Three-dimensional (3D) in vitro models capturing both the structural and dynamic complexity of the in vivo situation are in great demand as an alternative to animal models. Despite tremendous ...progress in engineering complex tissue/organ models in the past decade, approaches that support the required freedom in design, detail and chemistry for fabricating truly 3D constructs have remained limited. Here, we report a stereolithographic high-resolution 3D printing technique utilizing poly(ethylene glycol) diacrylate (PEGDA, MW 700) to manufacture diffusion-open and mechanically stable hydrogel constructs as self-contained chips, where confined culture volumes are traversed and surrounded by perfusable vascular-like networks. An optimized resin formulation enables printing of hydrogel chips holding perfusable microchannels with a cross-section as small as 100 μm × 100 μm, and the printed microchannels can be steadily perfused for at least one week. In addition, the integration of multiple independently perfusable and structurally stable channel systems further allows for easy combination of different bulk material volumes at exact relative spatial positions. We demonstrate this structural and material flexibility by embedding a highly compliant cell-laden gelatin hydrogel within the confines of a 3D printed resilient PEGDA hydrogel chip of intermediate compliance. Overall, our proposed strategy represents an automated, cost-effective and high resolution technique to manufacture complex 3D constructs containing microfluidic perfusion networks for advanced in vitro models.
Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1-labeled DNA in nanofluidic channels with a combination of formamide and ...local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.
Sufficient and controllable oxygen supply is essential for
3D cell and tissue culture at high cell densities, which calls for volumetric
oxygen analysis methods to quantitatively assess the oxygen ...distribution. This paper presents a general approach for accurate and precise non-contact 3D mapping of oxygen tension in high cell-density cultures
embedded commercially available oxygen microsensor beads read out by confocal phosphorescence lifetime microscopy (PLIM). Optimal acquisition conditions and data analysis procedures are established and implemented in a publicly available software package. The versatility of the established method is first demonstrated in model-assisted fluidic design of microperfused 3D printed hydrogel culture chips with the aim of full culture oxygenation, and subsequently for monitoring and maintenance of physiologically relevant spatial and temporal oxygen gradients in the 3D printed chips controlled by static or dynamic flow conditions during 3D culture.
We demonstrate a confinement spectroscopy technique capable of probing small conformational changes of unanchored single DNA molecules in a manner analogous to force spectroscopy, in the regime ...corresponding to femtonewton forces. In contrast to force spectroscopy, various structural forms of DNA can easily be probed, as indicated by experiments on linear and circular DNA. The extension of circular DNA is found to scale according to the de Gennes exponent, unlike for linear DNA.
Illness behaviour effects the quality of life of patients with chronic obstructive pulmonary disease (COPD) but is scarcely described in literature. The aim of this study was to explore self‐reported ...illness behaviour of patients with COPD, who have declined nonpharmacological rehabilitation. The study has a qualitative design using semi‐structured interviews. The subsequent analysis is a theory‐guided mapping of actions reported by the patients in order to manage symptoms. These actions are understood and categorised according to the styles of coping described by Alonzo. Four categories of illness behaviour are identified: containment of symptoms, coping with symptoms through formal and informal interventions, adjustment of situations through compensating and economising interventions and crisis coping by surrendering. The analysis shows that behaviour that may seem unhelpful from a healthcare perspective may seem rational in the everyday life perspective of the patient. Findings show that reluctance to participation in rehabilitation should not only be interpreted as lack of motivation or health literacy. In the patients’ perspective, nonpharmacological interventions might be perceived as a threat that could tip the delicate balance of everyday life with severe COPD.
We demonstrate that fabrication of well-defined nanofluidic systems can be greatly simplified by injection molding of thermoplastic polymers. Chips featuring nanochannel arrays, microchannels and ...integrated interconnects are produced in a single processing step by injection molding. The resulting open channel structures are subsequently sealed by facile plasma-enhanced thermal bonding of a polymer film. This fast, inexpensive and industry-compatible method thus provides a single-use all-polymer platform for nanofluidic lab-on-a-chip applications. Its applicability for nanofluidics is demonstrated by DNA stretching experiments performed on individual double-stranded DNA molecules confined in the injection molded nanochannels. The obtained results are consistent with measurements performed in costly state-of-the-art silica nanochannels, for both straight and tapered channel geometries.
Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modeling of atherosclerosis has been the mainstay for preclinical testing of genetic and ...pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity, and it is critical to monitor lipoprotein levels during preclinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during preclinical experiments limit analyses to measuring total cholesterol and triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple reaction monitoring (MRM) stable isotope dilution (SID) mass spectrometry assay for simultaneous relative quantification of nine apolipoproteins using a few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: the widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools, we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during preclinical investigations.
We demonstrate the transfer and immobilization of active antibodies from a low surface- energy mold surface to thermoplastic replica surfaces using injection molding, and we investigate the process ...at molecular scale. The transfer process is highly efficient, as verified by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) of the mold and replica surfaces. AFM analysis reveals partial nanometer-scale embedding of the protein into the polymer matrix as a possible mechanism of permanent immobilization. Replicas with rabbit anti-mouse IgG immobilized as capture antibody at the hot polymer melt surface during injection molding show similar affinity for their antigen (mouse IgG) in sandwich enzyme-linked immunosorbent assay (ELISA) as capture antibodies deposited by passive adsorption onto a bare thermoplastic replica. The transferred antibodies retain their functionality after incubation in serum-containing cell medium for >1 week. A mold coating time of 10 min prior to injection molding is sufficient for producing highly sensitive ELISA assays, thus enabling the short processing cycle times required for mass production of single-use biodevices relying on active immobilized antibodies.
We present a method for making polymer chips with a narrow-band near-infrared absorber layer that enables light-induced local heating of liquids inside fluidic micro- and nanochannels fabricated by ...thermal imprint in polymethyl methacrylate. We have characterized the resulting liquid temperature profiles in microchannels using the temperature dependent fluorescence of the complex Ru(bpy)(3)(2+). We demonstrate thermophoretic manipulation of individual YOYO-1 stained T4 DNA molecules inside micro- and nanochannels.
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