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Temperature-responsive hydrogel-grafted vessel-on-a-chip: Exploring cold-induced endothelial injury.Biotechnology and Bioengineering Jul 2024Cold-induced vasoconstriction is a significant contributor that leads to chilblains and hypothermia in humans. However, current animal models have limitations in...
Cold-induced vasoconstriction is a significant contributor that leads to chilblains and hypothermia in humans. However, current animal models have limitations in replicating cold-induced acral injury due to their low sensitivity to cold. Moreover, existing in vitro vascular chips composed of endothelial cells and perfusion systems lack temperature responsiveness, failing to simulate the vasoconstriction observed under cold stress. This study presents a novel approach where a microfluidic bioreactor of vessel-on-a-chip was developed by grafting the inner microchannel surface of polydimethylsiloxane with a thermosensitive hydrogel skin composed of N-isopropyl acrylamide and gelatin methacrylamide. With a lower critical solution temperature set at 30°C, the gel layer exhibited swelling at low temperatures, reducing the flow rate inside the channel by 10% when the temperature dropped from 37°C to 4°C. This well mimicked the blood stasis observed in capillary vessels in vivo. The vessel-on-a-chip was further constructed by culturing endothelial cells on the surface of the thermosensitive hydrogel layer, and a perfused medium was introduced to the cells to provide a physiological shear stress. Notably, cold stimulation of the vessel-on-a-chip led to cell necrosis, mitochondrial membrane potential (ΔΨ) collapse, cytoskeleton disaggregation, and increased levels of reactive oxygen species. In contrast, the static culture of endothelial cells showed limited response to cold exposure. By faithfully replicating cold-induced endothelial injury, this groundbreaking thermosensitive vessel-on-a-chip technology offers promising advancements in the study of cold-induced cardiovascular diseases, including pathogenesis and therapeutic drug screening.
PubMed: 38946677
DOI: 10.1002/bit.28779 -
The Journal of Heart and Lung... Jun 2024Many hearts offered for pediatric heart transplantation (HT) are not placed. In 2016, we initiated a quality improvement endeavor to increase heart offer acceptance....
BACKGROUND
Many hearts offered for pediatric heart transplantation (HT) are not placed. In 2016, we initiated a quality improvement endeavor to increase heart offer acceptance. This study assessed the effect of these interventions at our center.
METHODS
Evaluation of pre-/post-implementation cohorts (1/1/2008-12/31/2016 vs. 1/1/2017- 7/1/2023) comparing donor heart utilization. Six interventions were iterated over time to increase offer acceptance ("extended criteria"): ABO-incompatible transplant, ex vivo perfusion for distanced donors, 3-dimensional total cardiac volume (TCV) assessment, acceptance of Hepatitis-C or SARS-COV-2 infected donors, and institutional culture change favoring consideration of donors previously considered unacceptable (Public Health Service Risk, long CPR duration, etc.). Outcomes studied included annual HT volume, median waitlist duration, sequence number at acceptance, and post-transplant clinical outcomes.
RESULTS
From 1/2008-7/2023 annual transplant volume increased from 16/year to 25/year pre-/post-implementation. Three hundred-thirteen/389 (80%) listed patients were transplanted. Waitlist duration shortened post-implementation (P=0.01), as did the percentage of accepted heart offers utilizing at least one extended criterion (P<0.001). Institutional culture change and TCV assessment had the largest impact on donor heart utilization (P=0.04 &P<0.001). There was no difference in post-HT intubation or cardiovascular intensive care unit (CVICU) days (P= 0.05-0.9), though post-transplant hospitalization duration (P<0.001) increased. Post-transplant survival was unaffected by use of extended criteria hearts (P=0.3).
CONCLUSIONS
We report increased donor heart offer acceptance resulting from a longitudinal, multi-faceted effort to increase organ offer utilization, with institutional culture change and TCV assessments having the greatest impact. Use of extended criteria hearts was not associated with inferior survival.
PubMed: 38945282
DOI: 10.1016/j.healun.2024.06.015 -
Journal of Biomedical Materials... Jul 2024Despite recent advances in the field of tissue engineering, the development of complex tissue-like structures in vitro is compromised by the lack of integration of a...
Despite recent advances in the field of tissue engineering, the development of complex tissue-like structures in vitro is compromised by the lack of integration of a functioning vasculature. In this study, we propose a mesoscale three-dimensional (3D) in vitro vascularized connective tissue model and demonstrate its feasibility to prompt the self-assembly of endothelial cells into vessel-like structures. Moreover, we investigate the effect of perfusion on the organization of the cells. For this purpose, primary endothelial cells (HUVECs) and a cell line of human foreskin fibroblasts are cultivated in ECM-like matrices made up of freeze-dried collagen scaffolds permeated with collagen type I hydrogel. A tailored bioreactor is designed to investigate the effect of perfusion on self-organization of HUVECs. Immunofluorescent staining, two-photon microscopy, second-harmonic generation imaging, and scanning electron microscopy are applied to visualize the spatial arrangement of the cells. The analyses reveal the formation of hollow, vessel-like structures of HUVECs in hydrogel-permeated collagen scaffolds under both static and dynamic conditions. In conclusion, we demonstrate the feasibility of a 3D porous collagen scaffolding system that enables and maintains the self-organization of HUVECs into vessel-like structures independent of a dynamic flow.
Topics: Humans; Tissue Scaffolds; Human Umbilical Vein Endothelial Cells; Porosity; Tissue Engineering; Collagen; Fibroblasts; Hydrogels; Bioreactors
PubMed: 38923270
DOI: 10.1002/jbm.b.35444 -
Human Reproduction (Oxford, England) Jun 2024What is the pathological mechanism involved in a thin endometrium, particularly under ischaemic conditions?
Cytoskeletal and inter-cellular junction remodelling in endometrial organoids under oxygen-glucose deprivation: a new potential pathological mechanism for thin endometria.
STUDY QUESTION
What is the pathological mechanism involved in a thin endometrium, particularly under ischaemic conditions?
SUMMARY ANSWER
Endometrial dysfunction in patients with thin endometrium primarily results from remodelling in cytoskeletons and cellular junctions of endometrial epithelial cells under ischemic conditions.
WHAT IS KNOWN ALREADY
A healthy endometrium is essential for successful embryo implantation and subsequent pregnancy; ischemic conditions in a thin endometrium compromise fertility outcomes.
STUDY DESIGN, SIZE, DURATION
We recruited 10 patients with thin endometrium and 15 patients with healthy endometrium. Doppler ultrasound and immunohistochemical results confirmed the presence of insufficient endometrial blood perfusion in patients with thin endometrium. Organoids were constructed using healthy endometrial tissue and cultured under oxygen-glucose deprivation (OGD) conditions for 24 h. The morphological, transcriptomic, protein expression, and signaling pathway changes in the OGD organoids were observed. These findings were validated in both thin endometrial tissue and healthy endometrial tissue samples.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Endometrial thickness and blood flow were measured during the late follicular phase using transvaginal Doppler ultrasound. Endometrial tissue was obtained via hysteroscopy. Fresh endometrial tissues were used for the generation and culture of human endometrial organoids. Organoids were cultured in an appropriate medium and subjected to OGD to simulate ischemic conditions. Apoptosis and cell death were assessed using Annexin-V/propidium iodide staining. Immunofluorescence analysis, RNA sequencing, western blotting, simple westerns, immunohistochemistry, and electron microscopy were conducted to evaluate cellular and molecular changes.
MAIN RESULTS AND THE ROLE OF CHANCE
Patients with thin endometrium showed significantly reduced endometrial thickness and altered blood flow patterns compared to those with healthy endometrium. Immunohistochemical staining revealed fewer CD34-positive blood vessels and glands in the thin endometrium group. Organoids cultured under OGD conditions exhibited significant morphological changes, increased apoptosis, and cell death. RNA-seq identified differentially expressed genes related to cytoskeletal remodeling and stress responses. OGD induced a strong cytoskeletal reorganization, mediated by the RhoA/ROCK signaling pathway. Additionally, electron microscopy indicated compromised epithelial integrity and abnormal cell junctions in thin endometrial tissues. Upregulation of hypoxia markers (HIF-1α and HIF-2α) and activation of the RhoA/ROCK pathway were also observed in thin endometrial tissues, suggesting ischemia and hypoxia as underlying mechanisms.
LARGE SCALE DATA
none.
LIMITATIONS AND REASONS FOR CAUTION
The study was conducted in an in vitro model, which may not fully replicate the complexity of in vivo conditions.
WIDER IMPLICATIONS OF THE FINDINGS
This research provides a new three-dimensional in vitro model of thin endometrium, as well as novel insights into the pathophysiological mechanisms of endometrial ischaemia in thin endometrium, offering potential avenues for identifying therapeutic targets for treating fertility issues related to thin endometrium.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported by the National Natural Science Foundation of China (81925013); National Key Research and Development Project of China (2022YFC2702500, 2021YFC2700303, 2021YFC2700601); the Capital Health Research and Development Project (SF2022-1-4092); the National Natural Science Foundation of China (82288102, 81925013, 82225019, 82192873); Special Project on Capital Clinical Diagnosis and Treatment Technology Research and Transformation Application (Z211100002921054); the Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation(TFJC2023010001). The authors declare that no competing interests exist.
PubMed: 38915267
DOI: 10.1093/humrep/deae137 -
Molecular Biology Reports Jun 2024Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in... (Review)
Review
Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in morphology, inlining, and protecting blood vessel tissue, as well as maintaining homeostatic conditions. ECs are essential in vascularization and blood vessels formation. The differentiation process, generally carried out in 2D culture systems, were relied on growth factors induction. Therefore, an artificial extracellular matrix with relevant mechanical properties is essential to build 3D culture models. Various 3D fabrication techniques, such as hydrogel-based and fibrous scaffolds, scaffold-free, and co-culture to endothelial cells were reviewed and summarized to gain insights. The obtained MSCs-derived ECs are shown by the expression of endothelial gene markers and tubule-like structure. In order to mimicking relevant vascular tissue, 3D-bioprinting facilitates to form more complex microstructures. In addition, a microfluidic chip with adequate flow rate allows medium perfusion, providing mechanical cues like shear stress to the artificial vascular vessels.
Topics: Humans; Mesenchymal Stem Cells; Cell Differentiation; Endothelial Cells; Cell Culture Techniques, Three Dimensional; Tissue Scaffolds; Tissue Engineering; Animals; Hydrogels; Cell Culture Techniques; Coculture Techniques; Extracellular Matrix
PubMed: 38913199
DOI: 10.1007/s11033-024-09743-8 -
Resuscitation Plus Sep 2024Minimizing cardiac arrest times is critical in extracorporeal cardiopulmonary resuscitation (ECPR). Pre-primed extra corporeal membrane oxygenator (ECMO) is used for...
AIM
Minimizing cardiac arrest times is critical in extracorporeal cardiopulmonary resuscitation (ECPR). Pre-primed extra corporeal membrane oxygenator (ECMO) is used for this, but knowledge is limited to experimental studies. We prospectively investigated oxygenator function and sterility in dry plus wet pre-priming in a clinical setting.
METHODS
This prospective clinical study included 107 ECMO circuits used at Sahlgrenska University Hospital between October 2019 and December 2021. Circuits underwent dry set-up, followed by wet priming when the previous wet-primed circuit was used. Sterility was assessed by culturing the priming solution. Oxygenator function parameters, including sweep gas flow, fraction of oxygen (FiO), and oxygenator resistance, were analyzed at ECMO initiation and during treatment using linear mixed models.
RESULTS
Median total set-up time was 14 days (range 0-97), with a median wet prime time of 6 days (range 0-57). 103 of 105 circuits with culture results were negative, two showed bacterial growth (coagulase-negative staphylococci and ). Wet prime time did not significantly affect initial oxygenator function. Oxygenator resistance and FiO increased during ECMO treatment (0.035 mmHg/L min (95 % confidence interval (CI) 0.015-0.055) < 0.001, and 2.19 % (0.92-3.46) = 0.009), but these changes were unrelated to wet prime time.
CONCLUSION
Wet pre-priming of ECMO circuits for up to 57 days did not affect oxygenator function. The low incidence of bacterial growth (1.9 %) suggests that pre-primed ECMO generally maintain sterility and can facilitate rapid ECPR initiation. However, bacterial growth highlights the need for caution in non-urgent cases. Culturing the circuit at initiation can mitigate this risk.
PubMed: 38912528
DOI: 10.1016/j.resplu.2024.100680 -
Frontiers in Physiology 2024This study was undertaken to explore the potential therapeutic effects of Tongyang Huoxue Granules (TYHX) on sinoatrial node (SAN) dysfunction, a cardiac disorder...
INTRODUCTION
This study was undertaken to explore the potential therapeutic effects of Tongyang Huoxue Granules (TYHX) on sinoatrial node (SAN) dysfunction, a cardiac disorder characterized by impaired impulse generation or conduction. The research question addressed whether TYHX could positively influence SAN ion channel function, specifically targeting the sodium-calcium exchanger ( ) and L-type calcium channel ( ) of the SAN.
METHODS
Sinoatrial node cells (SANCs) were isolated and cultured from neonatal Japanese big-eared white rabbits within 24 h of birth. The study encompassed five groups: Control, H/R (hypoxia/reoxygenation), H/R+100 μg/mL TYHX, H/R+200 μg/mL TYHX, and H/R+400 μg/mL TYHX. The H/R model, simulating hypoxia/reoxygenation stress, was induced within 5 days of culture. Whole-cell patch clamp technique was employed to record currents following a 3-min perfusion and stabilization period with TYHX.
RESULTS
TYHX administration demonstrated improvements in the ignition phase of impaired SANCs. The half-maximal effective dose of TYHX, as determined by SANC beating frequency, was found to be 323.63 μg/mL. Inward current density of increased in response to TYHX (200 and 400 μg/mL), while TYHX enhanced current density in H/R SANCs, with 400 μg/mL exhibiting greater efficacy. Additionally, TYHX regulated the gating mechanisms of by right-shifting the steady-state inactivation curve and accelerating recovery from inactivation. Notably, TYHX increased the activation time constant under 200 and 400 μg/mL, prolonged the fast inactivation time constant τ1 with 400 μg/mL, and extended the slow inactivation time constant τ2 with 100 and 400 μg/mL.
DISCUSSION AND CONCLUSION
The findings suggest that TYHX may hold promise as a therapeutic intervention for sinus node dysfunction, offering potential avenues for drug development aimed at safeguarding SAN function.
PubMed: 38911325
DOI: 10.3389/fphys.2024.1402478 -
Cell Stem Cell Jun 2024The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues....
The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. In vivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2 weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.
PubMed: 38908380
DOI: 10.1016/j.stem.2024.05.011 -
Biomedicine & Pharmacotherapy =... Jun 2024Peptide-functionalized hydrogel is one of commonly used biomaterials to introduce hydrogel-induced vessel regeneration. Despite many reports about the discoveries of...
Peptide-functionalized hydrogel is one of commonly used biomaterials to introduce hydrogel-induced vessel regeneration. Despite many reports about the discoveries of high-active peptides (or ligands) for regeneration, the study on the conjugating methods for the hydrogel functionalization with peptides is limited. Here, we compared the vasculogenic efficacy of the peptide-functionalized hydrogels prepared by two commonly used conjugating methods, 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and Click methods, through cell models, organ-on-chips models, animal models, and RNA sequencing analysis. Two vascular-related cell types, the human umbilical vein endothelial cells (HUVECs) and the adipose-derived stem cells (ADSCs), have been cultured on the hydrogel surfaces prepared by EDC/Click methods. It showed that the hydrogels prepared by Click method supported the higher vasculogenic activities while the ones made by EDC method compromised the peptide activities on hydrogels. The vasculogenesis assays further revealed that hydrogels prepared by Click method promoted a better vascular network formation. In a critical ischemic hindlimb model, only the peptide-functionalized hydrogels prepared by Click method successfully salvaged the ischemic limb, significantly improved blood perfusion, and enhanced the functional recoveries (through gait analysis and animal behavior studies). RNA sequencing studies revealed that the hydrogels prepared by Click method significantly promoted the PI3K-AKT pathway activation compared to the hydrogels prepared by EDC method. All the results suggested that EDC method compromised the functions of the peptides, while Click method preserved the vascular regenerating capacities of the peptides on the hydrogels, illustrating the importance of the conjugating method during the preparation of the peptide-functionalized hydrogels.
PubMed: 38906023
DOI: 10.1016/j.biopha.2024.116959 -
Biotechnology Progress Jun 2024The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the...
The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and increasingly more common perfusion bioreactors have increased over last two decades through intensified upstream bioreactor production processes, the traditional primary clarification unit operations of centrifugation and/or microfiltration become more challenging with issues like frequent desludging, cell disruption due to shear damage and quick fouling of membranes. We have developed a novel compact cell settler device exploiting the enhanced sedimentation on inclined surfaces and demonstrated that this settler device can be adapted easily to remove and contain cells or cell clumps from the clarified supernatant collected via the top effluent of the settler. In this work, we present high product recovery results during primary clarification of mammalian cell culture supernatant using our novel single-use disposable BioSettler150 while processing about 10 L of cell culture broth within short processing times of about 4 h.
PubMed: 38898736
DOI: 10.1002/btpr.3489