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COmpound COatings NUrturing applications in Tissue Engineering

Periodic Reporting for period 1 - COCONUTE (COmpound COatings NUrturing applications in Tissue Engineering)

Reporting period: 2020-09-01 to 2022-08-31

Tissue engineering is a set of technologies aimed at producing artificial biological tissues that can be implanted into living organisms or used to test drugs or treatments on them, among other applications. In the long term, these technologies promise to eliminate the need for donors in organ transplants and to speed up the translation of drugs and medical treatments from the lab to the clinic. However, these long-term goals face several technical challenges whose solution require advances in fundamental fluid-mechanical processes. These advances encompass new techniques to deposit, grow or transport living cells at microscales. The overall objective of the COCONUTE project is to address fundamental multiphase fluid-mechanical problems with immediate application in the characterization and control of microflows relevant to tissue engineering. The more specific objectives we addressed were:

a) Developing an experimental non-invasive characterization technique for thin (micrometric) liquid coatings containing several immiscible phases.

b) Carrying out a theoretical and numerical study of the formation of compound thin films consisting of two layers.

c) Conducting an experimental and theoretical study of the periodic formation of floating liquid drops with volumes in the microliter range.

d) Characterizing experimentally and theoretically the inertia-driven flow of a viscous fluid in a porous fiber bundle.
We achieved the following progress towards the objectives of the COCONUTE project:

a) We designed, built and optimized an optical set-up able to characterize accurately the thickness of micrometric thin viscous liquid coatings deposited by dip-coating on a flat plate. The technique we developed is based on laser-induced fluorescence (LIF) and can be applied to films as thin as < 10 m, the characteristic size of eukaryotic cells. In the future, this technique may be exploited for the characterization of complex coatings (e.g. made of viscoelastic liquids) relevant to tissue engineering applications.

b) We developed a theory to describe the double coated film entrained on a flat plate pulled from a bath made of two immiscible liquid layers (e.g. oil on top of water). The model allows to compute the thicknesses of the coated films and predicts that one of them is always much thinner than the other. The results of our theoretical and numerical study were published in 2021 in the leading journal of our field, the Journal of Fluid Mechanics. They were also presented in two international and one national conferences.

c) We discovered a way to generate in a very controlled fashion floating drops (called liquid lenses) with volumes in the microliter scale, relevant to tissue engineering applications. By varying experimentally the injection flow rate and viscosity of the liquid forming the drops, we found scaling laws describing how the drop volume and their generation rate depend on these control parameters. Moreover, we have rationalized these experimental findings with a simple mathematical theory. We are currently preparing two scientific articles describing two different aspects of the problem.

d) We designed and optimized an experimental setup to produce an inertia-driven flow of a viscous fluid in a porous fiber bundle, by the means of short, well-controlled impacts. In our experiments we varied both the intensity of the impact and the viscosity of the liquids to determine in which region of the parameter space this inertia-driven transport mechanism could be achieved. The experimental data was rationalized using order-of-magnitude analyses. These results have been presented in an international conference in 2022.

During the project, we also organized three events to disseminate our results to less specialized audiences. Two of these events were scientific workshops targeting technical but broad audiences (soft matter scientists in general or even researchers from other scientific areas). Another event was addressed to the general public, in the context of the Madrid Science Week in 2021.
The project’s results lead to progress beyond the state-of-the-art and potential impact in the following ways:

a) The coating characterization technique we developed has several advantages compared to existing techniques: (i) it is comparatively inexpensive and fully non-invasive; (ii) it allows us to determine the time and space evolution of the coating thickness in real time; (iii) it is easy to apply to other geometries (e.g. coating on a fiber or rod) or fluids (e.g. cell suspensions akin to the bioinks used in 3D bioprinting) with minimal modifications. We expect our technique to speed up and reduce costs in the optimization of parameters in tissue engineering, which is usually a time-consuming and expensive part of the process.

b) The two-liquid dip-coating configuration we explored in the second objective of the project is totally novel. In particular, the idea of using a floating immiscible liquid layer to control the deposition of the coating has not been reported previously in the literature, to the best of our knowledge. This study endows researchers with a theoretical tool to accurately control the properties of cell-laden thin coating films by using a second immiscible liquid. We also expect this configuration to find applications beyond the tissue engineering processes that motivated the study, for example for the handling of liquid metals.

c) The liquid lens generation technique we discovered is analogous to the flow focusing technique widely used in microfluidics to produce micrometric drops. The main simplification brought by our method is the fact that drops (floating liquid lenses) are formed and evolve in a flow open to the ambient, thus avoiding limitations associated to the presence of walls in conventional microfluidics. We believe that its simplicity could make this novel technique competitive in situations where large flow rates are not needed (as is typically the case in tissue engineering applications). It may be used to produce drops containing micrometric tissue “seeds”, i.e. small clusters of cells that will form organoids, that need to be grown at the liquid/air interface (e.g. lung or skin tissue).

d) The flow of a viscous liquid inside a densely packed fiber bundle finds applications in the 3D-printing of tubular organs and tissues (such as blood vessels, muscles, etc.). One of the challenges in driving these flows is how to impose the necessary pressure gradients in a way that does not damage the incipient tissue precursors. Using the inertia-driven fluid-transport mechanism we studied may facilitate the removal of the scaffolds needed to grow tubular tissues and organs. It could also help to overcome some practical difficulties found in resin injection techniques used in the manufacturing of composite materials.