IMMUNITY-ON-A-CHIP Microfluidic Devices for Immunoengineering

Abstract

Immunitet-på-mikrobrikke Mikrofluidiske innretninger for immunologisk forskning og utvikling Sammendrag: Det siste tiåret har mikrofluidteknologi, dvs væskestrømmer i mikroskala basert på prosesser fra halvlederindustrien, gitt et betydelig løfte om forbedringer innen diagnostikk, biomedisinsk forskning og individ-tilpasset medisin. Det er også i ferd med å bli et bærekraftig alternativt verktøy for dyremodeller og dagens in vitromodeller for sykdom og helse for utprøving av nye medikamenter I dette prosjektet ble mikrofluidiske innretninger utviklet med det formål å gjenskape og etterligne komplekse immunologiske og inflammatoriske celle-prosesser som naturlig skjer i kroppene våre. Spesielt var vi interessert i de to sentrale immunologiske prosessene cellemigrasjon, hvordan celler finner veien mot ønskede mål, og celle:celle interaksjon, hvordan cellene snakker sammen gjennom direkte kontakt. Produksjonen av disse biochipene ble gjort på renrommet ved NTNU NanoLab mens celleforsøkene ble utført ved Institutt for Klinisk og Molekylær Medisin ved Fakultet for Medisin og Helsevitenskap. Resultatene viser at vi kan gjenskape fysiologiske hendelser inne i våre kunstige immunologiske mikromiljøer. Dette åpner for helt nye tilnærminsgmåter og helt nye muligheter for immunterapi mot kreft og andre sykdommer med immunologisk komponenter, og kan bli et helt nytt verktøy for uttesting av vaksiner og medikamentelle terapier.

The immune system plays an important role on the defense and regulation of the human body. To study human immunity and diseases, animal models and traditional cell cultures have been used for many years, and contributed significantly on the development of powerful new therapies. However, animal studies have evidenced limitations considering the differences with the human metabolism and disease. Additionally, traditional in vitro models have failed to mimic the normal microarchitecture environment of cells in tissues because of the absence of biochemicomechanical features (e.g., scaffold stiffness; controlled flow for the delivery of nutrients and gases; shear stress; haptotatic gradients). In this context, in the past decade microfluidic technology has emerged as an important alternative tool for translational immunology. Microfluidics offer the possibility to recreate in vivo compartmentalization of microphysiological models on in vitro platforms, allowing precise control of cellular biophysical and biochemical stimuli. These platforms capabilities further increase by coupling on-chip technologies for detection, sorting and 3D scaffold growth, or other needs/requirements to be explored in the future (e.g., the development of a recirculation system through a vascular network for a multi-organ-on-a-chip). Thus, this technology has a remarkable potential for medical applications, and with industrial scale up, especially as a diagnostic and research tool for point-of-care testing, drug screening tests and translational cancer and immunology. Microfluidic technology also allows the development of clinical tool towards in vitro immunization, personalized cancer vaccination, immunotherapeutic approaches for cancer and autoimmune diseases etc. In this context, the scope of this project was focused on the conception of microfluidic platforms, which allowed to mimic and to explore in vitro microphysiological models for translational immunology. These platforms allowed to study in real time on-a-chip several physiological events that occur during the initial stage of inflammation and adaptive immune response, such as the encounters and interactions between T cells with dendritic cells supported by a natural 3D fibroblast reticular scaffold, the immunological synapse formation and its cleavage, the immune cell migration and decision-making processes under inflammatory conditions. In this work, the fabrication of the microfluidic devices was performed at NTNU NanoLab cleanroom. Cell preparations and experiments were done at the Department of Clinical and Molecular Medicine, Faculty of Medicine NTNU (Trondheim, Norway). The microfabrication methodology consisted initially on the creation of designs and Multiphysics simulations to predict hemodynamic velocity and shear stress inside microchannels or microchambers. By using standard semiconductor photolithography and well-suited soft lithography for biomedical applications, to produce polydimethylsiloxane (PDMS)-based microfluidic devices. During this project, in order to mimic the immune environment conditions several cell types were used such as mouse lymph node fibroblast reticular cells (FRCs), primary bone marrow-derived dendritic cells (BMDC), enhanced green fluorescent protein (eGFP)+murine dendritic cell line “MutuDC 1940”, and non-adherent CD4+ and CD8+ T cells.

In this context, narrowing the focus to more specific sub-goals, during this PhD project three classes of microfluidic devices were developed: (i) Paper I, a microfluidic channel to promote the cognate physical interactions between T cells:APCs under varying shear stress, in order to study and to establish basic operational procedures associated with the immunological synapse formation; (ii) Paper II, a single-step and user friendly diffusion flow-free device to study the immune cell migration and decision-making process under inflammatory conditions; and (iii) Paper III, a microfluidic chamber that mimicked the microarchitecture of immune environment and cell behavior in the T cell zone of a lymph node (LN), being considered as a LN-T cell zone-on-the-chip. In this paper, the mechanisms and dynamics of the growth of FRC scaffold by induction of continuous flow was explored. Basic cell behavior were further performed namely, attachment and detachment of antigenspecific and unspecific T cells to active antigen-presenting or non-activated dendritic cells supported on FRC meshwork at different shear stresses. The studied immune platforms demonstrated microphysiological events very similar to the ones reported in vivo. As main results, in Paper I we observed random migration of antigen-specific T cells onto the antigenpresenting DC monolayer with a mean T cell:DC dwell time of 12.8min and a mean velocity of 6μm min-1 at a shear stress of 0.01Dyn cm-2. In this study, we further identified that the range of mechanical force associated with the immunological synapse formation was ~0.25-4.8nN. Through the Paper II, the developed platform allowed us to identify the directional migration of T cells and DCs towards the inflammation foci (with a mean speed 9μm min-1). In Paper III, we developed a microfluidic chamber that allowed the growth of FRC scaffold through the application of perfusion flow, which induces both chemical and mechanical stimuli. This was attained upon continuous perfusion during 48h at 100nl min-1 with a corresponding shear stress 0.001 to 0.004Dyn cm-2. By using this FRC scaffold, a second stage of cell attachment/detachment tests were performed with 2 types of cell interactions: DCs:FRC scaffold and T cells:FRC scaffold. From these tests, high cell motility was observed on DCs and T cells, both with a mean velocity of ~10μm min-1. At a third stage of attachment/detachment tests, 3 types of cells and interactions T cell:DC:FRC scaffold were analyzed: without cognate interactions, T cells and DCs presented a mean velocity of ~5μm min-1, random movements and “stop and go” interactions during 3min. With cognate interactions, it was visible a significant decrease of DC and T cell velocity to ~2.5μm min-1, where T cells moved in characteristic looping patterns making serial contacts with the same or with neighboring DCs, with interval of interactions around ~7min. In this sense, we believe that these microfluidic platforms can open new horizons for the investigation of intercellular signaling of immune synapses and therapeutic targets for immunotherapies.