Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior.
Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior.
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DOI：
10.1016/j.exer.2022.109102
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发表时间：
2022-07
影响因子：
3.4
通讯作者：
Ganapathy, Preethi S.
中科院分区：
文献类型：
--
作者：
Strat, Ana N.;Kirschner, Alexander;Yoo, Hannah;Singh, Ayushi;Bague, Tyler;Li, Haiyan;Herberg, Samuel;Ganapathy, Preethi S.
关键词：
在青光眼中，视神经乳头（ONH）内的星形胶质细胞重排其肌动蛋白细胞骨架，同时变得反应性并上调中间丝胶质细胞酸性蛋白（GFAP）。转化生长因子β 2（TGF β2）水平升高与脑胶质瘤ONH功能障碍有关。使用常规2D培养来研究ONH星形胶质细胞行为的关键限制是不能忠实地复制体内ONH微环境。在这里，我们设计了一种3D ONH星形胶质细胞水凝胶，以更好地模拟体内小鼠ONH星形胶质细胞（MONHA）形态，并使用TGF β2测试MONHA反应性的诱导。从C57 BL/6 J小鼠中分离原代MONHA并确认细胞纯度。为了设计3D细胞负载水凝胶，将MONHA与光敏细胞外基质组分（I型胶原蛋白，透明质酸）混合，并使用光引发剂（0.025%核黄素）和UV光（405-500 nm，10.3 mW/cm 2）交联5分钟。将MONHA包封的水凝胶培养3周，然后用TGF β2（2.5、5.0或10 ng/ml）处理7天以评估反应性。包封后，MONHA在水凝胶中保持了高细胞活力，并通过活/死染色和MTS测定法测定在4周内继续增殖。Sholl分析表明，随着时间的推移，随着工艺长度的增加，水凝胶中的MONHA开发出越来越多的工艺复杂性。细胞突起与相邻细胞相连，与星形胶质细胞突起内的连接蛋白43表达一致。通过改变F-肌动蛋白细胞骨架形态、增加GFAP表达以及升高纤连蛋白和胶原IV沉积确定，用TGF β2处理诱导MONHA包封的水凝胶中的反应性。我们的数据为将来使用这种3D仿生ONH星形胶质细胞包封的水凝胶研究星形胶质细胞对损伤的反应奠定了基础。
In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF β2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF β2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405–500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF β2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF β2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury.
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