Diese Arbeit wurde durch ein NIH Ey10540 zu PAT finanziert.

1. Iris Cell Culture <ol><li> Sammeln Augäpfel von 7 Molche (narkotisierten in 0,1% Ethyl-3-Aminobenzoesäure Methansulfonsäure in PBS) hergestellt und in Calcium-Magnesium-freier Hanks-Lösung (CMF). </li><li> Wechseln Sie die Handschuhe und die Arbeit in der Gewebekultur Kapuze. </li><li> Sterilisieren Augäpfel in Lugol-EtOH für 3 Sekunden. </li><li> Wash 2 Mal in CMF. </li><li> Transfer Augen Hanks und beginnen Dissektion. </li><li> Dissect Iris-Hornhaut-Komplex und entfernen neuronalen Netzhaut. </li><li> Transfer-Komplexe in ein neues Gericht von Hanks. </li><li> Mit Nr. 15 Skalpell, separate Komplexe in dorsalen und ventralen Hälften. </li><li> Entfernen Iris-Fragmente (ventral zuerst) und in eine Schüssel mit 1 ml L-15. </li><li> Fügen Sie 15% (150 ul) Volumen von Dispase (7,5 Einheiten / ml Konzentration). </li><li> Inkubation bei 27 ° C für 2 Stunden (wrap Schale mit Parafilm). </li><li> Isolieren IPE Zellen von Stroma (Place Trypsin-Lösung bei 27 ° C). </li><li> Sammeln IPE Zellen in ein 1,5 ml-Tube. </li><li> Zentrifugation bei 1000 rpm bei RT (Raumtemperatur) für 2 Minuten, entfernen Überstand. </li><li> Waschen mit 1 ml Hanks und Zentrifuge bei 1000 rpm bei RT für 2 Minuten, Überstand entfernen. </li><li> 1 ml Trypsin-Lösung; inkubieren für 2 Stunden bei 27 ° C. </li><li> Zentrifugation bei 1000 rpm bei RT für 2 Minuten, entfernen Trypsin. </li><li> 1 ml L-15 zu waschen, zentrifugieren bei 1000 rpm für 2 Minuten. </li><li> Add 800 ul L-15, Pipette langsam auf und ab ~ 10-mal (mehr als 12 Mal reduziert Haftung). </li><li> Platte der IPE Zellen auf einem 24-Well-Kollagen beschichtete Platte und inkubieren bei 27 ° C für 2 Wochen (in der Regel, 4 dorsalen und 4 ventralen Brunnen sind zwischen 7 und Molche erhalten). </li><li> In diesem Stadium Zellen eignen sich für die Gen-Transfektion oder die Behandlung mit Faktoren, um ihre Rolle bei der Induktion oder stören Objektiv Regeneration zu bestimmen. </li></ol> 2. Aggregation von Iris Cells <ol><li> Um die Platten mit Iris-Zellen add 20 &amp;mgr; l der Dispase-Lösung zu 400 ul Medium, vorsichtig schwenken. </li><li> Die Platten bei 27 ° C über Nacht. </li><li> Pipettieren vorsichtig nach oben und unten zu verdrängen Zellen </li><li> Legen Sie Zellen in Eppendorf-Röhrchen und Spin 2 Minuten bei 1000 rpm bei RT. </li><li> Nehmen Sie mittel-und waschen durch Zugabe von 1 ml komplette L-15, Spin 2 Minuten bei 1000 rpm bei RT, entfernen Medium. </li><li> Verwenden 2000-7000 Zellen pro Aggregat. Geben Sie 200 ul L-15 pro Aggregat in jedes Röhrchen. </li><li> Split-Zellen (200 ul) in neue Eppendorf-Röhrchen. </li><li> Spin bei 1000 rpm für 2 min bei RT. </li><li> Inkubieren für 48 Stunden bei 27 ° C. </li></ol> 3. Die Implantation von Aggregrated Cells <ol><li> Machen Sie einen Schlitz in die Hornhaut des Auges newt und entfernen Sie die Linse. </li><li> Nach Linse entfernt, legen Sie die IPE Zellverbandes knapp unterhalb der Hornhaut auf der Bauchseite des Auges. </li><li> Die Molche sind für einen Monat aufbewahrt, damit sie regenerieren die Linse. </li></ol> 4. Einbettung von Newt Eye <ol><li> Entfernen Sie den Molch Augen mit dem Aggregat implantiert und legen Sie sie in Phosphat-gepufferter Kochsalzlösung (PBS). </li><li> Fix in 4% Paraformaldehyd bei 4 ° C für mindestens 4 Stunden oder über Nacht. </li><li> Wash in PBS, 4 ° C für 30 Minuten. </li><li> Behandeln Sie es mit 0,85% iger Kochsalzlösung: 4 ° C, 30 Minuten. </li><li> Wash in Saline / Ethanol (1:1): RT für 15 Minuten. </li><li> Wash in 70% Ethanol: RT, 15 Minuten. Wiederholen Sie es einmal. </li><li> Wash in 85% Ethanol und 95% Ethanol bei RT, 30 Minuten </li><li> Wash in 100% Ethanol: RT, 30 Minuten. Wiederholen Sie es einmal. </li><li> Lagerung bei 4 ° C oder fortzusetzen. </li><li> Wash in 100% Xylol: RT, 30 Minuten. Wiederholen Sie es einmal. </li><li> Behandeln Sie mit Xylol / Paraffin (1:1): 60 ° C, 45 Minuten. </li><li> Behandeln Sie mit 100% Paraffin: 60 ° C, 20 Minuten. Wiederholen Sie es noch zwei weitere Male. </li><li> Embed in Einbettformen. </li></ol> 5. Sectioning <ol><li> Bereiten 15 um Abschnitte der embedded Auge mit Hilfe eines Mikrotoms. </li><li> Legen Sie das Auge Abschnitte auf einer Gelatine beschichtete Folie und lassen Sie es auf einer Folie wärmer. </li></ol> 6. Die Färbung <ol><li> Die Objektträger in Xylol für 10 Minuten. Wiederholen Sie es noch einmal. </li><li> Hydrate in: 100%, 95%, 80%, 70%, 30% Ethanol für jeweils 1 Minute </li><li> Spülen in DI-Wasser für eine Minute. </li><li> Wash in PBS für 15 Minuten. </li><li> Wash in PBST (0,2% Triton-X 100 in PBS) für 15 Minuten. </li><li> Wash in PBS für 15 Minuten. </li><li> Legen Sie in Blocking-Lösung (10% Ziegenmilch sekundärer Antikörper) für eine Stunde. </li><li> Legen Sie in primärer Antikörper zur Übernachtung. </li><li> Wash primärer Antikörper in PBS für 15 Minuten. </li><li> Wash in PBST für 15 Minuten. </li><li> Wash in PBS für 15 Minuten. </li><li> Legen Sie in sekundären Antikörper für 2 Stunden in dunklen (1:100 Verdünnung in PBS). </li><li> Wash in PBS, PBST und PBS für jeweils 15 Minuten und dann montieren. </li><li> Den Objektträger unter dem Fluoreszenzmikroskop. </li></ol> 7. Repräsentative Ergebnisse: Dieses Verfahren zur Kultivierung newt irist Zellen wurde verwendet, um die Regenerationsfähigkeit der dorsalen und der ventralen IPE Zellen zu untersuchen. Darüber hinaus ist es auch möglich, spezielle Gene, die in Richtung des Objektivs Regeneration Mechanismus in newt Auge beitragen zu studieren. Wenn die Zellen für 2 Wochen kultiviert wurden (Abbildung 1) können sie von den Genen transfiziert werden, um ihre Funktion in der Linse Regeneration zu untersuchen. Von besonderem Interesse sind Gene, die die ventralen Iris induzieren könnte. Da die ventralen Iris Zellen können nicht auf die Linse transdifferenzieren (Abbildung 2) die induktive Funktion eines Kandidatengen untersucht werden können. In der Vergangenheit mit dieser Technik haben wir gezeigt, dass, wenn sechs-3 über wurde in Anwesenheit von Retinsäure Objektiv Induktion exprimiert wurde von der ventralen Iris 6 beobachtet. In Abbildung 3 sehen wir, dass der ventralen Iris aggregierten Anstieg gab ein ausgewachsener und differenzierte Linse (Pfeilspitze), nicht anders als die Host-Objektiv aus dem dorsalen Iris (Pfeil). <img src="/files/ftp_upload/2713/2713fig1.jpg" alt="Abbildung 1" /> Abbildung 1. a) Dorsal Iris pigmentierten Epithelzellen in vitro für einen Zeitraum von 2 Wochen kultiviert. b) Ventrale Iris pigmentierten Epithelzellen in vitro für einen Zeitraum von 2 Wochen kultiviert. Beachten Sie, dass Pigmentierung dieser Phase besteht in beiden dorsalen und ventralen Iris-Zellen. <img src="/files/ftp_upload/2713/2713fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Regenerationsfähigkeit von kultivierten IPE Zellen. a) Objektiv Induktion von dorsal IPE Zellverband (Pfeilspitze). Host-Objektiv Induktion von dorsal Iris (Pfeil), di: dorsale Iris, vi: ventral Iris le: Linsenepithel, lf: Linsenfasern. b) Fehlen der Linse Induktion von ventral IPE Zellverband (Pfeilspitze). Host-Objektiv Induktion von dorsal Iris (Pfeil). <img src="/files/ftp_upload/2713/2713fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Objektiv Induktion von ventral Aggregat mit sechs-3 transfiziert und mit Retinsäure. Host-Objektiv Induktion von dorsal Iris (Pfeil). Objektiv Induktion von ventral Aggregat (Pfeilspitze).

<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>

Dieses Protokoll wurde ein in vitro System zur Linse Regeneration Mechanismen in Molche Studie etabliert. Da die Aggregate (entweder dorsal oder ventral treu zu folgen ihren in vivo-Verhalten während der Regeneration dieser Technik kann der enorme Aufwand für Transgenese in Molche erforderlich und kann für gain of function sowie Verlust der Funktion Experimente 7,8,9 verwendet werden zu lindern. Auch können die Aggregate oder die Iris als Ganzes leicht mit Wachstumsfaktoren behandelt und untersucht deren Auswirkungen, wie in diesem Protokoll beschrieben. Zum Beispiel die Rolle der BMP-Weg wurde untersucht mit dieser Technik 6.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
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a system for culturing iris pigment epithelial cells to study lens regeneration in newt

Salamander wie Newt Axolotl besitzen die Fähigkeit, viele seiner verlorenen Körperteile wie Beine, der Schwanz mit Rückenmark, Augen, Gehirn, Herz, die Backe 1 regenerieren. Insbesondere sind Molche einzigartig für seine Linse Regenerationsfähigkeit. Nach Linse entfernt, IPE Zellen der dorsalen Iris-Objektiv Zellen transdifferenzieren und bilden schließlich eine neue Linse in etwa einem Monat 2,3. Diese Eigenschaft der Regeneration wird nie von der ventralen Iris Zellen zeigten. Die Regenerationsfähigkeit der Iris-Zellen können, indem Transplantation von in vitro kultivierten Zellen IPE untersucht werden. Für die Kultur sind die dorsalen und ventralen Iris Zellen zunächst aus dem Auge isoliert und separat für einen Zeitraum von 2 Wochen (Abbildung 1) kultiviert. Diese gezüchteten Zellen werden neu zusammengefügt und wieder auf das Newt Auge implantiert. Frühere Studien haben gezeigt, dass die dorsale reaggregieren seiner Linse bilden Kapazität behält, während die ventralen Aggregat der keinen Linse, rekapitulieren, damit die in vivo-Verfahren (Abb. 2) 4,5. Dieses System zur Bestimmung Regenerationsfähigkeit der dorsalen und ventralen Iris-Zellen ist sehr nützlich bei der Untersuchung der Rolle von Genen und Proteinen in der Linse Regeneration beteiligt.

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, regeneriert die Linse immer von der dorsalen Iris durch Transdifferenzierung der Iris pigmentierten Epithelzellen (IPE). Hier beschreiben wir ein Verfahren zur Kultur dorsalen und ventralen newt IPE Zellen und deren Implantation der Molch Auge. Die implantierten Zellen werden dann durch Gewebe Schnitte und Immunhistochemie untersucht.

Ein System zur Kultivierung Iris Pigmentepithelzellen nach Lens Regeneration in Newt Study

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 Aufrufe.  University of Dayton. In newt, regeneriert die Linse immer von der dorsalen Iris durch Transdifferenzierung der Iris pigmentierten Epithelzellen (IPE). Hier beschreiben wir ein Verfahren zur Kultur dorsalen und ventralen newt IPE Zellen und deren Implantation der Molch Auge. Die implantierten Zellen werden dann durch Gewebe Schnitte und Immunhistochemie untersucht.

Serie: 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.

Laser Ablation des Zebrafisch Vorniere auf Study Renal Epithelregeneration

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 ...

Forschung

Biologie

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.

Ein 3D-System für Züchtung menschlichen Chondrozyten in der Synovialflüssigkeit

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 Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

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.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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.

Die Kultivierung der menschlichen Nasen Epithelzellen an der Air flüssig-Grenzfläche

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.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

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.

Regeneration des Darmepithels als Reaktion auf ionisierende Bestrahlung

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.

Primärkultur von retinalen Pigmentepithelzellen des Schweins

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