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1. Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes

   https://doi.org/10.1002/anbr.202100049  

   Wang, Alex J. ; Allen, Allysa ; Sofman, Marianna ; Sphabmixay, Pierre ; Yildiz, Ece ; Griffith, Linda G. ( November 2021 , Advanced NanoBiomed Research)

   In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influences phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHHs) cultured in a commercially available perfused bioreactor. A 3D printing‐based alginate microwell system is designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver are engineered to replace polystyrene scaffolds, and the biochemical microenvironment is modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increases tissue density, albumin secretion, and CYP3A4 activity but also upregulates inflammatory markers. Basal inflammatory markers are lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibit the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA‐Seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

    

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2. Effective Modulation of CNS Inhibitory Microenvironment using Bioinspired Hybrid‐Nanoscaffold‐Based Therapeutic Interventions

   https://doi.org/10.1002/adma.202002578  

   Yang, Letao ; Conley, Brian M. ; Cerqueira, Susana R. ; Pongkulapa, Thanapat ; Wang, Shenqiang ; Lee, Jae K. ; Lee, Ki‐Bum ( September 2020 , Advanced Materials)

   Central nervous system (CNS) injuries are often debilitating, and most currently have no cure. This is due to the formation of a neuroinhibitory microenvironment at injury sites, which includes neuroinflammatory signaling and non‐permissive extracellular matrix (ECM) components. To address this challenge, a viscous interfacial self‐assembly approach, to generate a bioinspired hybrid 3D porous nanoscaffold platform for delivering anti‐inflammatory molecules and establish a favorable 3D‐ECM environment for the effective suppression of the neuroinhibitory microenvironment, is developed. By tailoring the structural and biochemical properties of the 3D porous nanoscaffold, enhanced axonal growth from the dual‐targeting therapeutic strategy in a human induced pluripotent stem cell (hiPSC)‐based in vitro model of neuroinflammation is demonstrated. Moreover, nanoscaffold‐based approaches promote significant axonal growth and functional recovery in vivo in a spinal cord injury model through a unique mechanism of anti‐inflammation‐based fibrotic scar reduction. Given the critical role of neuroinflammation and ECM microenvironments in neuroinhibitory signaling, the developed nanobiomaterial‐based therapeutic intervention may pave a new road for treating CNS injuries.

    

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3. Differential Effects of Extracellular Vesicles of Lineage-Specific Human Pluripotent Stem Cells on the Cellular Behaviors of Isogenic Cortical Spheroids

   https://doi.org/10.3390/cells8090993  

   Marzano, Mark ; Bejoy, Julie ; Cheerathodi, Mujeeb R. ; Sun, Li ; York, Sara B. ; Zhao, Jing ; Kanekiyo, Takahisa ; Bu, Guojun ; Meckes, David G. ; Li, Yan ( September 2019 , Cells)

   Extracellular vesicles (EVs) contribute to a variety of signaling processes and the overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. There is growing interest in the use of EVs derived from hiPSCs as therapeutics, biomarkers, and drug delivery vehicles. However, little is known about the characteristics of EVs secreted by hiPSCs and paracrine signaling during tissue morphogenesis and lineage specification. Methods: In this study, the physical and biological properties of EVs isolated from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer’s-associated SY-UBH lines) were analyzed. Results: Nanoparticle tracking analysis and electron microscopy results indicate that hiPSC-derived EVs have an average size of 100–250 nm. Immunoblot analyses confirmed the enrichment of exosomal markers Alix, CD63, TSG101, and Hsc70 in the purified EV preparations. MicroRNAs including miR-133, miR-155, miR-221, and miR-34a were differently expressed in the EVs isolated from distinct hiPSC lineages. Treatment of cortical spheroids with hiPSC-EVs in vitro resulted in enhanced cell proliferation (indicated by BrdU+ cells) and axonal growth (indicated by β-tubulin III staining). Furthermore, hiPSC-derived EVs exhibited neural protective abilities in Aβ42 oligomer-treated cultures, enhancing cell viability and reducing oxidative stress. Our results demonstrate that the paracrine signaling provided by tissue context-dependent EVs derived from hiPSCs elicit distinct responses to impact the physiological state of cortical spheroids. Overall, this study advances our understanding of cell‒cell communication in the stem cell microenvironment and provides possible therapeutic options for treating neural degeneration. 

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4. 3D Printed Cartilage‐Like Tissue Constructs with Spatially Controlled Mechanical Properties

   https://doi.org/10.1002/adfm.201906330  

   de Melo, Bruna A. G. ; Jodat, Yasamin A. ; Mehrotra, Shreya ; Calabrese, Michelle A. ; Kamperman, Tom ; Mandal, Biman B. ; Santana, Maria H. A. ; Alsberg, Eben ; Leijten, Jeroen ; Shin, Su Ryon ( October 2019 , Advanced Functional Materials)

   Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage‐like tissue is fabricated using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a bath composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel (MPa order compressive modulus) is developed as an extracellular matrix (ECM) with self‐healing properties. Within this bath supplemented with thrombin, human mesenchymal stem cell (hMSC) spheroids embedded in fibrinogen are 3D bioprinted, creating a soft microenvironment composed of fibrin (kPa order compressive modulus) that simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids present high viability and chondrogenic‐like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to locally bioprint a soft and cell stimulating biomaterial inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro‐ and macromechanical properties of the 3D printed tissues such as cartilage.

    

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5. Methods for Investigating Corneal Cell Interactions and Extracellular Vesicles In Vitro

   https://doi.org/10.1002/cpcb.114  

   McKay, Tina B. ; Guo, Xiaoqing ; Hutcheon, Audrey E. K. ; Karamichos, Dimitrios ; Ciolino, Joseph B. ( September 2020 , Current Protocols in Cell Biology)

   Science and medicine have become increasingly “human‐centric” over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human‐derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post‐transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype in a pre‐designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue‐engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell‐cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step‐by‐step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial‐ and endothelial‐stromal co‐cultures and isolation of extracellular vesicles. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Isolating and culturing human corneal keratocytes and fibroblasts

   Basic Protocol 2: Isolating and culturing human corneal epithelial cells

   Basic Protocol 3: Isolating and culturing human corneal endothelial cells

   Basic Protocol 4: 3D corneal stromal construct assembly

   Basic Protocol 5: 3D corneal epithelial‐stromal construct assembly

   Basic Protocol 6: 3D corneal endothelial‐stromal construct assembly

   Basic Protocol 7: Isolating extracellular vesicles from corneal cell conditioned medium

   Support Protocol: Cryopreserving human corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells

    

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