Customized 3D-printed stackable cell culture inserts tailored with bioactive membranes

There is a high demand in various fields to develop complex cell cultures. Apart from titer plates, Transwell inserts are the most popular device because they are commercially available, easy to use, and versatile. While Transwell inserts are standardized, there are potential gains to customize inserts in terms of the number of layers, height between the layers and the size and composition of the bioactive membrane. To demonstrate such customization, we present a small library of 3D-printed inserts and a robust method to functionalize the inserts with hydrogel and synthetic membrane materials. The library consists of 24- to 96-well sized inserts as whole plates, strips, and singlets. The density of cultures (the number of wells per plate) and the number of layers was decided by the wall thickness, the capillary forces between the layers and the ability to support fluid operations. The highest density for a two-layer culture was 48-well plate format because the corresponding 96-well format could not support fluidic operations. The bottom apertures were functionalized with hydrogels using a new high-throughput dip-casting technique. This yielded well-defined hydrogel membranes in the apertures with a thickness of about 500 µm and a %CV (coefficient of variance) of < 10%. Consistent intestine barrier was formed on the gelatin over 3-weeks period. Furthermore, mouse intestinal organoid development was compared on hydrogel and synthetic filters glued to the bottom of the 3D-printed inserts. Condensation was most pronounced in inserts with filters followed by the gelatin membrane and the control, which were organoids cultured at the bottom of a titer plate well. This showed that the bottom of an insert should be chosen based on the application. All the inserts were fabricated using an easy-to-use stereolithography (SLA) printer commonly used for dentistry and surgical applications. Therefore, on demand printing of the customized inserts is realistic in many laboratory settings.