RESUMO
The on-demand printing of living cells using inkjet technologies has recently been demonstrated and allows for the controlled deposition of cells in microarrays. Here, we show that such arrays can be interrogated directly by robot-controlled liquid microextraction coupled with chip-based nanoelectospray mass spectrometry. Such automated analyses generate a profile of abundant membrane lipids that are characteristic of cell type. Significantly, the spatial control in both deposition and extraction steps combined with the sensitivity of the mass spectrometric detection allows for robust molecular profiling of individual cells.
Assuntos
Bioimpressão/métodos , Tinta , Metabolismo dos Lipídeos , Análise de Célula Única/métodos , Análise Serial de Tecidos/métodos , Animais , Bioimpressão/instrumentação , Linhagem Celular , Espectrometria de Massas , Camundongos , Nanotecnologia , Ratos , Robótica , Análise de Célula Única/instrumentação , Análise Serial de Tecidos/instrumentaçãoRESUMO
In the present work we have revisited the application of quantitative ultrasound imaging (QUI) to cellular hydrogels, by using the reference phantom method (RPM) in combination with a local attenuation compensation algorithm. The investigated biological samples consisted of cell-laden collagen hydrogels with PC12 neural cells. These cell-laden hydrogels were used to calibrate the integrated backscattering coefficient (IBC) as a function of cell density, which was then used to generate parametric images of local cell density. The image resolution used for QUI and its impact on the relative IBC error was also investigated. Another important contribution of our work was the monitoring of PC12 cell proliferation. The cell number estimates obtained via the calibrated IBC compared well with data obtained using a conventional quantitative method, the MTS assay. Evaluation of spectral changes as a function of culture time also provided additional information on the cell cluster size, which was found to be in close agreement with that observed by microscopy. Last but not least, we also applied QUI on a 3D printed cellular construct in order to illustrate its capabilities for the evaluation of bioprinted structures. STATEMENT OF SIGNIFICANCE: While there is intensive research in the areas of polymer science, biology, and 3D bio-printing, there exists a gap in available characterisation tools for the non-destructive inspection of biological constructs in the three-dimensional domain, on the macroscopic scale, and with fast data acquisition times. Quantitative ultrasound imaging is a suitable characterization technique for providing essential information on the development of tissue engineered constructs. These results provide a detailed and comprehensive guide on the capabilities and limitations of the technique.
Assuntos
Hidrogéis/química , Processamento de Imagem Assistida por Computador , Impressão Tridimensional , Alicerces Teciduais/química , Animais , Células PC12 , Ratos , UltrassonografiaRESUMO
3D bio-nanofibrous polypyrrole/poly(styrene-beta-isobutylene-beta-styrene) mats, prepared via a vapor-phase polymerisation modified electrospinning process, provide excellent platforms for PC12 cells attachment and growth, indicating potential applications in areas requiring good mass transport such as nerve growth guidance channels.
Assuntos
Técnicas de Cultura de Células/instrumentação , Estirenos/química , Animais , Materiais Biocompatíveis , Soluções Tampão , Adesão Celular , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanotecnologia , Oxidantes/química , Células PC12 , Ratos , Análise Espectral RamanRESUMO
Carbon nanotubes present a new material for the construction of electrodes for electrochemical devices such as batteries, capacitors, and actuators. Such electrodes require high conductivity, strength, and surface area. The latter two requirements are often incompatible. Electrodes composed entirely of carbon nanotubes (bucky paper) have high surface areas but are typically weak, and have insufficient conductivity for practical macroscopic applications. Here we report a technique that uses naturally occurring biopolymers to produce electrodes (free standing films) that exhibit conductivities of 300 S/cm. These composites also have considerable mechanical strength (up to 145 MPa) and sufficient specific capacitance of 19-27 F/g to enable them to be used as freestanding electrodes. One potential application that deserves special attention is that of biocompatible electrodes, where the binder is a biopolymer already used in a range of implants. Preliminary studies reported here show that the new carbon nanotube biopolymer electrodes can foster prolific L929 cell growth.
Assuntos
Quitosana/química , DNA de Cadeia Simples/química , Microeletrodos , Nanotubos de Carbono/química , Animais , Biopolímeros/química , Linhagem Celular , Proliferação de Células , Capacitância Elétrica , CamundongosRESUMO
Neuromuscular junctions (NMJ) are specialized synapses that link motor neurons with muscle fibers. These sites are fundamental to human muscle activity, controlling swallowing and breathing amongst many other vital functions. Study of this synapse formation is an essential area in neuroscience; the understanding of how neurons interact and control their targets during development and regeneration are fundamental questions. Existing data reveals that during initial stages of development neurons target and form synapses driven by biophysical and biochemical cues, and during later stages they require electrical activity to develop their functional interactions. The aim of this study was to investigate the effect of exogenous electrical stimulation (ES) electrodes directly in contact with cells, on the number and size of acetylcholine receptor (AChR) clusters available for NMJ formation. We used a novel in vitro model that utilizes a flexible electrical stimulation system and allows the systematic testing of several stimulation parameters simultaneously as well as the use of alternative electrode materials such as conductive polymers to deliver the stimulation. Functionality of NMJs under our co-culture conditions was demonstrated by monitoring changes in the responses of primary myoblasts to chemical stimulants that specifically target neuronal signaling. Our results suggest that biphasic electrical stimulation at 250Hz, 100µs pulse width and current density of 1mA/cm2 for 8h, applied via either gold-coated mylar or the conductive polymer PPy, significantly increased the number and size of AChRs clusters available for NMJ formation. This study supports the beneficial use of direct electrical stimulation as a strategic therapy for neuromuscular disorders. STATEMENT OF SIGNIFICANCE: The beneficial effects of electrical stimulation (ES) on human cells in vitro and in vivo have long been known. Although the effects of stimulation are clear and the therapeutic benefits are known, no uniform parameters exist with regard to the duration, frequency and amplitude of the ES. To this end, we are answering several important questions on the parameters for ES of nerve and muscle monocultures and co-cultures by probing the effects on the enhancement of acetylcholine receptors (AChR) clustering available for neuromuscular junction formation using a conductive platform. This work opens the possibility to combine electrical stimulus delivered via conductive polymer substrates, from which biomolecules could also be delivered, providing opportunities to further enhance the therapeutic effect.
Assuntos
Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Técnicas de Cocultura , Estimulação Elétrica , Eletrodos , Teste de Materiais , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Músculos/metabolismo , Tecido Nervoso/metabolismoRESUMO
The demands of multifunctional scaffolds have exceeded the passive biocompatible properties previously considered sufficient for tissue engineering. Herein, a novel and facile method used to fabricate a core-shell structure consisting of a conducting fiber core and an electrospun fiber shell is presented. This multifunctional structure simultaneously provides the high conductivity of conducting polymers as well as the enhanced interactions between cells and the sub-micron topographical environments provided by highly aligned cytocompatible electrospun fibers. Unlimited lengths of PEDOT:PSS-Chitosan-PLGA fibers loaded with an antibiotic drug, ciprofloxacin hydrochloride, were produced using this method. The fibers provide modulated drug release with excellent mechanical properties, electrochemical performance and cytocompatibility, which hold great promise for the application of conductive electrospun scaffolds in regenerative medicine.
RESUMO
The brain is an enormously complex organ structured into various regions of layered tissue. Researchers have attempted to study the brain by modeling the architecture using two dimensional (2D) in vitro cell culturing methods. While those platforms attempt to mimic the in vivo environment, they do not truly resemble the three dimensional (3D) microstructure of neuronal tissues. Development of an accurate in vitro model of the brain remains a significant obstacle to our understanding of the functioning of the brain at the tissue or organ level. To address these obstacles, we demonstrate a new method to bioprint 3D brain-like structures consisting of discrete layers of primary neural cells encapsulated in hydrogels. Brain-like structures were constructed using a bio-ink consisting of a novel peptide-modified biopolymer, gellan gum-RGD (RGD-GG), combined with primary cortical neurons. The ink was optimized for a modified reactive printing process and developed for use in traditional cell culturing facilities without the need for extensive bioprinting equipment. Furthermore the peptide modification of the gellan gum hydrogel was found to have a profound positive effect on primary cell proliferation and network formation. The neural cell viability combined with the support of neural network formation demonstrated the cell supportive nature of the matrix. The facile ability to form discrete cell-containing layers validates the application of this novel printing technique to form complex, layered and viable 3D cell structures. These brain-like structures offer the opportunity to reproduce more accurate 3D in vitro microstructures with applications ranging from cell behavior studies to improving our understanding of brain injuries and neurodegenerative diseases.
Assuntos
Encéfalo/fisiologia , Oligopeptídeos/farmacologia , Polissacarídeos Bacterianos/farmacologia , Impressão Tridimensional , Animais , Encéfalo/efeitos dos fármacos , Bovinos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Técnica de Fratura por Congelamento , Géis , Camundongos Endogâmicos BALB C , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Porosidade , Soroalbumina Bovina/metabolismoRESUMO
Drop-on-demand bioprinting allows the controlled placement of living cells, and will benefit research in the fields of tissue engineering, drug screening and toxicology. We show that a bio-ink based on a novel microgel suspension in a surfactant-containing tissue culture medium can be used to reproducibly print several different cell types, from two different commercially available drop-on-demand printing systems, over long printing periods. The bio-ink maintains a stable cell suspension, preventing the settling and aggregation of cells that usually impedes cell printing, whilst meeting the stringent fluid property requirements needed to enable printing even from many-nozzle commercial inkjet print heads. This innovation in printing technology may pave the way for the biofabrication of multi-cellular structures and functional tissue.
RESUMO
Polythiophenes are attractive candidate polymers for use in synthetic cell scaffolds as they are amenable to modification of functional groups as a means by which to increase biocompatibility. In the current study we analysed the physical properties and response of primary myoblasts to three thiophene polymers synthesized from either a basic bithiophene monomer or from one of two different thiophene monomers with alkoxy functional groups. In addition, the effect of the dopants pTS- and ClO4 - was investigated. In general, it was found that pTS- doped polymers were significantly smoother and tended to be more hydrophilic than their ClO4 - doped counterparts, demonstrating that the choice of dopant significantly affects the polythiophene physical properties. These properties had a significant effect on the response of primary myoblasts to the polymer surfaces; LDH activity measured from cells harvested at 24 and 48 h post-seeding revealed significant differences between numbers of cells attaching to the different thiophene polymers, whilst all of the polymers equally supported cell doubling over the 48 h period. Differences in morphology were also observed, with reduced cell spreading observed on polymers with alkoxy groups. In addition, significant differences were seen in the polymers' ability to support myoblast fusion. In general pTS- doped polymers were better able to support fusion than their ClO4 - doped counterparts. These studies demonstrate that modification of thiophene polymers can be used to promote specific cellular response (e.g. proliferation over differentiation) without the use of biological agents.
RESUMO
The characterization of a polyelectrolyte complex material comprised of two biopolymers, a chitosan upper layer and a gellan gum under layer, is reported. It is shown that the upper layer of chitosan with incorporated levofloxacin displays an antibacterial activity, while the under layer of a gellan gum/TiO(2) composite supports the growth of fibroblastic cells.
Assuntos
Materiais Biocompatíveis/síntese química , Quitosana/química , Levofloxacino , Ofloxacino/farmacologia , Polissacarídeos Bacterianos/química , Antibacterianos/química , Antibacterianos/farmacologia , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Composição de Medicamentos , Escherichia coli/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Ofloxacino/química , Titânio/químicaRESUMO
The purpose of this work was to investigate the potential biomedical application of novel aligned electrospun polypyrrole (PPy)/poly(styrene-beta-isobutylene-beta-styrene) (SIBS) fibers. After successfully aligning the electroactive PPy/SIBS fibers based on our modified electrospinning method, we demonstrated that neurite outgrowth from PC12 cells could be highly orientated parallel to the aligned PPy/SIBS fibers. Physical interactions between the nerve cells and PPy/SIBS fibers through filopodia "sensing" were observed using atomic force microscopy. These observations indicate a role of contact guidance as a mechanism for the observed alignment. This work highlights the capacity for electroactive PPy/SIBS fibers to support and guide nerve cell differentiation through topographic cues, which is a highly desirable characteristic in medical implants for neurological applications.
Assuntos
Teste de Materiais/métodos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Polímeros/farmacologia , Pirróis/farmacologia , Estireno/farmacologia , Estirenos/farmacologia , Alicerces Teciduais/química , Animais , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Células PC12 , RatosRESUMO
The purpose of this work was to investigate for the first time the potential biomedical applications of novel polypyrrole (PPy) composites incorporating a large polyelectrolyte dopant, poly (2-methoxy-5 aniline sulfonic acid) (PMAS). The physical and electrochemical properties were characterized. The PPy/PMAS composites were found to be smooth and hydrophilic and have low electrical impedance. We demonstrate that PPy/PMAS supports nerve cell (PC12) differentiation, and that clinically relevant 250 Hz biphasic current pulses delivered via PPy/PMAS films significantly promote nerve cell differentiation in the presence of nerve growth factor (NGF). The capacity of PPy/PMAS composites to support and enhance nerve cell differentiation via electrical stimulation renders them valuable for medical implants for neurological applications.
Assuntos
Compostos de Anilina/química , Técnicas de Cultura de Células/métodos , Estimulação Elétrica , Neurogênese , Neurônios/fisiologia , Polímeros/química , Pirróis/química , Ácidos Sulfônicos/química , Animais , Materiais Biocompatíveis/química , Células Cultivadas , Impedância Elétrica , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Fator de Crescimento Neural/metabolismo , Neuritos/fisiologia , Neuritos/ultraestrutura , Neurônios/citologia , Neurônios/ultraestrutura , Células PC12 , RatosRESUMO
Conducting polymers have been developed as substrates for in vitro studies with a range of cell types including electrically-excitable cells such as nerve and smooth muscle. The goal of this study was to optimise and characterise a range of polypyrrole materials to act as substrates for electrical stimulation of differentiating skeletal myoblasts. Although all of the polymer materials provided suitable substrates for myoblast adhesion and proliferation, significant differences became apparent under the low-serum conditions used for differentiation of primary myoblasts. The significance of the work lies in the design and control of polymer materials to facilitate different stages of skeletal muscle cell proliferation and/or differentiation, opening up opportunities for engineering of this tissue. This paper therefore constitutes not just a biocompatibility assessment but a comprehensive study of how synthesis conditions affect the final outcome in terms of cell response.