Design, Fabrication, and Validation of a Petri Dish-Compatible PDMS Bioreactor for the Tensile Stimulation and Characterization of Microtissues

Abstract

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.

Biocompatible micro tweezers for 3D hydrogel organoid array mechanical characterization

“…The molds were then sandwiched, vise clamped, and placed in a convection oven at 65 C° for 12 hours curing time. In this study, the elastic modulus of the PDMS cantilevers was calibrated and measured (2.46 MPa) [ 28 ]. Only the PDMS molds in ( Fig 3L ) were cured for 3 hours at 65 C°.…”

“…However, excessive crosslinking curing agent ratios were avoided to avert the potential of excess crosslinking agent leaching out into cell culture and maintain high PDMS elasticity, which also supports the PDMS actuation and bending with reduced to no hysteresis. Further relevant fabrication and curing condition details were previously reviewed and reported [ 28 ].…”

“…Based on the design dimensions and the elastic modulus E = 2.46 MPa we found in our previous study [ 28 ], the calculated PDMS cantilever spring constant was (0.0765 N/m), and COMSOL stationary solid mechanics’ simulation resulted in a spring constant (0.08 N/m). The spring constant calculated using the measured average cantilever thickness (148 ± 1.9 μm, n = 8) was (0.0725 N/m), and the updated COMSOL spring constant became (0.079 N/m).…”

“…To evaluate the systems’ biocompatibility without 3D cell culture effects such as necrosis and hypoxia, 2D monolayers of human dermal fibroblasts were cultured in 24 well-plates with the PDMS manipulators, starting at low seeding numbers and achieving high numbers over time. Our previous publication [ 28 ] tested PDMS characteristics under a cell culture condition and demonstrated biocompatibility using the human dermal fibroblast cell line. This study used the same cell line to compare with the previous well-tested PDMS device design and use the results as a reference.…”

“…The μTweezer system can mechanically characterize 3D tissue cultures of defined structures, including and not limited to hydrogel pillars, organoids, spheroids, tumoroids, chondrospheres. The system fabrication relied on the widely available milling and molding techniques [ 28 ] rather than previously reported spin-coating approaches [ 24 , 27 ]. The 3D elastomer molding technique allowed the fabrication of mechanically functional 3D PDMS devices with sub-millimeter-scale resolution.…”