Engineering 3D micro-compartments for highly efficient and scale-independent expansion of human pluripotent stem cells in bioreactors.

Human pluripotent stem cells (hPSCs) have emerged as the most promising cellular source for cell therapies. To overcome the scale-up limitations of classical 2D culture systems, suspension cultures have been developed to meet the need for large-scale culture in regenerative medicine. Despite constant improvements, current protocols that use microcarriers or generate cell aggregates only achieve moderate amplification performance. Here, guided by reports showing that hPSCs can self-organize in vitro into cysts reminiscent of the epiblast stage in embryo development, we developed a physio-mimetic approach for hPSC culture. We engineered stem cell niche microenvironments inside microfluidics-assisted core-shell microcapsules. We demonstrate that lumenized three-dimensional colonies significantly improve viability and expansion rates while maintaining pluripotency compared to standard hPSC culture platforms such as 2D cultures, microcarriers, and aggregates. By further tuning capsule size and culture conditions, we scale up this method to industrial-scale stirred tank bioreactors and achieve an unprecedented hPSC amplification rate of 277-fold in 6.5 days. In brief, our findings indicate that our 3D culture system offers a suitable strategy both for basic stem cell biology experiments and for clinical applications.

Interactions of Organosilanes with Fibrinogen and Their Influence on Muscle Cell Proliferation in 3D Fibrin Hydrogels.

Silanization of biomacromolecules has emerged as a fruitful approach to prepare hybrid biohydrogels. However, very little is known about interactions between organosilanes and biopolymers in solution. Here we focused on fibrin, a protein of interest in the biomedical field, whose self-assembly process and resulting gel structure are highly sensitive to experimental conditions. Three main silanes were selected to decipher the relative influence of the silanol groups and organic functions. Whereas no protein denaturation was observed, silanes bearing hydrophobic groups had a surfactant-like behavior and could improve the dispersion of fibrinogen molecules, impacting gel formation kinetics and rheological properties. 3D cultures of myoblasts evidenced that organosilanes could promote or impede cell proliferation, suggesting interactions of silanols with fibrin. These results demonstrate that the two sides of the coin of organosilane reactivity are relevant at different stages of fibrin gel formation and must be considered for future development of hybrid biomaterials.

Topotactic Fibrillogenesis of Freeze-Cast Microridged Collagen Scaffolds for 3D Cell Culture.

Type I collagen is the main component of the extracellular matrix (ECM). In vitro, under a narrow window of physicochemical conditions, type I collagen self-assembles to form complex supramolecular architectures reminiscent of those found in native ECM. Presently, a major challenge in collagen-based biomaterials is to couple the delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions. In this work, an ice-templating approach promoting the structuration of collagen into macroporous monoliths is used. Instead of common solvent removal procedures, a new topotactic conversion approach yielding self-assembled ordered fibrous materials is implemented. These collagen-only, non-cross-linked scaffolds exhibit uncommon mechanical properties in the wet state, with a Young's modulus of 33 ± 12 kPa, an ultimate tensile stress of 33 ± 6 kPa, and a strain at failure of 105 ± 28%. With the help of the ice-patterned microridge features, normal human dermal fibroblasts and C2C12 murine myoblasts successfully migrate and form highly aligned populations within the resulting three-dimensional (3D) collagen scaffolds. These results open a new pathway to the development of new tissue engineering scaffolds ordered across various organization levels from the molecule to the macropore and are of particular interest for biomedical applications where large-scale 3D cell alignment is needed such as for muscular or nerve reconstruction.

From Tendon Injury to Collagen-based Tendon Regeneration: Overview and Recent Advances.

Tendon injury is a clinical, societal and economical issue. Moreover, tendon repair represents an important clinical challenge, partly due to the mechanical constraints that occur at the junctions with muscle and bone. Several strategies have been developed for tendon repair. In this review, we first assess the importance of tendon injuries from different sites and their causes. After a short overview of tendon three-dimensional organization, the complexity of the perfect repair quest is presented ranging from current clinical procedures to new engineering scaffolds. We then sum up tendon engineering requirements and focus on new collagen-based scaffolds, which raise promising prospects to mimic and repair tendon. In particular, we survey quantitatively a large panel of techniques to produce these scaffolds, detailing their principle and recent improvements.

The role of water on the structure and mechanical properties of a thermoplastic natural block co-polymer from squid sucker ring teeth.

Hard biological polymers exhibiting a truly thermoplastic behavior that can maintain their structural properties after processing are extremely rare and highly desirable for use in advanced technological applications such as 3D-printing, biodegradable plastics and robust composites. One exception are the thermoplastic proteins that comprise the sucker ring teeth (SRT) of the Humboldt jumbo squid (Dosidicus gigas). In this work, we explore the mechanical properties of reconstituted SRT proteins and demonstrate that the material can be re-shaped by simple processing in water and at relatively low temperature (below 100 °C). The post-processed material maintains a high modulus in the GPa range, both in the dry and the wet states. When transitioning from low to high humidity, the material properties change from brittle to ductile with an increase in plastic deformation, where water acts as a plasticizer. Using synchrotron x-ray scattering tools, we found that water mostly influences nano scale structure, whereas at the molecular level, the protein structure remains largely unaffected. Furthermore, through simultaneous in situ x-ray scattering and mechanical tests, we show that the supramolecular network of the reconstituted SRT material exhibits a progressive alignment along the strain direction, which is attributed to chain alignment of the amorphous domains of SRT proteins. The high modulus in both dry and wet states, combined with their efficient thermal processing characteristics, make the SRT proteins promising substitutes for applications traditionally reserved for petroleum-based thermoplastics.

Stable Formation of Gold Nanoparticles onto Redox-Active Solid Biosubstrates Made of Squid Suckerin Proteins.

The use of biomolecules to synthesize inorganic nanomaterials, including metallic nanoparticles, offers the ability to induce controlled growth under mild environmental conditions. Here, recently discovered silk-like "suckerin" proteins are used to induce the formation of gold nanoparticles (AuNPs). Advantage is taken of the distinctive biological and physico-chemical characteristics of suckerins, namely their facile recombinant expression, their solubility in aqueous solutions, and their modular primary structure with high molar content of redox-active tyrosine (Tyr) residues to induce the formation of AuNPs not only in solution, but also from nanostructured solid substrates fabricated from suckerins.