bims-orenst 2021-07-04 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2021–07–04
ten papers selected by
Joram Mooiweer, University of Groningen

Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model.
Continuous Monitoring Reveals Protective Effects of N-Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit.
Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.
A Microfluidic Platform for Cavitation-Enhanced Drug Delivery.
Versatile Vessel-on-a-Chip Platform for Studying Key Features of Blood Vascular Tumors.
Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro.
A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses.
Mechanical Stimulation Modulates Osteocyte Regulation of Cancer Cell Phenotype.
Immuno-responsive tissue engineered oral mucosal equivalents containing macrophages.
Microfluidic Device Using Mouse Small Intestinal Tissue for the Observation of Fluidic Behavior in the Lumen.

ACS Biomater Sci Eng. 2021 Jun 29.

Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model.

Nicola Frenkel, Susanna Poghosyan, Carmen Rubio Alarcón, Silvia Bonilla García, Karla Queiroz, Lotte van den Bent, Jamila Laoukili, Inne Borel Rinkes, Paul Vulto, Onno Kranenburg, Jeroen Hagendoorn.

  The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel-tumor cell coculture assay.

Keywords:  3-lane OrganoPlate; BMI1; LEC; hTERT; organ-on-a-chip

DOI:  https://doi.org/10.1021/acsbiomaterials.0c01378
Small. 2021 Jun 26. e2101785

Continuous Monitoring Reveals Protective Effects of N-Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit.

Isabelle Matthiesen, Dimitrios Voulgaris, Polyxeni Nikolakopoulou, Thomas E Winkler, Anna Herland.

  Microphysiological systems mimic the in vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. In this study, the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2 min temporal resolution is reported. Its capabilities are showcased in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol-ene-epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell-substrate endothelial resistance monitoring allows for capturing the formation and breakdown of the BBB model, which consists of cocultured hiPSC-derived endothelial-like and astrocyte-like cells. Clear cellular disruption is observed when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide are reported. Using metabolomic network analysis reveals further drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like this opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.

Keywords:  continuous monitoring; human-induced pluripotent stem cells; microphysiological systems; neurovascular unit; oxidative and nitrosative stress

DOI:  https://doi.org/10.1002/smll.202101785
Int J Mol Sci. 2021 Jun 29. pii: 6997. [Epub ahead of print]22(13):

Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.

Gyeong-Ji Kim, Kwon-Jai Lee, Jeong-Woo Choi, Jeung Hee An.

  We developed a multi-channel cell chip containing a three-dimensional (3D) scaffold for horizontal co-culture and drug toxicity screening in multi-organ culture (human glioblastoma, cervical cancer, normal liver cells, and normal lung cells). The polydimethylsiloxane (PDMS) multi-channel cell chip (PMCCC) was based on fused deposition modeling (FDM) technology. The architecture of the PMCCC was an open-type cell chip and did not require a pump or syringe. We investigated cell proliferation and cytotoxicity by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and analysis of oleanolic acid (OA)-treated multi-channel cell chips. The results of the MTT and LDH assays showed that OA treatment in the multi-channel cell chip of four cell lines enhanced chemoresistance of cells compared with that in the 2D culture. Furthermore, we demonstrated the feasibility of the application of our multi-channel cell chip in various analysis methods through Annexin V-fluorescein isothiocyanate/propidium iodide staining, which is not used for conventional cell chips. Taken together, the results demonstrated that the PMCCC may be used as a new 3D platform because it enables simultaneous drug screening in multiple cells by single point injection and allows analysis of various biological processes.

Keywords:  3D culture; cell chip; drug screening; multi-channel; toxicity evaluation

DOI:  https://doi.org/10.3390/ijms22136997
Micromachines (Basel). 2021 Jun 03. pii: 658. [Epub ahead of print]12(6):

A Microfluidic Platform for Cavitation-Enhanced Drug Delivery.

Giulia Grisanti, Davide Caprini, Giorgia Sinibaldi, Chiara Scognamiglio, Giulia Silvani, Giovanna Peruzzi, Carlo Massimo Casciola.

  An endothelial-lined blood vessel model is obtained in a PDMS (Polydimethylsiloxane) microfluidic system, where vascular endothelial cells are grown under physiological shear stress, allowing -like maturation. This experimental model is employed for enhanced drug delivery studies, aimed at characterising the increase in endothelial permeability upon microbubble-enhanced ultrasound-induced (USMB) cavitation. We developed a multi-step protocol to couple the optical and the acoustic set-ups, thanks to a 3D-printed insonation chamber, provided with direct optical access and a support for the US transducer. Cavitation-induced interendothelial gap opening is then analysed using a customised code that quantifies gap area and the relative statistics. We show that exposure to US in presence of microbubbles significantly increases endothelial permeability and that tissue integrity completely recovers within 45 min upon insonation. This protocol, along with the versatility of the microfluidic platform, allows to quantitatively characterise cavitation-induced events for its potential employment in clinics.

Keywords:  drug delivery; endothelium permeabilization; microfluidics

DOI:  https://doi.org/10.3390/mi12060658
Bioengineering (Basel). 2021 Jun 09. pii: 81. [Epub ahead of print]8(6):

Versatile Vessel-on-a-Chip Platform for Studying Key Features of Blood Vascular Tumors.

Marina Llenas, Roberto Paoli, Natalia Feiner-Gracia, Lorenzo Albertazzi, Josep Samitier, David Caballero.

  Tumor vessel-on-a-chip systems have attracted the interest of the cancer research community due to their ability to accurately recapitulate the multiple dynamic events of the metastatic cascade. Vessel-on-a-chip microfluidic platforms have been less utilized for investigating the distinctive features and functional heterogeneities of tumor-derived vascular networks. In particular, vascular tumors are characterized by the massive formation of thrombi and severe bleeding, a rare and life-threatening situation for which there are yet no clear therapeutic guidelines. This is mainly due to the lack of technological platforms capable of reproducing these characteristic traits of the pathology in a simple and well-controlled manner. Herein, we report the fabrication of a versatile tumor vessel-on-a-chip platform to reproduce, investigate, and characterize the massive formation of thrombi and hemorrhage on-chip in a fast and easy manner. Despite its simplicity, this method offers multiple advantages to recapitulate the pathophysiological events of vascular tumors, and therefore, may find useful applications in the field of vascular-related diseases, while at the same time being an alternative to more complex approaches.

Keywords:  in vitro model; microfluidics; organ-on-chip; vascular tumor; vessel

DOI:  https://doi.org/10.3390/bioengineering8060081
Integr Biol (Camb). 2021 Jun 28. pii: zyab011. [Epub ahead of print]

Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro.

Prativa Das, Michael D DiVito, Jason A Wertheim, Lay Poh Tan.

  Alcohol injury induces hepatic fibrosis which gradually progresses to cirrhosis, sometimes may lead to liver cancer. Animal models are less efficient in mimicking responses of human liver cells, whereas in vitro models discussed so far are majorly based on rodent cells. In this work, a coculture of primary human hepatocytes (PHHs) with LX-2 cells was established on the unmodified (C:F_0:0), collagen-I modified (C:F_1:0), fibronectin modified (C:F_0:1) and 3:1 collagen-I to fibronectin modified (C:F_3:1) 3D electrospun fibrous scaffolds. The effect of alcohol injury was evaluated on this cell-scaffold model at 0-40 μl/ml alcohol concentrations over 14 days of culture period by using the gold standard sandwich culture as the control. Among all the culture groups, C:F_3:1 scaffold was able to maintain translational and transcriptional properties of human liver cells at all concentrations of alcohol treatment. The study reveals that, PHHs on C:F_3:1 were able to maintain ~4-fold and ~1.6-fold higher secretion of albumin than the gold standard sandwich culture on Day 3 and Day 7, respectively. When treated with alcohol, at concentrations of 20 and 40 μl/ml, albumin secretion was also observed to be higher (~2-fold) when compared to the gold standard sandwich culture. Again as expected, in C:F_3:1 culture group on 40 μl/ml alcohol treatment, albumin gene expression decreased by ~2-fold due to alcohol toxicity, whereas CYP2C9, CYP3A4, CYP2E1 and CYP1A2 gene expressions upregulated by ~3.5, ~~4, ~5 and ~15-fold, respectively in response to the alcohol injury. LX-2 cells also acquire more quiescent phenotype on C:F_3:1 scaffolds when compared to the gold standard sandwich culture upon alcohol treatment. Thus, C:F_3:1 scaffold with human liver cells was established as the potential platform to scan alcohol toxicity at varied alcohol concentrations. Thus, it can pave a promising path not only to support functional healthy human liver cells for liver tissue engineering but also to examine potential drugs to study the progression or inhibition of alcoholic liver fibrosis in vitro.

Keywords:  alcoholic liver disease model; collagen-I; electrospinning; fibronectin; liver tissue engineering; scaffolds

DOI:  https://doi.org/10.1093/intbio/zyab011
Biomedicines. 2021 Jun 02. pii: 631. [Epub ahead of print]9(6):

A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses.

Luis Soriano, Tehreem Khalid, Fergal J O'Brien, Cian O'Leary, Sally-Ann Cryan.

  Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.

Keywords:  3D in vitro models; air-liquid interface; bleomycin; co-culture; collagen; epithelium; lipopolysaccharide; respiratory tissue engineering; toxicology

DOI:  https://doi.org/10.3390/biomedicines9060631
Cancers (Basel). 2021 Jun 10. pii: 2906. [Epub ahead of print]13(12):

Mechanical Stimulation Modulates Osteocyte Regulation of Cancer Cell Phenotype.

Stefaan W Verbruggen, Clare L Thompson, Michael P Duffy, Sophia Lunetto, Joanne Nolan, Oliver M T Pearce, Christopher R Jacobs, Martin M Knight.

  Breast and prostate cancers preferentially metastasise to bone tissue, with metastatic lesions forming in the skeletons of most patients. On arriving in bone tissue, disseminated tumour cells enter a mechanical microenvironment that is substantially different to that of the primary tumour and is largely regulated by bone cells. Osteocytes, the most ubiquitous bone cell type, orchestrate healthy bone remodelling in response to physical exercise. However, the effects of mechanical loading of osteocytes on cancer cell behaviour is still poorly understood. The aim of this study was to characterise the effects of osteocyte mechanical stimulation on the behaviour of breast and prostate cancer cells. To replicate an osteocyte-controlled environment, this study treated breast (MDA-MB-231 and MCF-7) and prostate (PC-3 and LNCaP) cancer cell lines with conditioned media from MLO-Y4 osteocyte-like cells exposed to mechanical stimulation in the form of fluid shear stress. We found that osteocyte paracrine signalling acted to inhibit metastatic breast and prostate tumour growth, characterised by reduced proliferation and invasion and increased migration. In breast cancer cells, these effects were largely reversed by mechanical stimulation of osteocytes. In contrast, conditioned media from mechanically stimulated osteocytes had no effect on prostate cancer cells. To further investigate these interactions, we developed a microfluidic organ-chip model using the Emulate platform. This new organ-chip model enabled analysis of cancer cell migration, proliferation and invasion in the presence of mechanical stimulation of osteocytes by fluid shear stress, resulting in increased invasion of breast and prostate cancer cells. These findings demonstrate the importance of osteocytes and mechanical loading in regulating cancer cell behaviour and the need to incorporate these factors into predictive in vitro models of bone metastasis.

Keywords:  bone metastatic disease; breast cancer; cell co-culture; mechanical stimulation; microfluidic device; organ on a chip; osteocyte; prostate cancer; tumour metastasis

DOI:  https://doi.org/10.3390/cancers13122906
Tissue Eng Part C Methods. 2021 Jul 01.

Immuno-responsive tissue engineered oral mucosal equivalents containing macrophages.

Bethany Ollington, Helen Elizabeth Colley, Craig Murdoch.

Macrophages play a key role in orchestrating the host immune response towards invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME and to date none have contained primary human macrophages. Here we report the generation of an immuno-competent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of pro-inflammatory cytokine (IL-6, CXCL8 and TNF-α) gene expression and protein secretion as compared to culture as 2D monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate a MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased pro-inflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared to OME alone. Moreover, this pro-inflammatory response was inhibited by pre-treatment with dexamethasone, showing that MDM-OME are also amenable to drug-treatment. Dual-labelled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies.

DOI: https://doi.org/10.1089/ten.TEC.2021.0124
Micromachines (Basel). 2021 Jun 13. pii: 692. [Epub ahead of print]12(6):

Microfluidic Device Using Mouse Small Intestinal Tissue for the Observation of Fluidic Behavior in the Lumen.

Satoru Kuriu, Naoyuki Yamamoto, Tadashi Ishida.

  The small intestine has the majority of a host's immune cells, and it controls immune responses. Immune responses are induced by a gut bacteria sampling process in the small intestine. The mechanism of immune responses in the small intestine is studied by genomic or histological techniques after in vivo experiments. While the distribution of gut bacteria, which can be decided by the fluid flow field in the small intestinal tract, is important for immune responses, the fluid flow field has not been studied due to limits in experimental methods. Here, we propose a microfluidic device with chemically fixed small intestinal tissue as a channel. A fluid flow field in the small intestinal tract with villi was observed and analyzed by particle image velocimetry. After the experiment, the distribution of microparticles on the small intestinal tissue was histologically analyzed. The result suggests that the fluid flow field supports the settlement of microparticles on the villi.

Keywords:  embedded resin; ex vivo; histology; microfluidic device; sectioning; small intestine

DOI:  https://doi.org/10.3390/mi12060692