Dynamic acoustic field activated cell separation (DAFACS)

Advances in diagnostics, cell and stem cell technologies drive the development of application-specific tools for cell and particle separation. Acoustic micro-particle separation offers a promising avenue for highthroughput, label-free, high recovery, cell and particle separation and isolation in regenerative medicine. Here, we demonstrate a novel approach utilizing a dynamic acoustic field that is capable of separating an arbitrary size range of cells. We first demonstrate the method for the separation of particles with different diameters between 6 and 45 μm and secondly particles of different densities in a heterogeneous medium. The dynamic acoustic field is then used to separate dorsal root ganglion cells. The shearless, label-free and low damage characteristics make this method of manipulation particularly suited for biological applications. Advantages of using a dynamic acoustic field for the separation of cells include its inherent safety and biocompatibility, the possibility to operate over large distances (centimetres), high purity (ratio of particle population, up to 100%), and high efficiency (ratio of separated particles over total number of particles to separate, up to 100%)

Controlling fluid flow to improve cell seeding uniformity

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method–with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems

Particle separation by phase modulated surface acoustic waves

High efficiency isolation of cells or particles from a heterogeneous mixture is a critical processing step in lab-on-a-chip devices. Acoustic techniques offer contactless and label-free manipulation, preserve viability of biological cells, and provide versatility as the applied electrical signal can be adapted to various scenarios. Conventional acoustic separation methods use time-of-flight and achieve separation up to distances of quarter wavelength with limited separation power due to slow gradients in the force. The method proposed here allows separation by half of the wavelength and can be extended by repeating the modulation pattern and can ensure maximum force acting on the particles. In this work, we propose an optimised phase modulation scheme for particle separation in a surface acoustic wave microfluidic device. An expression for the acoustic radiation force arising from the interaction between acoustic waves in the fluid was derived. We demonstrated, for the first time, that the expression of the acoustic radiation force differs in surface acoustic wave and bulk devices, due to the presence of a geometric scaling factor. Two phase modulation schemes are investigated theoretically and experimentally. Theoretical findings were experimentally validated for different mixtures of polystyrene particles confirming that the method offers high selectivity. A Monte-Carlo simulation enabled us to assess performance in real situations, including the effects of particle size variation and non-uniform acoustic field on sorting efficiency and purity, validating the ability to separate particles with high purity and high resolution

Human platelet lysate as a potential clinical-translatable supplement to support the neurotrophic properties of human adipose-derived stem cells

Background: The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications. Methods: We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion. Results: We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-Added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties. Conclusions: The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research

Particle separation in surface acoustic wave microfluidic devices using reprogrammable, pseudo-standing waves

We report size and density/compressibility-based particle sorting using on-off quasi-standing waves based on the frequency difference between two ultrasonic transducers. The 13.3 MHz fundamental operating frequency of the surface acoustic wave microfluidic device allows the manipulation of particles on the micrometer scale. Experiments, validated by computational fluid dynamics, were carried out to demonstrate size-based sorting of 5–14.5 μm diameter polystyrene (PS) particles and density/compressibility-based sorting of 10 μm PS, iron-oxide, and poly(methyl methacrylate) particles, with densities ranging from 1.05 to 1.5 g/cm3. The method shows a sorting efficiency of >90% and a purity of >80% for particle separation of 10 μm and 14.5 μm, demonstrating better performance than similar sorting methods recently published (72%–83% efficiency). The sorting technique demonstrates high selectivity separation of particles, with the smallest particle ratio being 1.33, compared to 2.5 in previous work. Density/compressibility-based sorting of polystyrene and iron-oxide particles showed an efficiency of 97 ± 4% and a purity of 91 ± 5%. By varying the sign of the acoustic excitation signal, continuous batch acoustic sorting of target particles to a desired outlet was demonstrated with good sorting stability against variations of the inflow rates

Scattering length of the ground state Mg+Mg collision

We have constructed the X 1SIGMAg+ potential for the collision between two ground state Mg atoms and analyzed the effect of uncertainties in the shape of the potential on scattering properties at ultra-cold temperatures. This potential reproduces the experimental term values to 0.2 inverse cm and has a scattering length of +1.4(5) nm where the error is prodominantly due to the uncertainty in the dissociation energy and the C6 dispersion coefficient. A positive sign of the scattering length suggests that a Bose-Einstein condensate of ground state Mg atoms is stable.Comment: 15 pages, 3 figures, Submitted Phys. Rev.

Calculations of collisions between cold alkaline earth atoms in a weak laser field

We calculate the light-induced collisional loss of laser-cooled and trapped magnesium atoms for detunings up to 50 atomic linewidths to the red of the ^1S_0-^1P_1 cooling transition. We evaluate loss rate coefficients due to both radiative and nonradiative state-changing mechanisms for temperatures at and below the Doppler cooling temperature. We solve the Schrodinger equation with a complex potential to represent spontaneous decay, but also give analytic models for various limits. Vibrational structure due to molecular photoassociation is present in the trap loss spectrum. Relatively broad structure due to absorption to the Mg_2 ^1Sigma_u state occurs for detunings larger than about 10 atomic linewidths. Much sharper structure, especially evident at low temperature, occurs even at smaller detunings due to of Mg_2 ^1Pi_g absorption, which is weakly allowed due to relativistic retardation corrections to the forbidden dipole transition strength. We also perform model studies for the other alkaline earth species Ca, Sr, and Ba and for Yb, and find similar qualitative behavior as for Mg.Comment: 20 pages, RevTex, 13 eps figures embedde

Development of a novel 3D culture system for screening features of a complex implantable device for CNS repair

Tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (SCI). We aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three-dimensional (3D) scaffold and optimize the bioprocesses for tissue culture. Because of the complex 3D configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. Thus, we aim to develop scaled down mini-chambers as simplified in vitro simulation systems, to bridge the gap between two-dimensional (2D) cell cultures on structured substrates and three-dimensional (3D) tissue culture. The mini-chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in CNS repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. Since we use CNS myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. It was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. This research demonstrates that the mini-chambers represent a viable, novel, scale down approach for the evaluation of complex 3D scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of SCI

Fine Pathogen Discrimination within the APL1 Gene Family Protects Anopheles gambiae against Human and Rodent Malaria Species

Genetically controlled resistance of Anopheles gambiae mosquitoes to Plasmodium falciparum is a common trait in the natural population, and a cluster of natural resistance loci were mapped to the Plasmodium-Resistance Island (PRI) of the A. gambiae genome. The APL1 family of leucine-rich repeat (LRR) proteins was highlighted by candidate gene studies in the PRI, and is comprised of paralogs APL1A, APL1B and APL1C that share ≥50% amino acid identity. Here, we present a functional analysis of the joint response of APL1 family members during mosquito infection with human and rodent Plasmodium species. Only paralog APL1A protected A. gambiae against infection with the human malaria parasite P. falciparum from both the field population and in vitro culture. In contrast, only paralog APL1C protected against the rodent malaria parasites P. berghei and P. yoelii. We show that anti-P. falciparum protection is mediated by the Imd/Rel2 pathway, while protection against P. berghei infection was shown to require Toll/Rel1 signaling. Further, only the short Rel2-S isoform and not the long Rel2-F isoform of Rel2 confers protection against P. falciparum. Protection correlates with the transcriptional regulation of APL1A by Rel2-S but not Rel2-F, suggesting that the Rel2-S anti-parasite phenotype results at least in part from its transcriptional control over APL1A. These results indicate that distinct members of the APL1 gene family display a mutually exclusive protective effect against different classes of Plasmodium parasites. It appears that a gene-for-pathogen-class system orients the appropriate host defenses against distinct categories of similar pathogens. It is known that insect innate immune pathways can distinguish between grossly different microbes such as Gram-positive bacteria, Gram-negative bacteria, or fungi, but the function of the APL1 paralogs reveals that mosquito innate immunity possesses a more fine-grained capacity to distinguish between classes of closely related eukaryotic pathogens than has been previously recognized