bims-orenst Biomed News
on Organs-on-chips and engineered stem cell models
Issue of 2022‒04‒03
eight papers selected by
Joram Mooiweer
University of Groningen

  1. Toxicol Sci. 2022 Mar 31. pii: kfac036. [Epub ahead of print]
      Testing for acute inhalation hazards is conducted in animals; however, a number of robust in vitro human cell-based alternatives were developed and tested. These models range in complexity from cultures of cell lines or primary cells in air-liquid interface on trans-wells, to more complex and physiologically-relevant flow- and mechanical stimulation-enabled tissue chips. While the former models are relatively straightforward to establish and can be tested in medium/high-throughput, the latter require specialized equipment and lack in throughput. In this study, we developed a device that can be easily manufactured while allowing for the production of a differentiated lung tissue. This multilayered microfluidic device enables co-culture of primary human small airway epithelial cells and lung microvascular endothelial cells under physiological conditions for up to 14 days and recreates the parenchymal-vascular interface in the distal lung. To explore the potential of this airway-on-a-chip for applications in inhalation toxicology, we also devised a system that allows for direct gas/aerosol exposures of the engineered airway epithelium to noxious stimuli known to cause adverse respiratory effects, including dry flowing air, lipopolysaccharide, particulate matter, and iodomethane. This study generated quantitative, high-content data that were indicative of aberrant changes in biochemical (lactate dehydrogenase), barrier (dextran permeability), functional (ciliary beating), and molecular (imaging for various markers) phenotypes of the small airway epithelium due to inhalational exposures. This study is significant because it established an in vitro model of human small airway on a chip that can be used in medium/high-throughput studies of sub-acute effects of inhalation toxicants.
  2. Lab Chip. 2022 Mar 30.
      The multi-directional mechanical stimulation experienced by articular cartilage during motion is transferred to the chondrocytes through a thin layer of pericellular matrix around each cell; chondrocytes in turn respond by releasing matrix proteins and/or matrix-degrading enzymes. In the present study we investigated how different types of mechanical stimulation can affect a chondrocyte's phenotype and extracellular matrix (ECM) production. To this end, we employed a cartilage-on-chip system which allows exerting well-defined compressive and multi-directional mechanical stimulation on a 3D chondrocyte-laden agarose hydrogel using a thin deformable membrane and three individually addressed actuation chambers. First, the 3D chondrocyte culture in agarose responded to exposure to mechanical stimulation by an initial increase in IL-6 production and little-to-no change in IL-1β and TNF-α secretion after one day of on-chip culture. Exposure to mechanical stimulation enhanced COL2A1 (hyaline cartilage marker) and decreased COL1A1 (fibrotic cartilage) expression, this being more marked for the multi-directional stimulation. Remarkably, the production of glycosaminoglycans (GAGs), one of the main components of native cartilage ECM, was significantly increased after 15 days of on-chip culture and 14 days of mechanical stimulation. Specifically, a thin pericellular matrix shell (1-5 μm) surrounding the chondrocytes as well as an interstitial matrix, both reminiscent of the in vivo situation, were deposited. Matrix deposition was highest in chips exposed to multi-directional mechanical stimulation. Finally, exposure to mechanical cues enhanced the production of essential cartilage ECM markers, such as aggrecan, collagen II and collagen VI, a marker for the pericellular matrix. Altogether our results highlight the importance of mechanical cues, and using the right type of stimulation, to emulate in vitro, the chondrocyte microenvironment.
  3. Life Sci. 2022 Mar 28. pii: S0024-3205(22)00205-3. [Epub ahead of print] 120505
      AIMS: Recent studies show targeted therapy of new pyrazino[1,2-a]benzimidazole derivatives with COX-II inhibitory effects on different cancer cells. This study aimed to investigate 2D cell culture and 3D spheroid formation of glioblastoma multiforme (GBM) cells using a microfluidic device after exposure to these compounds.MAIN METHODS: After isolating astrocytes from human GBM samples, IC50 of 2,6-dimethyl pyrazino[1,2-a]benzimidazole (L1) and 3,4,5-trimethoxy pyrazino[1,2-a]benzimidazole (L2) were determined as 13 μM and 85 μM, respectively. Then, in all experiments, cells were exposed to subtoxic concentrations of L1 (6.5 μM) and L2 (42.5 μM), which were ½IC50. In the following, in two phases, cell cycle, migration, and gene expression through 2D cell culture and tumor spheroid formation ability using a 3D-printed microfluidic chip were assessed.
    KEY FINDINGS: The obtained results showed that both compounds have positive effects in reducing G2/M cell population and GBM cell migration. Furthermore, real-time gene expression data showed that L1 and L2 significantly impact the upregulation of P21 and P53 and down-regulation of cyclin D1, MMP2, and MMP9. On the other hand, GBM spheroids exposed to L1 and L2 become smaller with fewer live cells.
    SIGNIFICANCE: Our data on human isolated astrocyte cells in 2D and 3D cell culture conditions showed that L1 and L2 compounds could reduce GBM cells' invasion by controlling gene expressions associated with migration and proliferation. Moreover, designing microfluidic platform and related cell culture protocols facilitates the broad screening of 3D multicellular tumor spheroids derived from GBM tumor biopsies and provides effective drug development for brain gliomas.
    Keywords:  Glioblastoma multiforme; Microfluidic chip; Proliferation; Pyrazino[1,2-a]benzimidazole derivative; Spheroid
  4. Front Physiol. 2022 ;13 853317
      The past decade has witnessed tremendous endeavors to deliver novel preclinical in vitro lung models for pulmonary research endpoints, including foremost with the advent of organ- and lung-on-chips. With growing interest in aerosol transmission and infection of respiratory viruses within a host, most notably the SARS-CoV-2 virus amidst the global COVID-19 pandemic, the importance of crosstalk between the different lung regions (i.e., extra-thoracic, conductive and respiratory), with distinct cellular makeups and physiology, are acknowledged to play an important role in the progression of the disease from the initial onset of infection. In the present Methods article, we designed and fabricated to the best of our knowledge the first multi-compartment human airway-on-chip platform to serve as a preclinical in vitro benchmark underlining regional lung crosstalk for viral infection pathways. Combining microfabrication and 3D printing techniques, our platform mimics key elements of the respiratory system spanning (i) nasal passages that serve as the alleged origin of infections, (ii) the mid-bronchial airway region and (iii) the deep acinar region, distinct with alveolated airways. Crosstalk between the three components was exemplified in various assays. First, viral-load (including SARS-CoV-2) injected into the apical partition of the nasal compartment was detected in distal bronchial and acinar components upon applying physiological airflow across the connected compartment models. Secondly, nebulized viral-like dsRNA, poly I:C aerosols were administered to the nasal apical compartment, transmitted to downstream compartments via respiratory airflows and leading to an elevation in inflammatory cytokine levels secreted by distinct epithelial cells in each respective compartment. Overall, our assays establish an in vitro methodology that supports the hypothesis for viral-laden airflow mediated transmission through the respiratory system cellular landscape. With a keen eye for broader end user applications, we share detailed methodologies for fabricating, assembling, calibrating, and using our multi-compartment platform, including open-source fabrication files. Our platform serves as an early proof-of-concept that can be readily designed and adapted to specific preclinical pulmonary research endpoints.
    Keywords:  SARS-CoV-2; in vitro; inhalation; lungs; microfluidics; organ-on-chip; preclinical models; respiratory disease
  5. Nat Commun. 2022 Mar 30. 13(1): 1692
      Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.
  6. Acta Biomater. 2022 Mar 29. pii: S1742-7061(22)00183-0. [Epub ahead of print]
      Tumors, unlike normal tissue, have vascular anomalies and create interstitial flow (IF), which allows soluble substances from cancer cells to be transported directionally toward the tumor stroma. In the stroma, IF activates fibroblasts. Cancer-associated fibroblasts (CAFs) are formed from stimulated cells and aid cancer growth. A microfluidic device was designed to generate a one-directional flow of a small volume mimicking IF from donor cells to recipient at steady-state conditions only based on the medium evaporation from reservoirs with different diameter. The IF carried substances from donor cells, which stimulated the activation of fibroblasts on the receiving side, as well as their migration and stellate formation. Matrix metallopeptidases 9 and 14 as well as CAF markers such as fibroblast activation protein alpha, vimentin, and alpha-smooth muscle actin are abundantly expressed in the migrating fibroblasts. The created platform mimicked one-directional delivery in tumor stroma. This will allow researchers to investigate how cancer cells activate and differentiate stromal cells. STATEMENT OF SIGNIFICANCE: We show how to provide continuous one-directional interstitial flow (IF) in a microfluidic device without using any power source and instrumentation. This microfluidic technology was used to simulate the tumor microenvironment. Fibroblasts in the tumor stroma are activated and migrated toward cancer cells, as recapitulated by co-culture of cancer cells as donor and fibroblasts as recipient under the one-directional IF. We believe that soluble substances from cancerous cells delivered by the one-directional IF efficiently regulated the development of cancer-associated fibroblasts (CAFs), as shown by increasing roundness and decreased circularity, taking on a stellate morphology, and by enhanced invasion into a type I collagen hydrogel. Migrating fibroblasts into the hydrogel had significant levels of MMP-9, MMP-14, FAP, vimentin, and αSMA, all of which are CAF markers, bearing a capacity to form hot stroma affecting tumor malignancy.
    Keywords:  Interstitial flow; fibroblast; microfluidic device; tumor stroma
  7. Front Pharmacol. 2021 ;12 785851
      Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo-like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.
    Keywords:  drug testing; high-resolution imaging; microphysiological systems; pharmacokinetics; spheroids
  8. Nat Biomed Eng. 2022 Mar 28.
      Human spinal-cord-like tissues induced from human pluripotent stem cells are typically insufficiently mature and do not mimic the morphological features of neurulation. Here, we report a three-dimensional culture system and protocol for the production of human spinal-cord-like organoids (hSCOs) recapitulating the neurulation-like tube-forming morphogenesis of the early spinal cord. The hSCOs exhibited neurulation-like tube-forming morphogenesis, cellular differentiation into the major types of spinal-cord neurons as well as glial cells, and mature synaptic functional activities, among other features of the development of the spinal cord. We used the hSCOs to screen for antiepileptic drugs that can cause neural-tube defects. hSCOs may also facilitate the study of the development of the human spinal cord and the modelling of diseases associated with neural-tube defects.