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Research Square May 2024Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic...
Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. An amnion membrane (AM) organ-on-chip (OOC) was utilized to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 hours flow culture to mimic fluid motion. Static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control for pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to CK-18 ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a flow culture environment is not necessary to mimic any physiologic cellular conditions of fetal membrane-derived cells.
PubMed: 38798515
DOI: 10.21203/rs.3.rs-4372328/v1 -
Journal of Clinical Medicine May 2024: Since metabolic diseases and atherosclerotic vascular events are firmly associated, herein we investigate changes in central microcirculation and...
: Since metabolic diseases and atherosclerotic vascular events are firmly associated, herein we investigate changes in central microcirculation and atherosclerosis-related body fat distribution in patients with type 2 diabetes mellitus and obesity. Resting brain perfusion single-photon emission computed tomography (SPECT) imaging with Technetium-99m hexamethylpropylene amine oxime ([Tc]Tc-HMPAO SPECT) was performed, and the breath-holding index (BHI) and carotid intima-media thickness (cIMT) were measured to characterise central microcirculation. Besides CT-based abdominal fat tissue segmentation, C-peptide level, glycaemic and anthropometric parameters were registered to search for correlations with cerebral blood flow and vasoreactivity. : Although no significant difference was found between the resting cerebral perfusion of the two patient cohorts, a greater blood flow increase was experienced in the obese after the breath-holding test than in the diabetics ( < 0.05). A significant positive correlation was encountered between resting and provocation-triggered brain perfusion and C-peptide levels ( < 0.005). BMI and cIMT were negatively correlated (rho = -0.27 and -0.23 for maximum and mean cIMT, respectively), while BMI and BHI showed a positive association (rho = 0.31 and rho = 0.29 for maximum and mean BHI, respectively), which could be explained by BMI-dependent changes in fat tissue distribution. cIMT demonstrated a disproportional relationship with increasing age, and higher cIMT values were observed for the men. Overall, C-peptide levels and circulatory parameters seem to be strong applicants to predict brain microvascular alterations and related cognitive decline in such patient populations.
PubMed: 38792441
DOI: 10.3390/jcm13102900 -
Bioengineering (Basel, Switzerland) Apr 2024There is a growing interest in the production of bioinks that on the one hand, are biocompatible and, on the other hand, have mechanical properties that allow for the...
There is a growing interest in the production of bioinks that on the one hand, are biocompatible and, on the other hand, have mechanical properties that allow for the production of stable constructs that can survive for a long time after transplantation. While the selection of the right material is crucial for bioprinting, there is another equally important issue that is currently being extensively researched-the incorporation of the vascular system into the fabricated scaffolds. Therefore, in the following manuscript, we present the results of research on bioink with unique physico-chemical and biological properties. In this article, two methods of seeding cells were tested using bioink B and seeding after bioprinting the whole model. After 2, 5, 8, or 24 h of incubation, the flow medium was used in the tested systems. At the end of the experimental trial, for each time variant, the canals were stored in formaldehyde, and immunohistochemical staining was performed to examine the presence of cells on the canal walls and roof. Cells adhered to both ways of fiber arrangement; however, a parallel bioprint with the 5 h incubation and the intermediate plating of cells resulted in better adhesion efficiency. For this test variant, the percentage of cells that adhered was at least 20% higher than in the other analyzed variants. In addition, it was for this variant that the lowest percentage of viable cells was found that were washed out of the tested model. Importantly, hematoxylin and eosin staining showed that after 8 days of culture, the cells were evenly distributed throughout the canal roof. Our study clearly shows that neovascularization-promoting cells effectively adhere to ECM-based pancreatic bioink. Summarizing the presented results, it was demonstrated that the proposed bioink compositions can be used for bioprinting bionic organs with a vascular system formed by endothelial cells and fibroblasts.
PubMed: 38790306
DOI: 10.3390/bioengineering11050439 -
Kidney International May 2024Prolonged warm ischemic is the main cause discarding donated organs after cardiac death. Here, we identified that prolonged warm ischemic time induced disseminated...
Prolonged warm ischemic is the main cause discarding donated organs after cardiac death. Here, we identified that prolonged warm ischemic time induced disseminated intravascular coagulation and severe capillary vasospasm after cardiac death of rat kidneys. Additionally, we found a significant accumulation of fibrinogen in a hypoxic cell culture of human umbilical vein epithelial cells and in isolated kidneys exposed to prolonged warm ischemic following flushing out of blood. However, pre-flushing the kidney with snake venom plasmin in a 90-minute warm ischemic model maximized removal of micro thrombi and facilitated the delivery of oxygen and therapeutic agents. Application of carbon monoxide releasing CORM-401 during ex vivo hypothermic oxygenated perfusion achieved multipath protective effects in prolonged warm ischemic kidneys. This led to significant improvements in perfusion parameters, restoration of the microcirculation, amelioration of mitochondrial injury, oxidative stress, and apoptosis. This benefit resulted in significantly prolonged warm ischemic kidney recipient survival rates of 70%, compared with none in those receiving ex vivo hypothermic oxygenated perfusion alone. Significantly, ex vivo hypothermic oxygenated perfusion combined with cytoprotective carbon monoxide releasing CORM-401 treatment meaningfully protected the donated kidney after cardiac death from ischemia-reperfusion injury by reducing inflammation, oxidative stress, apoptosis, and pathological damage. Thus, our study suggests a new combination treatment strategy to potentially expand the donor pool by increasing use of organs after cardiac death and salvaging prolonged warm ischemic kidneys.
PubMed: 38789038
DOI: 10.1016/j.kint.2024.04.018 -
Gels (Basel, Switzerland) May 2024The bioprinting of high-concentrated collagen bioinks is a promising technology for tissue engineering and regenerative medicine. Collagen is a widely used biomaterial...
The bioprinting of high-concentrated collagen bioinks is a promising technology for tissue engineering and regenerative medicine. Collagen is a widely used biomaterial for bioprinting because of its natural abundance in the extracellular matrix of many tissues and its biocompatibility. High-concentrated collagen hydrogels have shown great potential in tissue engineering due to their favorable mechanical and structural properties. However, achieving high cell proliferation rates within these hydrogels remains a challenge. In static cultivation, the volume of the culture medium is changed once every few days. Thus, perfect perfusion is not achieved due to the relative increase in metabolic concentration and no medium flow. Therefore, in our work, we developed a culture system in which printed collagen bioinks (collagen concentration in hydrogels of 20 and 30 mg/mL with a final concentration of 10 and 15 mg/mL in bioink) where samples flow freely in the culture medium, thus enhancing the elimination of nutrients and metabolites of cells. Cell viability, morphology, and metabolic activity (MTT tests) were analyzed on collagen hydrogels with a collagen concentration of 20 and 30 mg/mL in static culture groups without medium exchange and with active medium perfusion; the influence of pure growth culture medium and smooth muscle cells differentiation medium was next investigated. Collagen isolated from porcine skins was used; every batch was titrated to optimize the pH of the resulting collagen to minimize the difference in production batches and, therefore, the results. Active medium perfusion significantly improved cell viability and activity in the high-concentrated gel, which, to date, is the most limiting factor for using these hydrogels. In addition, based on SEM images and geometry analysis, the cells remodel collagen material to their extracellular matrix.
PubMed: 38786233
DOI: 10.3390/gels10050316 -
BioRxiv : the Preprint Server For... May 2024Generation of tissue models with serially perfused hierarchical vasculature would allow greater control of fluid perfusion throughout the network and enable direct...
Generation of tissue models with serially perfused hierarchical vasculature would allow greater control of fluid perfusion throughout the network and enable direct mechanistic investigation of vasculogenesis, angiogenesis, and vascular remodeling. In this work, we have developed a method to produce a closed, serially perfused, multiscale vessel network embedded within an acellular hydrogel. We confirmed that the acellular and cellular gel-gel interface was functionally annealed without preventing or biasing cell migration and endothelial self-assembly. Multiscale connectivity of the vessel network was validated via high-resolution microscopy techniques to confirm anastomosis between self-assembled and patterned vessels. Lastly, using fluorescently labeled microspheres, the multiscale network was serially perfused to confirm patency and barrier function. Directional flow from inlet to outlet man-dated flow through the capillary bed. This method for producing closed, multiscale vascular networks was developed with the intention of straightforward fabrication and engineering techniques so as to be a low barrier to entry for researchers who wish to investigate mechanistic questions in vascular biology. This ease of use offers a facile extension of these methods for incorporation into organoid culture, organ-on-a-chip (OOC) models, and bioprinted tissues.
PubMed: 38766003
DOI: 10.1101/2024.05.03.592474 -
European Journal of Pharmaceutical... May 2024Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human...
Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research.
PubMed: 38763450
DOI: 10.1016/j.ejps.2024.106805 -
Cell Reports Methods May 2024Organoids, self-organizing three-dimensional (3D) structures derived from stem cells, offer unique advantages for studying organ development, modeling diseases, and... (Review)
Review
Organoids, self-organizing three-dimensional (3D) structures derived from stem cells, offer unique advantages for studying organ development, modeling diseases, and screening potential therapeutics. However, their translational potential and ability to mimic complex in vivo functions are often hindered by the lack of an integrated vascular network. To address this critical limitation, bioengineering strategies are rapidly advancing to enable efficient vascularization of organoids. These methods encompass co-culturing organoids with various vascular cell types, co-culturing lineage-specific organoids with vascular organoids, co-differentiating stem cells into organ-specific and vascular lineages, using organoid-on-a-chip technology to integrate perfusable vasculature within organoids, and using 3D bioprinting to also create perfusable organoids. This review explores the field of organoid vascularization, examining the biological principles that inform bioengineering approaches. Additionally, this review envisions how the converging disciplines of stem cell biology, biomaterials, and advanced fabrication technologies will propel the creation of increasingly sophisticated organoid models, ultimately accelerating biomedical discoveries and innovations.
PubMed: 38759654
DOI: 10.1016/j.crmeth.2024.100779 -
The Journal of Biological Chemistry May 2024OMT-28 is a metabolically robust small molecule developed to mimic the structure and function of omega-3 epoxyeicosanoids. However, it remained unknown to what extent...
OMT-28 is a metabolically robust small molecule developed to mimic the structure and function of omega-3 epoxyeicosanoids. However, it remained unknown to what extent OMT-28 also shares the cardio-protective and anti-inflammatory properties of its natural counterparts. To address this question, we analyzed the ability of OMT-28 to ameliorate hypoxia/reoxygenation (HR)-injury and lipopolysaccharide (LPS)-induced endotoxemia in cultured cardiomyocytes. Moreover, we investigated the potential of OMT-28 to limit functional damage and inflammasome activation in isolated perfused mouse hearts subjected to ischemia/reperfusion (IR) injury. In the HR model, OMT-28 (1 μM) treatment largely preserved cell viability (about 75 vs. 40 % with vehicle) and mitochondrial function as indicated by the maintenance of NAD+/NADH-, ADP/ATP- and respiratory control ratios. Moreover, OMT-28 blocked the HR-induced production of mitochondrial reactive oxygen species. Pharmacological inhibition experiments suggested that Gαi, PI3K, PPARα, and Sirt1 are essential components of the OMT-28 mediated pro-survival pathway. Counteracting inflammatory injury of cardiomyocytes, OMT-28 (1 μM) reduced LPS-induced increases in TNFα protein (by about 85 % vs vehicle) and NF-κB DNA binding (by about 70 % vs. vehicle). In the ex vivo model, OMT-28 improved post-IR myocardial function recovery to reach about 40 % of the baseline value compared to less than 20 % with vehicle. Furthermore, OMT-28 (1 μM) limited IR-induced NLRP3 inflammasome activation similarly like a direct NLRP3 inhibitor (MCC950). Overall, this study demonstrates that OMT-28 possesses potent cardio-protective and anti-inflammatory properties supporting the hypothesis that extending the bioavailability of omega-3 epoxyeicosanoids may improve their prospects as therapeutic agents.
PubMed: 38754781
DOI: 10.1016/j.jbc.2024.107372 -
Cell Stem Cell May 2024Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen...
Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen receptor (CAR)-T cell field. We developed a breast cancer-on-chip model with an integrated endothelial barrier that enables the transmigration of perfused immune cells, their infiltration into the tumor, and concomitant monitoring of cytokine release during perfused culture over a period of up to 8 days. Here, we exemplified its use for investigating CAR-T cell efficacy and the ability to control the immune reaction with a pharmacological on/off switch. Additionally, we integrated primary breast cancer organoids to study patient-specific CAR-T cell efficacy. The modular architecture of our tumor-on-chip paves the way for studying the role of other cell types in the TME and thus provides the potential for broad application in bench-to-bedside translation as well as acceleration of the preclinical development of CAR-T cell products.
PubMed: 38754430
DOI: 10.1016/j.stem.2024.04.018