Bioimprinting technologies for removal of myeloblasts from peripheral blood

Acute Myeloid Leukaemia (AML) is a malignancy occurring in the bone marrow and blood whereby immature and defective blast cells are overproduced. As a genetic condition, no cure is available. The condition is traditionally managed by treatment reliant on non-specific cytotoxic chemotherapy and bone marrow transplantation. Treatment is associated with causing discomfort and mortality and is ultimately ineffective; relapse is common and survival rates are poor.Bioimprinting is a technology whereby the size, shape and morphology of biological templates are recreated in polymer matrices. Studies aim to mimic and exploit specific binding reliant on complementary size and shape interactions as seen in a number of biological processes. The field has developed from the templating of rudimentary macromolecules to whole cells with extracellular features accurate on a nanometre scale.This study aimed to fabricate AML specific bioimprints able to discriminate neoplastic cells from patient aspirate. Myeloblasts provide an ideal target due to their inherent size difference and morphological irregularity. Bioimprints incorporated into a high throughput device could provide a vehicle for selectivity of myeloblasts, yielding an alternate treatment pathway in reducing the leukaemic burden in AML sufferers.Herein, methods were devised and evaluated to reliably fabricate high quality bioimprints, representative of the templated cell. Key in the protocol design is the control over the proportion of the cell surface exposed to the curing polymer matrix which dictates the size of the cavities produced and in-turn the ability of uptake of target cells to the bioimprint substrates. This method should be compatible with roll-to-roll nanoimprint lithography which has been highlighted as a viable method to upscale the imprints in order to deplete very high myeloblast cell populations in AML sufferers. Bioimprints of various cell types and polymer particles of similar size were made and further used to produce positive imprints and subsequent negative replica imprints. Ultimately, a methodology was devised and bioimprints of an AML in vitro cultured cell line were fabricated and reproduced into an area of hundreds of square metres.The success of bioimprinting technology was evaluated with high resolution microscopy and surface profiling; characterising bioimprinted cavities in comparison with the template cell type. Surface modifications were trialled in order to incur an attraction between substrates and incubated cell populations. A coating of weak cationic surface charge was introduced on the bioimprint surface, to attract the negative charges of extracellular groups. This interaction is amplified by an increased surface area contact, allowing binding of cells fitting flush into cavities. Cells unable to fit into cavities did not receive this attraction and remained unbound. With the intended use in mind, a method using materials approved for clinical use was found.Once produced and functionalised, the retention of incubated cell populations was examined under flow conditions. In doing, a bespoke microfluidic device was designed in order to control the hydrodynamics experienced by the bioimprint allowing for a comparison of retention per surface modification parameters. Retention of target cells to bioimprints made using the same cell type was measured as a function of incubated cell suspension concentration; analysis confirmed cells were retained and localised to the bioimprinted cavities. This was compared to cells incubated on bioimprints produced from microparticles of the same size distribution. Significantly poorer retention was observed, indicating the importance of cell shape and cellular surface properties in bioimprint capture.The preference of the bioimprints to the target cell type was assessed by exposure to binary cell mixtures of myeloblasts and PBMCs. Cell populations were characterised on account of size and shape and separately fluorescently labelled for identification and automated enumeration. Bioimprint selectivity towards the targeted cells (myeloblasts) was compared by the proportions of each cell type retained to the bioimprints. In each instance the bioimprint showed a preference for capture of the target cell type over the healthy control. It is anticipated that by reapplying or recirculating patient aspirate, myeloblasts can be completely depleted from samples due to the higher affinity. This effect was confirmed by comparison of the bioimprint path length on selectivity; using larger areas of bioimprint at fixed cell concentration to represent a recirculated population

Adhesion studies of T-lymphocytes: insights into the adhesion dynamics of integrin-mediated inside-out signaling in response to TNF

Integrin-mediated T-lymphocyte adhesion to endothelial cells is a crucial step in the mammalian inflammatory response and for the elimination of pathogens. Outside-in signaling is the well-known pathway in the integrin-mediated leukocyte adhesion in response to proinflammatory events, which is stimulated by an important proinflammatory cytokine, the tumor necrosis factor (TNF). Many studies have been reported that TNF upregulates the expression level of endothelial cell surface molecules. This in turn activates the extracellular domain of integrins and thus facilitates the adhesion of T-lymphocytes both regarding biomolecular interactions and cell adhesion strength. Recently, an inside-out signaling pathway of integrins in lymphocyte activation by TNF has been brought up. However, how this activation modulates T-lymphocyte adhesion strength and dynamics is still not understood. In the study presented here, T-lymphocyte (Jurkat E6-1) cell adhesion to fibronectin (FN)-coated surface was investigated. Such surfaces provide a biomimetic environment since FN is naturally present on top of endothelium and additional effects from the surface molecules, which are present on endothelial cells in vivo, can be excluded. In detail, phase contrast microscopy and photonic crystal slabs (PCS) were applied for the quantification of cell amount and cell size on fibronectin as a function of TNF stimulation. No difference in these parameters was found for the cells with TNF stimulation compared to those without. An advanced optical strategy, reflection interference contrast microscopy (RICM), was applied for the measurement of the real cell adhesion area and the length of microspikes projected from the cell body. With this technique, cell adhesion dynamics and the fluctuation of subcellular structures were visualized, and again no significant effect of TNF stimulation was detected. To quantify the cell adhesion strength, single-cell force spectroscopy (SCFS) was employed to measure cell detachment forces and single ruptures dynamics. TNF significantly increased cell detachment forces and detachment energies, as well as the number of molecular ruptures and the force associated with single rupture events. Meanwhile, the most pronounced effect was obtained at the shortest cell-surface contact time of about 0.2 sec compared to the longest contact time of 10 sec. To understand the behavior of T-lymphocyte cells in the initial capture and rolling phase, microfluidics, which mimics the shear stress in in vivo situations, was used to track and analyze the percent of adhering cells and the speed of rolling cells as a function of TNF stimulation. The preliminary data show that TNF facilitates more cells to adhere on the surface and decreases the rolling speed. To obtain a detailed understanding of the integrin distribution and the proteins close to the adhesion site in T-lymphocyte cells, functionalized gold nanopatterned structures were used as substrates. No significant effect of TNF stimulation on the cell number or morphology was observed. Our results show that the TNF-stimulated inside-out-signaling pathway directly enhances T-lymphocyte adhesion, particularly cell adhesion strength.Die integrinvermittelte Adhäsion von T-Lymphozyten an Endothelzellen ist sowohl ein wichtiger Bestandteil der Entzündungsreaktion von Säugetieren, als auch grundlegend für die Abwehr von Pathogenen. Der von außen nach innen gerichtete Signalweg ist bereits in der integrinvermittelten Adhäsion von Leukozyten als Reaktion auf entzündungsfördernde Ereignisse bekannt. Diese Ereignisse werden durch ein wichtiges proinflammatorische Zytokin, genannt Tumor-Nekrose-Faktor (TNF), stimuliert. Viele Studien haben gezeigt, dass TNF die Expression von Oberflächenmolekülen von Endothelzellen verstärkt, was wiederum den extrazellulären Teil von Integrinen aktiviert und damit die Adhäsion von T-Lymphozyten in Bezug auf biomolekulare Interaktionen und zelluläre Adhäsionskräfte begünstigt. Erst vor Kurzem wurde die Idee eines von innen nach außen gerichteten Signalweges in der Literatur erwähnt. Jedoch ist noch nicht bekannt, wie diese Art der Aktivierung die Adhäsionskräfte und die Dynamik von T-Lymphozyten reguliert. In der hier präsentierten Studie wurde die Adhäsion zwichen T-Lymphozyten (Jurkat E6-1) und Oberflächen, die mit Fibronektin (FN) beschichtet wurden, untersucht. Solche Oberflächen können als eine biomimetische Umgebung dienen, da das Endothel in der Natur von einer FN Schicht bedeckt ist und daher der Einfluss anderer Oberflächenmoleküle, die in vivo auf Endothezellen präsent sind, vernachlässigt werden kann. Phasenkontrastmikroskopie und planare photonische Kristalle (PCS) wurden in der vorliegenden Arbeit genutzt, um die Anzahl und die Größe von Zellen auf FN in Abhängigkeit von TFN Stimulation zu bestimmen. Es wurde kein Unterschied bezüglich dieser beiden Parameter zwischen TNF stimulierten und nicht stimulierten Zellen beobachtet. Interferenzreflexionsmikroskopie (RICM) wurde als hochentwickelte, optische Technik angewandt, um die reale zelluläre Adhäsionsfläche und die Länge der aus dem Zellkörper herausragenden Mikrostacheln zu messen. Diese Technik ermöglichte es, Zelladhäsionsdynamik sowie Fluktuationen von subzellulären Strukturen zu visualisieren. Wiederum wurde kein signifikanter Einfluss der TNF Stimulation gemessen. Mithilfe von Einzelzellkraftspektroskopie (SCFS) wurden Kräfte und Dynamiken von Zell- und Einzelabrissen untersucht. TNF erhöhte sowohl die Abrisskräfte und -energien der Zellen, als auch die Anzahl und Kräfte molekularer Einzelabrisseereignisse signifikant. Dieser Effekt wurde am stärksten für die kürzeste Kontaktzeit zwischen Zelle und Oberfläche von 0,2 s, verglichen mit einer Kontaktdauer von 10 s, beobachtet. Zum besseren Verständnis des Verhaltens von T-Lymphozyten während der anfänglichen Arretierungs- und Rollphase wurde die in vivo im Blutgefäß vorliegende Scherspannung mithilfe eines Mikrofluidikansatzes imitiert, um die prozentuale Menge adhärierender Zellen und deren Rollgeschwindigkeit in Abhängigkeit von der TNF Stimulation zu messen und zu analysieren. Die bisherigen Daten zeigen, dass TNF zu einer höheren Anzahl an adhärierenden Zellen und zu einer erhöhten Rollgeschwindigkeit führt. Für ein besseres Verständnis der Verteilungen von Integrinen und Proteinen nahe des Adhäsionskontaktes von T-Lymphozyten wurden funktionalisierte Gold-nano-Strukturen als Substrate genutzt. Es wurde kein signifikanter Effekt auf Zellanzahl oder -morphologie durch TNF Stimulation beobachtet. Unsere Resultate zeigen, dass TNF stimulierte, von innen nach außen gerichtete Signalwege die Adhäsion von T-Lymphozyten und insbesonders die zellulären Adhäsionskräfte direkt verstärken

A biophysical perspective on receptor-mediated virus entry with a focus on HIV

As part of their entry and infection strategy, viruses interact with specific receptor molecules expressed on the surface of target cells. The efficiency and kinetics of the virus-receptor interactions required for a virus to productively infect a cell is determined by the biophysical properties of the receptors, which are in turn influenced by the receptors' plasma membrane (PM) environments. Currently, little is known about the biophysical properties of these receptor molecules or their engagement during virus binding and entry. Here we review virus-receptor interactions focusing on the human immunodeficiency virus type 1 (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), as a model system. HIV is one of the best characterised enveloped viruses, with the identity, roles and structure of the key molecules required for infection well established. We review current knowledge of receptor-mediated HIV entry, addressing the properties of the HIV cell-surface receptors, the techniques used to measure these properties, and the macromolecular interactions and events required for virus entry. We discuss some of the key biophysical principles underlying receptor-mediated virus entry and attempt to interpret the available data in the context of biophysical mechanisms. We also highlight crucial outstanding questions and consider how new tools might be applied to advance understanding of the biophysical properties of viral receptors and the dynamic events leading to virus entry

The influence of adhesion molecules on binding and protein organization in cell contacts

Interactions between immune cells such as T cells and antigen-presenting cells (APCs) are integral for mounting an adaptive immune response. The interaction between the T cell receptor (TCR) and the antigen-presenting major histocompatibility complex (pMHC) on a contacting T cell and APC, is widely accepted to be the key interaction. If the interaction is favourable, then T cell activation occurs. A large pool of research has been aimed at characterizing this interaction by measuring the binding kinetics and relating it to the T cell response. A simplified model membrane system called a supported lipid bilayer (SLB) is often used to mimic the membrane of the APC. In many T cell activation studies, the SLB contains the nickel-chelating lipid DGS-NTA(Ni) to functionalize the SLB with histidine-tagged proteins. In the first part of this thesis I show that interactions between DGS-NTA(Ni) and the T cells can lead to, unwanted, T cell signaling. It was found that increasing the concentration of DGS-NTA(Ni) both increased cell adhesion and the fraction of signaling cells. Adding bovine serum albumin (BSA) functioned as a blocking agent, preventing unspecific cell adhesion and decreased the fraction of signaling cells down to a basal level. A low level of signaling was also obtained when functionalizing the blocked SLBs with adhesion molecules binding to receptors on the T cell. In contrast, without blocking these functionalized SLBs again signaled at a similar level to the unblocked, not functionalized DGS-NTA(Ni) SLBs. The DGS-NTA(Ni) signaling was argued to be due to TCR-DGS-NTA(Ni) interactions and stressed the importance of adequately blocking these interactions in T cell activation studies.In the second part of the thesis, a new method to measure the two-dimensional dissociation constant (2D Kd) of ligand-receptor interactions on single cells is presented. This is measured on individual cell-SLB contacts, providing an accurate new means of measuring binding affinity and to study differences in the 2D Kd in the cell population. In the final part of the thesis, the interaction of TCR-pMHC in the presence of adhesion molecules of different length and density is studied. Adhesion molecule pairs of similar height as TCR-pMHC have been argued to facilitate the TCR-pMHC interaction by physically keeping the opposing membranes at an optimal distance for binding. However, adhesion pairs of different height than that of TCR-pMHC are also important for cell-cell contact formation and have been shown to result in an impaired T cell response if removed. To better understand how, and if, adhesion molecules of different lengths influences TCR-pMHC binding the 2D Kd of TCR-pMHC in the presence of differently-sized adhesion molecules was studied. For this purpose, a SLB functionalized with TCR and an adhesion ligand, was allowed to bind cell with pMHC and the corresponding adhesion receptor. It was found that the 2D Kd of the TCR-pMHC interaction could be up to an order of magnitude higher (weaker) than the corresponding value for TCR-pMHC alone when having height-mismatched molecules. In addition, the TCR-pMHC distributed non-homogeneously in the cell-SLB contacts when having height-mismatched adhesion molecules, but homogeneously when having height-matched adhesion molecules. Furthermore, even for height-matched adhesion molecules the 2D Kd of the TCR-pMHC interaction was found to be dependent on the relative density fraction of TCR to adhesion molecules, with low fractions of TCR molecules giving 2-3 times weaker binding. This indicates that TCR-pMHC binding in cell contacts depends significantly on the local environment and not only on the protein-protein interaction per se

Investigations into Convective Deposition from Fundamental and Application-Driven Perspectives

Crystalline particle coatings can provide critical enhancement to wide-ranging energy and biomedical device applications. One method by which ordered particle arrays can be assembled is convective deposition. In convective deposition, particles flow to a surface via evaporation-driven convection, then order through capillary interactions. This thesis will serve to investigate convective deposition from fundamental and application-driven perspectives. Motivations for this work include the development of point-of-care diagnostic devices, macroporous membranes, and various energy applications. Immunoaffinity cell capture devices display enhanced diagnostic capabilities with intelligently varied surface roughness in the form of particle coatings. Relatedly, highly crystalline particle coatings can be used to template the fabrication of macroporous polymer membranes. These membranes display highly monodisperse pores at particle contact points. In addition, ordered areas of particles, acting as microlenses, can enhance LED performance by 2.66-fold and DSSC efficiency by 30%. Previous research has targeted the formation of crystalline monolayers of particles. However, much insight can be gleaned from imperfect coatings. The analysis of submonolayer coatings, exhibiting significant void spaces, provides insight as to the specific mechanisms and timescales for flow and crystallization. A pair of competing deposition modes, termed ballistic and locally-ordered, enables the intelligent design of experiments and enables significant enhancement in control of resultant thin film morphology. Surface tension-driven particle assembly is subject to a number of native instabilities and macroscale defects that can irreversibly compromise coating uniformity. These include the formation of three-dimensional streaks, where surface tension-driven flow spurs on the nucleation of large imperfections. These imperfections, once nucleated, exhibit a feedback loop of dramatically enhanced evaporation and resultant flow. In addition, thick nanoparticle coatings, subject to enormous drying stresses, exhibit highly uniform crack formation and spacing in an attempt to minimize system energy. Both these imperfections yield insight on convective deposition as a fundamental phenomenon, and intelligent design of experiments moving forward. Cracking can be suppressed through layer-by-layer particle assembly, whereas streaking can be controlled via several significant process enhancements. Process enhancements include the addition of smaller constituent, as packing aids, to suspension, the application of lateral vibration, and the reversal of relevant surface tension gradients. The transition from unary to binary suspensions represents a significant improvement to convective deposition as a process. Nanoparticles act as packing, and flow, aids, wholly suppress macroscale defects under ideal conditions. A relative deficiency or excess of nanoparticles can generate complex coating morphologies including multilayers and transverse stripes. The application of lateral vibration to convective deposition allows the assembly of monolayer particle coatings under a larger range of operating conditions and at a faster rate. Macroscale defect formation can increased through an enhancement of the natural condition, where evaporative cooling generates a thermal gradient in drying droplets. Conversely, these defects can be suppressed with a reversal of this gradient, which will reverse the direction of surface tension-driven recirculation. These fundamental developments in understanding, and associated process enhancements, are critical in current efforts to scale up convective deposition. As convective deposition evolves from laboratory-scale batch experiments to continuous, large scale, coatings, repeatability and robustness, as well as an ability to controllably change thin film morphology, will be essential

Quantification of propidium iodide delivery with millisecond electric pulses: A model study

A model study of propidium iodide delivery with millisecond electric pulses is presented; this work is a companion of the experimental efforts by Sadik et al. [1]. Both membrane permeabilization and delivery are examined with respect to six extra-cellular conductivities. The transmembrane potential of the permeabilized regions exhibits a consistent value, which corresponds to a bifurcation point in the pore-radius-potential relation. Both the pore area density and membrane conductance increase with an increasing extra-cellular conductivity. On the other hand, the inverse correlation between propidium iodide delivery and extra-cellular conductivity as observed in the experiments is quantitatively captured by the model. This agreement confirms that this behavior is primarily mediated by electrophoretic transport during the pulse. The results suggest that electrophoresis is important even for the delivery of small molecules such as propidium iodide. The direct comparison between model prediction and experimental data presented in this work helps validate the former as a robust predictive tool for the study of electroporation

New Types of Morpho-Physiological Changes in Cells Exposed to Nanosecond Pulsed Electric Field

Exposure of cells to a pulsed electric field (PEF) is the basis of multiple techniques and treatments. Nanosecond pulsed electric field (nsPEF) poses unique characteristics to induce subtle cellular effects while preserving cell integrity. Improving understanding of the mechanisms triggered by nsPEF in cells inspires new applications for the nanosecond pulse technology. Although many effects of nsPEF remain unknown, they can be inferred from morpho-physiologic changes, or cell reshaping, that accompany nsPEF exposure. During the exposure cells undergo reshaping that is manifested in swelling and diffuse blebbing. Recently we identified two new distinct forms of reshaping, pseudopod-like blebbing and microvesiculation, which are present in cells exposed to long trains of nsPEF with high pulse repetition frequency (PRF). Microvesiculation is known in activated and damaged cells, while pseudopod-like blebbing has not been described previously. The objective of this dissertation is to characterize and establish the key mechanisms involved in these new nsPEF-induced phenomena. Two specific aims of the dissertation are 1) to establish the factors involved in nsPEF-induced microvesiculation; and 2) to define pseudopod-like blebbing as a function of pulse parameters and establish the mechanisms underlying the formation of pseudopod-like blebs (PLBs). These aims are fulfilled through the analysis of microscopy data obtained from the nsPEF-exposed cells using fluorescent labeling and pharmacologic inhibition. The adapted labeling techniques take advantage of the nsPEF-induced cell permeabilization to induce staining of microvesicles and pseudopod-like blebs (PLB). The results show that microvesiculation develops in HL60 and U937 cells in response to Ca2+ presence during nsPEF exposure. Microvesiculation does not depend on colloid-osmotic swelling (COS). PLBs are produced in U937 cells due to active formation of actin cortex and require the absence of Ca2+. Extension of PLBs is triggered and guided by nanosecond pulses while bleb growth is fueled by water uptake through a COS mechanism. PLB retraction is produced by myosin contractility and can be coupled to cell translocation

RECEPTOR MOBILITY AND CYTOSKELETAL DYNAMICS AT THE IMMUNE SYNAPSE: THE ROLE OF ACTIN REGULATORY PROTEINS

Spatial and temporal regulation of actin and microtubule dynamics is of utmost importance for many cellular processes at different sub-cellular length scales. This is particularly relevant for cells of the immune system, which must respond rapidly and accurately to protect the host, where B cells and T cells are the main players during the adaptive immune response. An understanding of the biophysical principles underlying cytoskeletal dynamics and regulation of signaling will help elucidate the fundamental mechanisms driving B and T cell immune response. B cell receptor (BCR) diffusivity is modulated by signaling activation, however the factors linking mobility and signaling state are not completely understood. I used single molecule imaging to examine BCR mobility during signaling activation and a novel machine learning based method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors Arp2/3 and formins resulted decreased BCR mobility. Loss of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, leads to a predominance of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces diffusivity of the stimulatory co-receptor CD19, but not that of unstimulated FcγRIIB, an inhibitory co-receptor. Our results implicate the dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions, thereby modulating B cell signaling. Activation of T cells leads to the formation of the immunological synapse (IS) with an antigen presenting cell (APC). This requires T cell polarization and coordination between the actomyosin and microtubule cytoskeleton. The interactions between the different cytoskeletal components during T cell activation are not well understood. I use high-resolution fluorescence microscopy to study actin-microtubule crosstalk during IS formation. Microtubules in actin rich zones display more deformed shapes and higher dynamics compared to MTs at the actin-depleted region. Chemical inhibition of formin and myosin activation reduced MT deformations, suggesting that actomyosin contractility plays an important role in defining MT shapes. Interestingly MT growth was slowed by formin inhibition and resulting enrichment of Arp2/3 nucleated actin networks. These observations indicate an important mechanical coupling between the actomyosin and microtubule systems where different actin structures influence microtubule dynamics in distinct ways