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We evaluated the elasticity of live tissues of zebrafish embryos using label‐free optical elastography. We employed a pair of custom‐built elastic microcantilevers to gently compress a zebrafish embryo and used optical‐tracking analysis to obtain the induced internal strain. We then built a finite element method (FEM) model and matched the strain with the optical analysis. The elastic moduli were found by minimizing the root‐mean‐square errors between the optical and FEM analyses. We evaluated the average elastic moduli of a developing somite, the overlying ectoderm, and the underlying yolk of seven zebrafish embryos during the early somitogenesis stages. The estimation results showed that the average elastic modulus of the somite increased from 150 to 700 Pa between 4‐ and 8‐somite stages, while those of the ectoderm and the yolk stayed between 100 and 200 Pa, and they did not show significant changes. The result matches well with the developmental process of somitogenesis reported in the literature. This is among the first attempts to quantify spatially‐resolved elasticity of embryonic tissues from optical elastography.

 

Multicellular cancer spheroids are an in vitro tissue model that mimics the three-dimensional microenvironment. As spheroids grow, they develop the gradients of oxygen, nutrients, and catabolites, affecting crucial tumor characteristics such as proliferation and treatment responses. The measurement of spheroid stiffness provides a quantitative measure to evaluate such structural changes over time. In this report, we measured the stiffness of size-matched day 5 and day 20 tumor spheroids using a custom-built microscale force sensor and conducted transmission electron microscopy (TEM) imaging to compare the internal structures. We found that older spheroids reduce interstitial spaces in the core region and became significantly stiffer. The measured elastic moduli were 260±100 and 680±150 Pa, for day 5 and day 20 spheroids, respectively. The day 20 spheroids showed an optically dark region in the center. Analyzing the high-resolution TEM images of spheroid middle sections across the diameter showed that the cells in the inner region of the day 20 spheroids are significantly larger and more closely packed than those in the outer regions. On the other hand, the day 5 spheroids did not show a significant difference between the inner and outer regions. The observed reduction of the interstitial space may be one factor that contributes to stiffer older spheroids. 

This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (μTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 μl/sample using the μTweezers vs. ~157 μl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred μm-1500 μm) 3D cell cultures. The μTweezers applied and measured nano-millinewton forces through cantilever’ deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids’ applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young’s modulus of the control group (Control—day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The μTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators. 

In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live microtissues. The purpose of this study was to develop and validate an application-targeted sterile bioreactor that is accessible, inexpensive, adjustable, and easily fabricated. Our method relies on a simple polydimethylsiloxane (PDMS) molding technique for fabrication and is compatible with commonly-used laboratory equipment and materials. Our unique design includes a flexible thin membrane that allows for the transfer of an external actuation into the PDMS beam-based actuator and sensor placed inside a conventional 35 mm cell culture Petri dish. Through computational analysis followed by experimental testing, we demonstrated its functionality, accuracy, sensitivity, and tunable operating range. Through time-course testing, the actuator delivered strains of over 20% to biodegradable electrospun poly (D, L-lactide-co-glycolide) (PLGA) 85:15 non-aligned nanofibers (~91 µm thick). At the same time, the sensor was able to characterize time-course changes in Young’s modulus (down to 10–150 kPa), induced by an application of isopropyl alcohol (IPA). Furthermore, the actuator delivered strains of up to 4% to PDMS monolayers (~30 µm thick), simultaneously characterizing their elastic modulus up to ~2.2 MPa. The platform repeatedly applied dynamic (0.23 Hz) tensile stimuli to live Human Dermal Fibroblast (HDF) cells for 12 hours (h) and recorded the cellular reorientation towards two angle regimes, with averages of −58.85° and +56.02°. The device biocompatibility with live cells was demonstrated for one week, with no signs of cytotoxicity. We can conclude that our PDMS bioreactor is advantageous for low-cost tissue/cell culture micromanipulation studies involving mechanical actuation and characterization. Our device eliminates the need for an expensive experimental setup for cell micromanipulation, increasing the ease of live-cell manipulation studies by providing an affordable way of conducting high-throughput experiments without the need to open the Petri dish, reducing manual handling, cross-contamination, supplies, and costs. The device design, material, and methods allow the user to define the operational range based on their targeted samples/application.