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Human 2D Crypt Model for Assaying Intestinal Stem Cell Proliferation and Differentiation
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-23 , DOI: 10.1021/acs.analchem.2c00905 Yuli Wang 1 , Christopher E Sims 2 , Nancy L Allbritton 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-23 , DOI: 10.1021/acs.analchem.2c00905 Yuli Wang 1 , Christopher E Sims 2 , Nancy L Allbritton 1
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Intestine is a common site of adverse drug effects in clinical trials; thus, improved in vitro models for preclinical screening of pharmaceutical compounds are sought. A planar, self-renewing human intestinal monolayer platform based on primary adult gastrointestinal stem cells, termed the 2D crypt model, has been developed to screen for the effects of various compounds on the intestinal epithelium. The 2D crypt platform is based on a standard 12-well plate format and consists of cell culture inserts with a collagen film overlaying an impermeable film patterned with an array of micron-scale holes. This two-chamber format enables a gradient of growth factors to be applied such that the tissue self-organizes into spatially segregated stem and differentiated cell compartments. The patterned monolayer mimics a gut epithelium in possessing a stem cell niche, migrating proliferative and differentiated cells. Once established, the 2D crypts replicate many aspects of in vivo physiology, including cell migration, maturation, and apoptotic cell death. The planar geometry of the system simplifies dosing, sampling, and imaging during assay. An immunofluorescence-based assay was established to quantitatively assess cell density, proliferation, migration, viability, and the abundance and localization of postmitotic lineages as a function of time. The model was used to perform a small-scale screen of compounds, including signaling molecules, endogenous hormones/cytokines, and microbial metabolites, on tissue homeostasis. Hit compounds that significantly impacted proliferation and/or differentiation were readily identified. The 2D crypt platform represents a significant innovation in the development of microphysiological systems for emulating the gut epithelium for compound screens.
中文翻译：
用于测定肠道干细胞增殖和分化的人体二维隐窝模型
肠道是临床试验中药物不良反应的常见部位；因此，改善了体外寻找药物化合物临床前筛选的模型。基于原代成体胃肠道干细胞的平面、自我更新的人类肠道单层平台（称为二维隐窝模型）已被开发出来，用于筛选各种化合物对肠上皮的影响。2D 隐窝平台基于标准 12 孔板格式，由细胞培养插入物和胶原蛋白膜组成，该插入物上覆盖有微米级孔阵列图案的不渗透膜。这种两室格式能够应用生长因子的梯度，从而使组织自组织成空间隔离的干细胞和分化的细胞区室。图案化的单层模仿肠道上皮，拥有干细胞生态位，迁移增殖和分化的细胞。一旦成立，体内生理学，包括细胞迁移、成熟和细胞凋亡。系统的平面几何形状简化了测定过程中的给药、采样和成像。建立了基于免疫荧光的测定法，以定量评估细胞密度、增殖、迁移、活力以及有丝分裂后谱系的丰度和定位随时间的变化。该模型用于对组织稳态的化合物进行小规模筛选，包括信号分子、内源性激素/细胞因子和微生物代谢物。很容易鉴定出显着影响增殖和/或分化的命中化合物。2D 隐窝平台代表了微生理系统开发中的一项重大创新，用于模拟肠道上皮以进行化合物筛选。
更新日期：2022-06-23
中文翻译：
用于测定肠道干细胞增殖和分化的人体二维隐窝模型
肠道是临床试验中药物不良反应的常见部位；因此，改善了体外寻找药物化合物临床前筛选的模型。基于原代成体胃肠道干细胞的平面、自我更新的人类肠道单层平台（称为二维隐窝模型）已被开发出来，用于筛选各种化合物对肠上皮的影响。2D 隐窝平台基于标准 12 孔板格式，由细胞培养插入物和胶原蛋白膜组成，该插入物上覆盖有微米级孔阵列图案的不渗透膜。这种两室格式能够应用生长因子的梯度，从而使组织自组织成空间隔离的干细胞和分化的细胞区室。图案化的单层模仿肠道上皮，拥有干细胞生态位，迁移增殖和分化的细胞。一旦成立，体内生理学，包括细胞迁移、成熟和细胞凋亡。系统的平面几何形状简化了测定过程中的给药、采样和成像。建立了基于免疫荧光的测定法，以定量评估细胞密度、增殖、迁移、活力以及有丝分裂后谱系的丰度和定位随时间的变化。该模型用于对组织稳态的化合物进行小规模筛选，包括信号分子、内源性激素/细胞因子和微生物代谢物。很容易鉴定出显着影响增殖和/或分化的命中化合物。2D 隐窝平台代表了微生理系统开发中的一项重大创新，用于模拟肠道上皮以进行化合物筛选。
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