In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels.
In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels.
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DOI：
10.1038/s41596-020-00419-8
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发表时间：
2021-01
期刊：
影响因子：
14.8
通讯作者：
Allbritton NL
中科院分区：
文献类型：
--
作者：
Hinman SS;Wang Y;Kim R;Allbritton NL
大肠以其排列在上皮上的隐窝和不同的腔内容物调节整个身体的稳态。在三维形状的水凝胶支架上由原代人肠上皮干细胞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，沿着微加工和软光刻方案，所述微加工和软光刻方案是使这些水凝胶成形以匹配体内隐窝的尺寸和密度所必需的。此外，还提供了干细胞放大方案，以便即使是超小的原始样品也可以用作起始材料。最初，将这些细胞作为增殖单层接种在成形支架上，并作为干细胞/增殖细胞培养以扩增细胞数量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龛和管腔分化细胞区的完全极化的功能单元，在隐窝中形成稳定的线性梯度的生长因子。沿着隐窝长轴施加的这些梯度支持基底干细胞小生境，其中转运扩增细胞沿着沿着该轴向上迁移以形成如体内的管腔分化细胞区。该平台支持跨隐窝形成多种化学梯度，包括生长和分化因子、炎性化合物、胆汁和食物代谢物以及细菌产物的化学梯度。如果首次进行，所有微加工和装置组装步骤预计需要8天，原代细胞培养12天以形成成熟的体外隐窝。体外隐窝阵列与标准显微镜方法的兼容性，以及可接近的管腔和基底储库，有利于机制研究和筛选，具有足够的重复和统计能力来测试假设或调查大量化合物（和细菌）对上皮稳态的影响。
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 three-dimensional 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. Additionally, 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 the cell number 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. These gradients imposed along the crypt long axis support a basal stem cell niche with transit amplifying cells migrating upward along this axis to form a luminal differentiated cell zone as in vivo. This platform supports the formation of a multitude 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 days if performing for the first time, with the primary cells cultured for 12 days to form mature in vitro crypts. Compatibility of the in vitro crypt arrays with standard microscopy methods, as well as the accessible luminal and basal reservoirs, facilitate mechanistic studies and screenings with sufficient replicates and statistical power to test hypotheses or survey large numbers of compounds (and bacteria) for their impact on epithelial homeostasis.
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DOI：
10.1021/la502418s
发表时间：
2014-09-23
期刊：
Langmuir : the ACS journal of surfaces and colloids
影响因子：
--
作者：
Drzewiecki KE;Parmar AS;Gaudet ID;Branch JR;Pike DH;Nanda V;Shreiber DI
通讯作者：
Shreiber DI
影响因子：
7.4
作者：
Hu, SW;Ren, XQ;Allbritton, N
通讯作者：
Allbritton, N
影响因子：
14.8
作者：
Dekkers, Johanna F.;Alieva, Maria;Rios, Anne C.
通讯作者：
Rios, Anne C.
影响因子：
64.5
作者：
Hsu YC;Li L;Fuchs E
通讯作者：
Fuchs E
DOI：
10.1073/pnas.1713510115
发表时间：
2018-04-03
影响因子：
11.1
作者：
Greicius G;Kabiri Z;Sigmundsson K;Liang C;Bunte R;Singh MK;Virshup DM
通讯作者：
Virshup DM