Department of Mechanical Engineering, Vanderbilt University, Nashville, TN, USA.
Biomater Sci. 2019 Mar 26;7(4):1358-1371. doi: 10.1039/c8bm01199k.
The process of cell differentiation in a developing embryo is influenced by numerous factors, including various biological molecules whose presentation varies dramatically over space and time. These morphogens regulate cell fate based on concentration profiles, thus creating discrete populations of cells and ultimately generating large, complex tissues and organs. Recently, several in vitro platforms have attempted to recapitulate the complex presentation of extrinsic signals found in nature. However, it has been a challenge to design versatile platforms that can dynamically control morphogen gradients over extended periods of time. To address some of these issues, we introduce a platform using channels patterned in hydrogels to deliver multiple morphogens to cells in a 3D scaffold, thus creating a spectrum of cell phenotypes based on the resultant morphogen gradients. The diffusion coefficient of a common small molecule morphogen, retinoic acid (RA), was measured within our hydrogel platform using Raman spectroscopy and its diffusion in our platform's geometry was modeled using finite element analysis. The predictive model of spatial gradients was validated in a cell-free hydrogel, and temporal control of morphogen gradients was then demonstrated using a reporter cell line that expresses green fluorescent protein in the presence of RA. Finally, the utility of this approach for regulating cell phenotype was demonstrated by generating opposing morphogen gradients to create a spectrum of mesenchymal stem cell differentiation states.
胚胎发育过程中的细胞分化受到许多因素的影响，包括各种生物分子，其呈现方式在空间和时间上差异很大。这些形态发生素根据浓度分布来调节细胞命运，从而产生离散的细胞群体，并最终生成大型、复杂的组织和器官。最近，一些体外平台试图再现自然界中存在的复杂的外在信号呈现方式。然而，设计能够长时间动态控制形态发生素梯度的多功能平台一直是一个挑战。为了解决其中的一些问题，我们引入了一种使用通道在水凝胶中进行图案化的平台，将多种形态发生素递送到 3D 支架中的细胞中，从而根据产生的形态发生素梯度产生一系列细胞表型。使用拉曼光谱测量了我们的水凝胶平台内常见的小分子形态发生素视黄酸（RA）的扩散系数，并使用有限元分析对其在我们平台几何形状中的扩散进行建模。在无细胞水凝胶中验证了空间梯度的预测模型，然后使用表达 RA 存在时绿色荧光蛋白的报告细胞系证明了形态发生素梯度的时间控制。最后，通过生成相反的形态发生素梯度来产生一系列间充质干细胞分化状态，证明了这种方法在调节细胞表型方面的用途。
