Biology subjects

Castillo, E. A.

Publications and source records attributed to Castillo, E. A..

4 recordsLinked to original sources

bioRxiv2023

Spatial Patterning of Laminin and N-Cadherin for Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)

Controlling cellular shape with protein micropatterning can mimic physiological morphologies and has been shown to improve reproducibility, enhancing our ability to collect statistics on single-cell behaviors. It has also advanced efforts in developing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a promising human model for studies of heart structure and function. hiPSC-CMs have key physiological differences from primary human cardiomyocytes (CMs), including lower sarcomere alignment and contractility, smaller area and lower aspect ratio, and lower force production. Protein micropatterning has been demonstrated to make hiPSC-CMs behave more like primary human CMs across these metrics. However, these micropatterned models typically use only extracellular matrix (ECM) proteins and have not investigated whether providing a protein associated with CM-CM interactions, such as N-cadherin, further enhances hiPSC-CM structure and function. Here, we developed a novel dual-protein patterning process to geometrically control single-cell CM placement on deformable hydrogels suitable for traction force microscopy (TFM). The patterns were comprised of rectangular laminin islands for attachment across the majority of the cell area, with N-cadherin "end-caps" imitating cell-cell interactions. We first photopatterned two proteins on a glass coverslip using a two-step process with photomolecular adsorption of proteins. After both photopatterning steps were complete, we transferred the pattern from the coverslip to a physiologically relevant [~]10-kPa polyacrylamide hydrogel. We seeded -actinin-tagged hiPSC-CMs on the dual-protein-patterned hydrogels and verified interaction between the hiPSC-CMs and the N-cadherin end-caps via immunofluorescent staining. We found hiPSC-CMs on dual-protein patterns have a higher cell area and contractility in the direction of sarcomere organization than those on laminin-only patterns, but no difference in sarcomere organization or force production. While N-cadherin modestly improves the single-cell patterned hiPSC-CM model, it is not sufficient to replicate the role of cell-cell contacts in CM development for in vitro hiPSC-CM systems.

bioengineering↗

bioRxiv2021

Insights into single hiPSC-derived cardiomyocyte phenotypes and maturation using ConTraX, an efficient pipeline for tracking contractile dynamics

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in-vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. To understand how cellular mechanisms affect the heart, it is crucial to quantify the contractile function in single hiPSC-CMs over time, however, such measurements remain demanding and low-throughput, and are too seldom considered. We developed an open-access, versatile, streamlined, and highly automated pipeline to address these challenges and enable quantitative tracking of the contractile dynamics of single hiPSC- CMs over time: ConTraX. Three interlocking software modules enable: (i) parameter-based localization and selection of single hiPSC-CMs; (ii) automated video acquisition of >200 cells/hour; and (iii) streamlined measurements of the contractile parameters via traction force microscopy. Using ConTraX, we analyzed >2,753 hiPSC-CMs over time under orthogonal experimental conditions in terms of culture media and substrate stiffnesses. Using undirected high-dimensional clustering, we dissected the complex diversity of contractile phenotypes in hiPSC-CM populations and revealed converging maturation patterns. Our modular ConTraX pipeline empowers biologists with a potent quantitative analytic tool applicable to the development of cardiac therapies.

bioengineering↗

bioRxiv2020

A benchmarking model for validation and standardization of traction force microscopy analysis tools

Traction Force Microscopy (TFM) has become a well-established technique to assay the biophysical force produced by cells cultured on soft substrates of controlled stiffness. However, experimental conditions as well as computational implementations can have a large impact on the analysis results accuracy and reproducibility. While this can be alleviated using appropriate controls and a rigorous analytical approach, the comparison of results across studies remains difficult and there is a need for validation and benchmarking tools. To validate the accuracy of and compare various computational TFM analysis algorithms, we developed a virtual in silico model of a cell contracting on a soft substrate of controlled stiffness. The model utilizes user-defined parameters for the cell dimensions as well as for the strength and spatial distribution of a contraction dipole to calculate the deformation that would result on a soft substrate due to the cell contraction. The deformation is computed using the forward analytical stress-strain tensor calculation in the Fourier space. The resulting displacement field is used to apply, using image processing, a deformation on a real or simulated image of fluorescent microspheres embedded into a soft hydrogel, which is normally obtain experimentally by TFM imaging. The deformation field and resulting image then serve as input in the PIV and TFM analysis. The model also enables to create movies of a dynamic cell contraction, such as that of a cardiomyocyte, to validate the time accuracy of the TFM analysis after application of image processing algorithm, such as denoising. Our tool therefore addresses the need for validation and standardization of TFM analytical algorithms and its experimental implementations.

biophysics↗

bioRxiv2017

Using Mobile Phones As Acoustic Sensors For High-Throughput Surveillance Of Mosquito Ecology

The direct monitoring of mosquito populations in field settings is a crucial input for shaping appropriate and timely control measures for mosquito-borne diseases. Here, we demonstrate that commercially available mobile phones are a powerful tool for acoustically mapping mosquito species distributions worldwide. We show that even low-cost mobile phones with very basic functionality are capable of sensitively acquiring acoustic data on species-specific mosquito wingbeat sounds, while simultaneously recording the time and location of the human-mosquito encounter. We survey a wide range of medically important mosquito species, to quantitatively demonstrate how acoustic recordings supported by spatio-temporal metadata enable rapid, non-invasive species identification. As proof-of-concept, we carry out field demonstrations where minimally-trained users map local mosquitoes using their personal phones. Thus, we establish a new paradigm for mosquito surveillance that takes advantage of the existing global mobile network infrastructure, to enable continuous and large-scale data acquisition in resource-constrained areas.

ecology↗