Stacey C. Skaalure

Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high‐quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties.

Spatio-temporally tailoring cell–material interactions is essential for developing smart delivery systems and intelligent biointerfaces. Here we report new photo-activatable cell–material interfacing systems that trigger cellular uptake of various cargoes and cell adhesion towards surfaces. To achieve this, we designed a novel photo-caged peptide which undergoes a structural transition from an antifouling ligand to a cell-penetrating peptide upon photo-irradiation. When the peptide is conjugated to ligands of interest, we demonstrate the photo-activated cellular uptake of a wide range of cargoes, including small fluorophores, proteins, inorganic (e.g., quantum dots and gold nanostars) and organic nanomaterials (e.g., polymeric particles), and liposomes. Using this system, we can remotely regulate drug administration into cancer cells by functionalizing camptothecin-loaded polymeric nanoparticles with our synthetic peptide ligands. Furthermore, we show light-controlled cell adhesion on a peptide-modified surface and 3D spatiotemporal control over cellular uptake of nanoparticles using two-photon excitation. We anticipate that the innovative approach proposed in this work will help to establish new stimuli-responsive delivery systems and biomaterials.

Hydrogels are promising materials for mimicking the extra-cellular environment. Here, we present a simple methodology for the formation of a free-standing viscoelastic hydrogel from the abundant and low cost protein serum albumin. We show that the mechanical properties of the hydrogel exhibit a complicated behaviour as a function of the weight fraction of the protein component. We further use X-ray scattering to shed light on the mechanism of gelation from the formation of a fibrillary network at low weight fractions to interconnected aggregates at higher weight fractions. Given the match between our hydrogel elasticity and that of the myocardium, we investigated its potential for supporting cardiac cells in vitro. Interestingly, these hydrogels support the formation of several layers of myocytes and significantly promote the maintenance of a native-like gene expression profile compared to those cultured on glass. When confronted with a multicellular ventricular cell preparation, the hydrogels can support macroscopically contracting cardiac-like tissues with a distinct cell arrangement, and form mm-long vascular-like structures. We envisage that our simple approach for the formation of an elastic substrate from an abundant protein makes the hydrogel a compelling biomedical material candidate for a wide range of cell types.

Enzyme‐sensitive hydrogels are promising for cell encapsulation and tissue engineering, but result in complex spatiotemporal degradation behavior that is characteristic of reaction‐diffusion mechanisms. An experimental and theoretical approach is presented to identify dimensionless quantities that serve as a design tool for engineering enzyme‐sensitive hydrogels with controlled degradation patterns by tuning the initial hydrogel properties and enzyme kinetics.

A new cartilage‐specific degradable hydrogel based on photoclickable thiol‐ene poly(ethylene glycol) (PEG) hydrogels is presented. The hydrogel crosslinks are composed of the peptide, CRDTEGE‐ARGSVIDRC, derived from the aggrecanase‐cleavable site in aggrecan. This new hydrogel is evaluated for use in cartilage tissue engineering by encapsulating bovine chondrocytes from different cell sources (skeletally immature (juvenile) and mature (adult) donors and adult cells stimulated with proinflammatory lipopolysaccharide (LPS)) and culturing for 12 weeks. Regardless of cell source, a twofold decrease in compressive modulus is observed by 12 weeks, but without significant hydrogel swelling indicating limited bulk degradation. For juvenile cells, a connected matrix rich in aggrecan and collagen II, but minimal collagens I and X is observed. For adult cells, less matrix, but similar quality, is deposited. Aggrecanase activity is elevated, although without accelerating bulk hydrogel degradation. LPS further decreases matrix production, but does not affect aggrecanase activity. In contrast, matrix deposition in the nondegradable hydrogels consists of aggrecan and collagens I, II, and X, indicative of hypertrophic cartilage. Lastly, no inflammatory response in chondrocytes is observed by the aggrecanase‐sensitive hydrogels. Overall, it is demonstrated that this new aggrecanase‐sensitive hydrogel, which is degradable by chondrocytes and promotes a hyaline‐like engineered cartilage, is promising for cartilage regeneration.

Encapsulating chondrocytes in synthetic and degradable hydrogels for cartilage tissue engineering enables tuning of scaffold degradation, but provides no biological cues. Culture medium that recapitulates the physiological osmolarity of the interstitial fluid in cartilage can enhance matrix synthesis in the short term, but long‐term benefits remain to be determined. This study investigates the long‐term effect of culture medium osmolarity on tissue synthesis using chondrocytes isolated from three skeletally mature bovine donors encapsulated in degradable poly(ethylene glycol) hydrogels. The cell‐laden hydrogels were cultured up to 4 weeks in standard chondrocyte‐specific medium (330 mOsm) or medium adjusted by sucrose or salts (NaCl and KCl) to reach a physiological osmolarity (400 mOsm). Neocartilaginous matrix synthesis and matrix catabolism were evaluated by quantitative and immunofluorescence methods. Hydrogel degradation kinetics of acellular constructs were not affected by medium osmolarity or osmolyte. Matrix composition was predominantly aggrecan and collagen type II for all conditions. One day after encapsulation, total collagen accumulated in the constructs was increased by 80–90% in 400 mOsm medium, regardless of osmolyte. However, this effect did not persist, and at 4 weeks, total collagen synthesized and released to the medium was more than three times higher in 330 mOsm medium. Medium osmolarity had minimal effects on sulfated glycosaminoglycan content and did not affect catabolic activity. These findings suggest that culture medium at physiological osmolarities may not be beneficial for long‐term chondrocyte culture in degradable hydrogels, but that initially culturing chondrocytes at a higher osmolarity may enhance early tissue deposition. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2186–2192, 2015.

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform because their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then are encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator (SLM) illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm is enabled by a lithium acylphosphinate (LAP) salt photoinitiator. Use of a polyethylene glycol dimethacrylate (PEGDMA) based monomer is compared to that of polyethylene glycol (PEG) hydrogels formed by thiol-ene photo-click chemistry for patterning structures with cellular resolution, and for maintaining cell viability. Cells patterned in thiol-ene with RGD are shown to retain viability up to 4 days after the trapping and encapsulation procedure. Further, cells patterned in thiol-ene with RGD and a degradable ester link, are shown to fuse, indicating the initial stages of development of multi-nucleated cells.

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform since their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm was enabled by a lithium acylphosphinate (LAP) salt photoinitiator. These wavelengths, in addition to brightfield imaging with a white light LED, could be simultaneously focused by a single oil immersion objective. Large lateral dimensions of the patterned gel/cell structure are achieved by x and y step-and-repeat process. Large thickness is achieved through multi-layer stereolithography, allowing fabrication of precisely-arranged 3D live cell scaffolds with micron-scale structure and millimeter dimensions. Cells are shown to retain viability after the trapping and encapsulation procedure.