Preparing Chamber Slides With Pressed Collagen for Live Imaging Monolayers of Primary Human Intestinal Stem Cells

Primary human intestinal stem cells (ISCs) can be cultured and passaged indefinitely as two-dimensional monolayers grown on soft collagen. Culturing ISCs as monolayers enables easy access to the luminal side for chemical treatments and provides a simpler topology for high-resolution imaging compared to cells cultured as three-dimensional organoids. However, the soft collagen required to support primary ISC growth can pose a challenge for live imaging with an inverted microscope, as the collagen creates a steep meniscus when poured into wells. This may lead to uneven growth toward the center of the well, with cells at the edges often extending beyond the working distance of confocal microscopes. We have engineered a 3D-printed collagen mold that enables the preparation of chamber slides with flat, smooth, and reproducible thin collagen layers. These layers are adequate to support ISC growth while being thin enough to optimize live imaging with an inverted microscope. We present methods for constructing the collagen press, preparing chamber slides with pressed collagen, and plating primary human ISCs for growth and analysis. Key features • This protocol describes how to construct and use collagen presses for chamber slides, as demonstrated in Cotton et al. [1]. • The soft collagen and culture media presented are optimized for primary human intestinal stem cells. • The full protocol, including 3D printing, preparing collagen-coated chamber slides, and plating cells can be completed in under one week. • This protocol requires access to a 3D printer.