The large intestine, with its array of crypts lining the epithelium and diverse luminal contents, regulates homeostasis throughout the body. In vitro crypts formed from primary human intestinal epithelial stem cells on a 3D shaped hydrogel scaffold replicate the functional and architectural features of in vivo crypts. Collagen scaffolding assembly methods are provided, along with the microfabrication and soft lithography protocols necessary to shape these hydrogels to match the dimensions and density of in vivo crypts. In addition, stem-cell scale-up protocols are provided so that even ultrasmall primary samples can be used as starting material. Initially, these cells are seeded as a proliferative monolayer over the shaped scaffold and cultured as stem/proliferative cells to expand them and cover the scaffold surface with the crypt-shaped structures. To convert these immature crypts into fully polarized, functional units with a basal stem cell niche and luminal differentiated cell zone, stable, linear gradients of growth factors are formed across the crypts. This platform supports the formation of chemical gradients across the crypts, including those of growth and differentiation factors, inflammatory compounds, bile and food metabolites and bacterial products. All microfabrication and device assembly steps are expected to take 8 d, with the primary cells cultured for 12 d to form mature in vitro crypts.

大肠具有排列在上皮细胞内的隐窝和不同的肠腔内容物，调节整个身体的体内平衡。由原代人肠上皮干细胞在 3D 形状的水凝胶支架上形成的体外隐窝复制了体内隐窝的功能和结构特征。提供了胶原支架组装方法，以及塑造这些水凝胶以匹配体内隐窝的尺寸和密度所需的微加工和软光刻方案。此外，还提供了干细胞放大方案，因此即使是超小的初级样品也可以用作起始材料。最初，这些细胞作为增殖单层接种在成形支架上，并作为干/增殖细胞培养以扩展它们并用隐窝状结构覆盖支架表面。为了将这些未成熟的隐窝转化为具有基底干细胞生态位和管腔分化细胞区的完全极化的功能单元，在整个隐窝中形成稳定的线性生长因子梯度。该平台支持整个隐窝化学梯度的形成，包括生长和分化因子、炎症化合物、胆汁和食物代谢物以及细菌产物的化学梯度。所有微加工和装置组装步骤预计需要 8 天，其中原代细胞培养 12 天以形成成熟的体外隐窝。