Este trabalho foi apoiado por uma bolsa de investigação dos Institutos Nacionais de Saúde (HL068915 para DBC), uma nova Researcher Award do Fundo de Investigação Thrasher (a PAC), um Grant-in-Aid da American Heart Association (12GRNT11910008 para DBC) , uma bolsa de investigação da Fundação das Crianças do Coração (para DBC) e doações para Hospital Cardiológico Fundo das Crianças de Boston Condução, a família de Ryan Endowment, e por David Pullman.

Aqui vamos demonstrar o isolamento de COL1 de pele de cordeiro. Como escrito, este protocolo pode também ser utilizado com êxito para isolar COL1 de coelho e pele humana. 1. Prepare dérmica Amostra <ol><li> Equilibrar todos os reagentes a 4 ° C antes de usar. </li><li> Lavar amostra dérmica (25-50 g) em gelada dH 2 O e remover qualquer lã, pele, cabelo ou com creme depilatório. </li><li> Use uma lâmina de barbear de ponta única para raspar a limpo amostra de tecido conjuntivo e de gordura. </li><li> Lavar a amostra em gelada dH 2 O. </li><li> Corte a amostra de pele em 1 cm x 1 centímetro pedaços com uma lâmina de barbear de ponta única. </li></ol> 2. Remover noncollagenous solubilizado de materiais <ol><li> Pesar 5 g de amostra por tubo de 50 ml e adicionar 30 ml de 0,5 M de acetato de sódio gelada. </li><li> Misturar os tubos durante 1 minuto na configuração 6 m / seg utilizando uma placa de tubo de 50 ml no homogeneizador de bancada. </li><li> Descarte supernatant e repita para um total de 7 ciclos de lavagem de acetato de sódio. </li><li> Lavar a amostra em gelada dH 2 O e misture uma vez para remover o acetato de sódio residual. </li><li> Usar uma espátula para comprimir a amostra de encontro ao lado do tubo para remover o excesso de líquido e, em seguida, transferir para um tubo de 50 ml fresco. </li></ol> 3. Colágeno tipo I Extração <ol><li> Tampão citrato de lavar a amostra duas vezes em 2 ml / g de sódio 0,075 M, durante 1 min a 6 m / s na bancada homogeneizador comprimindo a amostra e descartando o sobrenadante depois de cada lavagem. </li><li> Adicionar a 2 ml / g nova porção de tampão de citrato de sódio 0,075 M à amostra. </li><li> Realizar 6 seqüencial 1 min, 6 m / s ciclos de bancada mix homogeneizador de agitação, sem retirar o tampão entre cada ciclo. </li><li> Transfira a espessura, o sobrenadante claro para um tubo de coleta. </li><li> Adicionar um tampão de citrato de sódio 0,075 M adicional de 1 ml / g para a amostra e realizar uma última bancada homogeneizador agitaciclo ção. </li><li> Adicionar este sobrenadante final para um tubo de recolha. </li><li> Centrifuga-se o tubo de colheita de 3.200 xg durante 10 min a 4 ° C. </li><li> Transferir o sobrenadante para o topo do compartimento de um peso molecular de 100.000 cortado dispositivo de filtração centrífuga. </li><li> Centrifugar a 3.200 xg durante 30 min a 4 ° C. </li><li> Transferir a COL1 purificada a partir do compartimento superior para um tubo cónico limpo e armazenar a 4 ° C. </li></ol>

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Os autores não têm concorrentes interesses financeiros para divulgar.

Um componente importante do presente método é a compressão eficaz repetida da amostra cutânea para remover o excesso de líquido. É essencial para remover tanto quanto possível, de acetato de sódio no passo de 2,5 por compressão da amostra. Usamos uma espátula para compactar a pele contra o lado do tubo, no entanto, gaze também pode ser usado, embora isto exigir a remoção da amostra do tubo e aumentar o tempo necessário para realizar estes passos. Da mesma forma, é importante para comprimir a amostra no pa...

Durante décadas, os investigadores e os fornecedores comerciais isolaram COL1 solubilizado a partir de uma variedade de fontes de tecidos, incluindo a pele e tendões usando uma variação de um protocolo de extracção de ácido simples, seguido de neutralização, o que resulta numa libertação de uma matriz de fibrilas COL1 organizados que podem ser utilizados para uma infinidade de aplicações biomédicas 1-4. Embora existam muitos exemplos de aplicações clínicas para COL1, alguns deles empregam COL1 autologously derivados porque a preparação desta proteína requer longas extrações levando dias ou semanas para realizar 5-7. Como resultado, os investigadores e médicos investigação utilizam geralmente caros, preparações comerciais de COL1 que são preparados usando os tecidos não-humanas, tais como rato, bovino, porcino ou cório ou usando pele retiradas de cadáveres ou depois da circuncisão masculina. Para aplicações regenerativas, muitos destes produtos apresentam variabilidade no que diz respeito à sua capacidade para formar sólidomatrizes ou construções de tecidos capazes de suportar a ligação de células e o crescimento. Por conseguinte, existe uma grande necessidade de um novo método normalizado concebido para extrair rapidamente COL1 a partir de fontes de tecidos autólogos acessíveis e abundantes. Tal método poderia economizar tempo e dinheiro no ambiente de pesquisa, onde COL1 poderia ser extraído do modelo animal de interesse e utilizado para testes pré-clínicos de tecidos artificiais montados em andaimes à base de colágeno. Ao mesmo tempo, ter a opção de usar um rapidamente isolada, COL1 autologously derivado na clínica seria aumentar a segurança geral dos pacientes que, de outra forma recebem alogénicas ou xenogénicas preparações. Para os pacientes que receberam uma preparação autogeneic, este método simplificado seria reduzir bastante o intervalo entre a coleta de biópsia e aplicação de colágeno. Por estas razões, buscamos aperfeiçoar um método de isolamento e purificação COL1 estabelecido há muito tempo usando ag de alta velocidadeacreditação e de exclusão de tamanho de centrifugação. Este método é simples de executar, usando soluções padrão e equipamentos encontrados em muitos laboratórios. Ao empregar a agitação de alta velocidade, o nosso protocolo reduz o tempo necessário para isolar solúvel, dérmica COL1 de aproximadamente 10 dias para menos de 3 horas 8. Importante, este método pode ser facilmente realizada no cenário clínico, a fim de preparar COL1 autologously-derivado para utilização em doentes durante um único conjunto de procedimentos.

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
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			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
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an improved method for the preparation of type i collagen from skin

Tipo 1 Solúvel colagénio (COL1) é amplamente utilizado como um substrato adesivo para culturas de células e como um andaime para aplicações de regeneração celular. Clinicamente, esta proteína é amplamente utilizado para a cirurgia estética, injeções dérmicas, enxerto ósseo e cirurgia reconstrutiva. As fontes de COL1 para estes processos são geralmente não-humanos, o que aumenta o potencial para a inflamação e a rejeição, bem como a transmissão da doença de xenobióticos. Em vista disto, um método eficiente e rápido para purificar COL1 de quantidades limitadas de tecidos autologously derivados iria contornar muitos destes problemas, no entanto, os protocolos de isolamento convencionais são demorados e muitas vezes necessitam de grandes quantidades de tecidos colagenosos. Aqui, demonstramos um método de extracção COL1 eficiente que reduz o tempo necessário para isolar e purificar a proteína a partir de cerca de 10 dias para menos de 3 horas. Escolhemos a derme como a fonte de tecido devido à sua disponibilidade durante muitos procedimentos cirúrgicos. Tseu método usa buffers de extração tradicionais combinados com agitação vigorosa e filtração centrífuga para obter altamente pura, COL1 solúvel de pequenas quantidades de cório. Resumidamente, as biópsias da derme são lavados em gelada dH2O após a remoção de gordura, tecido conectivo, e cabelo. As amostras de pele são retirados de proteínas e polissacarídeos noncollagenous usando 0,5 M de acetato de sódio e uma alta velocidade de bancada homogeneizador. O colagénio de sólidos residuais é subsequentemente extraído com um tampão de citrato de sódio 0,075 M com o homogeneizador. Estes extractos são purificados usando 100.000 MW de corte filtros centrífugos que produzem preparações COL1 de qualidade comparável ou superior aos produtos comerciais ou aquelas obtidas através de procedimentos tradicionais. Prevemos que este método vai proporcionar a utilização dos derivados de autologously COL1 para uma multiplicidade de pesquisa e aplicações clínicas.

Este protocolo de isolamento COL1 requer cerca de 2 horas e 15 minutos para ser concluído. A Figura 1 mostra um diagrama esquemático que representa as principais etapas deste processo. Preparação da amostra e realizando os 7 ciclos de agitações de alta velocidade e lavagens com acetato de sódio leva cerca de 35 minutos (Figuras 1A-C). Realizando uma agitação e enxágüe ciclo com dH2O demora cerca de 5 min (Figuras 1D e 1E). A adição de citrato de ...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

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

Procedimentos tradicionais para o isolamento do tipo solúvel 1 colágeno (col1) requerem cerca de 10 dias do início ao fim por causa de incubações tampão longas e trabalhosas resuspensions de fibrilas. Aqui, nós descrevemos um meio para purificar a partir de pequenas biópsias COL1 dérmicos em menos de 3 horas.

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. ...

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Research

JoVE Journal

Bioengineering

22.7K visualizações.  Boston Children's Hospital.  Procedimentos tradicionais para o isolamento do tipo solúvel 1 colágeno (col1) requerem cerca de 10 dias do início ao fim por causa de incubações tampão longas e trabalhosas resuspensions de fibrilas. Aqui, nós descrevemos um meio para purificar a partir de pequenas biópsias COL1 dérmicos em menos de 3 horas. 

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

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotípica Colágeno Ensaio I: Uma Plataforma maleável para Avaliar Comportamento celular em um contexto de 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Bioengenharia

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Melhoria da Metodologia para a Elaboração de um baseado em celular, Contact Modelo Humano da barreira hemato-encefálica

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Um método melhorado Ensaios Mecânicos para Avaliar osso-implante Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

O tecido triturado em Compressed Collagen: Um BioTransplant contendo celular para o reparo Reconstructive Single-encenado

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Avaliação da proliferação queratinócito em duas e três dimensões tipo I colágeno substratos

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantificação simultânea de kynureninas selecionadas analisadas por espectrometria de cromatografia líquida-massa em média coletada de culturas celulares cancerosas

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Um novo método de injeção lenta minimamente invasivo para reduzir a lesão tecidual

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Autor em destaque: um novo método de injeção de células com invasão mínima  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Um novo método de injeção de células com invasão mínima

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...