Este trabalho foi financiado por uma doação do Instituto Nacional de Artrite e Doenças Musculosqueléticas e da Pele: 3R01AR054458-02S2.

1. 1 dia 1) Placa de células <ol><li> HMSC placa em um frasco T75 em uma confluência de 80%, no mínimo, 18-24 horas antes da rotulagem. Consulte a Tabela 1 para a instrução para os navios alternativo. </li></ol> 2. Dia 2 2) Prepare solução de rotulagem. Esta preparação vai rotular um (1) T75 frasco a 80% confluência com uma concentração de 400 mcg Fe / ml. Consulte a Tabela 1 para a instrução para os navios alternativo. <ol><li> Criar uma solução (solução 1) através da mistura de 1 ml de soro livre de media e 93,1 mL de ferumoxytol (stock: 30 mg / ml) em um tubo de 15 ml. </li><li> Criar uma segunda solução (solução 2) através da mistura de 1 ml de soro livre de media e 7 mL de sulfato de protamina (stock: 10 mg / ml) em um tubo de 15 ml segundo cônico. </li><li> Permitir que cada solução em repouso por 5 minutos. </li><li> Adicionar soluções 1 e 2 juntos. Misture suavemente a solução de etiquetagem. Permitir que a solução descansar por 5 minutos para permitir USPIO pro-complexos sulfato de histamina a se formar. </li><li> Adicionar 5 ml de soro livre de mídia para o 2 ml misto de SPIO / solução de sulfato de protamina para um volume final de 7 ml. </li></ol> 3) Prepare células de rotulagem <ol><li> Aspirar a mídia a partir de células. </li><li> Lavar cuidadosamente as células uma vez com 2-3 ml de pré-aquecido Mg / Ca-livre D-PBS ou soro livre de mídia para enxaguar residual proteínas séricas e ou outros componentes de mídia que possam prejudicar a captação de contraste e eficiência rotulagem. Aspirar o líquido de lavagem. </li></ol> 4) células Etiqueta <ol><li> Adicione a 7 ml solução completa de rotulagem para as células. </li><li> Células lugar em uma incubadora (37 ° C / 5% CO 2) e permitir que as células para incubar na solução de rotulagem para 4 horas. </li><li> Adicionar 700 mL da FCS para a solução rotulagem das células para alcançar uma concentração final de soro de 10%. O soro é adicionado ao re-estabelecer o ambiente enriquecido do qual as células estão acostumados, e para ajudar na minimizing morte celular que poderia resultar de uma mudança abrupta para 24 horas de exposição a um ambiente sem soro. </li><li> Permitir que as células para incubar com a solução de etiquetagem por mais 20 horas. </li></ol> 3. Dia 3 5) Prepare células marcadas <ol><li> Aspirar a solução de rotulagem a partir de células. </li><li> Lave as células com 2-3 ml de pré-aquecido Mg / Ca-livre D-PBS. Aspirar o PBS. É importante o uso de Mg / Ca-free PBS como o Mg e Ca irá prejudicar a eficiência de Tripsina na próxima etapa. </li><li> Adicionar 2 ml de pré-aquecido tripsina 0,05% para as células e incline o frasco para trás e para garantir toda a superfície do recipiente é coberta com uma fina camada de Tripsina. Coloque as células em uma incubadora (37 ° C / 5% CO 2) e permitir que as células em repouso por 5-7 minutos ou até que as células começam a se destacar do prato. Pode ser necessário para confirmar o desapego sob um microscópio. Bata suavemente nas laterais do balão se necessário, para facilitar superfície celular desapego. </li><li> Adicionar 4 ml de pré-aquecido mídia completa para o frasco para neutralizar a tripsina. Gentilmente lavar o balão por pipetagem do Tripsina / media / solução célula para cima e para baixo várias vezes. Recolher toda a solução em um tubo de 15 ml. Centrifugar a solução de célula a 400 RCF por 5 minutos. </li><li> Aspirar cuidadosamente o sobrenadante sem perturbar o pellet celular. Ressuspender as células em 5 ml de mídia (soro contendo ou soro-free). Centrifugar a solução de célula a 400 RCF por 5 minutos. </li><li> Repita a lavagem das células descrito no passo 5.5. No total, as células serão lavadas três vezes para remover residual, agente de contraste livre na mídia e na superfície de células. </li><li> Contagem de células neste momento para atingir a contagem de células mais precisos, como as células serão perdidos durante a lavagem anterior. Realizar um teste de viabilidade das células. Células estão agora prontos para posterior análise e ou aplicação experimental. RM pode ser realizada com T1-w e<html> T2-w parâmetros, porque embora ferumoxytol é um agente de contraste T2-w, ferumoxytol também apresenta efeitos T1. Parâmetros representativos T2-w ressonância magnética que pode ser usado para a imagem ferumoxytol células marcadas incluem: FSE ou SE_Multislice seqüências com TR de 2000-2500 ms e um TE de 60-80 ms. Para adquirir uma imagem de T1-w, diminuir o valor do TE em uma seqüência FSE ou SE_Multislice ou usar uma seqüência GRE com uma TR de alta e baixa TE. A Figura 4 demonstra um potencial de aplicação vivo para imagens rotuladas com células-tronco. </li></ol> 4. Resultados representativos: Células marcadas demonstrar um efeito de contraste significativo escurecimento ou negativos em imagens T2-weighted MR e um efeito de contraste brilho ou positivo em imagens T1-weighted MR (Figura 2). Longitudinal ressonância magnética e testes de viabilidade realizados cinco dias de pós rotulagem demonstrou nenhum sinal de MR significativo e nenhum impacto significativo sobre a viabilidade em comparação com controles não identificados (dados não mostrados). jove_content &quot;&gt; células-tronco identificadas posteriormente pode ser diferenciada em vários tipos celulares ou por via intravenosa injetada na investigação vivo. <img alt="Figura 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> Figura 1. Linha do tempo Esquema de rotulagem células-tronco com nanopartículas de óxido de ferro, ferumoxytol. <img alt="Figura 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> Figura 2. As imagens de RM de secções sagitais thrrough eppendorf tubos com células em um pellet celular. A) Unlabeled controles. B) As células identificadas demonstrando uma significativa T2 ou efeito negativo agente de contraste em T2 FSE ou SE_Multislice de imagem e um efeito positivo ou T1 agente de contraste na seqüência GRE ponderada em T1. <img alt="Figura 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> Figura 3. Sagital seções de tubos de eppendorf com as células de pel celularpermite. Tão pouco quanto 10.000 ferumoxytol células marcadas podem ser detectadas através de ressonância magnética T2-w. <img alt="Figura 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> Figura 4. MR visualização de ferumoxytol marcado com células-tronco injetadas em camundongos ventrículos cerebrais. A) axial, imagem de MR barin murino sem injeção de USPIO marcado com células-tronco. B) axial, C) coronal, e D) imagens sagitais MR retratando o T2, efeito de contraste negativo de USPIO marcado com células-tronco (setas brancas) injetado em um cérebro de camundongos. <img alt="Tabela 1" src="/files/ftp_upload/3482/3482table1.jpg" /> Adaptações tabela 1. Quantidade de rotulagem células nos vasos alternativo.

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Todos os colaboradores deste estudo não oferecem divulgações.

Melhorar a eficácia da engraftments células-tronco é fundamental para o avanço da medicina regenerativa. A técnica de visualização não invasiva de células-tronco in vivo aumenta significativamente nossa capacidade de compreender os mecanismos que levam a resultados enxerto bem-sucedido. Rotulagem magnética para MR de visualização, como o procedimento que temos demonstrado, permite que no acompanhamento in vivo de células-tronco com ressonância magnética. Células-tronco magnetic...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> D-MEM glicose alta </td><td> Sigma </td><td> D5648 </td><td> Ou médio outra base para a linha desejada com células-tronco a ser utilizada </td></tr><tr><td> D-PBS (Ca + +, Mg + + livre) </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td> Tripsina-EDTA 0,05% </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> De soro fetal bovino (FBS) </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol (Feraheme) </td><td> AMAG </td><td> 59338-0775-01 </td><td></td></tr><tr><td> Sulfato de protamina </td><td> APP Pharm. </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

Terapias com células-tronco com base oferecem um potencial significativo para o campo da medicina regenerativa. No entanto, ainda há muito a ser entendido em relação ao vivo na cinética das células transplantadas. Um método não-invasivo para monitorar repetidamente células-tronco transplantadas in vivo permitiria que investigadores monitorar diretamente os transplantes de células-tronco e identificar resultados pega bem ou mal sucedidas. Uma ampla gama de células-tronco continua a ser investigada para inúmeras aplicações. Este protocolo centra-se em três populações de células-tronco diferentes: rim embrionário humano 293 (HEK293) células, as células-tronco mesenquimais (hMSC) e tronco pluripotentes induzidas (iPS) células. HEK 293 células são derivadas de células embrionárias do rim cresceram em cultura com sheared DNA do adenovírus 5. Estas células são amplamente utilizados nas pesquisas, pois são facilmente cultivadas, crescem rapidamente e são facilmente transfectadas. hMSCs são encontradas na medula adulta. Estas células can ser replicado como células indiferenciadas, mantendo multipotency ou o potencial de se diferenciar em um número limitado de destinos celular. hMSCs pode diferenciar a linhagens de tecidos mesenquimais, incluindo osteoblastos, adipócitos, condrócitos, tendão, músculo e estroma da medula. As células iPS são células adultas reprogramadas geneticamente que foram modificados para expressar genes e fatores semelhantes às propriedades definidoras das células-tronco embrionárias. Estas células são pluripotentes significado que eles têm a capacidade de se diferenciar em todas as linhagens de células 1. Ambos hMSCs e células iPS têm demonstrado capacidade regenerativa dos tecidos in vivo. A ressonância magnética (RM), juntamente com o uso de óxido de ferro superparamagnético (SPIO) rótulos de células de nanopartículas têm se mostrado eficazes no controle in vivo de células-tronco devido à resolução anatômica perto microscópica, um sangue mais meia-vida, que permite imagens longitudinal e sensitivit altay para a detecção de células fornecido por ressonância magnética de nanopartículas SPIO 2-4. Além disso, a RM com o uso de SPIOs é clinicamente traduzíveis. SPIOs são compostos de um núcleo de óxido de ferro com um revestimento superficial dextran, carboxydextran ou amido que serve para conter o núcleo de ferro a partir de componentes bioreactive plasma. Estes agentes locais criar inomogeneidades do campo magnético que levam a uma diminuição do sinal em imagens de T2-weighted MR 5. Infelizmente, SPIOs não são mais fabricados. De segunda geração, ultrasmall SPIOs (USPIO), no entanto, oferecer uma alternativa viável. Ferumoxytol (FerahemeTM) é uma USPIO composto por um núcleo de magnetita não estequiométrica rodeado por uma camada carboxymethylether polyglucose sorbitol. O tamanho coloidal de partículas de 17-30 nm ferumoxytol é determinado por espalhamento de luz. O peso molecular é 750 kDa, ea relaxação constante em 2T campo MRI é 58,609 mM-1 sec-1 de força 4. Ferumoxytol foi recentemente aprovado pelo FDA ums um suplemento de ferro para o tratamento da deficiência de ferro em pacientes com insuficiência renal 6. Nosso grupo tem aplicado este agente em um &quot;off label&quot; uso para aplicações de etiquetagem celular. Nossa técnica demonstra rotulagem eficiente de células-tronco com ferumoxytol que leva a um impacto significativo no sinal MR de células marcadas em imagens de RM. Esta técnica pode ser aplicada para não-invasivos de monitoramento de terapias com células-tronco em ambientes pré-clínicos e clínicos.

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Nós descrevemos uma técnica para rotulagem e rastreamento de células-tronco com FDA-approved, óxido de ferro superparamagnético (SPIO), ferumoxytol (Feraheme). Esta técnica de imagem celular que utiliza a ressonância magnética (RM) para visualização, é facilmente acessível a longo prazo de monitoramento e diagnóstico de engraftments tronco bem ou mal sucedidas de células em pacientes.

Rotulagem células-tronco com Ferumoxytol, uma FDA-Approved nanopartículas de óxido de ferro

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

labeling-stem-cells-with-ferumoxytol-an-fda-approved-iron-oxide

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20.7K visualizações.  Molecular Imaging Program at Stanford (MIPS). Nós descrevemos uma técnica para rotulagem e rastreamento de células-tronco com FDA-approved, óxido de ferro superparamagnético (SPIO), ferumoxytol (Feraheme). Esta técnica de imagem celular que utiliza a ressonância magnética (RM) para visualização, é facilmente acessível a longo prazo de monitoramento e diagnóstico de engraftments tronco bem ou mal sucedidas de células em pacientes.

Vídeo: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicina

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inchaço na Organóides intestinal induzida por forscolina: An<em&gt; In Vitro</em&gt; Ensaio para a Avaliação de Medicamentos Response na Fibrose Cística Os pacientes

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

Contínua transfusão de câmbio manual para pacientes com doença falciforme: um método eficiente para evitar a sobrecarga de ferro

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Síntese, Caracterização e Aplicação de Nanoprobes superparamagnéticos de óxido de ferro para detecção de tuberculose extrapulmonar

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Nanopartículas de óxido de ferro revestido de Polyethyleneimine como um veículo para a entrega de RNA de interferência pequeno para macrófagos In Vitro e In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Um método sem enzimas para isolamento e expansão de células-tronco mesenchymal derivadas do adiposo humano

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Rastreamento de células-tronco mesenchymal rotuladas por óxido de ferro superparamagnético usando ressonância magnética após parto intranasal em um modelo de urina de urina de lesão cerebral traumática

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Estimativa de nanocristais urinários em humanos usando rotulagem fluorófora de cálcio e análise de rastreamento de nanopartículas

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Medição do teor de ferro não-heme do tecido usando um ensaio colorimétrico baseado em banhontrolina

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...