Micro-cold-forming: a simple, rapid, and inexpensive method for the fabrication of microcavities for 3D cell culture.

Recently, microcavity arrays have been increasingly used to enable the controlled culture of 3D cellular aggregates, such as spheroids, organoids, and gastruloids. However, current fabrication techniques remain technically demanding and largely inaccessible. While micro(scale) thermoforming is already used for fabricating thin-walled microcavities in films from thermoplastic polymers, the process requires specialized equipment for the controlled application of forming temperature and pressure. Here, a new microfabrication method, referred to by us as "micro-cold-forming", is described. This method enables the simple, rapid, and inexpensive fabrication of microcavities (with diameters of 2 mm and depths ranging from 530 to 690 µm) in thin (25-30 µm) thermoplastic polymer films. The fabrication method is carried out at ambient or room temperature and only uses commercially available, easily affordable components. Here, we demonstrate that the microcavities can be applied for 3D cell culture by culturing adult human kidney organoids, called "tubuloids", in the microcavities. To demonstrate the ability of the microcavities to be applied for toxicity studies, the tubuloids were exposed to a high concentration of ascorbic acid. The new forming technique can provide widespread, low-barrier access to self-fabricated microcavities for researchers both in biological laboratories and in low-income countries.