li> Tratar con xileno / parafina (1:1): 60 ° C, 45 minutos. </li><li> Tratar con el 100% de parafina: 60 ° C, 20 minutos. Repítelo dos veces más. </li><li> Integrar al incorporar los moldes. </li></ol> 5. Seccionamiento <ol><li> Prepare 15 secciones micras de los ojos incrustados utilizando un microtomo. </li><li> Coloque las secciones de los ojos en un portaobjetos recubiertos de gelatina y se deja en una lámina caliente. </li></ol> 6. Tinción <ol><li> Coloque los portaobjetos en xileno durante 10 minutos. Repetir una vez más. </li><li> De hidratos en: 100%, 95%, 80%, 70%, 30% de etanol durante 1 minuto cada uno </li><li> Enjuague con agua DI por un minuto. </li><li> Se lavan con PBS durante 15 minutos. </li><li> Lave en PBST (0,2% Triton X-100 en PBS) durante 15 minutos. </li><li> Se lavan con PBS durante 15 minutos. </li><li> Lugar en solución de bloqueo (10% anticuerpo de cabra secundaria) durante una hora. </li><li> Lugar en el anticuerpo primario durante la noche. </li><li> Lavar el anticuerpo primario en PBS durante 15 minutos. </li><li> Lave en PBST durante 15 minutos. </li><li> Se lavan con PBS durante 15 minutos. </li><li> Lugar en el anticuerpo secundario durante 2 horas en la oscuridad (dilución 1:100 en PBS). </li><li> Se lavan con PBS, PBST, y PBS durante 15 minutos cada uno y luego montar. </li><li> Los portaobjetos con un microscopio de fluorescencia. </li></ol> 7. Los resultados representativos: Este procedimiento de cultivo tritón ires que las células se ha utilizado para estudiar el potencial de regeneración de la parte dorsal y ventral de las células IPE. Por otra parte, también es posible estudiar los genes específicos que contribuyen a la regeneración del mecanismo de la lente en el ojo de tritón. Cuando las células han sido cultivadas durante dos semanas (Figura 1) pueden ser transfectadas por los genes para examinar su función en la regeneración de la lente. De particular interés son los genes que pueden inducir el iris ventral. Dado que el ventral las células del iris no puede transdiferenciarse a la lente (Figura 2) la función de inducción de un gen candidato puede ser estudiado. En el pasado, con esta técnica se ha demostrado que, cuando seis-3 se expresó sobre la presencia de la inducción de la lente ácido retinoico se observó desde el ventral iris 6. En la figura 3 se puede observar que el ventral agregado iris dio lugar a una lente completamente desarrollado y diferenciado (cabeza de flecha), no diferente de la lente de host de la dorsal del iris (flecha). <img src="/files/ftp_upload/2713/2713fig1.jpg" alt="Figura 1" /> Figura 1. a) dorsal iris células epiteliales pigmentadas cultivados in vitro durante un periodo de 2 semanas. b) ventral iris células epiteliales pigmentadas cultivados in vitro durante un periodo de 2 semanas. Tenga en cuenta que persiste la pigmentación de esta etapa en las células del iris dorsal y ventral. <img src="/files/ftp_upload/2713/2713fig2.jpg" alt="Figura 2" /> Figura 2. La regeneración de la capacidad de los cultivos de células de IPE. a) la inducción de la lente de agregados celulares dorsal IPE (cabeza de flecha). Anfitrión de la lente de inducción dorsal iris (flecha), di: dorsal iris, vi: ventral iris, le: epitelio del cristalino, Si: las fibras del cristalino. b) La ausencia de inducción de la lente de agregados de células IPE ventral (flecha). Anfitrión de la lente de inducción dorsal iris (flecha). <img src="/files/ftp_upload/2713/2713fig3.jpg" alt="Figura 3" /> Figura 3. La inducción de la lente de agregado ventral transfectadas con seis-3 y se trata con ácido retinoico. Anfitrión de la lente de inducción dorsal iris (flecha). Lente de la inducción de agregado ventral (flecha).

<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>

Este protocolo se ha establecido un sistema in vitro para estudiar los mecanismos de regeneración en la lente de tritones. Dado que los agregados (ya sea dorsal o ventral siguen fielmente a sus comportamiento in vivo durante la regeneración de esta técnica puede aliviar el enorme esfuerzo necesario para la transgénesis en tritones y puede ser usado para ganancia de función, así como la pérdida de 7,8,9 experimentos de la función. También , los agregados o el iris en su conjunto se puede tratar fácilmente con factores de crecimiento y examinar sus efectos como se describe en este protocolo. Por ejemplo, el papel de la vía BMP ha sido estudiado con esta técnica 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>
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<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a system for culturing iris pigment epithelial cells to study lens regeneration in newt

Salamandras y tritones como ajolote poseen la capacidad de regenerar muchas de sus partes del cuerpo perdido, como las extremidades, la cola con la médula espinal, ojos, cerebro, corazón, la mandíbula 1. En concreto, los tritones son únicos por su capacidad de regeneración de la lente. Tras la eliminación de lentes, las células de la IPE dorsal iris transdiferenciarse a las células del cristalino y, finalmente, formar un nuevo objetivo dentro de un mes 2,3. Esta propiedad de la regeneración no es exhibida por la ventral las células del iris. El potencial de regeneración de las células del iris puede ser estudiado por lo que los trasplantes de la in vitro IPE células cultivadas. Para el cultivo, las células del iris dorsal y ventral se aisló por primera vez a los ojos y cultivados por separado durante un periodo de 2 semanas (Figura 1). Estas células cultivadas se reaggregated y se implanta de nuevo al ojo de tritón. Estudios anteriores han demostrado que el reaggregate dorsal mantiene su capacidad de formación de la lente, mientras que el agregado ventral no forma una lente, recapitulando, por lo tanto el proceso en vivo (Figura 2) 4,5. Este sistema de determinación del potencial de regeneración de las células del iris dorsal y ventral es muy útil para estudiar el papel de los genes y las proteínas implicadas en la regeneración de la lente.

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

En Tritón, la lente siempre se regenera a partir de la dorsal del iris por la transdiferenciación de las células epiteliales pigmentadas de iris (IPE). Aquí se describe un procedimiento para cultivar células tritón dorsal y ventral IPE y su implantación en el ojo de tritón. Las células implantadas se estudian por el corte de tejidos e inmunohistoquímica.

Un sistema para el cultivo de células epiteliales del pigmento del iris para el Estudio de Regeneración del objetivo de 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, ...

a-system-for-culturing-iris-pigment-epithelial-cells-to-study-lens

Research

JoVE Journal

Biology

11.7K vistas.  University of Dayton. En Tritón, la lente siempre se regenera a partir de la dorsal del iris por la transdiferenciación de las células epiteliales pigmentadas de iris (IPE). Aquí se describe un procedimiento para cultivar células tritón dorsal y ventral IPE y su implantación en el ojo de tritón. Las células implantadas se estudian por el corte de tejidos e inmunohistoquímica.

Video: 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.

La ablación con láser de la pronefros pez cebra para el Estudio de la regeneración epitelial renal

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 ...

Investigación

Biología

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.

Un sistema 3D para los condrocitos articulares humanos cultivo de líquido sinovial

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.

Do-It-Yourself de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

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.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

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.

Inducción y Análisis de epitelial a mesenquimal de transición

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.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

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.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

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.

Regeneración epitelial intestinal en respuesta a la irradiación ionizante

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.

Cultivo primario de células epiteliales pigmentarias de la retina porcina

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