Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5⁺ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP⁺ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5⁺ ISCs. Considering the key roles Lgr5⁺ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy).

中文翻译：

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朝着确定的基于ECM和小分子的单层培养系统扩展小鼠和人类肠道干细胞

当前的ISC培养系统面临着严峻的挑战，例如动物来源或不确定的基质组成，批次间的变异性（例如基于Matrigel的类器官培养）以及测定细胞聚集体（如类器官）的复杂性，这使得ISC的研究和临床翻译成为可能具有挑战性的。在这里，通过筛选合适的ECM成分，我们报告了定义的基于胶原的单层培养系统，该系统支持富含Lgr5 ⁺的小鼠和人肠上皮细胞（IEC）的生长与基于标准Matrigel的类器官培养物中发现的水平相当或更高的种群。该系统称为Bolstering Lgr5转化（BLT）夹心培养，包括胶原蛋白IV涂覆的多孔基质和胶原蛋白I凝胶覆盖层，它们之间夹有IEC单层。不同的胶原蛋白线索通过最大化不同细胞表面粘附受体（即整联蛋白α2β1，整联蛋白β4）和细胞极性的结合来协同调节IEC附着，增殖和Lgr5表达。此外，我们应用BLT三明治系统来确定骨形态发生蛋白（BMP）受体抑制剂（LDN-193189）的添加可改善Lgr5-GFP ⁺的扩增来自小鼠小肠隐窝的细胞几乎增加了2.5倍。值得注意的是，与传统的Matrigel培养相比，BLT三明治培养能够以更高的LGR5 mRNA水平扩增人源性IEC ，从而提供人LGR5 ⁺ ISC的优异扩增。考虑到Lgr5 ⁺ ISC在肠道上皮稳态和再生中所起的关键作用，我们设想BLT三明治培养系统在体外理解和操纵ISC生物学（例如，为ISC介导的肠道疾病建模）或扩大大量应用方面具有巨大潜力。用于临床用途的ISC（例如用于干细胞治疗）。