Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes

Investigating the Role of Mechanical Forces in the Catheter-Related Pathogenesis of Staphylococci, From Adhesion to Biofilm Formation

Intravenous catheter related blood stream infections (CRBSI) are the major cause of healthcare-associated infections to date, and result in both increased morbidity and mortality in patients with undeveloped and compromised immunity, as well as a significant cost burden on health systems. Staphylococcus epidermidis and S. aureus are both normal inhabitants of human skin and mucous membranes and also are the organisms most significantly cultured from these infections. CRBSIs from Staphylococci can be extremely harmful if left untreated even for a mater of a few days. These infections can result in serious conditions such as native valve endocarditis, and even bacteremia sepsis.The pathogenesis is complex, involving multicellular choreography and host immune evasion, however it is well accepted that two key steps are (i) adhesion to the catheter lumen by planktonic cells and (ii) subsequent biofilm formation to establish a stable source of bacterial cells for infection. Adhesion is largely mediated by surface exposed adhesins, targeting a number of soluble host plasma proteins and extracellular matrix components. Biofilm formation has been shown to occur through a number of pathways, however a commonly occurring theme is through secretion of polysaccharide intracellular adhesin (PIA) matrix, driven by expression of the chromosomal icaADBC operon.We have developed a novel toolset using microfluidics to recapitulate the pathogenic environment incorporating clinically relevant fluid shear stress. Using this microfluidic assay, we show that shear stress from fluid flow modulated the pathogenic potential of S. epidermidis, both in terms of increased adhesive capability as well as the induction of biofilm formation is normally quiescent strains. Further, we have developed a high-throughput, multidimensional microfluidic assay incorporating functional adhesive protein microarrays and large scale microfluidic networks. This assay will be used to generate quantitative `pathogenicity landscapes' in Staphylococci, towards the identification of novel therapeutic targets to mitigate and treat device related infections

Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments.

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes

A penta-daughter cell division may lead to aneuploid daughter cells.

<p>As a qualitative example, this cell is in a partially confined state between 3–7 µm. Extreme confinement conditions observed here can lead to aberrant mitotic orientation resulting in highly asymmetric divisions. These microscopic images show the time lapse of a cell undergoing a single 1→5 successful mitosis. Scale bar: 20 µm.</p

Quantitative effects of confinement on cell-cycle abnormalities.

<p>(a) Summary of experimental data in terms of abnormalities per cell cycle characterized by: multi-daughter divisions, asymmetric divisions, apoptotic divisions, and divisions lasting for more than 140 minutes. (b) Multi-daughter divisions per cell cycle characterized as divisions producing more than two daughter cells. Error bars are SEP. (c) Asymmetric divisions are characterized by daughter cells having a difference greater than 15% in area. Error bars are SEP. (d) Cell deaths per cell cycle are characterized by cells that cease activity and contract prior to completion of mitosis. Error bars are SEP. (e) Comparing the mitosis duration of the high confinement (Δy = 3 µm, <i>E</i> = 130 kPa) condition to control and both low confinement conditions. Error bars are SEM. (f) Summary of divisions in each case completing division within 140 minutes. For high confinement E = 1 MPa, no data (ND) was available as no completed mitosis events was observed. Error bars are SEP.</p

Microfluidic cell confinement device.

<p>(a) Device schematic with inset of confinement assay. The posts are 20×80 µm spaced equally 40 µm apart. (b) In the unconfined state, the posts are raised from the glass substrate (top), upon applying pressure the confinement chamber is compressed such that the posts are in contact with the glass substrate. (c) Seeding cells in the confinement chamber (left), when the posts are lowered the cell is confined to 3 or 7 µm height and is squeezed out from the post area (middle). Cells spread and attach in the confined volume (right).</p

Abnormal cell divisions under mechanical confinement.

<p>(a) In an unconfined device control, mitosis occurs within 150 minutes. (b) A confinement condition (Δy = 3 µm, <i>E</i> = 130 kPa) showing sequential tri-daughter divisions with viable daughter cells. The first tri-daughter division is seen at 100 minutes, with cell fusion labeled by black arrows. The second tri-polar division is seen at 1550 minutes along with the viable 2^nd daughter cell labeled in orange. (c) A confinement condition (Δy = 7 µm, <i>E</i> = 130 kPa) showing an asymmetric multi-daughter division of cytoplasm after mitosis (cell with arrow receives disproportionate cytoplasmic volume). (d) A confinement condition (Δy = 7 µm, <i>E</i> = 1 MPa) showing a tetra-daughter division with a cross geometry chromosome alignment (marked by black arrows at 0 minutes) and cell fusion at 60 minutes (labeled by black arrows). (e) A confinement condition (Δy = 7 µm, <i>E</i> = 1 MPa) showing a triangular chromosome arrangement (marked by black arrows) leading to cell death. All scale bars are 20 µm.</p