The Cellular Beacon: Three Dimensional Fabrication of Cell Encapsulated Scaffolds and Synthetic Receptors as an in-vitro model of acute phase inflammation.

Project leaders

Annalisa Tirella, Hung Yin Lin

Agreement

TAIWAN - NSTC - National Science and Technology Council

Call

CNR/NSC 2014-2015

Department

Biomedical sciences

Thematic area

Biomedical sciences

Status of the project

New

Research proposal

Recapitulating the innate immune response in-vitro is a challenging task as the immune system is highly complex, with a multitude of cell types and signaling molecules. Upon tissue damage or infection, circulating monocytes are recruited to the tissue where they differentiate to macrophages, and initiate cytokine production. Successively, a cascade of signals and pathways are activated, setting off a systemic response. From a simplified point of view, acute phase inflammation can be depicted as the initial responses of the immune system in which chemical factors (i.e. inflammatory mediators) are released by injured cells (e.g. resident macrophages, dendritic cells) to promote a cascade of events. Cytokines produced by macrophages and other cells of the immune system can thus be considered as the first molecular beacons signaling stress in the body. Reports show that macrophage signaling increases with substrate stiffness and that hepatic tissue stiffness is associated with monocyte activation. Indeed both the presence of a substrate as well as the biochemical nature of the microenvironment condition monocyte differentiation into tissue macrophages.
Recreating and interrogating this environment in a non-destructive manner not only poses an important technical challenge, but could also be used as a cellular beacon for probing cell response to environmental triggers such as pollutants, drugs, or nanomaterials.
In this context, the aim of this project is to construct a cellular beacon as an in-vitro model of the acute phase immune response. Adopting the liver as a starting point, we will seek to reconstruct a simplified model of the hepatic microenvironment in the form of gel microspheres containing encapsulated macrophages and monocytes and oxygen nanosensors. The cellular beacon will be triggered to activate stress signals either by modulation of substrate stiffness or through exposure to toxic nanoparticles. Smart molecular imprinted sensors for TNFa will enable monitoring of paracrine signals generated by the cells.
Workplan and methodology
Task1. Development of electrochemical sensors for TNFa (NUK@TWN)
Molecularly imprinted polymers (MIPs) offer the advantage of being relatively stable, unlike biological recognition elements such as enzymes or antibodies. In this project we will use patterned in situ polymerized hydrogels imprinted with TNFa as electrochemical sensors. We propose to prepare micro- and nanopatterned polypeptide hydrogels on conducting substrates by the formation of thiol-ene/yne bond or dityrosine by UV illumination. The sensors will be characterized for sensitivity and reversibility to ensure a picomolar detection limit and time response of a few minutes.

Task2. Develop magnetic fluorescent nanosensors (NS) for O2 (NUK@TWN)
Not only is hypoxia associated with inflammation, but the production of reactive oxidative species also plays a crucial role the modulation of the immune response. Furthermore, O2 is a limiting nutrient in 3D in-vitro cultures. In this proposal we will incorporate ruthenium into core-shell magnetic synthetic polymer nanoparticles to realize O2-NS. The performance of the NSs will be measured using standard commercial probes to ensure an acceptable dynamic range (0 to 20 mmHg of O2 partial pressure), sensitivity (0.1 mmHg) and time response (a few minutes).

Task3. Fabricate micro-sized hydrogel spheres with healthy and fibrotic hepatic stiffness (IT,NUK@TWN)
The Spherical Hydrogel Generator (SpHyGa) developed @IFC-CNR allows the fabrication of spherical sized controlled hydrogels with diameters ranging from 100 to 4000 um. Microspheres corresponding to the dimensions of hepatic lobules will be fabricated using alginate/collagen composites. By tuning the degree of cross-linking of the collagen component, we will generate porous spheres with elastic moduli corresponding to those of a normal liver (1 kPa) and a fibrotic liver (5 kPa). Optical Coherence Tomography (OCT, available @NCKU) will be used to map the stiffness distribution in the spheres (spatial resolution 8 um, stiffness resolution 0.1 kPa).

Task4. Fabricate cellular beacons by encapsulation of THP1 macrophages and O2-NS in soft and stiff micro-environments. (IT, NCKU&NUK@TWN)
Hydrogel microspheres including oxygen NSs developed in Task2 will be fabricated, tailoring biomaterial composition in order to obtain respectively healthy and fibrotic hepatic stiffness. Microsphere properties will be characterized using OCT. The ability to measure/monitor normoxia and hypoxia states will be then tested using confocal microscopy. Then differentiated THP1 macrophages will be co-encapsulated and characterized for vitality and function using vital tracker dyes (calcein), IL-1 expression and TNFa using both ELISA and the MIP sensor developed in Task1.

Task5. Cellular beacon fabrication by encapsulation of human monocytes in micro environment composed of synthetic scaffolds with different elastic moduli and measurement of systemic TNFa, oxygen content and stiffness (IT,NCKU&NUK@TWN)
Task4 will be repeated using human monocytes withdrawn from healthy volunteers. Here our aim is to assess the feasibility of using primary monocytes in our system, as the first step towards the establishment of an acute phase immune system for application in personalized medicine.

Task6. Exposure of the systemic environment to silver nanoparticles (NPs) in the presence of cellular beacons developed in Task4.
As demonstrated by the CNR group and others, silver nanoparticles are known to induce TNFa expression in a variety of cell types. Using sub-lethal NP concentrations already established in our previous studies, we will study the response of the system when exposed to different concentrations of Ag. Here our aim is to assess the feasibility of using the cellular beacon to detect and predict the pro-inflammatory potential of environmental materials.

Research goals

The aim of this project is to construct a cellular beacon as a model of the acute phase immune response. A simplified model of the hepatic microenvironment in the form of gel microspheres will be developed and used to trigger the activation of macrophages and monocytes encapsulated within the spheres. MIP electrochemical sensors for TNFa will be used to detect the degree of activation in the system, monitoring paracrine signaling. Optical nanosensors coupled with confocal microscopy and OCT will be used to monitor respectively local oxygen partial pressure and stiffness in the microspheres. Successively the system will be challenged with silver nanoparticles known to induce hepatic and systemic inflammation in order to assess the responsivity of the cellular beacon.
The proposal combines expertise of the two partners to recreate a smart, versatile cellular beacon for a myriad of applications such as toxicology, personalized medicine and environmental sensing. The knowledge of polymer chemistry, biosensing and microfabrication of the Taiwanese partners will be employed to develop amperometric MIP sensors for TNFa and ruthenium based magnetic oxygen fluorescent nanosensors. The Italian partners, experts in gel fabrication, hepatic tissue biomechanics, cell culture, nanotoxicology and imaging, will contribute by designing, fabricating and monitoring the monocyte encapsulated microspheres and conducting cell culture and nanomaterial exposure experiments.

Last update: 23/04/2024