bims-orenst Biomed News
on Organs-on-chips and engineered stem cell models
Issue of 2021‒02‒07
fourteen papers selected by
Joram Mooiweer
University of Groningen


  1. J Pharm Sci. 2021 Feb 02. pii: S0022-3549(21)00070-8. [Epub ahead of print]
    Vormann MK, Vriend J, Lanz HL, Gijzen L, van den Heuvel A, Hutter S, Joore J, Trietsch SJ, Stuut C, Nieskens TTG, Peters JGP, Ramp D, Caj M, Russel FGM, Jacobsen B, Roth A, Lu S, Polli JW, Naidoo AA, Vulto P, Masereeuw R, Wilmer MJ, Suter-Dick L.
      Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-β-D-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).
    Keywords:  Drug-screening; Drug-transporter interaction; Renal-proximal-tubule-on-a-chip; miRNA; microfluidics; pharmaceutical
    DOI:  https://doi.org/10.1016/j.xphs.2021.01.028
  2. Front Med Technol. 2020 Aug;pii: 2. [Epub ahead of print]2
    Shin W, Ambrosini YM, Shin YC, Wu A, Min S, Koh D, Park S, Kim S, Koh H, Kim HJ.
      Polydimethylsiloxane (PDMS) is a silicone polymer that has been predominantly used in a human organ-on-a-chip microphysiological system. The hydrophobic surface of a microfluidic channel made of PDMS often results in poor adhesion of the extracellular matrix (ECM) as well as cell attachment. The surface modification by plasma or UV/ozone treatment in a PDMS-based device produces a hydrophilic surface that allows robust ECM coating and the reproducible attachment of human intestinal immortalized cell lines. However, these surface-activating methods have not been successful in forming a monolayer of the biopsy-derived primary organoid epithelium. Several existing protocols to grow human intestinal organoid cells in a PDMS microchannel are not always reproducibly operative due to the limited information. Here, we report an optimized methodology that enables robust and reproducible attachment of the intestinal organoid epithelium in a PDMS-based gut-on-a-chip. Among several reported protocols, we optimized a method by performing polyethyleneimine-based surface functionalization followed by the glutaraldehyde cross linking to activate the PDMS surface. Moreover, we discovered that the post-functionalization step contributes to provide uniform ECM deposition that allows to produce a robust attachment of the dissociated intestinal organoid epithelium in a PDMS-based microdevice. We envision that our optimized protocol may disseminate an enabling methodology to advance the integration of human organotypic cultures in a human organ-on-a-chip for patient-specific disease modeling.
    Keywords:  cell attachment; extracellular matrix; gut-on-a-chip; hydrophobicity; organoids; polydimethylsiloxane; surface functionalization
    DOI:  https://doi.org/10.3389/fmedt.2020.00002
  3. Nano Converg. 2021 Feb 02. 8(1): 3
    Farooqi HMU, Kang B, Khalid MAU, Salih ARC, Hyun K, Park SH, Huh D, Choi KH.
      Hepatic fibrosis is a foreshadowing of future adverse events like liver cirrhosis, liver failure, and cancer. Hepatic stellate cell activation is the main event of liver fibrosis, which results in excessive extracellular matrix deposition and hepatic parenchyma's disintegration. Several biochemical and molecular assays have been introduced for in vitro study of the hepatic fibrosis progression. However, they do not forecast real-time events happening to the in vitro models. Trans-epithelial electrical resistance (TEER) is used in cell culture science to measure cell monolayer barrier integrity. Herein, we explored TEER measurement's utility for monitoring fibrosis development in a dynamic cell culture microphysiological system. Immortal HepG2 cells and fibroblasts were co-cultured, and transforming growth factor β1 (TGF-β1) was used as a fibrosis stimulus to create a liver fibrosis-on-chip model. A glass chip-based embedded TEER and reactive oxygen species (ROS) sensors were employed to gauge the effect of TGF-β1 within the microphysiological system, which promotes a positive feedback response in fibrosis development. Furthermore, albumin, Urea, CYP450 measurements, and immunofluorescent microscopy were performed to correlate the following data with embedded sensors responses. We found that chip embedded electrochemical sensors could be used as a potential substitute for conventional end-point assays for studying fibrosis in microphysiological systems.
    Keywords:  Embedded sensors; Liver fibrosis-on-chip; ROS sensor; TEER sensor; TGF-β1
    DOI:  https://doi.org/10.1186/s40580-021-00253-y
  4. Micromachines (Basel). 2021 Jan 26. pii: 132. [Epub ahead of print]12(2):
    Dabaghi M, Shahriari S, Saraei N, Da K, Chandiramohan A, Selvaganapathy PR, Hirota JA.
      Polydimethylsiloxane (PDMS) is a silicone-based synthetic material used in various biomedical applications due to its properties, including transparency, flexibility, permeability to gases, and ease of use. Though PDMS facilitates and assists the fabrication of complicated geometries at micro- and nano-scales, it does not optimally interact with cells for adherence and proliferation. Various strategies have been proposed to render PDMS to enhance cell attachment. The majority of these surface modification techniques have been offered for a static cell culture system. However, dynamic cell culture systems such as organ-on-a-chip devices are demanding platforms that recapitulate a living tissue microenvironment's complexity. In organ-on-a-chip platforms, PDMS surfaces are usually coated by extracellular matrix (ECM) proteins, which occur as a result of a physical and weak bonding between PDMS and ECM proteins, and this binding can be degraded when it is exposed to shear stresses. This work reports static and dynamic coating methods to covalently bind collagen within a PDMS-based microfluidic device using polydopamine (PDA). These coating methods were evaluated using water contact angle measurement and atomic force microscopy (AFM) to optimize coating conditions. The biocompatibility of collagen-coated PDMS devices was assessed by culturing primary human bronchial epithelial cells (HBECs) in microfluidic devices. It was shown that both PDA coating methods could be used to bind collagen, thereby improving cell adhesion (approximately three times higher) without showing any discernible difference in cell attachment between these two methods. These results suggested that such a surface modification can help coat extracellular matrix protein onto PDMS-based microfluidic devices.
    Keywords:  collagen; microfluidic; organ-on-a-chip; polydimethylsiloxane (PDMS); polydopamine
    DOI:  https://doi.org/10.3390/mi12020132
  5. Lab Chip. 2021 Feb 04.
    Margolis EA, Cleveland DS, Kong YP, Beamish JA, Wang WY, Baker BM, Putnam AJ.
      Supportive stromal cells of mesenchymal origins regulate vascular morphogenesis in developmental, pathological, and regenerative contexts, contributing to vessel formation, maturation, and long-term stability, in part via the secretion of bioactive molecules. In this work, we adapted a microfluidic lab-on-a-chip system that enables the formation and perfusion of microvascular capillary beds with connections to arteriole-scale endothelialized channels to explore how stromal cell (SC) identity influences endothelial cell (EC) morphogenesis. We compared and contrasted lung fibroblasts (LFs), dermal fibroblasts (DFs), and bone marrow-derived mesenchymal stem cells (MSCs) for their abilities to support endothelial morphogenesis and subsequent perfusion of microvascular networks formed in fibrin hydrogels within the microfluidic device. We demonstrated that while all 3 SC types supported EC morphogenesis, LFs in particular resulted in microvascular morphologies with the highest total network length, vessel diameter, and vessel interconnectivity across a range of SC-EC ratio and density conditions. Not only did LFs support robust vascular morphology, but also, they were the only SC type to support functional perfusion of the resultant capillary beds. Lastly, we identified heightened traction stress produced by LFs as a possible mechanism by which LFs enhance endothelial morphogenesis in 3D compared to other SC types examined. This study provides a unique comparison of three different SC types and their role in supporting the formation of microvasculature that could provide insights for the choice of cells for vascular cell-based therapies and the regulation of tissue-specific vasculature.
    DOI:  https://doi.org/10.1039/d0lc01092h
  6. Sci Rep. 2021 Feb 05. 11(1): 3234
    Kulthong K, Hooiveld GJEJ, Duivenvoorde L, Miro Estruch I, Marin V, van der Zande M, Bouwmeester H.
      Gut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.
    DOI:  https://doi.org/10.1038/s41598-021-82853-6
  7. Lab Chip. 2021 Feb 03.
    Ayuso JM, Sadangi S, Lares M, Rehman S, Humayun M, Denecke KM, Skala MC, Beebe DJ, Setaluri V.
      Melanoma evolution is a complex process. The role epidermal keratinocytes and dermal fibroblasts play in this process and the mechanisms involved in tumor-stroma interactions remain poorly understood. Here, we used a microfluidic platform to evaluate the cross-talk between human primary melanoma cells, keratinocytes and dermal fibroblasts. The microfluidic device included multiple circular chambers separated by a series of narrow connection channels. The microdevice design allowed us to develop a new cell patterning method based on air-walls, removing the need for hydrogel barriers, porous membranes, or external equipment. Using this method, we co-cultured melanoma cells in the presence of keratinocytes and/or dermal fibroblasts. The results demonstrated that the presence of dermal fibroblasts and keratinocytes led to changes in melanoma cell morphology and growth pattern. Molecular analysis revealed changes in the chemokine secretion pattern, identifying multiple secreted factors involved in tumor progression. Finally, optical metabolic imaging showed that melanoma cells, fibroblasts, and keratinocytes exhibited different metabolic features. Additionally, the presence of stromal cells led to a metabolic shift in melanoma cells, highlighting the role the skin microenvironment on melanoma evolution.
    DOI:  https://doi.org/10.1039/d0lc00988a
  8. Lab Chip. 2021 Feb 01.
    Crippa M, Bersini S, Gilardi M, Arrigoni C, Gamba S, Falanga A, Candrian C, Dubini G, Vanoni M, Moretti M.
      During metastatic progression multiple players establish competitive mechanisms, whereby cancer cells (CCs) are exposed to both pro- and anti-metastatic stimuli. The early metastatic niche (EMN) is a transient microenvironment which forms in the circulation during CC dissemination. EMN is characterized by the crosstalk among CCs, platelets, leukocytes and endothelial cells (ECs), increasing CC ability to extravasate and colonize secondary tissues. To better understand this complex crosstalk, we designed a human "EMN-on-a-chip" which involves the presence of blood cells as compared to standard metastases-on-chip models, hence providing a microenvironment more similar to the in vivo situation. We showed that CC transendothelial migration (TEM) was significantly increased in the presence of neutrophils and platelets in the EMN-on-a-chip compared to CC alone. Moreover, exploiting the EMN-on-chip in combination with multi-culture experiments, we showed that platelets increased the expression of epithelial to mesenchymal transition (EMT) markers in CCs and that the addition of a clinically approved antiplatelet drug (eptifibatide, inhibiting integrin β3) impaired platelet aggregation and decreased CC expression of EMT markers. Inhibition of integrin β3 in the co-culture system modulated the activation of the Src-FAK-VE-cadherin signaling axis and partially restored the architecture of inter-endothelial junctions by limiting VE-cadherinY658 phosphorylation and its nuclear localization. These observations correlate with the decreased CC TEM observed in the presence of integrin β3 inhibitor. Our EMN-on-a-chip can be easily implemented for drug repurposing studies and to investigate new candidate molecules counteracting CC extravasation.
    DOI:  https://doi.org/10.1039/d0lc01011a
  9. Front Bioeng Biotechnol. 2020 ;8 617364
    van Genderen AM, Jansen K, Kristen M, van Duijn J, Li Y, Schuurmans CCL, Malda J, Vermonden T, Jansen J, Masereeuw R, Castilho M.
      Introduction: To date, tubular tissue engineering relies on large, non-porous tubular scaffolds (Ø > 2 mm) for mechanical self-support, or smaller (Ø 150-500 μm) tubes within bulk hydrogels for studying renal transport phenomena. To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties. Materials and Methods: A custom-built melt-electrowriting (MEW) device was used to fabricate tubular fibrous scaffolds with small diameter sizes (Ø = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the tubular scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality. Results: Tubular fibrous scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 1 mm and pore sizes of 0.2 mm were achieved and used for subsequent cell experiments. While HUVEC were unable to bridge the pores, ciPTEC formed tight monolayers in all scaffold microarchitectures tested. Well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp). Discussion and Conclusion: Here, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.
    Keywords:  3D culture; bioartificial kidney; contact guidance; melt-electrowriting; tissue engineering
    DOI:  https://doi.org/10.3389/fbioe.2020.617364
  10. Toxicol In Vitro. 2021 Feb 02. pii: S0887-2333(21)00032-1. [Epub ahead of print] 105107
    Ter Braak B, Niemeijer M, Boon R, Parmentier C, Baze A, Richert L, Huppelschoten S, Wink S, Verfaillie C, van de Water B.
      Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to other in vitro liver models. Here, we systematically compared the transcriptomic profiles upon chemical activation in HLCs, hiPSC, primary human hepatocytes (PHH) and HepG2 liver cancer cells. We used targeted RNA-sequencing to map concentration transcriptional response using benchmark concentration modeling for the various stress responses in the different test systems. We found that HLC are very sensitive towards oxidative stress and inflammation conditions as corresponding genes were activated at over 3 fold lower concentrations of the corresponding pathway inducing compounds as compared to PHH. PHH were the most sensitive model when studying UPR related effects. Due to the non-proliferative nature of PHH and HLC, these do not pose a good/sensitive model to pick up DNA damage responses, while hiPSC and HepG2s were more sensitive in these conditions. We envision that this study contributes to a better understanding on how HLCs can contribute to the assessment of cell physiological stress response activation to predict hepatotoxic events.
    Keywords:  DNA damage; Induced pluripotent stem cell derived hepatocytes; Inflammation; Oxidative stress; Transcriptomics; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.tiv.2021.105107
  11. Commun Biol. 2021 Feb 05. 4(1): 168
    Zamprogno P, Wüthrich S, Achenbach S, Thoma G, Stucki JD, Hobi N, Schneider-Daum N, Lehr CM, Huwer H, Geiser T, Schmid RA, Guenat OT.
      The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.
    DOI:  https://doi.org/10.1038/s42003-021-01695-0
  12. Biofabrication. 2021 Feb 01.
    Surendran V, Rutledge D, Colmon R, Chandrasekaran A.
      Neutrophils are the most abundant type of leukocytes in the blood, traditionally regarded as the first immune responders to infections and inflammations. In the context of tumors, neutrophils have been shown to possess both tumor-promoting and tumor-limiting properties. A better understanding of the inter-cellular dynamics between the neutrophils and aggregated tumors could possibly shed light on the different modalities of neutrophil involvement in tumor progression. To study in-vitro the interactional dynamics of neutrophils and growing tumor aggregates, in this work, we engineered a novel, microfluidics-integrated, three-dimensional (3D) Tumor-Immune Microenvironment (TIME)-on-Chip device, and we investigated the effect of neutrophils on the inception of collective 3D invasion of ovarian tumor cells. Herein, tumor spheroids generated and cultured on hydrogel based multi-microwell plates, and embedded within collagen matrix of defined thickness, were magnetically hybrid-integrated with a 3D bioprinting enabled microfluidic system fabricated on a porous membrane and carrying neutrophils. This setting recreated a typical TIME in-vitro to model dynamic neutrophil migration and 3D tumor invasion. Using this device, we observed that neutrophils respond to the growing tumor spheroids through both chemotaxis and generation of Neutrophil Extracellular Traps (NETs). The formation of NETs stimulated the reciprocation of tumor cells from their aggregated state to collectively invade into the surrounding collagen matrix, in a manner more significant compared to their response to known tumor-derived stimulants such as TGF-β and IL-8. This effect was reversed by drug-induced inhibition of NETs formation, suggesting that induction of NETs by cancer cells could be a pro-migratory tumor behavior. Further, we additionally report a previously unidentified, location-dictated mechanism of NETosis, in which NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of the ovarian tumor cells, thus providing a rationale for new anti-tumor therapeutics research.
    Keywords:  Biomimetics; Hybrid Integration; Microfluidics; Neutrophil Extracellular Traps; Neutrophils; Tissue Engineering; Tumor Immune Microenvironment
    DOI:  https://doi.org/10.1088/1758-5090/abe1cf
  13. Polymers (Basel). 2021 Feb 03. pii: 480. [Epub ahead of print]13(4):
    Moore CA, Siddiqui Z, Carney GJ, Naaldijk Y, Guiro K, Ferrer AI, Sherman LS, Guvendiren M, Kumar VA, Rameshwar P.
      Translational medicine requires facile experimental systems to replicate the dynamic biological systems of diseases. Drug approval continues to lag, partly due to incongruencies in the research pipeline that traditionally involve 2D models, which could be improved with 3D models. The bone marrow (BM) poses challenges to harvest as an intact organ, making it difficult to study disease processes such as breast cancer (BC) survival in BM, and to effective evaluation of drug response in BM. Furthermore, it is a challenge to develop 3D BM structures due to its weak physical properties, and complex hierarchical structure and cellular landscape. To address this, we leveraged 3D bioprinting to create a BM structure with varied methylcellulose (M): alginate (A) ratios. We selected hydrogels containing 4% (w/v) M and 2% (w/v) A, which recapitulates rheological and ultrastructural features of the BM while maintaining stability in culture. This hydrogel sustained the culture of two key primary BM microenvironmental cells found at the perivascular region, mesenchymal stem cells and endothelial cells. More importantly, the scaffold showed evidence of cell autonomous dedifferentiation of BC cells to cancer stem cell properties. This scaffold could be the platform to create BM models for various diseases and also for drug screening.
    Keywords:  alginate; bioprinting; bone marrow; breast cancer; hydrogel; methylcellulose; stem cells
    DOI:  https://doi.org/10.3390/polym13040480
  14. PLoS One. 2021 ;16(2): e0245805
    Calcagno TM, Zhang C, Tian R, Ebrahimi B, Mirsaeidi M.
      Sarcoidosis is a multi-system disorder of granulomatous inflammation which most commonly affects the lungs. Its etiology and pathogenesis are not well defined in part due to the lack of reliable modeling. Here, we present the development of an in vitro three-dimensional lung-on-chip biochip designed to mimic granuloma formation. A lung on chip fluidic macrodevice was developed and added to our previously developed a lung-on-membrane model (LOMM). Granulomas were cultured from blood samples of patients with sarcoidosis and then inserted in the air-lung-interface of the microchip to create a three-dimensional biochip pulmonary sarcoidosis model (3D BSGM). Cytokines were measured after 48 hours. ELISA testing was performed to measure cytokine response difference between LOMM with 3D BSGM. There were statistically significant differences in IL-1ß (P = 0.001953), IL-6 (P = 0.001953), GM-CSF (P = 0.001953), and INF-γ expressions (P = 0.09375) between two groups. The current model represents the first 3D biochip sarcoidosis model created by adding a microfluidics system to a dual-chambered lung on membrane model and introducing developed sarcoid-granuloma to its air-lung-interface.
    DOI:  https://doi.org/10.1371/journal.pone.0245805