RESUMO
Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.
Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Microfluídica , TransfecçãoRESUMO
Blood plasma is a source of biomarkers in blood and a simple, fast, and easy extraction method is highly required for point-of-care testing (POCT) applications. This paper proposes a membrane filter integrated microfluidic device to extract blood plasma from whole blood, without any external instrumentation. A commercially available membrane filter was integrated with a newly designed dual-cover microfluidic device to avoid leakage of the extracted plasma and remaining blood cells. Nano-interstices installed on both sides of the microfluidic channels actively draw the extracted plasma from the membrane. The developed device successfully supplied 20 µL of extracted plasma with a high extraction yield (~45%) in 16 min.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Microfluídica , Plasma , Testes ImediatosRESUMO
The protozoan parasite Toxoplasma gondii (T. gondii) causes one of the most common human zoonotic diseases and infects approximately one-third of the global population. T. gondii infects nearly every cell type and causes severe symptoms in susceptible populations. In previous laboratory animal studies, T. gondii movement and transmission were not analyzed in real time. In a three-dimensional (3D) microfluidic assay, we successfully supported the complex lytic cycle of T. gondii in situ by generating a stable microvasculature. The physiology of the T. gondii-infected microvasculature was monitored in order to investigate the growth, paracellular and transcellular migration, and transmission of T. gondii, as well as the efficacy of T. gondii drugs.
Assuntos
Toxoplasma , Toxoplasmose , Animais , Microfluídica , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Migração Transendotelial e Transepitelial , ZoonosesRESUMO
Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.
Assuntos
Endotélio Vascular/metabolismo , Técnicas Analíticas Microfluídicas , Neovascularização Fisiológica/efeitos dos fármacos , Técnicas de Cultura de Células , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Hidrogéis/químicaRESUMO
Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.
Assuntos
Células-Tronco Hematopoéticas/citologia , Linfócitos/metabolismo , Microfluídica , Células-Tronco/citologia , Transfecção/métodos , Antígenos CD34/biossíntese , Transporte Biológico , Sobrevivência Celular , Eletroporação , Citometria de Fluxo , Terapia Genética , Humanos , Células Matadoras Naturais/citologia , Dispositivos Lab-On-A-Chip , Engenharia de Proteínas , RNA Mensageiro/metabolismo , Linfócitos T/citologiaRESUMO
Engineering physiologically relevant in vitro models of human organs remains a fundamental challenge. Despite significant strides made within the field, many promising organ-on-a-chip models fall short in recapitulating cellular interactions with neighboring cell types, surrounding extracellular matrix (ECM), and exposure to soluble cues due, in part, to the formation of artificial structures that obstruct >50% of the surface area of the ECM. Here, a 3D cell culture platform based upon hydrophobic patterning of hydrogels that is capable of precisely generating a 3D ECM within a microfluidic channel with an interaction area >95% is reported. In this study, for demonstrative purposes, type I collagen (COL1), Matrigel (MAT), COL1/MAT mixture, hyaluronic acid, and cell-laden MAT are formed in the device. Three potential applications are demonstrated, including creating a 3D endothelium model, studying the interstitial migration of cancer cells, and analyzing stem cell differentiation in a 3D environment. The hydrophobic patterned-based 3D cell culture device provides the ease-of-fabrication and flexibility necessary for broad potential applications in organ-on-a-chip platforms.
Assuntos
Técnicas de Cultura de Células , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Movimento Celular , Humanos , Células MCF-7 , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodosRESUMO
The mechanical properties of cells change with their differentiation, chronological age, and malignant progression. Consequently, these properties may be useful label-free biomarkers of various functional or clinically relevant cell states. Here, we demonstrate mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell-analysis method that utilizes a four-terminal measurement of the current across a microfluidic channel to quantify simultaneously cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We define a new parameter, the whole-cell deformability index (wCDI), which provides a quantitative mechanical metric of the resistance to compressive deformation that can be used to discriminate among different cell types. The wCDI and the transverse deformation under constant strain show malignant MCF-7 and A549 cell lines are mechanically distinct from non-malignant, MCF-10A and BEAS-2B cell lines, and distinguishes between cells treated or untreated with cytoskeleton-perturbing small molecules. We categorize cell recovery time, ΔTr, as instantaneous (ΔTr ~ 0 ms), transient (ΔTr ≤ 40ms), or prolonged (ΔTr > 40ms), and show that the composition of recovery types, which is a consequence of changes in cytoskeletal organization, correlates with cellular transformation. Through the wCDI and cell-recovery time, mechano-NPS discriminates between sub-lineages of normal primary human mammary epithelial cells with accuracy comparable to flow cytometry, but without antibody labeling. Mechano-NPS identifies mechanical phenotypes that distinguishes lineage, chronological age, and stage of malignant progression in human epithelial cells.
RESUMO
Quantitative microfluidic point-of-care testing has been translated into clinical applications to support a prompt decision on patient treatment. A nanointerstice-driven filling technique has been developed to realize the fast and robust filling of microfluidic channels with liquid samples, but it has failed to provide a consistent filling time owing to the wide variation in liquid viscosity, resulting in an increase in quantification errors. There is a strong demand for simple and quick flow control to ensure accurate quantification, without a serious increase in system complexity. A new control mechanism employing two-beam refraction and one solenoid valve was developed and found to successfully generate digitized filling flow, completely free from errors due to changes in viscosity. The validity of digitized filling flow was evaluated by the immunoassay, using liquids with a wide range of viscosity. This digitized microfluidic filling flow is a novel approach that could be applied in conventional microfluidic point-of-care testing.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Animais , Anticorpos Imobilizados/química , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Imunoensaio/instrumentação , Imunoglobulina G/química , Camundongos , Testes Imediatos , ViscosidadeRESUMO
Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix.
Assuntos
Bacteriófagos/metabolismo , Colágeno Tipo I/farmacologia , Matriz Extracelular/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Recombinação Genética/genética , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/efeitos dos fármacos , Imunofluorescência , Humanos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Nanofibras/química , Engenharia de Proteínas , RatosRESUMO
Aptamers have recently emerged as reliable and promising targeting agents in the field of biology. However, their therapeutic potential has yet to be completely assessed due to their poor pharmacokinetics for systemic administration. Here, we describe a novel aptamer-antibody complex, designated an "oligobody" (oligomer+antibody) that may overcome the therapeutic limitations of aptamers. To provide proof-of-principle study, we investigated the druggability of oligobody in vivo using cotinine conjugated t44-OMe aptamer, which is specific for the sequence of pegaptanib, and an anti-cotinine antibody. The antibody part of oligobody resulted in extended in vivo pharmacokinetics of the aptamer without influencing its binding affinity. Moreover, the aptamer of oligobody penetrated deeply into the tumor tissues whereas the anti-VEGF antibody did not. Finally, the systemic administration of this oligobody reduced the tumor burden in a xenograft mouse model. Together, these results suggested that our oligobody strategy may represent a novel platform for rapid, low-cost and high-throughput cancer therapy.
Assuntos
Anticorpos Monoclonais , Aptâmeros de Nucleotídeos , Cotinina , Neoplasias Pulmonares/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Células A549 , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/farmacocinética , Aptâmeros de Nucleotídeos/uso terapêutico , Cotinina/química , Cotinina/imunologia , Sistemas de Liberação de Medicamentos , Feminino , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo MolecularRESUMO
The construction of well-controllable in vitro models of physiological and pathological vascular endothelium remains a fundamental challenge in tissue engineering and drug development. Here, we present an approach for forming a synthetic endothelial extracellular matrix (ECM) that closely resembles that of the native structure by locally depositing basement membrane materials onto type 1 collagen nanofibers only in a region adjacent to the endothelial cell (EC) monolayer. Culturing the EC monolayer on this synthetic endothelial ECM remarkably enhanced its physiological properties, reducing its vascular permeability, and promoting a stabilized, quiescent phenotype. We demonstrated that the EC monolayer on the synthetic endothelial ECM neither creates non-physiological barriers to cell-cell or cell-ECM interactions, nor hinders molecular diffusion of growth factors and other molecules. The synthetic endothelial ECM and vascular endothelium on it may help us enter in a new phase of research in which various models of the biological barrier behavior can be tested experimentally.
Assuntos
Técnicas de Cultura de Células/métodos , Endotélio Vascular/crescimento & desenvolvimento , Matriz Extracelular/fisiologia , Engenharia Tecidual , Membrana Basal/citologia , Membrana Basal/crescimento & desenvolvimento , Adesão Celular/fisiologia , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Endotélio Vascular/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microfluídica/métodos , Nanofibras/química , PermeabilidadeRESUMO
Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.
Assuntos
Técnicas de Cultura de Células/instrumentação , Dispositivos Lab-On-A-Chip , Células-Tronco Mesenquimais/citologia , Células-Tronco Neurais/citologia , Neurogênese , Comunicação Parácrina , Animais , Lesões Encefálicas/terapia , Desenho de Equipamento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Regulação para CimaRESUMO
We present a novel in vitro breast tumor model to mimic intratumoral phenotypic heterogeneity based on a microfluidic system incorporating ECM scaffolds capable of providing a physiologically relevant tumor microenvironment. To study the regulation of invasive potentials by intratumoral subpopulation conditions, we developed heterogeneous cancer cell subpopulations by co-culturing two breast cancer cell types with distinct phenotypes, specifically, highly invasive and epithelial-like cancer cells. Our results indicate that intratumoral phenotypic heterogeneity acts as an encourager of cancer cell invasion through a 3D matrix depending on the neighboring ECM, with highly invasive cancer cells acting as the 'leader' and epithelial-like cancer cells as the 'follower', therefore enhancing the metastatic potential.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Matriz Extracelular/patologia , Microambiente Tumoral/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Epiteliais , Feminino , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Microscopia de Fluorescência , FenótipoRESUMO
Pre-concentration methods are essential for detecting low concentrations of influenza virus in biological samples from patients. Here, we describe a new method for draining buffer from solution in the reservoir of a microfluidic device to increase the concentration of virus in the reservoir. Viruses were captured in the reservoir by an ion depletion barrier from connected ion selective microfluidic channels. 75 µl of buffer was successfully drained from a 100 µl sample, resulting in a 4-fold increase in influenza hemagglutinin concentration in the reservoir. The volume of the final concentrated sample was suitable for detection of influenza hemagglutinin by the enzyme-linked immunosorbent assay, demonstrating the usefulness of the developed platform for enhanced sensitivity of virus detection in a conventional analysis.
Assuntos
Métodos Analíticos de Preparação de Amostras/instrumentação , Polímeros de Fluorcarboneto/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Membranas Artificiais , Técnicas Analíticas Microfluídicas/instrumentação , Dimetilpolisiloxanos/química , Técnicas Eletroquímicas , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Limite de Detecção , Teste de Materiais , Concentração Osmolar , Permeabilidade , Impressão Tridimensional , Kit de Reagentes para Diagnóstico , Propriedades de Superfície , Fatores de TempoRESUMO
Neural stem cells (NSCs) reside in a vascular microenvironment termed the "NSC niche." Blood vessels in the NSC niche play an important role in maintaining an appropriate balance between NSC self-renewal and differentiation that serves to maintain homeostasis. Understanding the role of brain vessels in the NSC niche will facilitate basic research in neurogenesis and vasculogenesis as well as aid the development of potential therapies for degenerative disorders. Here, an in vitro-reconstituted NSC-vascular niche consisting of a 3D brain vasculature and extracellular matrix (ECM) microenvironment that allows NSCs to adopt physiologically relevant phenotypes through the combined effects of ECM components, chemical gradients, and signaling effectors from the brain vasculature is described. The reconstituted niche can provide precise spatiotemporal control of the NSC niche, regulating self-renewal, proliferation and colony formation of NSCs, and suppressing neuronal generation but promoting NSC differentiation into astrocytes and oligodendrocytes. It is proved that Notch effectors regulate both the astrocyte differentiation and NSC self-renewal, but the astrocyte differentiation is more active in NSCs in close proximity to brain vasculature. A potential role of the other vascular microenvironmental factor of pigment epithelium-derived factor from brain vasculature in the regulation of NSC self-renewal is also proved.
Assuntos
Técnicas de Cultura de Células/instrumentação , Proteínas da Matriz Extracelular/química , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Nicho de Células-Tronco/fisiologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Encéfalo/irrigação sanguínea , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Técnicas Analíticas Microfluídicas , Microvasos/fisiologiaRESUMO
Plasticity and reciprocity of breast cancer cells to various extracellular matrice (ECMs) are three-dimensionally analyzed in quantitative way in a novel and powerful microfluidic in vitro platform. This successfully demonstrates the metastatic potential of cancer cells and their effective strategies of ECM proteolytic remodeling and morphological change, while interacting with other cells and invading into heterogeneous ECMs.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Matriz Extracelular/diagnóstico por imagem , Matriz Extracelular/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , UltrassonografiaRESUMO
In aquatic environments, microorganisms tend to form biofilms on surfaces to protect them from harsh conditions. The biofilms then accumulate into multilayered mat-like structures. In this study, we evaluated the effects of the hydrodynamic conditions on the ecology of biofilms produced by Pseudomonas aeruginosa (PA14). In microfluidic channels, we found that the development of biofilms was regulated by hydrodynamic conditions, but the developed biofilms also changed flow velocity by narrowing flow width. The coupled growing conditions were simplified by a new concept of consequent variables, and the dimensionless biofilm development (Ab/h(2) & Ab/w(cs)(2)) was successfully expressed by the Reynolds number (Re) and the dimension of the channel (r). At low Re, higher flow rates encouraged growth of biofilms, while higher flow rates with high Re suppressed growth of biofilms. These results provide a simple model as a theoretical basis for understanding development of biofilms in microfluidic channels.
Assuntos
Biofilmes/crescimento & desenvolvimento , Técnicas Analíticas Microfluídicas , Pseudomonas aeruginosa/fisiologia , Hidrodinâmica , Modelos BiológicosRESUMO
We report a microfluidic array for investigating and quantitatively analyzing human neural stem cell (hNSC) self-renewal and differentiation in an in vivo-like microenvironment. NSC niche conditions, including three-dimensional (3D) extracellular matrices and low oxygen tension, were effectively reconstituted in the microfluidic array in a combinatorial manner. The array device was fabricated to be detachable, rendering it compatible with quantitative real-time polymerase chain reaction for quantifying the effects of the biomimetic conditions on hNSC self-renewal and differentiation. We show that throughput of 3D cell culture and quantitative analysis can be increased. We also show that 3D hypoxic microenvironments maintain hNSC self-renewal capacity and direct neuronal commitment during hNSC differentiation.
Assuntos
Microfluídica/métodos , Células-Tronco Neurais/citologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Células Cultivadas , Humanos , Modelos BiológicosRESUMO
This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.
Assuntos
Técnicas de Cultura de Células/métodos , Fenômenos Fisiológicos Celulares/fisiologia , Hidrogéis , Técnicas Analíticas Microfluídicas/métodos , Nanotecnologia/métodos , Dimetilpolisiloxanos , NylonsRESUMO
Here, we report a unique method to quantify the effects of in vivo-like extracellular matrix (ECM) for guiding differentiation of neural stem cells (NSCs) in three-dimensional (3D) microenvironments using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully monitored and quantified differentiation of NSCs in small volume ECMs and found that differentiation of NSCs, especially those differentiating towards neuronal and oligodendrocytic lineages, is significantly enhanced by 3D microenvironments reconstituted in the microfluidic channels.