Abstract

Cells in tissues in vivo face a very different microenvironment than typical cultured cells plated on a plastic dish. Already several decades ago, cell biologists observed that cell lines show dramatically different morphology and growth characteristics when embedded into three-dimensional (3D) substrates or standard tissue culture plates (Montesano R, Schaller G, Orci L, Cell. 66:697–711, 1991; Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ, Development. 105:223–235, 1989; Simian M, Bissell MJ, J Cell Biol. 216:31–40, 2017). Despite its imminent benefit for cell biological studies, suspicion and prejudice toward more complicated sample preparation requirements limited the popularity of 3D culture techniques until recently, when it was shown that soft 3D gels made of basement membrane extracts (BME) allow prolonged culture of many types of primary epithelial cells (Clevers H, Cell. 165:1586–1597, 2016; Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al., Nature. 459:262–265, 2009). These observations have brought 3D organoid culture systems into the mainstream. Here we describe two protocols for culturing epithelial cells in 3D substrates, the “blob culture” setup where cells are fully embedded into BME gel and the “overlay setup” where cells are seeded on top of BME gel and then overlaid with a thin layer of BME (Debnath J, Brugge JS, Nat Rev Cancer. 5:675–688, 2005; Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, Martin-Belmonte F, Mostov KE, Nat Cell Biol. 12:1035–1045, 2010).