bims-orenst 2021-05-02 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2021–05–02
nine papers selected by
Joram Mooiweer, University of Groningen

Establishment of Colorectal Cancer Organoids in Microfluidic-Based System.
Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alveolar Epithelium Barrier Function under Various Stimulations.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model.
Calcium carbonate nanoparticles stimulate cancer cell reprogramming to suppress tumor growth and invasion in an organ-on-a-chip system.
A perfusable vascularized full-thickness skin model for potential topical and systemic applications.
A multilayered blood vessel/tumor tissue chip to investigate T cell infiltration into solid tumor tissues.
Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization.
Study of Stem Cells Influence on Cardiac Cells Cultured with a Cyanide-P-Trifluoromethoxyphenylhydrazone in Organ-on-a-Chip System.
Analyzing Developing Brain-On-Chip Cultures with the CALIMA Calcium Imaging Tool.

Micromachines (Basel). 2021 Apr 28. pii: 497. [Epub ahead of print]12(5):

Establishment of Colorectal Cancer Organoids in Microfluidic-Based System.

Diana Pinho, Denis Santos, Ana Vila, Sandra Carvalho.

  Colorectal cancer is the second leading cause of cancer death worldwide. Significant advances in the molecular mechanisms underlying colorectal cancer have been made; however, the clinical approval of new drugs faces many challenges. Drug discovery is a lengthy process causing a rapid increase in global health care costs. Patient-derived tumour organoids are considered preclinical models with the potential for preclinical drug screening, prediction of patient outcomes, and guiding optimized therapy strategies at an individual level. Combining microfluidic technology with 3D tumour organoid models to recapitulate tumour organization and in vivo functions led to the development of an appropriate preclinical tumour model, organoid-on-a-chip, paving the way for personalized cancer medicine. Herein, a low-cost microfluidic device suitable for culturing and expanding organoids, OrganoidChip, was developed. Patient-derived colorectal cancer organoids were cultured within OrganoidChip, and their viability and proliferative activity increased significantly. No significant differences were verified in the organoids' response to 5-fluorouracil (5-FU) treatment on-chip and on-plate. However, the culture within the OrganoidChip led to a significant increase in colorectal cancer organoid-forming efficiency and overall size compared with conventional culture on a 24-well plate. Interestingly, early-stage and late-stage organoids were predominantly observed on-plate and within the OrganoidChip, respectively. The OrganoidChip thus has the potential to generate in vivo-like organotypic structures for disease modelling and drug screening applications.

Keywords:  3D model; colorectal cancer; drug screening; microfluidics; patient-derived organoids

DOI:  https://doi.org/10.3390/mi12050497
Micromachines (Basel). 2021 Apr 06. pii: 406. [Epub ahead of print]12(4):

Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alveolar Epithelium Barrier Function under Various Stimulations.

Shu-Han Chang, Ping-Liang Ko, Wei-Hao Liao, Chien-Chung Peng, Yi-Chung Tung.

  This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air-liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies.

Keywords:  air-liquid interface (ALI) cell culture; alveolar epithelium; atmospheric exposure; microfluidics; trans-epithelial electrical resistance (TEER); transwell insert

DOI:  https://doi.org/10.3390/mi12040406
EMBO Rep. 2021 Apr 28. e52744

Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model.

Vivek V Thacker, Kunal Sharma, Neeraj Dhar, Gian-Filippo Mancini, Jessica Sordet-Dessimoz, John D McKinney.

  Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune-cell mediated cytokine storm remain unknown. Using a vascularised lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity, and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with Tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2 mediated endothelial cell damage thus occurs independently of cytokine storm.

Keywords:  COVID-19; alveolar models; interleukin 6; organ-on-chip; vasculitis

DOI:  https://doi.org/10.15252/embr.202152744
Sci Rep. 2021 Apr 29. 11(1): 9246

Calcium carbonate nanoparticles stimulate cancer cell reprogramming to suppress tumor growth and invasion in an organ-on-a-chip system.

Sandra F Lam, Kevin W Bishop, Rachel Mintz, Lei Fang, Samuel Achilefu.

The acidic microenvironment of solid tumors induces the propagation of highly invasive and metastatic phenotypes. However, simulating these conditions in animal models present challenges that confound the effects of pH modulators on tumor progression. To recapitulate the tumor microenvironment and isolate the effect of pH on tumor viability, we developed a bifurcated microfluidic device that supports two different cell environments for direct comparison. RFP-expressing breast cancer cells (MDA-MB-231) were cultured in treatment and control chambers surrounded by fibrin, which received acid-neutralizing CaCO3 nanoparticles (nanoCaCO3) and cell culture media, respectively. Data analysis revealed that nanoCaCO3 buffered the pH within the normal physiological range and inhibited tumor cell proliferation compared to the untreated control (p < 0.05). Co-incubation of cancer cells and fibroblasts, followed by nanoCaCO3 treatment showed that the nanoparticles selectively inhibited the growth of the MDA-MB-231 cells and reduced cellular migration of these cells with no impact on the fibroblasts. Sustainable decrease in the intracellular pH of cancer cells treated with nanoCaCO3 indicates that the extracellular pH induced cellular metabolic reprogramming. These results suggest that the nanoCaCO3 can restrict the aggressiveness of tumor cells without affecting the growth and behavior of the surrounding stromal cells.

DOI: https://doi.org/10.1038/s41598-021-88687-6
Biofabrication. 2021 Apr 28.

A perfusable vascularized full-thickness skin model for potential topical and systemic applications.

Sacha Salameh, Nicolas Tissot, Kevin Caché, Joaquim Lima, Itaru Suzuki, Paulo Andé Marinho, Maité Rielland, Jérémie Soeur, Shoji Takeuchi, Stéphane Germain, Lionel Breton.

  Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance of in vitro applications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We here combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing i) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, ii) 3 perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, iii) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and Benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

Keywords:  Angiogenesis; Perfusion; Reconstructed skin; Tissue Engineering; Vascularization; Vasculogenesis

DOI:  https://doi.org/10.1088/1758-5090/abfca8
Lab Chip. 2021 Apr 29.

A multilayered blood vessel/tumor tissue chip to investigate T cell infiltration into solid tumor tissues.

Jaehyun Lee, Seong-Eun Kim, Dowon Moon, Junsang Doh.

Cancer immunotherapies based on the ability of T cells to recognize and kill tumor cells (TCs), including immune checkpoint blockade (ICB) therapy and chimeric antigen receptor (CAR) T cell therapy, have been greatly successful recently, but they are applicable for only a fraction of patients. One of the main challenges in cancer immunotherapy is the improvement of T cell infiltration into solid tumor tissues, as T cells can exert cytotoxicity against TCs only when they are in contact with TCs. T cells in the bloodstream infiltrate into solid tumor tissues by following two steps known as extravasation and interstitial migration. Herein, we developed a multilayered blood vessel/tumor tissue chip (MBTC) that allows systematic investigation on T cell tumor infiltration. The MBTC is composed of a top fluidic chamber, a porous membrane covered with an endothelial cell (EC) monolayer, and a collagen gel block encapsulating TCs. The full sequence of T cell tumor infiltration, including extravasation and interstitial migration, required for TC killing is demonstrated in the MBTCs: T cells applied through the top fluidic chamber of the MBTCs exhibited dynamic interactions with ECs for extravasation, including intraluminal crawling and transendothelial migration (TEM). After extravasation, T cells migrate toward TCs located at the bottom of a collagen block to kill them. Key characteristics of T cell dynamics in tumor microenvironments are recapitulated in the MBTCs: the vascular endothelial growth factor (VEGF) produced by TCs suppressed EC activation by inflammatory cytokines, or induced EC anergy, thereby significantly reducing T cell extravasation, whereas chemokines produced by TCs triggered T cell chemotaxis toward TCs. Anti-VEGF treatment in the MBTCs reverts EC anergy and promotes T cell infiltration, similar to the clinical effects of anti-VEGF. The MBTC is a useful model for pre-clinical evaluation of immunotherapeutics and the fundamental study of tumor immunology.

DOI: https://doi.org/10.1039/d1lc00182e
Front Bioeng Biotechnol. 2021 ;9 664094

Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization.

Federico Sala, Carlotta Ficorella, Rebeca Martínez Vázquez, Hannah Marie Eichholz, Josef A Käs, Roberto Osellame.

  The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.

Keywords:  cell migration; femtosecond laser microfabrication; lab-on-a-chip; neuronal cell; two-photon polymerization

DOI:  https://doi.org/10.3389/fbioe.2021.664094
Biosensors (Basel). 2021 Apr 23. pii: 131. [Epub ahead of print]11(5):

Study of Stem Cells Influence on Cardiac Cells Cultured with a Cyanide-P-Trifluoromethoxyphenylhydrazone in Organ-on-a-Chip System.

Anna Kobuszewska, Dominik Kolodziejek, Michal Wojasinski, Tomasz Ciach, Zbigniew Brzozka, Elzbieta Jastrzebska.

  Regenerative medicine and stem cells could prove to be an effective solution to the problem of treating heart failure caused by ischemic heart disease. However, further studies on the understanding of the processes which occur during the regeneration of damaged tissue are needed. Microfluidic systems, which provide conditions similar to in vivo, could be useful tools for the development of new therapies using stem cells. We investigated how mesenchymal stem cells (MSCs) affect the metabolic activity of cardiac cells (rat cardiomyoblasts and human cardiomyocytes) incubated with a potent uncoupler of mitochondrial oxidative phosphorylation under microfluidic conditions. A cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to mimic disfunctions of mitochondria of cardiac cells. The study was performed in a microfluidic system integrated with nanofiber mats made of poly-l-lactid acid (PLLA) or polyurethane (PU). The microsystem geometry allows four different cell cultures to be conducted under different conditions (which we called: normal, abnormal-as both a mono- and co-culture). Metabolic activity of the cells, based on the bioluminescence assay, was assessed in the culture's performed in the microsystem. It was proved that stem cells increased metabolic activity of cardiac cells maintained with FCCP.

Keywords:  cardiovascular diseases; heart-on-a-chip; microfluidics; stem cells

DOI:  https://doi.org/10.3390/bios11050131
Micromachines (Basel). 2021 Apr 08. pii: 412. [Epub ahead of print]12(4):

Analyzing Developing Brain-On-Chip Cultures with the CALIMA Calcium Imaging Tool.

Elles A L Raaijmakers, Nikki Wanders, Rob M C Mestrom, Regina Luttge.

  Brain-on-chip (BoC) models are tools for reproducing the native microenvironment of neurons, in order to study the (patho)physiology and drug-response of the brain. Recent developments in BoC techniques focus on steering neurons in their activity via microfabrication and via computer-steered feedback mechanisms. These cultures are often studied through calcium imaging (CI), a method for visualizing the cellular activity through infusing cells with a fluorescent dye. CAlciumImagingAnalyser 2.0 (CALIMA 2.0) is an updated version of a software tool that detects and analyzes fluorescent signals and correlates cellular activity to identify possible network formation in BoC cultures. Using three previous published data sets, it was demonstrated that CALIMA 2.0 can analyze large data sets of CI-data and interpret cell activity to help study the activity and maturity of BoC cultures. Last, an analysis of the processing speed shows that CALIMA 2.0 is sufficiently fast to process data sets with an acquisition rate up to 5 Hz in real-time on a medium-performance computer.

Keywords:  brain-on-chip culture; calcium fluorescence imaging; neuronal network; software tool

DOI:  https://doi.org/10.3390/mi12040412