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1. In Situ Super‐Resolution Imaging of Organoids and Extracellular Matrix Interactions via Phototransfer by Allyl Sulfide Exchange‐Expansion Microscopy (PhASE‐ExM)

   https://doi.org/10.1002/adma.202109252  

   Blatchley, Michael R.; Günay, Kemal Arda; Yavitt, F. Max; Hawat, Elijah M.; Dempsey, Peter J.; Anseth, Kristi S. (March 2022, Advanced Materials)

   Abstract 3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high‐resolution microscopes and depth‐dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell‐extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange‐expansion microscopy (PhASE‐ExM) is developed, enabling optical clearance and super‐resolution imaging of organoids and their ECM in 3D. PhASE‐ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE‐ExM enables 3.25× expansion and super‐resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl‐sulfide groups (PEG‐AlS) through simultaneous photopolymerization and radical‐mediated chain‐transfer reactions that complete in <70 s. Further, PEG‐AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super‐resolution imaging platform both for pre‐ and post‐differentiated organoids. Overall, PhASE‐ExM is a useful tool to decipher organoid behavior by enabling sub‐micrometer scale, 3D visualization of proteins and signal transduction pathways. 

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2. Stretchable Mesh Nanoelectronics for 3D Single‐Cell Chronic Electrophysiology from Developing Brain Organoids

   https://doi.org/10.1002/adma.202106829  

   Le Floch, Paul; Li, Qiang; Lin, Zuwan; Zhao, Siyuan; Liu, Ren; Tasnim, Kazi; Jiang, Han; Liu, Jia (March 2022, Advanced Materials)

   Abstract Human induced pluripotent stem cell derived brain organoids have shown great potential for studies of human brain development and neurological disorders. However, quantifying the evolution of the electrical properties of brain organoids during development is currently limited by the measurement techniques, which cannot provide long‐term stable 3D bioelectrical interfaces with developing brain organoids. Here, a cyborg brain organoid platform is reported, in which “tissue‐like” stretchable mesh nanoelectronics are designed to match the mechanical properties of brain organoids and to be folded by the organogenetic process of progenitor or stem cells, distributing stretchable electrode arrays across the 3D organoids. The tissue‐wide integrated stretchable electrode arrays show no interruption to brain organoid development, adapt to the volume and morphological changes during brain organoid organogenesis, and provide long‐term stable electrical contacts with neurons within brain organoids during development. The seamless and noninvasive coupling of electrodes to neurons enables long‐term stable, continuous recording and captures the emergence of single‐cell action potentials from early‐stage brain organoid development. 

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3. Biocompatible micro tweezers for 3D hydrogel organoid array mechanical characterization

   https://doi.org/10.1371/journal.pone.0262950  

   Alhudaithy, Soliman; Hoshino, Kazunori (January 2022, PLOS ONE)

   Jabbari, Esmaiel (Ed.)

   This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (μTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 μl/sample using the μTweezers vs. ~157 μl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred μm-1500 μm) 3D cell cultures. The μTweezers applied and measured nano-millinewton forces through cantilever’ deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids’ applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young’s modulus of the control group (Control—day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The μTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators. 

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4. Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture

   https://doi.org/10.1002/adma.202101966  

   Indana, Dhiraj; Agarwal, Pranay; Bhutani, Nidhi; Chaudhuri, Ovijit (September 2021, Advanced Materials)

   Abstract Organoids are lumen‐containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine–glycine–aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3D culture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes‐associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids. 

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5. Microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes for salivary gland tissue engineering

   https://doi.org/10.1016/j.bioadv.2023.213588  

   Yin, Yu; Vázquez-Rosado, Ephraim J.; Wu, Danielle; Viswananthan, Vignesh; Farach, Andrew; Farach-Carson, Mary C.; Harrington, Daniel A. (November 2023, Biomaterials Advances)

   Replacement therapy for the salivary gland (SG) remains an unmet clinical need. Xerostomia (“dry mouth”) due to hyposalivation can result from injury or disease to the SG, such as salivary acinar death caused by radiation therapy (RT) for head and neck squamous cell carcinoma (HNSCC). Currently, only palliative treatments exist for xerostomia, and many patients endure deteriorated oral health and poor quality of life. Tissue engineering could offer a permanent solution for SG replacement by isolating healthy SG tissues prior to RT, expanding its cells in vitro, and recreating a functional salivary neogland for implantation post-RT. 3D bioprinting methods potentiate spatial cell deposition into defined hydrogel-based architectures, mimicking the thin epithelia developed during the complex branching morphogenesis of SG. By leveraging a microfluidics-based bioprinter with coaxial polymer and crosslinker streams, we fabricated thin, biocompatible, and reproducible hydrogel features that recapitulate the thin epithelia characteristics of SG. This flexible platform enabled two modes of printing: we produced solid hydrogel fibers, with diameters <100 μm, that could be rastered to create larger mm-scale structures. By a second method, we generated hollow tubes with wall thicknesses ranging 45-80 μm, total tube diameters spanning 0.6 – 2.2 mm, and confirmed tube patency. In both cases, SG cells could be printed within the thin hydrogel features, with preserved phenotype and high viability, even at high density (5.0 × 10^6 cells/mL). Our work demonstrates hydrogel feature control across multiple length scales, and a new paradigm for addressing SG restoration by creating microscale tissue engineered components. 

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