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© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)., Blood-vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs-sprouting angiogenesis and vascular remodeling-is established by a shift of collective front-to-rear polarity of endothelial cells in the mouse retina. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood-flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns., Funding: European Research Council: C.A.F. (679368); X.T. (883739). European Commission: C.A.F. and M.O.B. (801423); X.T. and P.R.-C. (731957). H2020-MSCA-PF grants to M.G.-G. (797621) and M.O. (842498). Fondation LeDucq: C.A.F., A.E., and M.O.B. (17CVD03). EPSRC: M.O.B. (EP/T008806/1; EP/R029598/1). Fundação para a Ciência e Tecnologia: C.A.F. (PTDC/MED-PAT/31639/2017; PTDC/BIA-CEL/32180/2017; CEECIND/04251/2017). Spanish Ministry of Science and Innovation: P.R.-C. (PID2019-110298GB-I00); X.T. (PGC2018-099645-B-I00). Generalitat de Catalunya: X.T. and P.R.-C. (2017-SGR-1602). La Caixa Foundation: X.T. and P.R.-C. (LCF/PR/HR20/52400004). Fundació la Marató de TV3: X.T. (201903-30-31-32). EMBO: L.M.F. (ALTF 2-2018)

We present here a screening method based on a microfluidic platform, which can generate four orthogonal and overlapping concentration gradients of soluble compounds over a monolayer of cells, in combination with automated and in situ image analysis, for use in regenerative medicine research. The device includes a square chamber in which cells are grown, and four independent supply channels along the sides of the chamber, which are connected through an array of small diffusion channels. Compounds flown through the supply channels diffuse through diffusion channels into the chamber to create a gradient over the cell culture area. Further, the chamber is connected to two channels intended for introduction of cells and in situ staining. In this study, the dimensions of the different channels were optimized through finite element modeling to yield stable gradients, and two designs were used with gradients spanning 2.9-2.4M and 3.4-2.0M. Next, overlapping gradients were generated using four rhodamine-derived fluorescent dyes, and imaged using confocal microscopy. Finally, the platform was applied to assess the concentration-dependent response of an osteoblastic cell line exposed to a hypoxia-mimicking molecule phenanthroline, using an in situ fluorescent staining assay in combination with image analysis, applicable to closed microfluidic devices. The on-chip assay yielded results comparable to those observed in conventional culture, where a range of concentrations was tested in independent microwells. In the future, we intend to use this method to complement or replace current research approaches in screening soluble compounds for regenerative medicine, which are often based on one-sample-for-one-experiment principle.

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia The present work focuses the optimization of the production of human Leukemia Inhibitory Factor (hLIF) by human embryonic kidney 293 (HEK293) - EBNA1 cell line cultured as suspension aggregates in spinner flask. The effects of initial cell density and feeding-regime in cellular growth and productivity were evaluated. A first phase occurs until the end of exponential growth in medium containing serum, being followed by puromycin selection of cells containing hLIF plasmid. A second phase, the production phase, is developed under serum-free conditions. Three initial cell densities (2´104 cells.mL-1, 2´105 cells.mL-1 and 2´106 cells.mL-1) were tested and the effect on maximum cell density and cell aggregate size distribution was evaluated. The inoculum of 2´105 cells.mL-1 led to final higher cellular densities around 7´106 cells.mL-1 and homogeneous aggregates around 278 μm were observed. The effect of the feeding-regime was then studied for the production of recombinant hLIF with an initial cell density of 2´105 cells.mL-1 by performing metabolite analysis Metabolite analysis revealed the occurrence of glucose and glutamine starvation upon 9 and 7 days, respectively, in 25% FR. Therefore, glutamine acted as an alternative carbon, energy and aminoacid source in HEK293-EBNA1 growth. Observed lactate levels were bellow the inhibition limit concentrations (30 mM 1).HEK293-EBNA1 did not seem to be affected by the produced levels of ammonia. In conclusion, the 25% feeding regime at 2´105 cells.mL-1 initial cell density lead to the higher viable cell densities, either along culture time at growth and at production phase, with aggregates in suspension below necrotic diameters. This experiment conditions also allowed achieving the higher LIF volumetric productivity, 125 ± 2.2 ng.mL-1.

In the field of tissue regeneration, polymers are widely used for the manufacturing of scaffolds. Instructive structures, based on crosslinked polymers are expected to provide a proper environment for proliferation, migration and differentiation of the embedded cells/tissue. In order to mimic the natural extracellular matrix (ECM), the scale of the scaffolding needs to be in the micron range. Unlike conventional printing techniques, two photon polymerization (2PP) is capable of creating micro- (and even nano-) sized three-dimensional features with high resolution. In general, commercially available resins for 2PP lack the ability to be chemically post modified. Also, mechanical properties are difficult to tune. Especially, post-modification reactions might be very useful in the coupling of growth factors onto the scaffolds, which can lead to an even higher control over cell behavior. In this master thesis, the synthesis of photocurable resins for two photon polymerization was investigated. Besides crosslinker molecules and a photoinitiator, polymers were added to the mixture. These polymers were synthesized via an iniferter mechanism in a flow-setup. This variant of RAFT polymerization provide high end-group fidelity of the polymers. Resins, which include such polymers, were crosslinked in the 2PP process yielding microstructures with the end-groups onto the surface. These end groups can be modified to control cell behavior.

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