Daniel Stark

Three-dimensional tissue culture based on magnetic cell levitation

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

SDF‐1α stiffens myeloma bone marrow mesenchymal stromal cells through the activation of RhoA‐ROCK‐Myosin II

Multiple myeloma (MM) is a B lymphocyte malignancy that remains incurable despite extensive research efforts. This is due, in part, to frequent disease recurrences associated with the persistence of myeloma cancer stem cells (mCSCs). Bone marrow mesenchymal stromal cells (BMSCs) play critical roles in supporting mCSCs through genetic or biochemical alterations. Previously, we identified mechanical distinctions between BMSCs isolated from MM patients (mBMSCs) and those present in the BM of healthy individuals (nBMSCs). These properties of mBMSC contributed to their ability to preferentially support mCSCs. To further illustrate mechanisms underlying the differences between mBMSCs and nBMSCs, here we report that (i) mBMSCs express an abnormal, constitutively high level of phosphorylated Myosin II, which leads to stiffer membrane mechanics, (ii) mBMSCs are more sensitive to SDF‐1α‐induced activation of MYL2 through the G(i./o)‐PI3K‐RhoA‐ROCK‐Myosin II signaling pathway, affecting Youngs modulus in BMSCs and (iii) activated Myosin II confers increased cell contractile potential, leading to enhanced collagen matrix remodeling and promoting the cell–cell interaction between mCSCs and mBMSCs. Together, our findings suggest that interfering with SDF‐1α signaling may serve as a new therapeutic approach for eliminating mCSCs by disrupting their interaction with mBMSCs.

A microfabricated magnetic force transducer-microaspiration system for studying membrane mechanics.

The application of forces to cell membranes is a powerful method for studying membrane mechanics. To apply controlled dynamic forces on the piconewton scale, we designed and characterized a microfabricated magnetic force transducer (MMFT) consisting of current-carrying gold wires patterned on a sapphire substrate. The experimentally measured forces applied to paramagnetic and ferromagnetic beads as a function of applied current agree well with theoretical models. We used this device to pull tethers from microaspirated giant unilamellar vesicles and measure the threshold force for tether formation. In addition, the interlayer drag coefficient of the membrane was determined from the tether-return velocity under magnetic force-free conditions. At high levels of current, vesicles expanded as a result of local temperature changes. A finite element thermal model of the MMFT provided absolute temperature calibration, allowing determination of the thermal expansivity coefficient of stearoyl-oleoyl-phosphatidycholine vesicles (1.7 ± 0.4 × 10(-3) K(-1)) and characterization of the Joule heating associated with current passing through the device. This effect can be used as a sensitive probe of temperature changes on the microscale. These studies establish the MMFT as an effective tool for applying precise forces to membranes at controlled rates and quantitatively studying membrane mechanical and thermo-mechanical properties.

The Threshold Force for Membrane Tether Formation Depends Strongly on Loading Rate

Tethers are thin tubes of lipids (∼20-200 nm in diameter) that form when membranes are subjected to a point force. Tether dynamics are important to a myriad of biological processes including white blood cell adhesion and transport of intracellular material between neighboring cells. To understand the dynamics of tether formation more fully, we investigated the dependence of the force needed to create a tether on the rate of force change (loading rate). To conduct these experiments, a microfabricated magnetic force transducer was used to generate well-controlled and localized magnetic force profiles. Tethers were formed off the surface of microaspirated giant unilamellar vesicles (GUVs) attached to magnetic beads. We discovered a strong correlation between the threshold force of tether formation and the applied force ramp, with the force changing from <10 pN at low loading rates to ∼50 pN at high loading rates. At slow loading rates, the threshold force changes weakly with ln (loading rate), while at high loading rates a steeper dependence is observed. The experimental data can be fit to a energetic model based on Kramers theory, similar to models used to describe membrane rupture. The model predits that tether formation involves passage over two energy barriers and enbales characterization of the characteristic forces and timescales associated with these barriers. This new tool for dynamic studies of membrane mechanics may further be extended to study how tethers form off of flowing cells or how phase regimes, induced by the presence of cholesterol, influence membrane dynamics.

Large scale generation of genetically modified T-cells using micro-electroporators for cancer treatments

To address B-lineage acute lymphoblastic leukemia (B-ALL), we use a bioengineering approach to achieve high-efficient non-viral gene transfer of tumor-specific CAR+ T cells for on-demand use. Specifically, we have developed CD19-specific chimeric antigen receptors (CARs) to redirect the specificity of T cells for B-cell malignancies. We introduce CARs via a non-viral gene transfer: electroporation. We have fabricated and tested a series of high throughput micro-electroporation devices (HitMeDs) with the end result that a HitMeD can electroporate a large number (≫109) human T cells within short time period (≪4 hours). We connect the HitMeDs to a BTX pulse generator and a monitoring system. We have adapted HitMeDs to the electro-transfer of CAR mRNA which avoids the potential genotoxicity associated with vector integration. After electroporation, 70% to 80% of the T cells express CD19 -specific CAR.