bims-orenst Biomed News
on Organs-on-chips and engineered stem cell models
Issue of 2021‒02‒21
nine papers selected by
Joram Mooiweer
University of Groningen

  1. Lab Chip. 2021 Feb 19.
      Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.
  2. Lab Chip. 2021 Feb 19.
      The stem cell niche at the perivascular space in human tissue plays a pivotal role in dictating the overall fate of stem cells within it. Mesenchymal stem cells (MSCs) in particular, experience influential microenvironmental conditions, which induce specific metabolic profiles that affect processes of cell differentiation and dysregulation of the immunomodulatory function. Reports focusing specifically on the metabolic status of MSCs under the effect of pathophysiological stimuli - in terms of flow velocities, shear stresses or oxygen tension - do not model heterogeneous gradients, highlighting the need for more advanced models reproducing the metabolic niche. Organ-on-a-chip technology offers the most advanced tools for stem cell niche modelling thus allowing for controlled dynamic culture conditions while profiling tuneable oxygen tension gradients. However, current systems for live cell detection of metabolic activity inside microfluidic devices require the integration of microsensors. The presence of such microsensors poses the potential to alter microfluidics and their resolution does not enable intracellular measurements but rather a global representation concerning cellular metabolism. Here, we present a metabolic toolbox coupling a miniaturised in vitro system for human-MSCs dynamic culture, which mimics microenvironmental conditions of the perivascular niche, with high-resolution imaging of cell metabolism. Using fluorescence lifetime imaging microscopy (FLIM) we monitor the spatial metabolic machinery and correlate it with experimentally validated intracellular oxygen concentration after designing the oxygen tension decay along the fluidic chamber by in silico models prediction. Our platform allows the metabolic regulation of MSCs, mimicking the physiological niche in space and time, and its real-time monitoring representing a functional tool for modelling perivascular niches, relevant diseases and metabolic-related uptake of pharmaceuticals.
  3. Biomaterials. 2021 Feb 06. pii: S0142-9612(20)30887-5. [Epub ahead of print]270 120640
      In cancer metastasis, extravasation refers to the process where tumor cells exit the bloodstream by crossing the endothelium and invade the surrounding tissue. Tumor cells engage in complex crosstalk with other active players such as the endothelium leading to changes in functional behavior that exert pro-extravasation effects. Most in vitro studies to date have only focused on the independent effects of molecular targets on the functional changes of cancer cell extravasation behavior. However, singular targets cannot combat complex interactions involved in tumor cell extravasation that affects multiple cell types and signaling pathways. In this study, we employ an organotypic microfluidic model of human vasculature to investigate the independent and combined role of multiple upregulated secreted factors resulting from cancer-vascular interactions during cancer cell extravasation. The device consists of a tubular endothelial vessel generated from induced pluripotent stem cell derived endothelial cells within a collagen-fibrinogen matrix with breast cancer cells injected through and cultured along the lumen of the vessel. Our system identified cancer-vascular crosstalk, involving invasive breast cancer cells, that results in increased levels of secreted IL-6, IL-8, and MMP-3. Our model also showed that upregulation of these secreted factors correlates with invasive/metastatic potential of breast cancer cells. We also used therapeutic inhibitors to assess the independent and combined role of multiple signaling factors on the overall changes in functional behavior of both the cancer cells and the endothelium that promote extravasation. Taken together, these results demonstrate the potential of our organotypic model in elucidating mechanisms through which cancer-vascular interactions can promote extravasation, and in conducting functional assessment of therapeutic drugs that prevent extravasation in cancer metastasis.
    Keywords:  Cancer cell extravasation; Endothelial vessels; Induced pluripotent stem cell-derived endothelial cells; Microfluidic in vitro model; Therapeutic drug testing platform
  4. Sci Adv. 2021 Feb;pii: eabc2331. [Epub ahead of print]7(8):
      Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.
  5. ACS Biomater Sci Eng. 2021 Feb 14.
      Fas ligand (FasL, CD178) is known to bind to its receptor (Fas, CD95) and mediate cellular apoptosis to maintain immune homeostasis. Recently, it has been recognized that tumor cells and their microenvironments allow an adjacent vascular endothelium to express the FasL on its cell membrane, utilizing the endothelium as an immune barrier to kill antitumor cytotoxic T cells. Here, a microfluidic tumor vasculature model is presented, which enables the recapitulation of an endothelial immune barrier expressing FasL. The in vitro three-dimensional model replicates enhanced endothelial FasL expression under the hypoxic tumor microenvironment. Apoptosis rates of FasL-susceptible target cells are augmented under the microenvironment with upregulated FasL but are consequently abrogated by administrations of pharmacological inhibitions, FasL-Fas blockades. The microfluidic system suggests its promising applications in elucidating complex immunosuppressive mechanisms of the tumor microenvironment and screening of cell-mediated immunotherapies as a preclinical model.
    Keywords:  3D tumor vascular network; Fas ligand endothelium; immune cell apoptosis; in vitro disease model; microfluidic coculture platform
  6. ACS Biomater Sci Eng. 2021 Jan 11. 7(1): 350-359
      Animal models are frequently used in drug discovery because they represent a mammalian in vivo model system, they are the closest approximation to the human brain, and experimentation in humans is not ethical. Working with postmortem human brain samples is challenging and developing human in vitro systems, which mimic the in vivo human brain, has been challenging. However, the use of animal models in drug discovery for human neurological diseases is currently under scrutiny because data from animal models has come with variations due to genetic differences. Evidence from the literature suggests that techniques to reconstruct multiple neurotransmission projections, which characterize neurological disease circuits in humans, in vitro, have not been demonstrated. This paper presents a multicompartment microdevice for patterning neurospheres and specification of neural stem cell fate toward networks of multiple neuronal phenotypes. We validated our design by specification of human neural stem cells to dopaminergic and GABAergic neurons in different compartments of the device, simultaneously. The neurospheres formed unrestricted robust neuronal circuits between arrays of neurospheres in all compartments of the device. Such a device design may provide a basis for formation of multineurotransmission circuits to model functional connectivity between specific human brain regions, in vitro, using human-derived neural stem cells. This work finds relevance in neurological disease modeling and drug screening using human cell-based assays and may provide the impetus for shifting from animal-based models.
    Keywords:  3D neuronal tissue; brain-on-a-chip; drug discovery; microfabrication; neural stem cells; neurological disease models
  7. Biofabrication. 2021 Feb 13. 13(1): 015016
      'Organ-on-chip' devices which integrate three-dimensional (3D) cell culture techniques with microfluidic approaches have the capacity to overcome the limitations of classical 2D platforms. Although several different strategies have been developed to improve the angiogenesis within hydrogels, one of the main challenges in tissue engineering remains the lack of vascularization in the fabricated 3D models. The present work focuses on the high-definition (HD) bioprinting of microvascular structures directly on-chip using two-photon polymerization (2PP). 2PP is a nonlinear process, where the near-infrared laser irradiation will only lead to the polymerization of a very small volume pixel (voxel), allowing the fabrication of channels in the microvascular range (10-30 µm in diameter). Additionally, 2PP not only enables the fabrication of sub-micrometer resolution scaffolds but also allows the direct embedding of cells within the produced structure. The accuracy of the 2PP printing parameters were optimized in order to achieve high-throughput and HD production of microfluidic vessel-on-chip platforms. The spherical aberrations stemming from the refractive index mismatch and the focusing depth inside the sample were simulated and the effect of the voxel compensation as well as different printing modes were demonstrated. Different layer spacings and their dependency on the applied laser power were compared both in terms of accuracy and required printing time resulting in a 10-fold decrease in structuring time while yielding well-defined channels of small diameters. Finally, the capacity of 2PP to create vascular structures within a microfluidic chip was tested with two different settings, by direct embedding of a co-culture of endothelial- and supporting cells during the printing process and by creating a supporting, cell-containing vascular scaffold barrier where the endothelial cell spheroids can be seeded afterwards. The functionality of the formed vessels was demonstrated with immunostaining of vascular endothelial cadherin (VE-Cadherin) endothelial adhesion molecules in both static and perfused culture.
  8. Adv Healthc Mater. 2021 Feb 15. e2100031
      Engineering functional human tissues in vitro is currently limited by difficulty replicating the small caliber, complex connectivity, cellularity, and 3D curvature of the native microvasculature. Multiphoton ablation has emerged as a promising technique for fabrication of microvascular structures with high resolution and full 3D control, but cellularization and perfusion of complex capillary-scale structures has remained challenging. Here, multiphoton ablation combined with guided endothelial cell growth from pre-formed microvessels is used to successfully create perfusable and cellularized organ-specific microvascular structures at anatomic scale within collagen hydrogels. Fabrication and perfusion of model 3D pulmonary and renal microvascular beds is demonstrated, as is replication and perfusion of a brain microvascular unit derived from in vivo data. Successful endothelialization and blood perfusion of a kidney-specific microvascular structure is achieved, using laser-guided angiogenesis. Finally, proof-of-concept hierarchical blood vessels and complex multicellular models are created, using multistep patterning with multiphoton ablation techniques. These successes open new doors for the creation of engineered tissues and organ-on-a-chip devices.
    Keywords:  biomaterials; microphysiological systems; multiphoton ablation; organ-on-a-chip; tissue engineering
  9. FASEB J. 2021 Feb;35(2): e21291
      The intestinal environment is unique because it supports the intestinal epithelial cells under a normal oxygen environment and the microbiota under an anoxic environment. Due to importance of understanding the interactions between the epithelium and the microbiota, there is a strong need for developing representative and simple experimental models. Current approaches do not capture the partitioned oxygen environment, require external anaerobic chambers, or are complex. Another major limitation is that with the solutions that can mimic this oxygen environment, the oxygenation level of the epithelial cells is not known, raising the question whether the cells are hypoxic or not. We report standalone microfluidic devices that form a partitioned oxygen environment without the use of an external anaerobic chamber or oxygen scavengers to coculture intestinal epithelial and bacterial cells. By changing the thickness of the device cover, the oxygen tension in the chamber was modulated. We verified the oxygen levels using several tests: microscale oxygen sensitive sensors which were integrated within the devices, immunostaining of Caco-2 cells to determine hypoxia levels, and genetically encoded bacteria to visualize the growth. Collectively, these methods monitored oxygen concentrations in the devices more comprehensively than previous reports and allowed for control of oxygen tension to match the requirements of both intestinal cells and anaerobic bacteria. Our experimental model is supported by the mathematical model that considered diffusion of oxygen into the top chamber. This allowed us to experimentally determine the oxygen consumption rate of the intestinal epithelial cells under perfusion.
    Keywords:  anaerobe; barrier function; colon; hypoxic intestine; intestine-bacteria interaction; microfluidics; organ-on-chip; oxygen sensor; partitioned oxygen environment