RESEARCH IN APPLIED MICROBIOENGINEERING

  • Noncanonical Wnt Activation Promotes the Mechanotransduction of Cytotoxic CD8+ T-Cells

  • Mechanical Force Regulation of Asymmetric Vascular Cell Alignment

MICRO-MECHANO BIOENGINEERING INTRODUCTORY VIDEO

I spent the summers of 2020 and 2021 conducting research for New York University’s Applied Micro-Bioengineering Laboratory. My team and I collaborated on this video to introduce our field of research and the laboratory’s motivations. 

In this video, we discuss how micro-mechano bioengineering is a multidisciplinary field that intersects the usage of mechanics, electronics, and biological processes. Its applications allow for disease modeling though controlled environmental advancements. Our research specifically sought to bridge the fields of mechanobiology, tissue engineering, regenerative medicine, and cancer biology.

From 1:30 to 2:05, I discuss micro-fabrication protocols and their pertinence to our research objectives.

ABSTRACT #1

  • Noncanonical Wnt Activation Promotes the Mechanotransduction of Cytotoxic CD8+ T-Cells

    This project utilized force analyzation devices and algorithms for the tracking and subsequent computational analysis of force generation during the activation of CD8+ cytotoxic T-Cells. Stronger immune responses exhibited positive correlation with higher levels of total cellular forces during CD8+ T-Cell activation. The potential signaling pathways to augment the mechanotransduction and immune responses remain largely unexplored. Here, we explored the effects of noncanonical Wnt activation on T-Cell mechano-transduction to find an effective molecular pathway to elevate the cytotoxicity of CD8+ T-Cells and increase the efficiency of immunotherapy by conjugating the designated activating antibodies and noncanonical WNT- activating ligand, Foxy5 peptides, on the PDMS micropillar force measure device. Results showed that Foxy5-mediated noncanonical Wnt activation effectively increased the force feedback in activated CD8+ T-Cells. This research furthers the field of mechanoimmunology by providing a novel signaling pathway to modulate the CD8+ T-Cell activation.

    Research poster
NYU TANDON 2021 RESEARCH JOURNAL highlight

ILLUSTRATED METHODOLOGY

  • Step 1.1: Pillar Fabrication

    Make negative molds from silicon micro-pillar array detectors (mPADs) so they may be used to produce more 6um and 8um mPADs.

  • Step 1.2: Pillar Fabrication

    Attach the negative molds to glass slides using PDMS to form positive molds of micro-pillars.

  • Step 1.3: Pillar Fabrication

    Remove negative molds and dry the new mPADS made from PDMS.

  • Step 2: Antibody Protein Coating

    Conjugate the designated activating antibodies and noncanonical WNT-activating ligand, Foxy5 peptides, onto the PDMS micropillar force measurement devices.

    Transfer CD8+ T-Cell cultures to assorted PDMS micropillar force measurement devices to initiate antibody-to-cell interactions.

  • Step 3.1: Dynamic Cell Force Analysis

    Following mechanotransduction and monitored activation of CD8+ cells, photograph pillars under microscope. Images of pillar displacement are used to develop all.json files for Cellogram analysis.

  • Step 3.2: Dynamic Cell Force Analysis

    Run all.json files though Cellogram to calculate displacement metrics for each micro-pillar array.

  • Step 3.3: Dynamic Cell Force Analysis

    Run Cellogram results through MATLAB to perform computational analysis on displacement metrics. Comparisons against micro-pillar locations prior to activation allow for direct quantification of force magnitude and direction during T-Cell mechanotransduction.

  • Step 4.1: Cell Fixation and Staining

    Fix cells with Triton-X. Stain cells with DAPI to visualize nuclei under fluorescent microscopy.

  • Step 4.2: Cell Fixation and Staining

    Stain cells with Vinculin and Alexa Fluor-488 to visualize actin-integrin binding sites under fluorescent microscopy.

  • Step 4.3: Cell Fixation and Staining

    Stain cells with Phalloidin and Alexa Fluor-555 to visualize actin under fluorescent microscopy.

  • Step 4.4: Cell Fixation and Staining

    Stain cells with FAK (Focal Adhesion Kinase) and Alexa Fluor-647 to visualize integrin using fluorescent microscopy.

  • Step 4.5: Cell Fixation and Staining

    Overlay fluorescent images to produce final visualization of CD8+ T-Cells after activation and mechanotransduction.

SUMMARY OF FINDINGS

Micro-pillar displacement metrics and fluorescent imaging were both critical to our investigation into how foxy5 peptides modulate cytotoxic CD8+ T-cell activation and mechanotransduction. The metrics quantified the direction and magnitude of forces involved, while the staining and imaging techniques provided a visualization of the cells after activation. Together, these methods offered a comprehensive approach to understanding the underlying mechanisms of T-cell activation and the cytotoxic immune response. By elucidating these mechanisms at the cellular level, this research can contribute to the development of novel strategies for enhancing immune function against infection and disease.

Force direction dynamics data shows clear spreading movement (positive) after Foxy5 activation, and minimal spreading movement after Src (control) activation. These contrast to squeezing movement (negative).

Force dynamics data shows clear differences in the strengths of forces produced by T-Cells when activated by Foxy5 versus Scr (control).

My team’s research on this project was published to The Royal Society of Chemistry’s Chemical Communications Journal: Issue 94, 2021.

DOI: 10.1039/D1CC05194F

ABSTRACT #2

  • Mechanical Force Regulation of Asymmetric Vascular Cell Alignment

    This study investigates the role of mechanobiology in vascular endothelial cells by comparing the mechanical characteristics and multicellular coordination abilities of healthy and diabetic aortic-endothelial cells (ECs). Cells experience mechanical forces and external stressors in their microenvironment that require appropriate cellular responses to maintain physiological functioning. The cultures were grown on mechanical force detecting substrates with pharmacological treatments and varying glucose concentrations to simulate and regulate morphogenesis in vitro.

    To investigate the impact of varying glucose conditions and simulated disease states, traction force microscopy (TFM) was employed to visualize the cell alignments and forces across different tissue geometries. The resulting data showed that healthy and diseased ECs exhibit unique mechanical properties and coordination abilities, as indicated by particle displacement in the substrate. These findings suggest that certain patterns of asymmetry may be related to cardiovascular diseases, highlighting the importance of mechanobiology in the adaptation of vascular systems. Overall, this study sheds light on the role of mechanical forces and external stressors in cellular responses and their impact on physiological functioning, providing valuable insights into the interplay between mechanical forces, cellular responses, and vascular physiology that could have important implications for the development of treatments for cardiovascular diseases.

    Research poster
NYU TANDON 2020 RESEARCH JOURNAL highlight

SUMMARY OF FINDINGS

The study aimed to investigate the role of mechanobiology in disease states of vascular endothelial cells. The results showed that diabetic cells experienced greater traction forces than healthy cells when greater mechanical stress was applied, indicating specific force patterns are exhibited by diseased cells. The study also revealed that high glucose levels impact multicellular cooperation, reflected through their average orientation. Additionally, negative control, positive control, and diabetic cells, respectively, showed increasing degrees of either clockwise or counterclockwise orientation in a multicellular system, indicating an impact of disease on coordination abilities. Overall, this study offers valuable insights into the interplay between mechanical forces, cellular responses, and vascular physiology, which could have significant implications for early diagnostics of cardiovascular diseases.

Micro-contact printing (fibronectin stamping) allows cells to grow in specified areas of the petri dish, producing different tissue geometries. The four types shown here (Type 1 to 4) result from fibronectin stamping and simulate varying levels of mechanical stress placed on vascular endothelial cells in the human body (increasing from left to right)."

  • Chirality Analysis

    The average degrees of clockwise or counterclockwise orientation of vascular endothelial cells were plotted across the four tissue geometries. In comparing diabetic cells to normal glucose (negative control) and high glucose (positive control) cells, greater degrees of orientation were observed in the disease state in three of the four geometries. These findings suggest higher levels of multicellular coordination in diseased cells where low to moderate levels of mechanical stress is applied.

  • Type 1: Traction Force Analysis

    In comparing normal glucose cells (negative control) to high glucose cells (positive control) and diabetic cells (disease state), normal glucose cells exhibited greater traction force at external boundaries. These findings suggest glucose concentration impacts force patterns were mechanical stress is applied.

  • Type 2: Traction Force Analysis

    In comparing normal glucose (negative control), high glucose (positive control), and diabetic cells (disease state), similar traction force patterns were observed, with the exception of diabetic cells exhibiting higher traction forces at internal and external boundaries. These findings suggest disease state impacts force patterns where mechanical stress is applied.

  • Type 3: Traction Force Analysis

    In comparing diabetic cells (disease state) to normal glucose (negative control) and high glucose (positive control) cells, notably higher traction forces were observed in the disease state. Type 3 is distinguished by higher levels of mechanical stress throughout the tissue culture. These findings further suggest disease state impacts force patterns where mechanical stress is applied.

  • Type 4: Traction Force Analysis

    In comparing diabetic cells (disease state) to normal glucose (negative control) and high glucose (positive control) cells, notably higher traction forces were observed in the disease state. Type 4 is distinguished by the highest levels of mechanical stress throughout the tissue culture. These findings further suggest disease state impacts force patterns where mechanical stress is applied.