An Ovarian Bioreactor for In Vitro Culture of the Whole Bovine Ovary: a Preliminary Report

Background: Improved cancer therapeutics and enhanced cancer survivorship have emphasized the severe long-term side effects of chemotherapy. Specifically, studies have linked many chemotherapy agents with primary ovarian insufficiency, although an exact insult model has not yet been determined. To investigate and ultimately solve this problem, a novel device for extended study of mammalian ovaries in vitro was developed. Methods: A bioreactor was fabricated for bovine ovarian culture that provides intravascular delivery of media to the ovary through isolation and cannulation of a main ovarian artery branch. Whole ovaries were cultured in vitro using three methods: (1) continuously supplied fresh culture media, (2) recirculated culture media, or (3) continuously supplied fresh culture media supplemented with 500 nM doxorubicin for 24 or 48 h. TUNEL assay was used to assess apoptotic cell percentages in the three groups as compared to uncultured baseline ovaries. Results: The ovary culture method was shown to maintain cell viability by effectively delivering nutrient-enriched pH-balanced media at a constant flow rate. Lower apoptosis observed in ovaries cultured in continuously supplied fresh culture media illustrates that this culture device and method are the first to sustain whole bovine ovary viability for 48 h. Meanwhile, the increase in the percentage of cell apoptosis with doxorubicin treatment indicates that the device can provide an alternative model for testing chemotherapy and chemoprotection treatments to prevent primary ovarian insufficiency in cancer patients. Conclusions: An ovarian bioreactor with consistent culture media flow through an ovarian vasculature-assisted approach maintains short-term whole bovine ovary viability

An Ovarian Bioreactor for In Vitro Culture of the Whole Bovine Ovary: a Preliminary Report

Background: Improved cancer therapeutics and enhanced cancer survivorship have emphasized the severe long-term side effects of chemotherapy. Specifically, studies have linked many chemotherapy agents with primary ovarian insufficiency, although an exact insult model has not yet been determined. To investigate and ultimately solve this problem, a novel device for extended study of mammalian ovaries in vitro was developed. Methods: A bioreactor was fabricated for bovine ovarian culture that provides intravascular delivery of media to the ovary through isolation and cannulation of a main ovarian artery branch. Whole ovaries were cultured in vitro using three methods: (1) continuously supplied fresh culture media, (2) recirculated culture media, or (3) continuously supplied fresh culture media supplemented with 500 nM doxorubicin for 24 or 48 h. TUNEL assay was used to assess apoptotic cell percentages in the three groups as compared to uncultured baseline ovaries. Results: The ovary culture method was shown to maintain cell viability by effectively delivering nutrient-enriched pH-balanced media at a constant flow rate. Lower apoptosis observed in ovaries cultured in continuously supplied fresh culture media illustrates that this culture device and method are the first to sustain whole bovine ovary viability for 48 h. Meanwhile, the increase in the percentage of cell apoptosis with doxorubicin treatment indicates that the device can provide an alternative model for testing chemotherapy and chemoprotection treatments to prevent primary ovarian insufficiency in cancer patients. Conclusions: An ovarian bioreactor with consistent culture media flow through an ovarian vasculature-assisted approach maintains short-term whole bovine ovary viability

Bioenergetic regulation of metastatic cell migration and tumor angiogenesis by matrix mechanics

278 pagesCell migration during many fundamental biological processes including metastasis and angiogenesis requires cells to traverse tissue with heterogeneous biophysical cues. During the invasion-metastasis cascade, cancer cells must navigate a structurally and mechanically complex microenvironment that significantly impacts behavior and directs migrating cells. While significant research has been performed to understand the cellular and molecular mechanisms guiding migration, less is understood about bioenergetic regulation and metabolism during migration. Here, I utilize in vitro models of the tumor-associated matrix to study the effect of mechanical cues on energetic costs associated with migration and their influence on motility. In high density collagen, where migration is impaired, intracellular bioenergetics increased and energy state decreased in aligned collagen matrices, where migration is facilitated. Motility in confined collagen microtracks impose high energetic demands on migrating cells and cells migrate in the direction that minimizes energetic costs. The pro-invasive cues collagen fiber alignment and fiber tension were next decoupled to study their individual impact on migration. Applying tension perpendicular to fiber alignment increases potential energy stored within collagen fibers, lowering requirements for cell-induced matrix deformation and energy usage during migration compared to motility in the direction of fiber alignment. Collagen density and pore size were then altered to change the level of physical constraint on migrating cells and individual cells were sorted based on their level of migration. The metabolic activity of each gel reflected the number of motile cells present and energetics were only a function of matrix properties for highly motile cells, not cells with low motility. Conditions most permissive to migration required less energy usage during movement, indicating efficient migration facilitates increased motility. Energy costs associated with migration were also demonstrated to play a role in determining endothelial cell phenotype during angiogenic sprouting. Tip cell lifetime decreased with increasing collagen density, as tip cells required more energy compared to stalk cells and this difference increased with increasing density. Together, this work provides a conceptual understanding of how mechanical cues influence bioenergetics during migration and demonstrates energy minimalization directs migration.2023-06-0

Fiber alignment drives changes in architectural and mechanical features in collagen matrices.

Aligned collagen architecture is a characteristic feature of the tumor extracellular matrix (ECM) and has been shown to facilitate cancer metastasis using 3D in vitro models. Additional features of the ECM, such as pore size and stiffness, have also been shown to influence cellular behavior and are implicated in cancer progression. While there are several methods to produce aligned matrices to study the effect on cell behavior in vitro, it is unclear how the alignment itself may alter these other important features of the matrix. In this study, we have generated aligned collagen matrices and characterized their pore sizes and mechanical properties at the micro- and macro-scale. Our results indicate that collagen alignment can alter pore-size of matrices depending on the polymerization temperature of the collagen. Furthermore, alignment does not affect the macro-scale stiffness but alters the micro-scale stiffness in a temperature independent manner. Overall, these results describe the manifestation of confounding variables that arise due to alignment and the importance of fully characterizing biomaterials at both micro- and macro-scales

Simvastatin Ameliorates Matrix Stiffness-Mediated Endothelial Monolayer Disruption.

Arterial stiffening accompanies both aging and atherosclerosis, and age-related stiffening of the arterial intima increases RhoA activity and cell contractility contributing to increased endothelium permeability. Notably, statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors whose pleiotropic effects include disrupting small GTPase activity; therefore, we hypothesized the statin simvastatin could be used to attenuate RhoA activity and inhibit the deleterious effects of increased age-related matrix stiffness on endothelial barrier function. Using polyacrylamide gels with stiffnesses of 2.5, 5, and 10 kPa to mimic the physiological stiffness of young and aged arteries, endothelial cells were grown to confluence and treated with simvastatin. Our data indicate that RhoA and phosphorylated myosin light chain activity increase with matrix stiffness but are attenuated when treated with the statin. Increases in cell contractility, cell-cell junction size, and indirect measurements of intercellular tension that increase with matrix stiffness, and are correlated with matrix stiffness-dependent increases in monolayer permeability, also decrease with statin treatment. Furthermore, we report that simvastatin increases activated Rac1 levels that contribute to endothelial barrier enhancing cytoskeletal reorganization. Simvastatin, which is prescribed clinically due to its ability to lower cholesterol, alters the endothelial cell response to increased matrix stiffness to restore endothelial monolayer barrier function, and therefore, presents a possible therapeutic intervention to prevent atherogenesis initiated by age-related arterial stiffening

Simvastatin decreases endothelial cell focal adhesion length and area.

<p>Endothelial cell focal adhesions increase in length and area with matrix stiffness but decrease with 1 μM simvastatin treatment demonstrating that statins alter cell-matrix interactions. (A) Representative images of vinculin stained focal adhesions in individual endothelial cells with increasing matrix stiffness and 1 μM simvastatin treatment. Inset shows individual vinculin stained focal adhesions. (B) Focal adhesion (FA) length and (C) area increase with substrate stiffness but decrease with the statin treatment (n = 3, 38–56 cells per condition). Data are presented as means ± standard error of the mean,**p<0.01, ***p<0.001.</p

Simvastatin alters actin organization, cell morphology, and Rac1 activity in endothelial monolayers.

<p>Cytoskeletal organization and Rac1 activity in endothelial monolayers is altered by simvastatin treatment. (A) Representative images of endothelial monolayers showing prominent actin stress fibers in control cells and a barrier enhancing cortical actin ring that forms with increasing simvastatin concentration. Cortactin changes from puncta to organized linear segments around the cell periphery and localizes with actin with increasing statin treatment. (B) Cortactin organization, measured by quantifying linear segments at cell-cell junctions, increases with simvastatin (n = 3, 30 fields of view per condition). (C) Endothelial cells adopt an elongated morphology as actin stress fibers diminish with increasing simvastatin treatment. Cell circularity, where a perfectly circular cell has a value of 1, decreases with increasing simvastatin concentration (n = 3, 50–54 cells per condition). (D) Rac1-GTP activity normalized to total protein of lysate increases across all stiffness levels with simvastatin treatment (n = 5, performed in duplicate or triplicate). (E) Representative Western blot probing for total cellular Rac1 expression and alpha tubulin (α-tub) loading control. (F) Quantification of total Rac1 normalized to alpha tubulin loading control demonstrating no significant change in expression with stiffness or statin treatment (n = 5). Data are presented as means ± standard error of the mean, ***p<0.001.</p

Simvastatin attenuates matrix stiffness-mediated traction forces.

<p>Cellular traction forces in endothelial cells increase with matrix stiffness but are attenuated with 1 μM simvastatin treatment. (A) Representative traction stress maps for endothelial cells at each level of matrix stiffness with and without simvastatin treatment. Inset shows corresponding phase image. (B) Cell contractility of endothelial cells increases with substrate stiffness but is attenuated by simvastatin (n = 3, 50–80 cells per condition). Data are presented as means ± standard error of the mean,*p<0.05, **p<0.01.</p

Simvastatin reduces vinculin localization at cell-cell adhesions in endothelial monolayers.

<p>Endothelial monolayers treated with control or 1 μM simvastatin are stained for vinculin and VE-cadherin, and the vinculin volume overlapping with VE-cadherin is quantified. Representative images of endothelial cell-cell junctions within a confluent monolayer fluorescently stained for vinculin and VE-cadherin on a 10 kPa polyacrylamide gel after 24 hour (A) control or (B) 1 μM simvastatin treatment demonstrating vinculin positive and vinculin negative junctions, respectively. (C) Vinculin localization per monolayer at cell-cell adhesions, a readout of intercellular junction tension, is quantified and increases with matrix stiffness but is significantly decreased with the statin treatment at higher matrix stiffnesses (n = 3, 70–90 fields of view per condition). Data are presented as means ± standard error of the mean, ###p<0.001 compared to matrix stiffness, *p<0.05 compared to the untreated control.</p