Gostaríamos de agradecer a todos os membros do Laboratório Kong e Yan Dr. Deng no Gabinete de Microscopia Core, da Ciência Colaborativa, NYUMC por sua assistência e suporte técnico. Este trabalho foi financiado por uma doação de piloto da New York University Center for AIDS Research (CFAR, NIH concessão AI027742).

1. A purificação do ADN plasmídico <ol><li> Transformar o plasmídeo de DNA eucariótico que codifica EGFP (PCX-EGFP) em células de E. coli DH5a competentes. O vector PCX vazio pode também ser usado como um controlo negativo. </li><li> Cultura e colheita das células de E. coli DH5a e purifica-se o pDNA de acordo com o Qiagen EndoFree Plasmid Purification Handbook. </li><li> Filtrar através de uma solução de pDNA PVDF de 0,22 um filtro esterilizado, e armazená-lo a -20 ° C até à sua utilização. </li></ol> 2. Preparação do Sistema tatuagem <ol><li> Ligar a unidade portátil e o pedal de controlo para a unidade de fonte de alimentação com as instruções do fabricante. </li><li> Definir a freqüência de oscilação da agulha para cerca de 100 Hz. Para o dispositivo de tatuagem que escolhemos (Stealth Sistema Tattoo Rotary), a marcação sobre o fornecimento de energia deve ser definido para 4 volts. Consulte o manual do usuário ou suporte técnico para sistemas de tatuagem outros. </li><li> Esterilizar o ar agulharay antes do uso. Recomendamos o uso de uma matriz de agulhas por animal. As agulhas podem ser reutilizadas após a limpeza com água e sabão e autoclavados. </li><li> Instale o conjunto agulha na unidade portátil e vagamente anexar a pega de plástico. </li><li> Ajustar a profundidade da agulha para uma definição adequada, movendo a pega de plástico para cima ou para baixo. Nós usamos uma profundidade agulha de aproximadamente 0,5 mm para o nosso experimento mouse. </li><li> Apertar a pega de plástico, quando a profundidade da agulha está correctamente ajustado, e o sistema de tatuagem está pronto para uso. </li></ol> 3. Raspar animais <ol><li> Escolher o local para o tratamento de tatuagem. O local deve ser uma área relativamente firme e plana, e carnuda da pele, por exemplo, o lado da perna traseira do animal. </li><li> Anestesiar um ratinho BALB / c com uma mistura de cetamina (90 mg / kg) e xilazina (5 mg / kg) de acordo com o peso corporal do animal. Para outros animais, consulte as diretrizes de anestesia da sua instituição. </li><li> Verifique oreflexos animal por beliscar o seu pé para confirmar que o animal é anestesiado adequadamente. </li><li> Use o cortador elétrico para aparar o cabelo em posteriores do animal devagar e com cuidado. Remova o cabelo a partir do site tatuagem planejada e área circundante. </li><li> Remover o cabelo mais curto residual com o aparelho de barbear descartável. Tenha cuidado para não cortar a pele do animal. Note-se que o creme depilatório pode ser utilizada como um método de remoção de pêlos alternativa, no entanto, preferimos barbear sobre o creme depilatório. </li><li> Remover pêlos cortados dispersos a partir da pele com ar comprimido, se necessário. </li></ol> 4. Entrega de ADN plasmídico por Tattooing <ol><li> Se um protocolo de imunização foi estabelecido anteriormente, a dose e volume de solução de DNA a ser aplicado deve ser calculada em conformidade. Recomendamos utilizando um volume tão pequeno quanto prático para tornar o processo mais controlável tatuagem. Nesta demonstração EGFP, utilizou-se 7,5 uL de solução de ADN a uma concentração de 0,25 mg / ml, o qual foi aplicado sobre uma área de cerca de 1 a tatuagem cm 2. Para as experiências de imunização, as concentrações mais elevadas pDNA pode ser necessária para induzir respostas imunes fortes a 1,2. </li><li> Aplicar a solução de DNA através de pipetagem directamente sobre o local de tatuagem. Em alternativa, carregue a solução de ADN para a pega de plástico por pipetagem, de modo que o líquido é suspensa entre a agulha e a ponta de aperto de plástico. Consulte o vídeo para uma demonstração. Temos observado nenhuma diferença notável entre os dois métodos. No entanto, carregar a solução para o aperto de plástico pode ser vantajoso em superfícies verticais da pele, por exemplo. </li><li> Iniciar a oscilação da agulha e colocar a matriz de agulha com uma leve pressão sobre a pele do animal no local de tatuagem. Em seguida, passar a agulha de oscilação suave e lentamente de uma forma linear para entregar o ADN do plasmídeo durante a área inteira tatuagem. Manter um ângulo de 90 graus entre a agulha e a pele para evitarlacerações da pele. </li><li> Observe a pele. Abrasão e inflamação são normais. Se o sangramento acontece, pare o processo de tatuagem e diminuir a profundidade da agulha. </li><li> Continue o movimento agulha por cerca de 1 minuto, e depois parar o processo de tatuagem. </li><li> Usar um cotonete de algodão limpo para aplicar uma fina camada de analgésico tópico no local de tatuagem. Analgésicos tópicos, como a sulfadiazina de prata (SSD creme) ou pomada Neosporin pode ajudar a aliviar a dor ou o sofrimento dos animais, e, portanto, recomenda-se aplicá-los antes da anestesia desgasta fora. Observar os animais no dia seguinte para sinais de dor ou sofrimento (mudança de inatividade marcha). Reaplicar o creme analgésico se os sinais persistirem. </li></ol> 5. A confirmação da expressão do antigénio <ol><li> 48 horas após o tratamento tatuagem (note que as construções diferentes pDNA pode exigir tempos de expressão diferentes), o mouse é sacrificados por CO 2 narcose. Por outroanimais, consulte as diretrizes de eutanásia de sua instituição. </li><li> Dissecar a pele no local da tatuagem, e fixar o tecido em paraformaldeído a 4% a 4 ° C durante a noite. </li><li> Enxaguar o tecido com 70% de etanol, e transferi-lo em 1X PBS para imagiologia. </li><li> Examine todo o tecido usando um microscópio confocal para procurar sinais EGFP. Alternativamente, o tecido da pele pode ser embebido em cera de parafina e examinado em secções com um microscópio fluorescente. </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>

Não há conflitos de interesse declarados.

Vacinação de DNA é considerada mais segura do que as estratégias de vacinação tradicional, uma vez que não requer manipulação de, ou expor as vacinas, agentes patogénicos vivos ou atenuados 4. No entanto, o resultado da vacinação de DNA depende fortemente da via de administração. A pele é abundante em células apresentadoras de antigénio, tais como células de Langerhans e células dendríticas 1, e, portanto, um local ideal para a imunização em termos de imunogenicidade e facilid...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> PCX-DNA do plasmídeo EGFP </td><td> Clontech </td><td></td><td></td></tr><tr><td> STEALTH Sistema Tattoo Rotary </td><td> Fonte Tattoo Mundial </td><td> STEALTH L- </td><td></td></tr><tr><td> Agulhas de tatuagem </td><td> Fonte Tattoo Mundial </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> PVDF de 0,22 um filtro estéril </td><td> Millipore </td><td> SLGV013SL </td><td></td></tr><tr><td> máquina de cortar cabelo elétrica </td><td> Comercialmente disponível </td><td></td><td></td></tr><tr><td> descartável segurança navalhas </td><td> Comercialmente disponível </td><td></td><td></td></tr><tr><td> PrataSulfadiazina </td><td> Watson </td><td> NDC 0591-0810-55 </td><td></td></tr><tr><td> Cetamina HCL </td><td></td><td> NDC 0856-2012-01 </td><td></td></tr><tr><td> Solução Estéril Zylazine </td><td></td><td> NADA 139-236 </td><td></td></tr></tbody></table>

skin tattooing as a novel approach for dna vaccine delivery

Vacinação à base de ácido nucléico é um tema de interesse crescente, especialmente DNA plasmídeo (pDNA) codificando antígenos imunologicamente importantes. Após o pDNA engenharia é administrado às vacinas, é transcrita e traduzida em proteínas de imunogénio, que pode eliciar respostas do sistema imunológico. Muitas formas de entrega de vacinas de DNA têm sido investigados, mas cada rota de entrega tem suas próprias vantagens e armadilhas. Tatuagem da pele é uma nova técnica que é seguro, eficaz e conveniente. Além disso, as perfurações provocadas pela agulha pode também servir como um adjuvante potente. Aqui, a) demonstrar a entrega intradérmica de codificação de ADN do plasmídeo melhorada da proteína verde fluorescente (EGFP-PCX) num modelo de ratinho utilizando um dispositivo de tatuagem e b) confirmar a expressão eficaz de GFP em células da pele, utilizando microscopia confocal.

A expressão de EGFP, com um pico de excitação a 488 nm e pico de emissão a 509 nm pode ser observado nas células da pele do rato. A partir de uma dose de 1,875 ug de DNA, contendo cerca de 3 x 10 17 cópias do plasmídeo, que tipicamente observada 10-20 sinais EGFP em de 1 cm 2 área tatuada. Este número relativamente baixo de células transfectadas é consistente com os resultados de um estudo anterior 3. A expressão de EGFP (Figura 1) proporciona a evidência de...

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

Tatuagem pele é um modo poderoso e seguro para a entrega de vacinas de ADN por via intradérmica. Aqui, um DNA que codifica EGFP plasmídeo é entregue por tatuagem na pele de um rato de laboratório, e a expressão de GFP em células da pele é então inspeccionado por microscopia confocal.

Pele a tatuagem como uma nova abordagem para a Campanha de Vacinação de DNA

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 visualizações.  New York University School of Medicine. Tatuagem pele é um modo poderoso e seguro para a entrega de vacinas de ADN por via intradérmica. Aqui, um DNA que codifica EGFP plasmídeo é entregue por tatuagem na pele de um rato de laboratório, e a expressão de GFP em células da pele é então inspeccionado por microscopia confocal.

Vídeo: 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.

Espremendo celular como uma plataforma de entrega intracelular Robust Microfluidic

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

Bioengenharia

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.

Um método aperfeiçoado para a preparação de colágeno tipo I Da Pele

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.

Caracterização de Sistemas Complexos Usando o Projeto de Experimentos Abordagem: Transient Protein Expression em tabaco como um Estudo de Caso

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.

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<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.

Microescala Electroporator para entrega Sequential Molecular assistida por Vortex

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.

Desenvolvimento de um modelo organotípicas multicelular tridimensional da Mucosa Intestinal Humano cultivadas sob microgravidade

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.

Estromais mesenquimais cultura de células e Entrega em condições autólogas: Uma abordagem inteligente para aplicações ortopédicas

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.

Nanotubos de DNA como uma ferramenta versátil para estudar semiflexível polímeros

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 da cartilagem e Analogs de tecido de pele utilizando um romance passiva unidade técnica de mistura para 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.