Materials Science and Engineering and Bioengineering, Stanford University, 476 Lomita Mall, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22067-72. doi: 10.1073/pnas.0904851106. Epub 2009 Dec 10.
Current protocols to encapsulate cells within physical hydrogels require substantial changes in environmental conditions (pH, temperature, or ionic strength) to initiate gelation. These conditions can be detrimental to cells and are often difficult to reproduce, therefore complicating their use in clinical settings. We report the development of a two-component, molecular-recognition gelation strategy that enables cell encapsulation without environmental triggers. Instead, the two components, which contain multiple repeats of WW and proline-rich peptide domains, undergo a sol-gel phase transition upon simple mixing and hetero-assembly of the peptide domains. We term these materials mixing-induced, two-component hydrogels. Our results demonstrate use of the WW and proline-rich domains in protein-engineered materials and expand the library of peptides successfully designed into engineered proteins. Because both of these association domains are normally found intracellularly, their molecular recognition is not disrupted by the presence of additional biomolecules in the extracellular milieu, thereby enabling reproducible encapsulation of multiple cell types, including PC-12 neuronal-like cells, human umbilical vein endothelial cells, and murine adult neural stem cells. Precise variations in the molecular-level design of the two components including (i) the frequency of repeated association domains per chain and (ii) the association energy between domains enable tailoring of the hydrogel viscoelasticity to achieve plateau shear moduli ranging from approximately 9 to 50 Pa. Because of the transient physical crosslinks that form between association domains, these hydrogels are shear-thinning, injectable, and self-healing. Neural stem cells encapsulated in the hydrogels form stable three-dimensional cultures that continue to self-renew, differentiate, and sprout extended neurites.
目前将细胞封装在物理水凝胶中的方法需要对环境条件（pH 值、温度或离子强度）进行实质性改变才能引发凝胶化。这些条件可能对细胞有害，并且通常难以重现，因此在临床环境中使用它们会变得复杂。我们报告了一种两成分、分子识别凝胶化策略的开发，该策略无需环境触发即可进行细胞封装。相反，这两种成分包含多个 WW 和富含脯氨酸的肽结构域重复，在简单混合和肽结构域的杂化组装后经历溶胶-凝胶相转变。我们将这些材料称为混合诱导的两成分水凝胶。我们的结果表明 WW 和富含脯氨酸的结构域可用于蛋白质工程材料，并扩展了成功设计成工程蛋白的肽文库。由于这两个缔合结构域通常存在于细胞内，因此它们的分子识别不会被细胞外环境中其他生物分子的存在所破坏，从而能够重复封装多种细胞类型，包括 PC-12 神经元样细胞、人脐静脉内皮细胞和鼠成年神经干细胞。两种成分的分子水平设计的精确变化，包括（i）每个链上重复缔合结构域的频率和（ii）结构域之间的缔合能，可实现水凝胶粘弹性的定制，以获得约 9 至 50 Pa 的平台剪切模量。由于缔合结构域之间形成的瞬态物理交联，这些水凝胶具有剪切稀化、可注射和自修复的特性。封装在水凝胶中的神经干细胞形成稳定的三维培养物，继续自我更新、分化和长出延伸的神经突。
