A novel culture system for modulating single cell geometry in 3D

Dedifferentiation of chondrocytes during in vitro expansion remains an unsolved challenge for repairing serious articular cartilage defects. In this study, a novel culture system was developed to modulate single cell geometry in 3D and investigate its effects on the chondrocyte phenotype. The approach uses 2D micropatterns followed by in situ hydrogel formation to constrain single cell shape and spreading. This enables independent control of cell geometry and extracellular matrix. Using collagen I matrix, we demonstrated the formation of a biomimetic collagenous “basket” enveloping individual chondrocytes cells. By quantitatively monitoring the production by single cells of chondrogenic matrix (e.g. collagen II and aggrecan) during 21-day cultures, we found that if the cell’s volume decreases, then so does its cell resistance to dedifferentiation (even if the cells remain spherical). Conversely, if the volume of spherical cells remains constant (after an initial decrease), then not only do the cells retain their differentiated status, but previously de-differentiated redifferentiate and regain a chondrocyte phenotype. The approach described here can be readily applied to pluripotent cells, offering a versatile platform in the search for niches toward either self-renewal or targeted differentiation

Highly efficient spatially offset Raman spectroscopy to profile molecular composition in bone

Spatially offset Raman spectroscopy (SORS) offers the prospect of collecting spectral information detailing the molecular composition of biomaterials at greater depths below the surface layers than are normally probed by conventional Raman spectroscopy. By collecting off-axial scattered light, the technique overcomes the large background from in-line light within scattering media. In this paper we present a configuration which enables the highly efficient collection of spectral markers, indicative of bone health, including Raman signatures to assess phosphate, collagen and carbonate content, at millimeter depths. We demonstrate the effectiveness of the technique by performing spectral decompositions to analyze the molecular distribution of these markers non-invasively, using in vitro model systems, comprising bone and tissue, in situ

Cell proliferation and migration inside single cell arrays

Cell proliferation and migration are fundamental processes in determining cell and tissue behaviour. In this study we show the design and fabrication of a new single cell microfluidic structure, called a “vertically integrated array” or “VIA” trap to explore quantitative functional assays including single cell attachment, proliferation and migration studies. The chip can be used in a continuous (flow-through) manner, with a continuous supply of new media, as well as in a quiescent mode. We show the fabrication of the device, together with the flow characteristics inside the network of channels and the single cell traps. The flow patterns inside the device not only facilitate cell trapping, but also protect the cells from mechanical flow-induced stress. MDA-MB-231 human breast cancer cells were used to study attachment and detachment during the cell cycle as well as explore the influences of the chemokine SDF-1 (enabling the quantification of the role of chemokine gradients both on pseudopod formation and directional cell migration)

Hybrid localized surface plasmon resonance and quartz crystal microbalance sensor for label free biosensing

We report on the design and fabrication of a hybrid sensor that integrates transmission-mode localized surface plasmonic resonance (LSPR) into a quartz crystal microbalance (QCM) for studying biochemical surface reactions. The coupling of LSPR nanostructures and a QCM allows optical spectra and QCM resonant frequency shifts to be recorded simultaneously and analyzed in real time for a given surface adsorption process. This integration simplifies the conventional combination of SPR and QCM and has the potential to be miniaturized for application in point-of-care (POC) diagnostics. The influence of antibody-antigen recognition effect on both the QCM and LSPR has been analyzed and discussed.`

Spatial heterodyne offset Raman spectroscopy enabling rapid, high sensitivity characterisation of materials’ interfaces

Spatially offset Raman spectroscopy is integrated with a fiber-coupled spatial heterodyne spectrometer to collect Raman spectra from deep within opaque or scattering materials. The method, named spatial heterodyne offset Raman spectroscopy generates a wavenumber-dependent spatial phase shift of the optical signal as a “spectral” image on a charge-coupled device detector. The image can be readily processed from the spatial domain using a single, simple, and “on-the-fly” Fourier transform to generate Raman spectra, in the frequency domain. By collecting all of the spatially offset Raman scattered photons that pass through the microscope's collection objective lens, the methodology gives an improvement in the Raman sensitivity by an order of magnitude. The instrumentation is both mechanically robust and “movement-free,” which when coupled with the associated advantages of highly efficient signal collection and ease of data processing, enables rapid interfacial analysis of complex constructs based on established biomaterials models

Fabrication and characterization of SU8 Polymeric microring resonators for biosensors

In this paper, SU8 polymer was used to fabricate a waveguide and microring resonator for optical biosensing applications in liquid mediums. Microring resonators offer the advantage of reducing the device size by orders of magnitude without scarifying the interaction length. The strength of the interaction can be measured by their resonance wavelength shift at high quality-factor (Q) resonance. High Q factor enables the achievement of high sensitivity due to the long residence times of the photons that propagate in the ring, which increases the probability of photons interacting with the analytes in solution. The refractive index contrast between liquid and polymer waveguide controls the resonance wavelength and strength of coupling between the waveguide and resonator. Preliminary simulation and theoretical calculation using Rsoft Photonics CAD are presented. Fabrication steps and results obtained by using UV photolithography and electron beam lithography showing the fabrication of microring with 200μm of radius, 200nm of critical coupling gap are presented. Characterization of the SU8 waveguide including propagation and bending losses were performed by using HeNe laser 632.8 nm wavelengths. Future work on microring resonator characterization such as Q factor, resonance wavelength shift using visible wavelength are currently in progress. Initial work on the application of biosensing will also be shown

Single-cell microfluidics to study the effects of genome deletion on bacterial growth behavior

By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population

Branched hybridization chain reaction – using highly dimensional DNA nanostructures for label-free, reagent-less multiplexed molecular diagnostics

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring

A 3D hydrodynamic flow-focusing device for cell sorting

Optical-based microfluidic cell sorting has become increasingly attractive for applications in life and environmental sciences due to its ability of sophisticated cell handling in flow. The majority of these microfluidic cell sorting devices employ two-dimensional fluid flow control strategies, which lack the ability to manipulate the position of cells arbitrarily for precise optical detection, therefore resulting in reduced sorting accuracy and purity. Although three-dimensional (3D) hydrodynamic devices have better flow-focusing characteristics, most lack the flexibility to arbitrarily position the sample flow in each direction. Thus, there have been very few studies using 3D hydrodynamic flow focusing for sorting. Herein, we designed a 3D hydrodynamic focusing sorting platform based on independent sheath flow-focusing and pressure-actuated switching. This design offers many advantages in terms of reliable acquisition of weak Raman signals due to the ability to precisely control the speed and position of samples in 3D. With a proof-of-concept demonstration, we show this 3D hydrodynamic focusing-based sorting device has the potential to reach a high degree of accuracy for Raman activated sorting