RESUMO
Chemical surface patterning can be an incredibly powerful tool in a variety of applications, as it enables precise spatial control over surface properties. But the equipment required to create functional surface patterns-especially "grayscale" patterns where independent control over species placement and density are needed-is often expensive and inaccessible. In this work, we leveraged equipment and methods readily available to many research labs, namely 3D printing and electroblotting, to generate controlled grayscale surface patterns. Three-dimensional-printed molds were used to cast polyacrylamide hydrogels with regions of variable polymer density; regions of low polymer density within the hydrogels served as reservoirs for proteins that were later driven onto a target surface using electrophoresis. This mechanism was used to deposit grayscale patterns of fluorescently labeled proteins, and the fluorescent intensity of these patterns was measured and compared to a theoretical analysis of the deposition mechanism.
Assuntos
Eletroforese/instrumentação , Hidrogéis/análise , Propriedades de Superfície , Desenho de Equipamento , Impressão Tridimensional , Proteínas/químicaRESUMO
3D cell culture and microfluidics both represent powerful tools for replicating critical components of the cell microenvironment; however, challenges involved in the integration of the two and compatibility with standard tissue culture protocols still represent a steep barrier to widespread adoption. Here we demonstrate the use of engineered surface roughness in the form of microfluidic channels to integrate 3D cell-laden hydrogels and microfluidic fluid delivery. When a liquid hydrogel precursor solution is pipetted onto a surface containing open microfluidic channels, the solid/liquid/air interface becomes pinned at sharp edges such that the hydrogel forms the "fourth wall" of the channels upon solidification. We designed Cassie-Baxter microfluidic surfaces that leverage this phenomenon, making it possible to have barrier-free diffusion between the channels and the hydrogel; in addition, sealing is robust enough to prevent leakage between the two components during fluid flow, but the sealing can also be reversed to facilitate recovery of the cell/hydrogel material after culture. This method was used to culture MDA-MB-231 cells in collagen, which remained viable and proliferated while receiving media exclusively through the microfluidic channels over the course of several days.
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 , Linhagem Celular Tumoral , HumanosRESUMO
HYPOTHESIS: Waxy hydrocarbons diffuse freely in polydimethylsiloxane (PDMS), and this capability can be leveraged to generate inexpensive surface micropatterns that modify adhesion and wetting. EXPERIMENTS: Patterns are created by placing a waxy Parafilm sheet on the back of a PDMS stamp containing microscale surface features. When heated, the paraffin liquefies and diffuses through the stamp, creating a thin liquid layer on the micropatterned stamp surface; when placed in contact with a target surface, the layer solidifies and is retained on the target when the stamp is removed. Micropatterns were generated on different materials and surface topographies; pattern geometry was evaluated using optical profilometry and changes in wetting were evaluated using contact angle goniometry. Diffusion of paraffin through PDMS was evaluated using XPS. FINDINGS: Wax micropatterns have submicron lateral resolution and thickness ranging from 85 to 380 nm depending on contact time. By using XPS analysis to track paraffin diffusion within the PDMS stamp during this process, we estimate the diffusion coefficient to be 5.3 × 10-7 cm2/s at 65 °C. This means that the paraffin layer at the stamp surface replenishes in less than a second after stamping, so it can be used multiple times without re-inking to deposit complex, multi-layer paraffin patterns.
Assuntos
Propriedades de SuperfícieRESUMO
BACKGROUND: Mechanical power is a promising new metric to assess energy transfer from a mechanical ventilator to a patient, which combines the contributions of multiple parameters into a single comprehensive value. However, at present, most ventilators are not capable of calculating mechanical power automatically, so there is a need for a simple equation that can be used to estimate this parameter at the bedside. For volume-controlled ventilation (VCV), excellent equations exist for calculating power from basic ventilator parameters, but for pressure-controlled ventilation (PCV), an accurate, easy-to-use equation has been elusive. RESULTS: Here, we present a new power equation and evaluate its accuracy compared to the three published PCV power equations. When applied to a sample of 50 patients on PCV with a non-zero rise time, we found that our equation estimated power within an average of 8.4% ± 5.9% (mean ± standard deviation) of the value obtained by numerical integration of the P-V loop. The other three equations estimated power with an error of 19.4% ± 12.9% (simplified Becher equation), 10.0% ± 6.8% (comprehensive Becher equation), and 16.5% ± 14.6% (van der Meijden equation). CONCLUSIONS: Our equation calculates power more accurately than the other three published equations, and is much easier to use than the only previously published equation with similar accuracy. The proposed new mechanical power equation is accurate and simple to use, making it an attractive option to estimate power in PCV cases at the bedside.
RESUMO
Tumor development and progression require chemical and mechanical cues derived from cellular and non-cellular components in the tumor microenvironment, including the extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells. Therefore, it is crucial to develop tissue culture models that can mimic in vivo cancer cell-ECM and cancer-stromal cell interactions. Three-dimensional (3D) tumor culture models have been widely utilized to study cancer development and progression. A recent advance in 3D culture is the development of patient-derived tumor organoid (PDO) models from primary human cancer tissue. PDOs maintain the heterogeneity of the primary tumor, which makes them more relevant for identifying therapeutic targets and verifying drug response. Other significant advances include development of 3D co-culture assays to investigate cell-cell interactions and tissue/organ morphogenesis, and microfluidic technology that can be integrated into 3D culture to mimic vasculature and blood flow. These advances offer spatial and temporal insights into cancer cell-stromal interactions and represent novel techniques to study tumor progression and drug response. Here, we summarize the recent progress in 3D culture and tumor organoid models, and discuss future directions and the potential of utilizing these models to study cancer-stromal interactions and direct personalized treatment.
Assuntos
Neoplasias , Medicina de Precisão , Comunicação Celular , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Organoides/patologia , Células Estromais/patologia , Células Tumorais Cultivadas/patologia , Microambiente TumoralRESUMO
Mechanical ventilation is a potentially life-saving therapy for patients with acute lung injury, but the ventilator itself may cause lung injury. Ventilator-induced lung injury (VILI) is sometimes an unfortunate consequence of mechanical ventilation. It is not clear however how best to minimize VILI through adjustment of various parameters including tidal volume, plateau pressure, driving pressure, and positive end expiratory pressure (PEEP). No single parameter provides a clear indication for onset of lung injury attributable exclusively to the ventilator. There is currently interest in quantifying how static and dynamic parameters contribute to VILI. One concept that has emerged is the consideration of the amount of energy transferred from the ventilator to the respiratory system per unit time, which can be quantified as mechanical power. This review article reports on recent literature in this emerging field and future roles for mechanical power assessments in prospective studies.
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Síndrome do Desconforto Respiratório , Lesão Pulmonar Induzida por Ventilação Mecânica , Humanos , Estudos Prospectivos , Respiração Artificial/efeitos adversos , Volume de Ventilação Pulmonar , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controleRESUMO
In vitro analysis of primary isolated adult cardiomyocyte physiological processes often involves optical imaging of dye-loaded cells on a glass substrate. However, when exposed to rapid solution changes, primary cardiomyocytes often move to compromise quantitative measures. Improved immobilization of cells to glass would permit higher throughput assays. Here, we engineer the peripheral membrane of cardiomyocytes with biotin to anchor cardiomyocytes to borosilicate glass coverslips functionalized with streptavidin. We use a rat cardiac myoblast cell line to determine general relationships between processing conditions, ligand density on the cell and the glass substrate, cellular function, and cell retention under shear flow. Use of the streptavidin-biotin system allows for more than 80% retention of cardiac myoblasts under conventional rinsing procedures, while unmodified cells are largely rinsed away. The adhesion system enables the in-field retention of cardiac cells during rapid fluid changes using traditional pipetting or a modern microfluidic system at a flow rate of 160 mL/min. Under fluid flow, the surface-engineered primary adult cardiomyocytes are retained in the field of view of the microscope, while unmodified cells are rinsed away. Importantly, the engineered cardiomyocytes are functional following adhesion to the glass substrate, where contractions are readily observed. When applying this adhesion system to cardiomyocyte functional analysis, we measure calcium release transients by caffeine induction at an 80% success rate compared to 20% without surface engineering.
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The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.
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Extracellular matrix (ECM), a major component of the cellular microenvironment, plays critical roles in normal tissue morphogenesis and disease progression. Binding of ECM to membrane receptor proteins, such as integrin, discoidin domain receptors, and dystroglycan, elicits biochemical and biomechanical signals that control cellular architecture and gene expression. These ECM signals cooperate with growth factors and hormones to regulate cell migration, differentiation, and transformation. ECM signaling is tightly regulated during normal mammary gland development. Deposition and alignment of fibrillar collagens direct migration and invasion of mammary epithelial cells during branching morphogenesis. Basement membrane proteins are required for polarized acinar morphogenesis and milk protein expression. Deregulation of ECM proteins in the long run is sufficient to promote breast cancer development and progression. Recent studies demonstrate that the integrated biophysical and biochemical signals from ECM and soluble factors are crucial for normal mammary gland development as well as breast cancer progression.
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Neoplasias da Mama/metabolismo , Matriz Extracelular/metabolismo , Glândulas Mamárias Humanas/embriologia , Glândulas Mamárias Humanas/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Matriz Extracelular/patologia , Feminino , Humanos , Transdução de Sinais/fisiologiaRESUMO
Microcontact printing (µCP) is a rapid, inexpensive way to create microscale chemical or biochemical patterns on a target surface. This microstamping method can be used to selectively modify a wide array of surface properties, from wettability and protein adsorption to chemical etch susceptibility. However, controlling the absolute location of features created with microcontact printing is difficult; this lack of precision makes it challenging to integrate with other microfabrication methods or to create complex, multi-chemical patterns on a single surface. In this research, we demonstrate a novel method of controlling the placement of microcontact printing stamps by using an integrated kinematic coupling device. This technique relies on mechanical reference points for rapid, optics-free registry of the stamp and allows µCP stamps to be quickly removed and replaced or even exchanged with submicron repeatability.