Cellular and molecular bioengineering最新文献

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.].

Purpose: Optogenetics is an invaluable tool to study brain circuits, but typical systems rely on tethered approaches to deliver light to the brain that hinder natural behavior. With the increasing prevalence of complex behavioral phenotyping in neuroscience experiments, wireless devices for optical stimulation offer great promise to overcome these limitations.

Methods: In this work we critically review recent systems engineering and device design approaches to deliver light to the brain with wireless operation for optogenetic experiments.

Results: We describe strategies used for wireless control and communication, wireless power transfer, and light delivery to the brain with a focus on device integration for in vivo operation in freely behaving mice.

Conclusion: Recent advances in optoelectronic systems, material science, and microtechnology have enabled the design and realization of miniaturized wirelessly-controlled optical stimulators for true untethered experiments in rodent models.

Introduction: Colorectal cancer (CRC) is a major cause of cancer related deaths in the United States, with CRC metastasis to the liver being a common occurrence. The development of an optimal metastatic environment is essential process prior to tumor metastasis. This process, called pre-metastatic niche (PMN) formation, involves activation of key resident liver cells, including fibroblast-like stellate cells and macrophages such as Kupffer cells. Tumor-mediated factors introduced to this environment transform resident cells that secrete additional growth factors and remodel the extracellular matrix (ECM), which is thought to promote tumor colonization and metastasis in the secondary environment.

Methods: To investigate the underlying mechanisms of these dynamics, we developed a multicellular computational model to characterize the spatiotemporal dynamics of the PMN formation in tissue. This modeling framework integrates intracellular and extracellular signaling, and traction and junctional forces into a Cellular Potts model, and represents multiple cell types with varying levels of cellular activation. We perform numerical experiments to investigate the role of key factors in PMN formation and tumor invasiveness, including growth factor concentration, timing of tumor arrival, relative composition of resident cells, and the size of invading tumor cluster.

Results: These parameter studies identified growth factor availability and ECM concentration in the environment as two of the key determinants of tumor invasiveness. We further predict that both the ECM concentration potential and growth factor sensitivity of the stellate cells are key drivers of the PMN formation and associated ECM concentration.

Conclusions: Overall, this modeling framework represents a significant step towards simulating cancer metastasis and investigating the role of key factors on PMN formation.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00831-0.

Introduction: Progressive aging, or senescence, of mesenchymal stem/stromal cells (MSCs) is a major obstacle faced when trying to culture potent stem cells for use in therapy. Senescent cells are irreversibly nondividing cells that cease performing critical functional effects. Elimination of senescent cells using biochemical means, such as the use of senolytic drugs like dasatinib, may be useful in retaining the viable and proliferating populations of the cells.

Methods: An in vitro approach was used to investigate the effect of dasatinib on phenotypic, genotypic, and immunomodulatory functionality of osteogenic and adipogenic differentiated MSCs. Replicative senescence was achieved through multiple sub-culturing in vitro, then senescent and non-senescent cultures were treated with a standard dosage of dasatinib. MSCs were then differentiated into osteogenic, adipogenic or chondrogenic cultures using conditioned media to be tested for the three criteria being investigated.

Results: Significant changes were observed in these criteria, indicated by evidence gathered from proliferation and indoleamine 2,3 dioxygenase activity assays. Phenotypic results of dasatinib were shown to reduce the population of senescent MSCs while allowing non-senescent MSCs to continue differentiating and proliferating without interference from senescent cells. Genotypic results showed no change to upregulation in markers associated with osteogenic and adipogenic cells when exposed to dasatinib. Indoleamine Dioxygenase activity showed insignificant differences in cells exposed to dasatinib versus control groups, providing evidence against compromised cellular immune function.

Conclusion: This investigation provides insight into how dasatinib effects MSCs functional ability and provides a better understanding of the function of senolytic agents.