Bioinspired Precision Engineering of Three‐Dimensional Epithelial Stem Cell Microniches

Maintenance of the epithelium relies on stem cells residing within specialized microenvironments, known as epithelial crypts. Two‐photon polymerization (2PP) is a valuable tool for fabricating 3D micro/nanostructures for stem cell niche engineering applications. Herein, biomimetic gelatin methacrylate‐based constructs, replicating the precise geometry of the limbal epithelial crypt structures (limbal stem cell “microniches”) as an exemplar epithelial niche, are fabricated using 2PP. Human limbal epithelial stem cells (hLESCs) are seeded within the microniches in xeno‐free conditions to investigate their ability to repopulate the crypts and the expression of various differentiation markers. Cell proliferation and a zonation in cell phenotype along the z‐axis are observed without the use of exogenous signaling molecules. Significant differences in cell phenotype between cells located at the base of the microniche and those situated towards the rim are observed, demonstrating that stem cell fate is strongly influenced by its location within a niche and the geometrical details of where it resides. This study provides insight into the influence of the niche’s spatial geometry on hLESCs and demonstrates a flexible approach for the fabrication of biomimetic crypt‐like structures in epithelial tissues. This has significant implications for regenerative medicine applications and can ultimately lead to implantable synthetic “niche‐based” treatments

Direct Observation of a Photochemical Alkyne–Allene Reaction and of a Twisted and Rehybridized Intramolecular Charge-Transfer State in a Donor–Acceptor Dyad

The excited-state dynamics of an aniline–triazine electron donor–acceptor dyad with an alkyne spacer has been investigated using a combination of ultrafast broadband mid-IR and visible transient absorption and fluorescence spectroscopies. The transient IR data reveal the occurrence of an efficient alkyne to allene isomerization of the spacer with a time constant increasing from a few hundreds of femtoseconds to a few picoseconds with solvent viscosity. This process is faster than the vibrational cooling of the Franck–Condon excited state, indicative of nonequilibrium dynamics. The transient electronic absorption and fluorescence data evidence that this transformation is accompanied by a charge separation between the donor and the acceptor subunits. The allene character of the spacer implies an orthogonal orientation of the donor and acceptor moieties, similar to that proposed for twisted intramolecular charge-transfer states. Such states are often invoked in the excited-state dynamics of donor–acceptor dyads, but their involvement could never be unambiguously evidenced spectroscopically. The alkyne–allene isomerization involves not only a torsional motion but also a bending of the molecule due to the sp to sp2 rehybridization of one of the alkyne carbon atoms. This twisted and rehybridized intramolecular charge transfer (“TRICT”) state decays back to the planar and linear alkyne ground state on a time scale decreasing from a few hundred to ten picoseconds upon going from weakly to highly polar solvents. The different solvent dependencies reveal that the dynamics of the allene buildup are controlled by the structural changes, whereas the decay is limited by the charge recombination step

3D high-resolution hydrogel structures for biological studies

Hydrogels are widely used as matrices for cell growth due to the similarity of their mechanical and diffusivity properties to the extracellular matrix. The microfabrication of hydrogels into arbitrarily complex 3D structures is becoming essential for numerous biological applications. Among the possible additive manufacturing techniques, two-photon lithography stands out for its unique capacity of a direct 3D writing. We report fabrication of 3D high resolution hydrogel gelatin structures by Nanoscribe GT Photonic Professional. According to the degree of polymerization, variable swelling and structure deformation is observed. As a preliminary investigation to any biological application, the developed 3D hydrogels are tested against the requirements of cytocompatibility, promotion of cell adhesion and diffusion using BJ cell lines