bims-orenst 2022-02-06 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2022–02–06
eight papers selected by
Joram Mooiweer, University of Groningen

3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert.
Evaluating the Role of IL-1β in Transmigration of Triple Negative Breast Cancer Cells Across the Brain Endothelium.
Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies.
Rapid 3D bioprinting of a multicellular model recapitulating pterygium microenvironment.
Influence of Substrate Stiffness on Barrier Function in an iPSC-Derived In Vitro Blood-Brain Barrier Model.
Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model.
Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system.
Engineer a pre-metastatic niched microenvironment to attract breast cancer cells by utilizing a 3D printed polycaprolactone/nano-hydroxyapatite osteogenic scaffold - An in vitro model system for proof of concept.

Nat Protoc. 2022 Feb 02.

3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert.

Woojung Shin, Hyun Jung Kim.

Human intestinal morphogenesis establishes 3D epithelial microarchitecture and spatially organized crypt-villus characteristics. This unique structure is necessary to maintain intestinal homeostasis by protecting the stem cell niche in the basal crypt from exogenous microbial antigens and their metabolites. Also, intestinal villi and secretory mucus present functionally differentiated epithelial cells with a protective barrier at the intestinal mucosal surface. Thus, re-creating the 3D epithelial structure is critical to building in vitro intestine models. Notably, an organomimetic gut-on-a-chip can induce spontaneous 3D morphogenesis of an intestinal epithelium with enhanced physiological function and biomechanics. Here we provide a reproducible protocol to robustly induce intestinal morphogenesis in a microfluidic gut-on-a-chip as well as in a Transwell-embedded hybrid chip. We describe detailed methods for device fabrication, culture of Caco-2 or intestinal organoid epithelial cells in conventional setups as well as on microfluidic platforms, induction of 3D morphogenesis and characterization of established 3D epithelium using multiple imaging modalities. This protocol enables the regeneration of functional intestinal microarchitecture by controlling basolateral fluid flow within 5 d. Our in vitro morphogenesis method employs physiologically relevant shear stress and mechanical motions, and does not require complex cellular engineering or manipulation, which may be advantageous over other existing techniques. We envision that our proposed protocol may have a broad impact on biomedical research communities, providing a method to regenerate in vitro 3D intestinal epithelial layers for biomedical, clinical and pharmaceutical applications.

DOI: https://doi.org/10.1038/s41596-021-00674-3
Cell Mol Bioeng. 2022 Feb;15(1): 99-114

Evaluating the Role of IL-1β in Transmigration of Triple Negative Breast Cancer Cells Across the Brain Endothelium.

Pedram Motallebnejad, Vinayak V Rajesh, Samira M Azarin.

   Introduction: In vivo, breast cancer cells spend on average 3-7 days adhered to the endothelial cells inside the vascular lumen before entering the brain. IL-1β is one of the highly upregulated molecules in brain-seeking triple negative breast cancer (TNBC) cells. In this study, the effect of IL-1β on the blood-brain barrier (BBB) and astrocytes and its role in transmigration of TNBC cells were evaluated.
Methods: The effect of IL-1β on transendothelial electrical resistance, gene and protein expression of human induced pluripotent stem cell-derived brain-specific microvascular endothelial-like cells (iBMECs) was studied. Transport of IL-1β across the iBMEC layer was investigated and the effect of IL-1β treatment of astrocytes on their cytokine and chemokine secretome was evaluated with a cytokine membrane array. Using BBB-on-a-chip devices, transmigration of MDA-MB-231 cells and their brain-seeking variant (231BR) across the iBMECs was studied, and the effect of an IL-1β neutralizing antibody on TNBC cell transmigration was investigated.
Results: We showed that IL-1β reduces BBB integrity and induces endothelial-to-mesenchymal transition in iBMECs. IL-1β crosses the iBMEC layer and induces secretion of multiple chemokines by astrocytes, which can enhance TNBC cell transmigration across the BBB. Transmigration assays in a BBB-on-a-chip device showed that 231BR cells have a higher rate of transmigration across the iBMECs compared to MDA-MB-231 cells, and IL-1β pretreatment of BBB-on-a-chip devices increases the number of transmigrated MDA-MB-231 cells. Finally, we demonstrated that neutralizing IL-1β reduces the rate of 231BR cell transmigration.
Conclusion: IL-1β plays a significant role in transmigration of brain-seeking TNBC cells across the BBB.
Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-021-00710-y.

Keywords:  Astrocytes; BBB-on-a-chip; Blood–brain barrier; Brain metastasis; Breast cancer; Chemokines; Endothelial-to-mesenchymal transition; Pluripotent stem cells

DOI:  https://doi.org/10.1007/s12195-021-00710-y
Lab Chip. 2022 Feb 02.

Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies.

Luca Docci, Nicolò Milani, Thomas Ramp, Andrea A Romeo, Patricio Godoy, Daniela Ortiz Franyuti, Stephan Krähenbühl, Michael Gertz, Aleksandra Galetin, Neil Parrott, Stephen Fowler.

Microphysiological systems (MPS) are complex and more physiologically realistic cellular in vitro tools that aim to provide more relevant human in vitro data for quantitative prediction of clinical pharmacokinetics while also reducing the need for animal testing. The PhysioMimix liver-on-a-chip integrates medium flow with hepatocyte culture and has the potential to be adopted for in vitro studies investigating the hepatic disposition characteristics of drug candidates. The current study focusses on liver-on-a-chip system exploration for multiple drug metabolism applications. Characterization of cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT) and aldehyde oxidase (AO) activities was performed using 15 drugs and in vitro to in vivo extrapolation (IVIVE) was assessed for 12 of them. Next, the utility of the liver-on-a-chip for estimation of the fraction metabolized (fm) via specific biotransformation pathways of quinidine and diclofenac was established. Finally, the metabolite identification opportunities were also explored using efavirenz as an example drug with complex primary and secondary metabolism involving a combination of CYP, UGT and sulfotransferase enzymes. A key aspect of these investigations was the application of mathematical modelling for improved parameter calculation. Such approaches will be required for quantitative assessment of metabolism and/or transporter processes in systems where medium flow and system compartments result in non-homogeneous drug concentrations. In particular, modelling was used to explore the effect of evaporation from the medium and it was found that the intrinsic clearance (CLint) might be underestimated by up to 40% for low clearance compounds if evaporation is not accounted for. Modelling of liver-on-a-chip in vitro data also enhanced the approach to fm estimation allowing objective assessment of metabolism models of different complexity. The resultant diclofenac fm,UGT of 0.64 was highly comparable with values reported previously in the literature. The current study demonstrates the integration of mathematical modelling with experimental liver-on-a-chip studies and illustrates how this approach supports generation of high quality of data from complex in vitro cellular systems.

DOI: https://doi.org/10.1039/d1lc01161h
Biomaterials. 2022 Jan 28. pii: S0142-9612(22)00030-8. [Epub ahead of print]282 121391

Rapid 3D bioprinting of a multicellular model recapitulating pterygium microenvironment.

Zheng Zhong, Jing Wang, Jing Tian, Xiaoqian Deng, Alis Balayan, Yazhi Sun, Yi Xiang, Jiaao Guan, Jacob Schimelman, Henry Hwang, Shangting You, Xiaokang Wu, Chao Ma, Xiaoao Shi, Emmie Yao, Sophie X Deng, Shaochen Chen.

  Pterygium is an ocular surface disorder with high prevalence that can lead to vision impairment. As a pathological outgrowth of conjunctiva, pterygium involves neovascularization and chronic inflammation. Here, we developed a 3D multicellular in vitro pterygium model using a digital light processing (DLP)-based 3D bioprinting platform with human conjunctival stem cells (hCjSCs). A novel feeder-free culture system was adopted and efficiently expanded the primary hCjSCs with homogeneity, stemness and differentiation potency. The DLP-based 3D bioprinting method was able to fabricate hydrogel scaffolds that support the viability and biological integrity of the encapsulated hCjSCs. The bioprinted 3D pterygium model consisted of hCjSCs, immune cells, and vascular cells to recapitulate the disease microenvironment. Transcriptomic analysis using RNA sequencing (RNA-seq) identified a distinct profile correlated to inflammation response, angiogenesis, and epithelial mesenchymal transition in the bioprinted 3D pterygium model. In addition, the pterygium signatures and disease relevance of the bioprinted model were validated with the public RNA-seq data from patient-derived pterygium tissues. By integrating the stem cell technology with 3D bioprinting, this is the first reported 3D in vitro disease model for pterygium that can be utilized for future studies towards personalized medicine and drug screening.

Keywords:  3D bioprinting; Disease model; Epithelial mesenchymal transition; Hydrogels; Pterygium; Stem cells; Tissue engineering

DOI:  https://doi.org/10.1016/j.biomaterials.2022.121391
Cell Mol Bioeng. 2022 Feb;15(1): 31-42

Influence of Substrate Stiffness on Barrier Function in an iPSC-Derived In Vitro Blood-Brain Barrier Model.

Allison M Bosworth, Hyosung Kim, Kristin P O'Grady, Isabella Richter, Lynn Lee, Brian J O'Grady, Ethan S Lippmann.

   Introduction: Vascular endothelial cells respond to a variety of biophysical cues such as shear stress and substrate stiffness. In peripheral vasculature, extracellular matrix (ECM) stiffening alters barrier function, leading to increased vascular permeability in atherosclerosis and pulmonary edema. The effect of ECM stiffness on blood-brain barrier (BBB) endothelial cells, however, has not been explored. To investigate this topic, we incorporated hydrogel substrates into an in vitro model of the human BBB.
Methods: Induced pluripotent stem cells were differentiated to brain microvascular endothelial-like (BMEC-like) cells and cultured on hydrogel substrates of varying stiffness. Cellular changes were measured by imaging, functional assays such as transendothelial electrical resistance (TEER) and p-glycoprotein efflux activity, and bulk transcriptome readouts.
Results: The magnitude and longevity of TEER in iPSC-derived BMEC-like cells is enhanced on compliant substrates. Quantitative imaging shows that BMEC-like cells form fewer intracellular actin stress fibers on substrates of intermediate stiffness (20 kPa relative to 1 and 150 kPa). Chemical induction of actin polymerization leads to a rapid decline in TEER, agreeing with imaging readouts. P-glycoprotein activity is unaffected by substrate stiffness. Modest differences in RNA expression corresponding to specific signaling pathways were observed as a function of substrate stiffness.
Conclusions: iPSC-derived BMEC-like cells exhibit differences in passive but not active barrier function in response to substrate stiffness. These findings may provide insight into BBB dysfunction during neurodegeneration, as well as aid in the optimization of more complex three-dimensional neurovascular models utilizing compliant hydrogels.
Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-021-00706-8.

Keywords:  Blood-brain barrier; Human induced pluripotent stem cell; Hydrogel; In vitro model; Mechanobiology

DOI:  https://doi.org/10.1007/s12195-021-00706-8
Adv Sci (Weinh). 2022 Feb 01. e2103939

Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model.

Benjamin T Wilks, Elisabeth B Evans, Andrew Howes, Caitlin M Hopkins, Morcos N Nakhla, Geoffrey Williams, Jeffrey R Morgan.

  Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three-dimensional (3D) multi-scale hierarchical architecture of collagen-rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non-adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring-shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly-aligned, collagen-rich matrix, a multi-modal approach integrating histology, multiphoton second-harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell-derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho-kinase inhibitor, and pro- and anti-fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.

Keywords:  3D tissue engineering; TGF-β1; collagen; connective tissue; extracellular matrix; fibroblast; fibrosis; mechanics; mechanophenotype

DOI:  https://doi.org/10.1002/advs.202103939
Cell Prolif. 2022 Jan 31. e13190

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system.

Thao Thi Thanh Ngo, Bella Rossbach, Isabelle Sébastien, Julia C Neubauer, Andreas Kurtz, Krithika Hariharan.

   OBJECTIVE: To provide a standardized protocol for large-scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC).
METHODS: The hPSC were expanded and differentiated into PTEC on matrix-coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin-treatment was assessed to check the potential use of PTEC to model drug-induced nephrotoxicity.
RESULTS: hPSC expansion and PTEC differentiation could be performed directly on matrix-coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10-fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico-basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM-1 after Cisplatin treatment.
CONCLUSION: We demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix-coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.

Keywords:  Pluripotent stem cells; alginate; bioreactor; functional tubular epithelial cells; kidney differentiation

DOI:  https://doi.org/10.1111/cpr.13190
J Biomed Mater Res B Appl Biomater. 2022 Feb 03.

Engineer a pre-metastatic niched microenvironment to attract breast cancer cells by utilizing a 3D printed polycaprolactone/nano-hydroxyapatite osteogenic scaffold - An in vitro model system for proof of concept.

Qisheng Xiong, Ningze Zhang, Miaomiao Zhang, Meng Wang, Lizhen Wang, Yubo Fan, Chia-Ying Lin.

  Breast cancer bone metastasis is not a random process. It is affected by the local microenvironment which determines the propensity of cancer cells to invade and colonize into the secondary sites. This microenvironment is termed a pre-metastatic niche. With the flexibility to incorporate different biofactors, tissue-engineering scaffolds provide an advantageous environment to promote "designed" osteogenesis that may mimic the bony pre-metastatic niche. In the current study, designed polycaprolactone (PCL) scaffolds enriched with nano-hydroxyapatite (nHA) were fabricated through three-dimensional (3D) printing. Subsequently, human mesenchymal stem cells (hMSCs) were seeded onto PCL-nHA scaffolds for osteogenic differentiation to establish the pre-metastatic niched microenvironment. Furthermore, transwell migration assay was used to investigate recruitment of MDA-MB-231, MCF-7, and MDA-MB-453 breast cancer cells to the osseous PCL-nHA scaffolds. Our results showed that the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN) of hMSCs on the PCL-nHA scaffolds were dramatically increased compared those with the PCL scaffolds (control) at day 7, 14, and 28. Meanwhile, the migration analysis showed that the higher maturation of osteogenesis and bone metabolism collectively contributed to the creation of a more favorable niched site for the cancerous invasion. Moreover, one of the hypothesized key mediators for the promoted migration, CXCL12, was confirmed using an assay of antagonist LIT-927. This early study demonstrated that a designed tissue engineering scaffold can be utilized to create a bone-mimicking environment that serves as a novel platform to recapitulate the pre-metastatic niche and help interrogate the scheme of bone metastasis by breast cancer.

Keywords:  CXCL12/stromal cell-derived factor 1; breast cancer; polycaprolactone; pre-metastatic niche; three-dimensional printing

DOI:  https://doi.org/10.1002/jbm.b.35021