Speaker
Description
Introduction
One of the persistent challenges in the development of reliable in vitro tissue models is the inability to precisely and reproducibly control microarchitectures. This is especially critical for vascularized constructs, where network formation often relies on the stochastic self-assembly of randomly distributed cells, which leads to batch-to-batch variability, limited predictability, and need for high-powered studies to draw statistically meaningful conclusions[1]. To address this, we present a deterministic bioprinting strategy based on laser induced forward transfer (LIFT), enabling single-cell resolution and control over spatial cell patterning[2].
Material and Methods
We leveraged LIFT’s versatility to systematically map the printability of a cell medium-like aqueous solution (BSA in PBS) and a polymer solutions (alginate) across a wide viscosity range (0–1000 mPa·s). We analysed printed outcomes (e.g., droplet diameter, circularity, pattern fidelity, and satellite formation) as well as captured jetting dynamics with high-speed imaging. Through a parametric study, we identified optimal transfer conditions by tuning laser energy, donor–receiver distance, ink volume, and composition.
As a model system, we targeted the reproducible biofabrication of vascular-like networks using GFP-labelled human umbilical vein endothelial cells (HUVECs). By tuning droplet spacing and cell density, we printed a series of branching vascular patterns with controlled droplet composition (e.g. cell count).
To further explore the functionalization of pre-vascular designs, we engineered bioprinted micro-architectures composed by co-cultures of HUVECs and mesenchymal stem cells (MSCs) using spatially resolved deposition and distinct ink-substrate combinations (cells printed on fibrin or embedded in fibrinogen and printed onto Matrigel/thrombin with a 3:1 ratio HUVEC/MSC).
Results
We established optimal LIFT biofabrication conditions, enabling the reproducible printing of branching vascular-like networks with precise control over droplet composition and pattern geometry. More precisely, we achieved the transfer of aqueous and polymer droplets with hundreds of picolitre-scale precision. Further, Poisson distribution predictions confirmed controlled cell-per-droplet deposition.
In branching constructs, we observed that patterns with identical total cell number but higher initial spatial density exhibited superior vascular organization and connectivity compared to those with more widely spaced droplets, underscoring the importance of positional control.
Additionally, we demonstrated robust post-printing viability of HUVECs and MSC. The spatial orchestration of HUVEC-MSC co-cultures enhanced construct stability over time and promoted early pre-vascularization, demonstrating that LIFT can serve as a modular platform to engineer microenvironments with tailored cell–cell and cell–matrix interactions.
Discussion and conclusion
Our results establish LIFT as a versatile technology that enables deterministic patterning at single-cell resolution. By addressing the limitations of random cell placement, our approach paves the way for more robust in vitro models for drug screening, disease modelling, and tissue engineering. Future work will focus on enhancing the functionality of the pre-vascularized constructs, such as perfusability to support nutrient supply in large tissue constructs.
References
[1] Fleischer, S., et al., From Arteries to Capillaries: Approaches to Engineering Human Vasculature (2020). Advanced Functional Materials.
[2] Bosmans, C., Ginés Rodriguez, N., et al., Towards single-cell bioprinting: micropatterning tools for organ-on-chip development (2023). Trends in Biotechnology.
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