RESUMO
Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.
Assuntos
Bioimpressão , Células Progenitoras Endoteliais , Engenharia Tecidual , Alicerces Teciduais , Humanos , Bioimpressão/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Hidrogéis/química , Capilares/fisiologia , Alginatos/química , Impressão TridimensionalRESUMO
Primary hepatocytes are essential cellular resources for drug screening and medical transplantation. While culture systems have already succeeded in reconstituting the biomimetic microenvironment of primary hepatocytes, acquiring additional capabilities to handle them easily as well as to expand them remains unmet needs. This paper describes a culture system for primary rat hepatocytes, based on cell fiber technology, that brings scalability and handleability. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium via the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated their proliferation while maintaining their viability and their hepatic specific functions for up to thirty days of subsequent culture. We then demonstrated the efficiency of proliferating primary rat hepatocytes in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Our culture system could therefore be included in innovative strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.
Assuntos
Hepatócitos , Hidrogéis , Animais , Proliferação de Células , Células Cultivadas , Meios de Cultura , RatosRESUMO
Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate, and organize into branched tubular structures with walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space, and show markers of epithelial maturation such as primary cilia and continuous tight junctions. The resulting structures have dimensions typically found in the intralobular and intrahepatic biliary tree and are stable for weeks, without any requirement of bulk supporting material, thereby offering total access to the external side of these biliary epithelial constructs.
Assuntos
Sistema Biliar , Estereolitografia , Animais , Sistema Biliar/diagnóstico por imagem , Células Epiteliais , Hidrogéis , RatosRESUMO
In esophageal pathologies, such as esophageal atresia, cancers, caustic burns, or post-operative stenosis, esophageal replacement is performed by using parts of the gastrointestinal tract to restore nutritional autonomy. However, this surgical procedure most often does not lead to complete functional recovery and is instead associated with many complications resulting in a decrease in the quality of life and survival rate. Esophageal tissue engineering (ETE) aims at repairing the defective esophagus and is considered as a promising therapeutic alternative. Noteworthy progress has recently been made in the ETE research area but strong challenges remain to replicate the structural and functional integrity of the esophagus with the approaches currently being developed. Within this context, 3D bioprinting is emerging as a new technology to facilitate the patterning of both cellular and acellular bioinks into well-organized 3D functional structures. Here, we present a comprehensive overview of the recent advances in tissue engineering for esophageal reconstruction with a specific focus on 3D bioprinting approaches in ETE. Current biofabrication techniques and bioink features are highlighted, and these are discussed in view of the complexity of the native esophagus that the designed substitute needs to replace. Finally, perspectives on recent strategies for fabricating other tubular organ substitutes via 3D bioprinting are discussed briefly for their potential in ETE applications.
Assuntos
Bioimpressão , Esôfago/cirurgia , Impressão Tridimensional , Qualidade de Vida , Engenharia Tecidual , Alicerces TeciduaisRESUMO
This review summarizes recent advances in micro- and nanopore technologies with a focus on the functionalization of pores using a promising method named contactless electro-functionalization (CLEF). CLEF enables the localized grafting of electroactive entities onto the inner wall of a micro- or nano-sized pore in a solid-state silicon/silicon oxide membrane. A voltage or electrical current applied across the pore induces the surface functionalization by electroactive entities exclusively on the inside pore wall, which is a significant improvement over existing methods. CLEF's mechanism is based on the polarization of a sandwich-like silicon/silicon oxide membrane, creating electronic pathways between the core silicon and the electrolyte. Correlation between numerical simulations and experiments have validated this hypothesis. CLEF-induced micro- and nanopores functionalized with antibodies or oligonucleotides were successfully used for the detection and identification of cells and are promising sensitive biosensors. This technology could soon be successfully applied to planar configurations of pores, such as restrictions in microfluidic channels.
Assuntos
Técnicas Biossensoriais , Silício/química , Impedância Elétrica , Técnicas Eletroquímicas , Membranas Artificiais , NanoporosRESUMO
Technical progress in materials science and bioprinting has for the past few decades fostered considerable advances in medicine. More recently, the understanding of the processes of self-organization of cells into three-dimensional multicellular structures and the study of organoids have opened new perspectives for tissue engineering. Here, we review microengineering approaches for building functional tissues, and discuss recent progress in the understanding of morphogenetic processes and in the ability to steer them in vitro. On the basis of biological and technical considerations, we emphasize the achievements and remaining challenges of bringing together microengineering and morphogenesis. Our viewpoint underlines the importance of cellular self-organization for the success of tissue engineering in therapeutic applications. We reason that directed self-organization, at the convergence of microengineering and cellular self-organization, is a promising direction for the manufacturing of complex functional tissues.
Assuntos
Bioimpressão/métodos , Morfogênese , Organoides/fisiologia , Engenharia Tecidual/métodos , Animais , Humanos , Tamanho do Órgão , Células-Tronco/fisiologiaRESUMO
Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 µm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.
Assuntos
Microscopia de Vídeo/métodos , Imagem com Lapso de Tempo/métodos , Adesão Celular , Técnicas de Cultura de Células , Movimento Celular , Proliferação de Células , Células Cultivadas , HumanosRESUMO
To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.
Assuntos
Técnicas Citológicas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Actinas/antagonistas & inibidores , Actinas/metabolismo , Adesão Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Miosinas/antagonistas & inibidores , Miosinas/metabolismo , Coloração e Rotulagem/métodosRESUMO
Planar patch-clamp is a two-dimensional variation of traditional patch-clamp. By contrast to classical glass micropipette, the seal quality of silicon patch-clamp chips (i.e. seal resistance and seal success rate) have remained poor due to the planar geometry and the nature of the substrate and thus partially obliterate the advantages related to planar patch-clamp. The characterization of physical parameters involved in seal formation is thus of major interest. In this paper, we demonstrate that the physical characterization of surfaces by a set of techniques (Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), surface energy (polar and dispersive contributions), drop angles, impedance spectroscopy, combined with a statistical design of experiments (DOE)) allowed us discriminating chips that provide relevant performances for planar patch-clamp analysis. Analyses of seal quality demonstrate that dispersive interactions and micropore size are the most crucial physical parameters of chip surfaces, by contrast to surface roughness and dielectric membrane thickness. This multi-scale study combined with electrophysiological validation of chips on a diverse set of cell-types expressing various ion channels (IRK1, hERG and hNa(v)1.5 channels) unveiled a suitable patch-clamp chip candidate. This original approach may inspire novel strategies for selecting appropriate surface parameters dedicated to biochips.
Assuntos
Microeletrodos , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos , Silício/química , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Canais Iônicos/metabolismo , Teste de Materiais , Propriedades de SuperfícieRESUMO
Customized pores are smart components that find challenging applications in a variety of fields including purification membranes and biosensing systems. The incorporation of recognition probes within pores is therefore a challenge, due to the technical difficulty of delimiting the area functionalized and obtaining the localized, specific chemical modification of pore walls. An innovative approach, named contactless electrofunctionalization (CLEF), is presented to overcome this problem. CLEF allows easy, one-step modification of the inner surface of a pore etched in a dielectric membrane. The pore wall is coated under the influence of an electric field created by the application of a voltage between two electrodes, located near but not in contact with the pore openings. This specific localization of the deposited material within the pore is extremely rapid. Coatings were reliably and reproducibly obtained using polypyrrole co-polymers bearing oligonucleotides, demonstrating that this technology has a promising future in the design of biosensors. Moreover, the versatility of this process allows the deposition of various electroactive entities such as iridium oxide and therefore indicates a strong potential for diverse applications involving porous materials.
Assuntos
Membranas/química , Nanotecnologia/métodos , Modelos Teóricos , PorosidadeAssuntos
Inibidores Enzimáticos , Análise Serial de Proteínas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Hidrazinas/química , Estrutura Molecular , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tensão Superficial , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The original micropatterning technique on gold, although very efficient, is not accessible to most biology labs and is not compatible with their techniques for image acquisition. Other solutions have been developed on silanized glass coverslips. These methods are still hardly accessible to biology labs and do not provide sufficient reproducibility to become incorporated in routine biological protocols. Here, we analyzed cell behavior on micro-patterns produced by various alternative techniques. Distinct cell types displayed different behavior on micropatterns, while some were easily constrained by the patterns others escaped or ripped off the patterned adhesion molecules. We report methods to overcome some of these limitations on glass coverslips and on plastic dishes which are compatible with our experimental biological applications. Finally, we present a new method based on UV crosslinking of adhesion proteins with benzophenone to easily and rapidly produce highly reproducible micropatterns without the use of a microfabricated elastomeric stamp.
Assuntos
Técnicas de Cultura de Células/métodos , Vidro/química , Nanotecnologia , Polímeros/química , Silanos/química , Raios Ultravioleta , Benzofenonas/química , Adesão Celular , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Células HeLa , Humanos , Polietilenoglicóis/química , Polilisina/química , Poliestirenos/química , Proteínas/química , Propriedades de Superfície , TemperaturaRESUMO
BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework.
Assuntos
Bioensaio/métodos , Nanoestruturas , Testes de Toxicidade/métodos , Animais , Arseniatos/farmacologia , Bioensaio/instrumentação , Cloreto de Cádmio/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Poluentes Ambientais/farmacologia , Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Herbicidas/farmacologia , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Paraquat/farmacologia , Regiões Promotoras Genéticas , Testes de Toxicidade/instrumentaçãoRESUMO
Successful development of cell-on-chip microsystems where living cells are deposited and grown in microfabricated structures is highly dependent on the control of cell/substrate interactions. In this study, several materials of interest were tested for CHO cell growth and morphology: (i) glass, fibronectin-, poly-L-lysine- and 3-aminopropyltriethoxysilane (APTES)--treated glass and UV/O(3)-modified PDMS coating on glass as well as (ii) silicon, poly-L-lysine-, APTES-, O(2) plasma-treated and oxide-coated silicon. In addition, we quantitatively characterized cell adhesion to these substrates using a radial flow detachment assay. Lack of correlation between cell adhesion and cell morphology was systematically observed for all substrates. In particular, we show that PDMS coatings on glass can be finely tuned by UV/O(3) treatment to enhance cell adhesion and induce elongated morphology. Moreover, we observed a low shear stress cell detachment mechanism on silicon oxide coatings on silicon wafers. It is therefore possible with these coatings to selectively influence either cell adhesion or morphology.
Assuntos
Células CHO/citologia , Materiais Revestidos Biocompatíveis , Vidro , Polímeros , Animais , Células CHO/fisiologia , Adesão Celular/fisiologia , Cricetinae , Cricetulus , SilícioRESUMO
The synthesis of a new type of fluorogenic ester substrates is described. Prepared from fluorescein in three steps with common commercially available precursors, they all generate bright green fluorescence upon proteolysis. Their particular structure allows the same substrate be used to report enzymatic activity of various proteases from serine and cysteine superfamilies. The substrate cleavage is sensitive to specific protease inhibitors providing a tool for inhibitor screening.
Assuntos
Ésteres/química , Ésteres/metabolismo , Fluoresceína/química , Peptídeo Hidrolases/metabolismo , Ésteres/síntese química , Cinética , Estrutura Molecular , Especificidade por SubstratoRESUMO
Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.
Assuntos
Canais Iônicos/fisiologia , Técnicas de Patch-Clamp/métodos , Dióxido de Silício/química , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Canais Iônicos/genética , Potenciais da Membrana/fisiologia , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Técnicas de Patch-Clamp/instrumentação , Plasmídeos/genética , Reprodutibilidade dos Testes , TransfecçãoRESUMO
An efficient synthesis of new type fluorescent amino acids is described. The Fmoc-protected dyes can be prepared in a four-step procedure with approximately 30% overall yield from aminofluoresceins and other inexpensive commercially available precursors. The dyes are much more photostable compared to fluorescein and exhibit constant pH-independent fluorescence that is advantageous in biological applications. The Fmoc-protected fluorescent amino acids are ready for use in solid phase peptide synthesis. As a proof of concept, a fluorogenic papain substrate was synthesized and employed for on-bead detection of the protease activity. By using a novel technique for quantitative analysis of bead fluorescence, a approximately 2.7-fold increase in mean bead brightness was measured and was attributed to substrate cleavage by papain. The new type fluorescent amino acids seem to be a promising tool for the synthesis of fluorescent peptide ligands and fluorogenic protease substrates.
Assuntos
Aminoácidos/metabolismo , Fluoresceína/química , Peptídeo Hidrolases/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Especificidade por SubstratoRESUMO
Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.
Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Eletroforese em Microchip/métodos , Proliferação de Células , Sobrevivência Celular , Tamanho da AmostraRESUMO
A simple way for photochemical patterning of biological molecules onto the inner wall of fused-silica capillary is described. The method is based on a modification of the inner capillary surface with photoactive benzophenone (BP) derivative. The UV irradiation at 365 nm of the capillary filled with a sample solution results in cross-linking of the solutes to the BP moiety via a stable covalent bond. As a proof of concept, oligonucleotides and proteins were arrayed inside the capillary using an inverted microscope as an irradiation device. We demonstrated that the capillary arrays produced in this way are functional and could be used in different bioassays including DNA hybridization, protein interaction studies, and immunoassays. Having a sensitivity comparable to the fluorophore-based assays in a planar format, the capillary array possesses several advantages including submicroliter sample volume and a short assay time. The capillary format should therefore be considered as a possible alternative to a planar format in a number of low-density array applications such as mutation detection and diagnostic immunoassays.