Quick and easy microfabrication of T-shaped cantilevers to generate arrays of microtissues

Abstract

Over the past decade, a major effort was made to miniaturize engineered tissues, as to further improve the throughput of such approach. Most existing methods for generating microtissues thus rely on T-shaped cantilevers made by soft lithography and based on the use of negative SU-8 photoresist. However, photopatterning T-shaped microstructures with these negative photoresists is fastidious and time-consuming. Here we introduce a novel method to quickly generate T-shaped cantilevers dedicated to generation of cellular microtissues, based on the use of positive photoresist. With only two layers of photoresist and one photomask, we were able to fabricate arrays of microwells in less than 3 h, each containing two T-shaped cantilevers presenting either a rectangular or a circular geometry. As a proof of concept, these arrays were then replicated in poly(dimethylsiloxane) and microtissues composed of NIH 3T3 fibroblasts encapsulated in collagen I were generated, while the two cantilevers simultaneously constrain and report forces generated by the microtissues. Immunostainings showed longitudinally aligned and elongated fibroblasts over the whole microtissue after 8 days of culture. The method described here opens the potential to quick prototyping platforms for high-throughput, low-volume screening applications.

3D culture models of tissues under tension

“…However, new protocols to facilitate the fabrication process of silicon masters are being developed (Kalman et al, 2016). Furthermore, evolving 3D printing techniques are approaching the resolution needed to accurately print the molds of microtissue devices and could thus replace current microfabrication methods in the near future.…”

“…As cells compact the matrix around the pillars, the tissue tension increases and causes the tissue to move upwards along the pillar until it slips off. By manufacturing T-shaped pillars, the tissue is prevented from slipping off and thus the tissue boundaries are outlined by the spacing of the pillars (Kalman et al, 2016;Legant et al, 2009). However, by introducing conical shaped pillars, one can control where and when the tissue is released from a pillar, and thus change the shape of the microtissue over time (Svoronos et al, 2014).…”

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

“…[ 1 ] Unsurprisingly, more and more research groups started investing resources and expertise in the generation of 3D in vitro models for various tissues. [ 2–10 ] Adopting this paradigm is particularly significant for contractile and load‐bearing tissues, such as tendons, cardiac, and skeletal muscle. [ 11–14 ] However, since the criteria for a transition from monolayer cultures to 3D systems were first outlined more than ten years ago, [ 15,16 ] cost‐effective ways for easy production of 3D cell culture systems are still lacking.…”

“…[ 17 ] Downscaling the size of engineered tissues is also possible by producing pillars in a T‐shape: “caps” on top of each pillar help the retention of smaller tissues under tension. [ 5–7,17 ] Multistep photolithography has been used to generate negative molds for polydimethylsiloxane (PDMS) T‐shaped pillars. [ 5–7 ] However, this approach carries a limitation in the production of features larger than few hundreds of micrometers and lacks versatility: producing a single master mold is time‐consuming and expensive, requiring most of the time dedicated facilities such as clean rooms.…”

“…[ 5–7,17 ] Multistep photolithography has been used to generate negative molds for polydimethylsiloxane (PDMS) T‐shaped pillars. [ 5–7 ] However, this approach carries a limitation in the production of features larger than few hundreds of micrometers and lacks versatility: producing a single master mold is time‐consuming and expensive, requiring most of the time dedicated facilities such as clean rooms. To avoid multistep photolithography, molds have been generated using single‐step lithography [ 8–10 ] or milled Teflon.…”