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ACS Applied Materials & Interfaces Mar 2024Most in vitro models use culture medium to apply fluid shear stress to endothelial cells, which does not capture the interaction between blood and endothelial cells....
Most in vitro models use culture medium to apply fluid shear stress to endothelial cells, which does not capture the interaction between blood and endothelial cells. Here, we describe a new system to characterize whole blood flow through a 3D-printed, endothelialized vascular topology that induces flow separation at a bifurcation. Drag-reducing polymers, which have been previously studied as a potential therapy to reduce the pressure drop across the vascular bed, are evaluated for their effect on mitigating the disturbed flow. Polymer concentrations of 1000 ppm prevented recirculation and disturbed flow at the wall. Proteomic analysis of plasma collected from whole blood recirculated through the vascularized channel with and without drag-reducing polymers provides insight into the effects of flow regimes on levels of proteins indicative of the endothelial-blood interaction. The results indicate that blood flow alters proteins associated with coagulation, inflammation, and other processes. Overall, these proof-of-concept experiments demonstrate the importance of using whole blood flow to study the endothelial response to perfusion.
Topics: Polymers; Endothelial Cells; Proteomics; Hemodynamics; Printing, Three-Dimensional; Stress, Mechanical
PubMed: 38488736
DOI: 10.1021/acsami.3c17099 -
The Journal of Extra-corporeal... Mar 2024This article advocates for an open communication culture in the perfusion and cardiothoracic community to enhance patient safety during surgery. All team members,...
This article advocates for an open communication culture in the perfusion and cardiothoracic community to enhance patient safety during surgery. All team members, including nurses, anesthesiologists, and perfusionists, should actively contribute their insights. Empowering perfusionists to voice concerns without fear of repercussions is crucial. Involvement in debriefs, root cause analyses and data management systems aids continuous improvement. A robust speak-up culture prevents unsafe practices and elevates perfusion care standards, leading to better patient outcomes.
Topics: Humans; Perfusion; Patient Safety; Communication
PubMed: 38488716
DOI: 10.1051/ject/2023041 -
Chemosphere Apr 2024Microalgal-based treatment of anaerobic digestate food effluent (ADFE) has been found to be efficient and effective. However, turbidity and high total ammonia nitrogen...
Microalgal-based treatment of anaerobic digestate food effluent (ADFE) has been found to be efficient and effective. However, turbidity and high total ammonia nitrogen (TAN)) content of ADFE is a major setback, requiring significant dilution. Although the possibility of growing microalgae in a high-strength ADFE with minimal dilution has been demonstrated in suspension cultures, such effluents remain highly turbid and affect the light path in suspension cultures. Here, the feasibility of growing Chlorella sp.MUR 269 in biofilm to treat ADFE with high TAN concentrations was investigated. Six different TAN concentrations in ADFE were evaluated for their effects on biofilm growth and nutrient removal by Chlorella sp. MUR 269 using the perfused biofilm technique. Biomass yields and productivities of this alga at various TAN concentrations (mg N NH L) were 55 (108 g m and 9.80 g m d)>100 > 200 = 300 = 500 > 1000. Growth was inhibited, resulting in a 28% reduction in yield of Chlorella biofilm when this alga was grown at 1000 mg N NH L. A survey of the photosynthetic parameters reveals evidence of stress occurring in the following sequence: 55 < 100<200 < 300<1000. A significant nutrient removal was observed across various TAN concentrations. The removal pattern also followed the concentration gradients except COD, where the highest removal occurred at 500 mg N NH L. Higher removal rates were seen at higher nutrient concentrations and declined gradually over time. In general, our results indicated that the perfused biofilm strategy is efficient, minimizes water consumption, offers easy biomass harvesting, and better exposure to light. Therefore, it can be suitable for treating turbid and concentrated effluent with minimal treatment to reduce the TAN concentration.
Topics: Chlorella; Ammonia; Anaerobiosis; Food; Microalgae; Biomass; Nitrogen
PubMed: 38484996
DOI: 10.1016/j.chemosphere.2024.141688 -
STAR Protocols Jun 2024Organ-on-a-chip technologies enable the fabrication of endothelial tissues, so-called microvessels (MVs), which emulate the endothelial barrier function in healthy or...
Organ-on-a-chip technologies enable the fabrication of endothelial tissues, so-called microvessels (MVs), which emulate the endothelial barrier function in healthy or disease conditions. In this protocol, we describe the fabrication of perfusable open-chamber style MVs embedded in collagen gels. We then report a simple technology to characterize the MV barrier properties in static or under pressure based on fluorescence confocal imaging. Finally, we provide quantification techniques that enable us to infer the structure of MV paracellular pores. For complete details on the use and execution of this protocol, please refer to Cacheux et al..
Topics: Humans; Human Umbilical Vein Endothelial Cells; Microvessels; Lab-On-A-Chip Devices; Microscopy, Confocal
PubMed: 38483899
DOI: 10.1016/j.xpro.2024.102950 -
BioRxiv : the Preprint Server For... Feb 2024The precise molecular drivers of abdominal aortic aneurysm (AAA) remain unclear. Thymidine phosphorylase (TYMP) contributes to increased platelet activation, thrombosis,...
AIMS
The precise molecular drivers of abdominal aortic aneurysm (AAA) remain unclear. Thymidine phosphorylase (TYMP) contributes to increased platelet activation, thrombosis, and inflammation, all of which are key factors in AAA development. Additionally, TYMP suppresses the proliferation of vascular smooth muscle cells (VSMCs), which are central to the development and progression of AAA. We hypothesize that TYMP plays a key role in AAA development.
METHODS AND RESULTS
We conducted a histological study using human AAA samples and normal abdominal aortas, revealing heightened levels of TYMP in human AAA vessel walls. To validate this observation, we utilized an Ang II perfusion-induced AAA model in wild-type C57BL/6J (WT) and mice, feeding them a Western diet (TD.88137) starting from 4 weeks of age. We found that mice were protected from Ang II perfusion-induced AAA formation. Furthermore, by using TYMP-expressing VSMCs as well as primarily cultured VSMCs from WT and mice, we elucidated the essential role of TYMP in regulating MMP2 expression and activation. TYMP deficiency or inhibition by tipiracil, a selective TYMP inhibitor, led to reduced MMP2 production, release, and activation in VSMCs. Additionally, TYMP was found to promote pro-inflammatory cytokine expression systemically, and its absence attenuates TNF-α-stimulated activation of MMP2 and AKT. By co-culturing VSMCs and platelets, we observed that TYMP-deficient platelets had a reduced inhibitory effect on VSMC proliferation compared to WT platelets. Moreover, TYMP appeared to enhance the expression of activated TGFβ1 in cultured VSMCs in vitro and in human AAA vessel walls in vivo. TYMP also boosted the activation of thrombospondin-1 type 1 repeat domain-enhanced TGFβ1 signaling, resulting in increased connective tissue growth factor production.
CONCLUSION
Our findings collectively demonstrated that TYMP serves as a novel regulatory force in vascular biology, exerting influence over VSMC functionality and inflammatory responses that promote the development of AAA.
PubMed: 38464026
DOI: 10.1101/2024.02.27.582208 -
Journal of Cellular and Molecular... Mar 2024Senescent kidney can lead to the maladaptive repairment and predispose age-related kidney diseases. Here, we explore the renal anti-senescence effect of a known kind of...
Senescent kidney can lead to the maladaptive repairment and predispose age-related kidney diseases. Here, we explore the renal anti-senescence effect of a known kind of drug, sodium-dependent glucose transporters 2 inhibitor (SGLT2i). After 4 months intragastrically administration with dapagliflozin on senescence-accelerated mouse prone 8 (SAMP8) strain mice, the physiologically effects (lowering urine protein, enhancing glomerular blood perfusion, inhibiting expression of senescence-related biomarkers) and structural changes (improving kidney atrophy, alleviating fibrosis, decreasing glomerular mesangial proliferation) indicate the potential value of delaying kidney senescence of SGLT2i. Senescent human proximal tubular epithelial (HK-2) cells induced by H O also exhibit lower senescent markers after dapagliflozin treatment. Further mechanism exploration suggests LTBP2 have the great possibility to be the target for SGLT2i to exert its renal anti-senescence role. Dapagliflozin down-regulate the LTBP2 expression in kidney tissues and HK-2 cells with senescent phenotypes. Immunofluorescence staining show SGLT2 and LTBP2 exist colocalization, and protein-docking analysis implies there is salt-bridge formation between them; these all indicate the possibility of weak-interaction between the two proteins. Apart from reducing LTBP2 expression in intracellular area induced by H O , dapagliflozin also decrease the concentration of LTBP2 in cell culture medium. Together, these results reveal dapagliflozin can delay natural kidney senescence in non-diabetes environment; the mechanism may be through regulating the role of LTBP2.
Topics: Mice; Humans; Animals; Sodium-Glucose Transporter 2 Inhibitors; Kidney; Glucosides; Benzhydryl Compounds; Kidney Diseases; Latent TGF-beta Binding Proteins
PubMed: 38454800
DOI: 10.1111/jcmm.18176 -
Biofabrication Mar 2024As bioprinting advances into clinical relevance with patient-specific tissue and organ constructs, it must be capable of multi-material fabrication at high resolutions...
As bioprinting advances into clinical relevance with patient-specific tissue and organ constructs, it must be capable of multi-material fabrication at high resolutions to accurately mimick the complex tissue structures found in the body. One of the most fundamental structures to regenerative medicine is microvasculature. Its continuous hierarchical branching vessel networks bridge surgically manipulatable arteries (∼1-6 mm) to capillary beds (∼10m). Microvascular perfusion must be established quickly for autologous, allogeneic, or tissue engineered grafts to survive implantation and heal in place. However, traditional syringe-based bioprinting techniques have struggled to produce perfusable constructs with hierarchical branching at the resolution of the arterioles (∼100-10m) found in microvascular tissues. This study introduces the novel CEVIC bioprinting device (i.e.ontinuouslyxtrudedariablenternalhanneling), a multi-material technology that breaks the current extrusion-based bioprinting paradigm of pushing cell-laden hydrogels through a nozzle as filaments, instead, in the version explored here, extruding thin, wide cell-laden hydrogel sheets. The CEVIC device adapts the chaotic printing approach to control the width and number of microchannels within the construct as it is extruded (i.e. on-the-fly). Utilizing novel flow valve designs, this strategy can produce continuous gradients varying geometry and materials across the construct and hierarchical branching channels with average widths ranging from 621.5 ± 42.92%m to 11.67 ± 14.99%m, respectively, encompassing the resolution range of microvascular vessels. These constructs can also include fugitive/sacrificial ink that vacates to leave demonstrably perfusable channels. In a proof-of-concept experiment, a co-culture of two microvascular cell types, endothelial cells and pericytes, sustained over 90% viability throughout 1 week in microchannels within CEVIC-produced gelatin methacryloyl-sodium alginate hydrogel constructs. These results justify further exploration of generating CEVIC-bioprinted microvasculature, such as pre-culturing and implantation studies.
Topics: Humans; Endothelial Cells; Bioprinting; Tissue Engineering; Hydrogels; Printing, Three-Dimensional; Tissue Scaffolds
PubMed: 38447217
DOI: 10.1088/1758-5090/ad30c8 -
Engineering in Life Sciences Mar 2024Process Analytical Technologies (PATs) are taking a key role in the run for automatization in the biopharmaceutical industry. Spectroscopic methods such as Raman...
Process Analytical Technologies (PATs) are taking a key role in the run for automatization in the biopharmaceutical industry. Spectroscopic methods such as Raman spectroscopy or mid-infrared (MIR) spectroscopy are getting more recognition in the recent years for inline monitoring of bioprocesses due to their ability to measure various molecules simultaneously. However, their dependency on laborious model calibration making them a challenge to implement. In this study, a novel one-point calibration that requires a single reference point prior to the inline monitoring of glucose and lactate in bioprocesses with MIR spectroscopy is assessed with 22 mammalian cell perfusion (PER) processes in two different scales and four different products. Concentrations are predicted over all PERs runs with a root mean square error (RMSE) of 0.29 g/L for glucose and 0.24 g/L for lactate, respectively. For comparison conventional partial least square regression (PLSR) models were used and trained with spectroscopic data from six bioreactor runs in two different scales and three products. The general accuracy of those models (RMSE of 0.41 g/L for glucose and 0.16 g/L for lactate) are in the range of the accuracy of the one-point calibration. This shows the potential of the one-point calibration as an approach making spectroscopy more accessible for bioprocess development.
PubMed: 38444619
DOI: 10.1002/elsc.202300237 -
Molecular Therapy. Methods & Clinical... Mar 2024Continuous manufacturing of lentiviral vectors (LVs) using stable producer cell lines could extend production periods, improve batch-to-batch reproducibility, and...
Continuous manufacturing of lentiviral vectors (LVs) using stable producer cell lines could extend production periods, improve batch-to-batch reproducibility, and eliminate costly plasmid DNA and transfection reagents. A continuous process was established by expanding cells constitutively expressing third-generation LVs in the iCELLis Nano fixed-bed bioreactor. Fixed-bed bioreactors provide scalable expansion of adherent cells and enable a straightforward transition from traditional surface-based culture vessels. At 0.5 vessel volume per day (VVD), the short half-life of LVs resulted in a low total infectious titer at 1.36 × 10 TU cm. Higher perfusion rates increased titers, peaking at 7.87 × 10 TU cm at 1.5 VVD. The supernatant at 0.5 VVD had a physical-to-infectious particle ratio of 659, whereas this was 166 ± 15 at 1, 1.5, and 2 VVD. Reducing the pH from 7.20 to 6.85 at 1.5 VVD improved the total infectious yield to 9.10 × 10 TU cm. Three independent runs at 1.5 VVD and a culture pH of 6.85 showed low batch-to-batch variability, with a coefficient of variation of 6.4% and 10.0% for total infectious and physical LV yield, respectively. This study demonstrated the manufacture of high-quality LV supernatant using a stable producer cell line that does not require induction.
PubMed: 38435128
DOI: 10.1016/j.omtm.2024.101209 -
Biomicrofluidics Mar 2024The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional model of the...
The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional model of the heart wall, are a useful tool to study cardiac physiology and pathology. However, cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow , whilst models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media, which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner, we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques, prevents flow blockage issues, and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids, resulting in large fluctuations in this metabolite under static culture. Despite this, neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact, RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together, we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems.
PubMed: 38434908
DOI: 10.1063/5.0169143