Cellular and molecular bioengineering最新文献

Purpose: The purpose of this study was to boost the therapeutic effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) by overexpressing the gene TSG-6 through CRISPR activation, and assess the biological activity of EVs from these modified MSCs in vitro on human intervertebral disc (IVD) cells.

Methods: An immortalized human MSC line was transduced with a CRISPR activation lentivirus system targeting TSG-6. MSC-EVs were harvested by ultracentrifugation and particle number/size distribution was determined by nanoparticle tracking analysis. The efficiency of transduction activation was assessed by analyzing gene and protein expression. EV proteomic contents were analyzed by mass spectrometry. Human IVD cells from patients undergoing spinal surgery were isolated, expanded, exposed to IL-1β pre-stimulation and co-treated with MSC-EVs.

Results: MSC-EVs presented size distribution, morphology, and molecular markers consistent with common EV characteristics. The expression level of TSG-6 was significantly higher (> 800 fold) in transduced MSCs relative to controls. Protein analysis of MSCs and EVs showed higher protein expression of TSG-6 in CRISPR activated samples than controls. Proteomics of EVs identified 35 proteins (including TSG-6) that were differentially expressed in TSG-6 activated EVs vs control EVs. EV co-Treatment of IL-1β pre-Stimulated IVD cells resulted in a significant downregulation of IL-8 and COX-2.

Conclusions: We successfully generated an MSC line overexpressing TSG-6. Furthermore, we show that EVs isolated from these modified MSCs have the potential to attenuate the pro-inflammatory gene expression in IVD cells. This genomic engineering approach hence holds promise for boosting the therapeutic effects of EVs.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00843-4.

Purpose: Obesity is associated with poor lymphatic solute drainage. It is unclear whether the chronic inflammation, hypoxia, and hyperlipidemia that are together associated with obesity cause impaired drainage function, and if so, whether these conditions act directly on lymphatic endothelial cells (LECs) or are indirectly mediated by the mechanical properties or cellular composition of the surrounding tissue.

Methods: We engineered blind-ended lymphatic vessels in type I collagen gels and simulated the obese microenvironment with a cocktail of tumor necrosis factor (TNF)-α, cobalt chloride (CoCl₂), and oleate, which model inflammation, hypoxia, and hyperlipidemia, respectively. We compared the solute drainage rate and leakage of lymphatics that were exposed to simulated obesity or not. We performed similar assays with lymphatics in stiffened gels, in adipocyte-laden gels, or in the presence of conditioned medium (CM) from adipose cells treated with the same cocktail.

Results: Lymphatics that were exposed to simulated obesity exhibited more gaps in endothelial junctions, leaked more solute, and drained solute less quickly than control lymphatics did, regardless of matrix stiffness. CM from adipose cells that were exposed to simulated obesity did not affect lymphatics. Lymphatics in adipocyte-laden gels did not exhibit worse drainage function when exposed to simulated obesity.

Conclusions: The combination of obesity-associated inflammation, hypoxia, and hyperlipidemia impairs lymphatic solute drainage and does so by acting directly on LECs. Surprisingly, adipocytes may play a protective role in preventing obesity-associated conditions from impairing lymphatic solute drainage.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00840-z.

Purpose: Cervical and endometrial cancers pose significant challenges in women's healthcare due to their high mortality rates and limited treatment options. High throughput screening (HTS) of cervical and endometrial cancer in vitro models offers a promising avenue for drug repurposing and broadening patient treatment options. Traditional two-dimensional (2D) cell-based screenings have limited capabilities to capture crucial multicellular interactions, that are improved upon in three dimensional (3D) multicellular tissue engineered models. However, manual fabrication of the 3D platforms is both time consuming and subject to variability. Thus, the goal of this study was to utilize automated cell dispensing to fabricate 3D cell-based HTS platforms using the HP D100 Single Cell Dispenser to dispense cervical and endometrial cancer cells.

Methods: We evaluated the effects of automated dispensing of the cancer cell lines by tuning the dispensing protocol to align with cell size measured in solution and the minimum cell number for acceptable cell viability and proliferation. We modified our previously reported coculture models of cervical and endometrial cancer to be in a 384 well plate format and measured microvessel length and cancer cell invasion.

Results: Automatically and manually dispensed cells were directly compared revealing minimal differences between the dispensing methods. These findings suggest that automated dispensing of cancer cells minimally affects cell behavior and can be deployed to decrease in vitro model fabrication time.

Conclusions: By streamlining the manufacturing process, automated dispensing holds promise for enhancing efficiency and scalability of 3D in vitro HTS platforms, ultimately contributing to advancement in cancer research and treatment.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00841-y.

Purpose: We previously demonstrated that hyperosmotic stress, which acts as mechanical stress, induces autophagy of tubular epithelial cells. This study aims to elucidate the molecular mechanisms of hyperosmolarity-induced autophagy. The research question addresses how hyperosmotic stress activates autophagy through transcription factor EB (TFEB) and Ca²⁺ signaling pathways, contributing to understanding cellular responses to mechanical stress.

Methods: NRK-52E normal rat kidney cells were subjected to hyperosmotic stress using mannitol-containing medium. Fluorescence microscopy was utilized to observe TFEB nuclear translocation, a crucial event in autophagy regulation. An intracellular Ca²⁺ chelator, BAPTA-AM, and a calcineurin inhibitor were used to dissect the Ca²⁺ signaling pathway involved in TFEB translocation. The phosphorylation of p70S6K, a substrate of the mammalian target of rapamycin complex 1 kinase, was analyzed to explore its role in TFEB localization. Additionally, the function of transient receptor potential mucolipin 1 (TRPML1), an intracellular Ca²⁺ channel, was assessed using pharmacological inhibition to determine its impact on TFEB translocation and autophagy marker LC3-II levels.

Results: Mannitol-induced hyperosmotic stress promoted the nuclear translocation of TFEB, which was completely abolished by treatment with BAPTA-AM. Inhibition of calcineurin suppressed TFEB nuclear translocation under hyperosmolarity, indicating that a signaling pathway governed by intracellular Ca²⁺ is involved in TFEB's nuclear translocation. In contrast, hyperosmotic stress did not significantly alter p70S6K phosphorylation. Pharmacological inhibition of TRPML1 attenuated both TFEB nuclear translocation and LC3-II upregulation in response to hyperosmotic stress.

Conclusions: Hyperosmotic stress promotes TFEB nuclear localization, and TRPML1-induced activation of calcineurin is involved in the mechanism of hyperosmolarity-induced autophagy.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00839-6.

Purpose: Altered tissue mechanics is a prominent feature of many pathological conditions including cancer. As such, much work has been dedicated to understanding how mechanical features of tissues contribute to pathogenesis. Interestingly, previous work has demonstrated that the tumor vasculature acquires pathological features in part due to enhanced tumor stiffening. To further understand how matrix mechanics may be translated into altered cell behavior and ultimately affect tumor vasculature function, we have investigated the effects of substrate stiffening on endothelial epigenetics. Specifically, we have focused on DNA methylation as recent work indicates DNA methylation in endothelial cells can contribute to aberrant behavior in a range of pathological conditions.

Methods: Human umbilical vein endothelial cells (HUVECs) were seeded on stiff and compliant collagen-coated polyacrylamide gels and allowed to form monolayers over 5 days. DNA methylation was assessed via 5-methylcytosine ELISA assays and immunofluorescent staining. Gene expression was assessed via qPCR on RNA isolated from HUVECs seeded on collagen-coated polyacrylamide gels of varying stiffness.

Results: Our work demonstrates that endothelial cells cultured on stiffer substrates exhibit lower levels of global DNA methylation relative to endothelial cells cultured on more compliant substrates. Interestingly, gene expression and DNA methylation dynamics suggest stiffness-mediated gene expression may play a role in establishing or maintaining differential DNA methylation levels in addition to enzyme activity. Additionally, we found that the process of passaging induced higher levels of global DNA methylation.

Conclusions: Altogether, our results underscore the importance of considering cell culture substrate mechanics to preserve the epigenetic integrity of primary cells and obtain analyses that recapitulate the primary environment. Furthermore, these results serve as an important launching point for further work studying the intersection tissue mechanics and epigenetics under pathological conditions.

Purpose: Cytoskeletal protein ensembles exhibit emergent mechanics where behavior in teams is not necessarily the sum of the components' single molecule properties. In addition, filaments may act as force sensors that distribute feedback and influence motor protein behavior. To understand the design principles of such emergent mechanics, we developed an approach utilizing QCM-D to measure how actomyosin bundles respond mechanically to environmental variables that alter constituent myosin II motor behavior.

Methods: QCM-D is used for the first time to probe alterations in actin-myosin bundle viscoelasticity due to changes in skeletal myosin II concentration and motor nucleotide state. Actomyosin bundles were constructed on a gold QCM-D sensor using a microfluidic setup, and frequency and dissipation change measurements were recorded for each component addition to decipher which assay constituents lead to changes in bundle structural compliancy.

Results: Lowering myosin concentration is detected as lower shifts in frequency and dissipation, while the relative changes in frequency and dissipation shifts for both the first and second actin additions are relatively similar. Strikingly, buffer washes with different nucleotides (ATP vs. ADP) yielded unique signatures in frequency and dissipation shifts. As myosin II's ADP-bound state tightly binds actin filaments, we observe an increase in frequency and decrease in dissipation change, indicating a decrease in viscoelasticity, likely due to myosin's increased affinity for actin, conversion from an active motor to a static crosslinker, and ability to recruit additional actin filaments from the surface, making an overall more rigid sensor coating. However, lowering the ADP concentration results in increased system compliancy, indicating that transient crosslinking and retaining a balance of motor activity perhaps results in a more cooperative and productive force generating system.

Conclusions: QCM-D can detect changes in actomyosin viscoelasticity due to molecular-level alterations, such as motor concentration and nucleotide state. These results provide support for actin's role as a mechanical force-feedback sensor and demonstrate a new approach for deciphering the feedback mechanisms that drive emergent cytoskeletal ensemble crosstalk and intracellular mechanosensing. This approach can be adapted to investigate environmental influences on more complex cytoskeletal ensemble mechanics, including addition of other motors, crosslinkers, and filament types.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00835-w.

Introduction: The interaction between endothelial cells can regulate hemostasis, vasodilation, as well as immune and inflammatory responses. Excessive loading on the endothelial cells leads to endothelial damage and endothelial barrier dysfunction. Understanding and mastering the dynamic nature of cell-cell rupture plays a crucial role in exploring the practical applications related to tumor destruction, vascular remodeling, and drug delivery by employing cavitation-induced damage to soft tissues.

Methods: To investigate the damage mechanisms of endothelial cellular networks under ultrasound cavitation, we developed a model of junction rupture in cellular networks based on the assumption that the process of intercellular rupture is irreversible when ultrasound-mediated forces exceed the damage threshold, whereas intercellular junctions have reversible behavior before rupture. Simulations using the strain accumulation method show that stress and strain exhibit complex nonlinear dynamic behavior. Ultrasonic cavitation damage was tested and evaluated on human umbilical vein endothelial cells.

Results: The results indicated that the cellular network damage was positively correlated with force amplitude and pulse frequency and was negatively correlated with driving frequency. The time lag and the internal force of cellular junctions have an important influence on the resistance to damage of the cellular network due to external forces. The damage experiment based on ultrasonic cavitation confirmed the effectiveness of the proposed model.

Conclusions: The model provided a platform for understanding the damage mechanism of endothelial tissues and ultimately improving options for their prevention and treatment.

[This corrects the article DOI: 10.1007/s12195-024-00830-1.].