A Multimaterial and multicellular Bioprinting platform to elaborate advanced models of microenvironments – PrinTiss

To achieve the objectives of the PrinTiss project, an interdisciplinary approach was adopted, combining microfluidics, laser photopolymerization, and tissue biology. The FlowPrint platform, jointly developed by LAAS-CNRS and Fluigent, is based on a microfluidic printhead integrating injection and aspiration channels for localized material delivery, as well as optical fibers for targeted polymerization. This design uses a hydrodynamic confinement principle that maintains a localized material flow while preventing cross-contamination through an integrated aspiration system. The channel geometry was optimized using numerical simulations to ensure high resolution and reproducibility of printed structures.

The microfluidic instrumentation developed by Fluigent enabled synchronization of material injection with photopolymerization by precisely controlling critical parameters such as flow rates, pressures, and printhead movement speed. Dedicated software was designed to automate the printing process by generating optimized trajectories from 3D files and dynamically adjusting parameters based on the materials used. Concurrently, the RESTORE team developed biological protocols to validate the relevance of printed tissue models. Mesenchymal stem cell spheroids were encapsulated in GelMA hydrogels, a biocompatible and photopolymerizable biomaterial whose mechanical properties were adjusted to replicate those of native adipose tissue. Cell cultures were conducted under optimized conditions to promote differentiation into functional adipocytes while preserving vascular network integrity.

A library of photopolymerizable biomaterials was established at LAAS-CNRS. These materials, whose rheological and mechanical properties can be controlled through photopolymerization, are now routinely used in multiple projects.

The results obtained within the PrinTiss project confirmed the success of the methodological approaches implemented while demonstrating the scientific and technological relevance of the Flowprint platform. From the early stages of the project, the consortium successfully validated a microfluidic printhead capable of combining microfluidics and photopolymerization for high-resolution bioprinting. Thanks to the integration of optical fibers and an optimized hydrodynamic confinement system, the platform achieved a resolution below twenty micrometers as initially planned, while enabling multimaterial printing with rapid transitions between materials.

The platform demonstrated its ability to print complex two-dimensional and three-dimensional structures using PEGDA and GelMA hydrogels, with a minimum resolution of twenty micrometers. Tests confirmed cellular viability exceeding ninety percent after encapsulation in GelMA hydrogels, as well as successful differentiation into functional adipocytes. The technology also enabled the printing of multimaterial structures with rapid material transitions, validating its flexibility for tissue engineering applications.

The RESTORE team validated the biological relevance of printed tissue models by demonstrating vascular network formation in encapsulated spheroids, confirmed through immunostaining. These models reproduced key characteristics of native adipose tissues, such as inducible UCP1 expression and increased mitochondrial respiration, confirming their relevance for studying metabolic diseases. A library of photopolymerizable biomaterials was developed and is now routinely used in multiple projects. These materials allow spatial control of mechanical and rheological properties, essential for tissue engineering applications.

Industrial collaborations were initiated with partners like Alvéole to explore the coupling between photopatterning and bioprinting. Discussions with Cellink and Gilson are ongoing to evaluate potential applications.

The perspectives opened by the PrinTiss project revolve around three main axes: extending technological applications toward fundamental biological questions, strengthening industrial and academic collaborations, and consolidating scientific advances for clinical applications. In the short term, the FlowPrint platform will focus on more fundamental biophysics applications aimed at creating two-dimensional and three-dimensional microenvironments with heterogeneous mechanical and compositional properties. These developments will particularly target epithelial and endothelial models, with a specific focus on adipose tissue vascularization mechanisms.

The initiated industrial collaborations, notably with Alvéole, offer perspectives for exploring the coupling between microscope-based photopatterning and FlowPrint technology. These partnerships could demonstrate the complementarity of these approaches for generating controlled cellular microenvironments. Furthermore, ongoing discussions with companies like Cellink and Gilson could lead to research collaborations aimed at evaluating the integration of developed biomaterials into their existing platforms.

In the longer term, clinical perspectives represent a major challenge. Prevascularized beige adipose tissue models could be optimized for therapeutic applications, particularly in the treatment of obesity and type 2 diabetes. This will require validating the metabolic functionality and stability of printed tissues in vivo, in collaboration with clinical partners. Current work on characterizing the mechanobiological properties of hydrogels and organoid encapsulation will provide a solid foundation for these developments.

A final aspect of the perspectives concerns continuing fundamental research on understanding photopolymerization mechanisms and their impact on hydrogel properties. Preliminary results showing that photopolymerization enables spatial control of hydrogel architecture and rheological properties will be further explored in Sandra Perez's work. This research could lead to significant advances in mastering the mechanical properties of biomaterials, essential for tissue engineering applications.

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This project aims at developing a multi-material bioprinting platform involving microfluidics and integrated optics concepts to create heterogenous 3D models of tissues. The system will be adapted for the 3D assembly of different biological building blocks consisting of biomaterials mimicking the extracellular matrix, individual cells, and spheroids or organoids with a placement accuracy down to the cell scale. Our ambition is to demonstrate the ability of the Printiss approach to fabricate controlled and reproducible models of tissues reproducing physiological or physiopathological features of the in vivo microenvironment, permitting their evolution towards a functional state. These models will allow one to explore cell-spheroid interactions as well as tissue-material interactions from the millimeter to the centimeter scales. We propose to apply this technology to fundamental questions related to the pre-vascularization of adipose tissue as a soft tissue model.