Wir möchten alle Mitglieder der Kong Lab und Dr. Yan Deng am Microscopy Core, Office of Collaborative Wissenschaft, NYUMC danken für ihre Unterstützung und technischen Support. Diese Arbeit wurde von einem Piloten Zuschuss von der New York University Center for AIDS Research (CFAR, NIH AI027742) unterstützt.

Ein. Plasmid DNA Purification <ol><li> Wandeln Sie die eukaryontischen Plasmid DNA, EGFP (pCX-EGFP) in DH5 E.coli kompetenten Zellen. Der leere pCX Vektor kann auch als negative Kontrolle verwendet werden. </li><li> Kultur und Ernte die DH5a E.coli Zellen und reinigen die pDNA nach dem Qiagen EndoFree Plasmid Purification Handbook. </li><li> Filter pDNA Lösung durch einen 0,22 um PVDF Sterilfilter, und speichern Sie sie bei -20 ° C bis zur Verwendung. </li></ol> 2. Tattoo System Preparation <ol><li> Schließen Sie das Handheld-Einheit und die Kontrolle Pedal Netzteil pro Anweisungen des Herstellers. </li><li> Die Nadel Schwingungsfrequenz auf etwa 100 Hz. Für die Tätowierung Gerät, das wir wählten (Stealth Rotary Tattoo System), sollte das Rad an der Stromversorgung bis 4 Volt eingestellt werden. Siehe Bedienungsanleitungen oder technische Unterstützung für andere Tätowierung Systeme. </li><li> Sterilisieren der Nadel array vor Gebrauch. Wir empfehlen die Verwendung einer Nadel array pro Tier. Die Nadeln können, nachdem sie mit Seifenwasser gereinigt und autoklaviert wiederverwendet werden. </li><li> Installieren Sie die Nadel Array in der tragbaren Einheit und lose befestigen Sie den Kunststoffgriff. </li><li> Passen Sie die Nadel Tiefe eine entsprechende Einstellung, indem Sie den Kunststoffgriff oben oder unten. Wir verwendeten eine Nadel Tiefe von etwa 0,5 mm für unseren Maus-Experiment. </li><li> Ziehen Sie die Kunststoff-Griff, wenn die Nadel Tiefe richtig eingestellt ist, und die Tätowierung System ist bereit für den Einsatz. </li></ol> 3. Tierische Shaving <ol><li> Wählen Sie den Standort für das Tätowieren Behandlung. Der Standort sollte eine relativ feste, flache und fleischige Bereich der Haut, zum Beispiel die Seite des Tieres Hinterbein sein. </li><li> Anästhesieren einer Balb / c-Maus mit einer Mischung aus Ketamin (90 mg / kg) und Xylazin (5 mg / kg) nach dem Körper des Tieres beträgt. Für andere Tiere, die Anästhesie Richtlinien Ihrer Einrichtung beziehen. </li><li> Überprüfen Sie dieTier Reflexe durch Kneifen seinen Fuß, um das Tier zu bestätigen ist richtig betäubt. </li><li> Verwenden Sie die elektrische Trimmer trimmen sich die Haare des Tieres Hinterhand langsam und vorsichtig. Entfernen Sie die Haare aus dem geplanten Tätowierung Ort und die Umgebung. </li><li> Entfernen Sie die verbleibende kürzere Haare mit dem Einweg-Rasierapparat. Achten Sie darauf, die Haut des Tieres geschnitten. Beachten Sie, dass Enthaarungscreme als Alternative Haarentfernung Verfahren verwendet werden kann, aber wir lieber über Enthaarungscreme Rasur. </li><li> Entfernen streunenden geschnittenen Haare aus der Haut mit Druckluft, wenn nötig. </li></ol> 4. Lieferung von Plasmid-DNA durch Tätowierung <ol><li> Wenn ein Immunisierungsprotokoll zuvor festgestellt worden, sollte die Dosis und das Volumen der DNA-Lösung aufgebracht werden entsprechend berechnet werden. Wir empfehlen die Verwendung als kleines Volumen als praktische, um die Tätowierung Prozess mehr überschaubar. In diesem EGFP Demonstration, verwendeten wir 7,5 &amp;mgr; l DNA-Lösung bei einer conzentration von 0,25 mg / ml, die auf ein Tätowieren Fläche von ca. 1 cm 2 aufgebracht wurde. Für die Immunisierung Experimente können höhere Konzentrationen erforderlich pDNA starke Immunantworten 1,2 induzieren. </li><li> Übernehmen Sie die DNA-Lösung mit einer Pipette direkt auf die Tätowierung Website. Alternativ laden die DNA-Lösung in den Kunststoffgriff durch Pipettieren, so wird die Flüssigkeit zwischen der Nadel und der Spitze der Kunststoffgriff suspendiert. Siehe das Video für eine Demonstration. Wir haben keine merkliche Differenz zwischen den zwei Verfahren beobachtet. Allerdings kann das Laden der Lösung in den Kunststoffgriff werden auf vertikalen Hautflächen vorteilhaft, zum Beispiel. </li><li> Starten Sie die Nadel Schwingung und die Nadel Array mit leichtem Druck auf die Haut des Tieres an der Tätowierung Website. Dann bewegen Sie den oszillierenden Nadel vorsichtig und langsam in einer linearen Weise, Plasmid-DNA über die gesamte Tätowierung Bereich liefern. Aufrechterhaltung eines 90-Grad-Winkel zwischen der Nadel und der Haut zu vermeiden,Schnittwunden auf der Haut. </li><li> Beachten Sie die Haut. Abrasion und Entzündungen sind normal. Wenn die Blutung auftritt, halten Sie die Tätowierung Prozess und verringern die Nadel Tiefe. </li><li> Fahren Sie mit der Nadel Bewegung für ca. 1 min, und dann stoppen die Tätowierung Prozess. </li><li> Verwenden Sie ein sauberes Wattestäbchen, um eine dünne Schicht aktuelles Analgetikum auf dem Tätowieren Website gelten. Topical Analgetika wie Silbersulfadiazin-Creme (SSD Cream) oder Neosporin Salbe kann Linderung der Schmerzen oder Leiden der Tiere, und deshalb ist es ratsam, sie anzuwenden, bevor die Betäubung nachlässt. Beobachten Sie die Tiere am nächsten Tag auf Anzeichen von Schmerzen oder Leiden (Änderung der Gangart, Inaktivität). Wenden schmerzstillende Creme, wenn die Zeichen anhalten. </li></ol> 5. Bestätigung der Antigen Expression <ol><li> 48 Stunden nach dem Tätowieren Behandlung (beachten Sie, dass verschiedene pDNA Konstrukte können unterschiedliche Expression Zeiten erfordern), wird die Maus durch CO 2 Narkose getötet. Für andereTiere, die Euthanasie Richtlinien Ihrer Einrichtung beziehen. </li><li> Sezieren die Haut an der Tätowierung Website, und befestigen Sie das Gewebe in 4% Paraformaldehyd bei 4 ° C über Nacht. </li><li> Spülen des Gewebes mit 70% Ethanol, und gibt sie in 1X PBS zur Bildgebung. </li><li> Untersuchen Sie das ganze Gewebe mit einem konfokalen Mikroskop für EGFP-Signale überprüfen. Alternativ kann das Hautgewebe in Paraffinwachs eingebettet und untersucht werden abschnittsweise unter einem Fluoreszenzmikroskop. </li></ol>

<ol>
	<li>Bins, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+potent+DNA+vaccination+strategy+defined+by+in+vivo+monitoring+of+antigen+expression.">A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression.</a> Nat Med. 11, 899-904 (2005).</li><li>Pokorna, D., Rubio, I., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-vaccination+via+tattooing+induces+stronger+humoral+and+cellular+immune+responses+than+intramuscular+delivery+supported+by+molecular+adjuvants.">DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants.</a> Genet. Vaccines Ther. 6, 4 (2008).</li><li>van den Berg, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intradermal+vaccination+by+DNA+tattooing+in+human+skin.">Optimization of intradermal vaccination by DNA tattooing in human skin.</a> Hum. Gene Ther. 20, 181-189 (2009).</li><li>Liu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines:+a+review.">DNA vaccines: a review.</a> J. Intern. Med. 253, 402-410 (2003).</li><li>Koide, Y., Nagata, T., Yoshida, A., Uchijima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines.">DNA vaccines.</a> Jpn. J. Pharmacol. 83, 167-174 (2000).</li><li>Peachman, K. K., Rao, M., Alving, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+DNA+through+the+skin.">Immunization with DNA through the skin.</a> Methods. 31, 232-242 (2003).</li><li>Verstrepen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+HIV-1+specific+T-cell+responses+by+short-interval+DNA+tattooing+as+compared+to+intramuscular+immunization+in+non-human+primates.">Improved HIV-1 specific T-cell responses by short-interval DNA tattooing as compared to intramuscular immunization in non-human primates.</a> Vaccine. 26, 3346-3351 (2008).</li><li>Potthoff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+and+efficacy+of+intradermal+tattoo+immunization+with+adenoviral+vector+vaccines.">Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines.</a> Vaccine. 27, 2768-2774 (2009).</li><li>Quaak, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+tattoo+vaccination:+effect+on+plasmid+purity+and+transfection+efficiency+of+different+topoisoforms.">DNA tattoo vaccination: effect on plasmid purity and transfection efficiency of different topoisoforms.</a> J. Control Release. 139, 153-159 (2009).</li><li>Pokorna, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+with+human+papillomavirus+type+16-derived+peptides+using+a+tattoo+device.">Vaccination with human papillomavirus type 16-derived peptides using a tattoo device.</a> Vaccine. 27, 3519-3529 (2009).</li></ol>

Keine Interessenskonflikte erklärt.

DNA-Impfung gilt als sicherer als herkömmliche Impfstrategien, da sie nicht verlangen, Manipulation oder aussetzen Impfstoffe, live oder abgeschwächte Erreger 4. Jedoch hängt das Ergebnis der DNA-Vakzinierung stark von der Lieferroute. Haut ist reichlich in Antigen-präsentierende Zellen, wie Langerhans-Zellen und dendritischen Zellen 1 und damit eine ideale Ort für die Immunisierung im Hinblick auf Immunogenität und leichte Zugänglichkeit 5,6. Als Ergebnis ist intradermale Impfstr...

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> pCX-EGFP-Plasmid-DNA </td><td> Clontech </td><td></td><td></td></tr><tr><td> STEALTH Rotary Tattoo-System </td><td> Weltweit Tattoo Supply </td><td> STEALTH-L </td><td></td></tr><tr><td> Tattoo Nadeln </td><td> Weltweit Tattoo Supply </td><td> 1207RSB </td><td></td></tr><tr><td> EndoFree Plasmid Maxi Kit </td><td> Qiagen </td><td> 12362 </td><td></td></tr><tr><td> 0,22 um PVDF Sterilfilter </td><td> Millipore </td><td> SLGV013SL </td><td></td></tr><tr><td> elektrischen Haarschneider </td><td> Kommerziell erhältliche </td><td></td><td></td></tr><tr><td> Einweg-Rasierapparate </td><td> Kommerziell erhältliche </td><td></td><td></td></tr><tr><td> SilberSulfadiazin-Creme </td><td> Watson </td><td> NDC 0591-0810-55 </td><td></td></tr><tr><td> Ketamin HCL </td><td></td><td> NDC 0856-2012-01 </td><td></td></tr><tr><td> Zylazine sterilen Lösung </td><td></td><td> NADA 139-236 </td><td></td></tr></tbody></table>

skin tattooing as a novel approach for dna vaccine delivery

Nukleinsäure-basierte Impfungen ist ein Thema von wachsendem Interesse, insbesondere Plasmid-DNA (pDNA) Codieren immunologisch wichtigen Antigenen. Nachdem sich das manipulierte pDNA den Impfstoffen verabreicht wird, wird es transkribiert und übersetzt in Immunogen Proteine, die Antworten des Immunsystems auslösen kann. Viele Wege zur Umsetzung der DNA-Impfstoffe untersucht worden, aber jede Lieferung Strecke hat seine eigenen Vorteile und Fallstricke. Haut Tätowieren ist eine neuartige Technik, die sicher, kostengünstig und bequem ist. Darüber hinaus könnten die Einstiche von der Nadel zugefügt auch als potente Adjuvans dienen. Hier haben wir a) zeigen die intradermalen Verabreichen von Plasmid DNA, die verstärkt grün fluoreszierende Protein (EGFP pCX-) in einem Maus-Modell unter Verwendung eines Geräts Tätowieren und b) bestätigen die effektive Expression von EGFP in den Hautzellen mittels konfokaler Mikroskopie.

Die Expression von EGFP mit einem Anregungsfilter Peak bei 488 nm und Emissionspeak bei 509 nm kann in Hautzellen von Mäusen beobachtet werden. Aus einer 1,875 ug Dosis von DNA, die etwa 3 × 10 17 Kopien des Plasmids, wir typischerweise beobachtet 10-20 EGFP Signale in den 1 cm 2 tätowierten Bereich. Diese relativ geringe Anzahl von transfizierten Zellen ist konsistent mit den Ergebnissen einer früheren Studie 3. Das EGFP-Expression (Abbildung 1) liefert den Beweis, ...

Watch this Scientific Journal Video about Skin Tattooing As A Novel Approach For DNA Vaccine Delivery at JoVE.com

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Haut Tätowieren ist ein potenter und sicheren Weg zur Auslieferung DNA-Impfstoff intradermal. Hier wird ein DNA-Plasmid kodiert EGFP durch Tätowierung auf der Haut eines Labormaus geliefert, und die Expression von EGFP in den Hautzellen wird dann durch konfokale Mikroskopie untersucht.

Haut Tätowieren als einen neuartigen Ansatz für DNA Vaccine Lieferung

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

skin-tattooing-as-a-novel-approach-for-dna-vaccine-delivery

Research

JoVE Journal

Bioengineering

15.8K Aufrufe.  New York University School of Medicine. Haut Tätowieren ist ein potenter und sicheren Weg zur Auslieferung DNA-Impfstoff intradermal. Hier wird ein DNA-Plasmid kodiert EGFP durch Tätowierung auf der Haut eines Labormaus geliefert, und die Expression von EGFP in den Hautzellen wird dann durch konfokale Mikroskopie untersucht.

Serie: Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Zellzusammendrücken als Robust, Mikrofluidik Intrazelluläre Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Forschung

Biotechnik

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Ein verbessertes Verfahren zur Herstellung von Typ I-Kollagen aus der Haut

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Charakterisierung komplexer Systeme Mit dem Design of Experiments Ansatz: Transient Protein Expression in Tabak als Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Ein Vollhautdefekt Modell zur Vaskularisierung von Biomaterialien Bewerten<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microscale Vortex-unterstützte Electroporator für Sequential Molekulare Liefer

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Entwicklung eines unter Schwerelosigkeits Vielzellige Dreidimensionale Organotypischen Modell des menschlichen Darmschleimhaut Grown

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Mesenchymale Stromazellen Zellkultur und Lieferung in Autologe Bedingungen: Ein intelligenter Ansatz für orthopädische Anwendungen

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA-Nanoröhren als ein vielseitiges Werkzeug, semiflexiblen Polymere zu studieren

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting von Knorpel und Haut-Gewebe-Analoga nutzen eine neuartige Passive Einheit Mischtechnik für Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.