Vroemen Pascal A M M, Seijas-Gamardo Adrián, Palmen Roy, Wieringa Paul A, Webers Carroll A B, Moroni Lorenzo, Gorgels Theo G M F
Maastricht UMC+, Maastricht University Medical Centre+, University Eye Clinic, Maastricht, the Netherlands.
MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, the Netherlands.
J Biomed Mater Res A. 2025 May;113(5):e37919. doi: 10.1002/jbm.a.37919.
Cell culture of neuronal cells places high demands on the surface for these cells to adhere to and grow on. Native extracellular matrix (ECM) proteins are often applied to the cell culture surface. The substrate is even more important when mechanical strain is applied to the cells in culture. These cells will easily detach and die, precluding the study of how mechanical factors affect these cells. Mechanical factors are, for example, important in the eye disorder glaucoma, which is characterized by the loss of the retinal ganglion cells (RGCs), the retinal neurons that transfer the visual information from the retina via the optic nerve to the brain. High intraocular pressure is the main risk factor of glaucoma. Here, we aimed to find an optimal coating formulation for mechanical testing of the two cell types that are often used for in vitro studies on glaucoma: primary rat retinal ganglion cells (RGCs) and the neuronal PC-12 cell line. Glass and polymer coverslips as well as well plate wells were coated with various substrates: fibronectin, collagen 1, RGD peptide, polyethyleneimine (PEI), poly-D-lysine (PDL), and laminin. We used a thermomixer for 1 min at 500RPM and 37°C to apply mechanical strain and test cell attachment in medium throughput during mechanical stimulation. Cell density, morphology, and cell death were measured to evaluate the coatings. First, a screen of various surfaces and coatings was performed using PC-12 cells, after which a selection of coating strategies was tested with RGCs. For PC-12 cells, the best results were obtained using a coating with a mixture of 10 μg/mL PDL with 2 or 50 μg/mL laminin in PBS (M2). This resulted in the highest cell density, with and without mechanical stimulation. Many other coating strategies failed to provide an effective substrate for adherence and growth of PC-12 cells. Coating composition as well as coating strategy influenced cell attachment and survival. Contrary to PC-12 cells, RGCs performed better in a sequential coating of first 10 μg/mL PDL and then 2 μg/mL laminin (S2). With this protocol, RGCs showed best neurite growth and highest cell density. Based on this difference between PC-12 cells and RGCs, we conclude that the optimal coating depends on the cell type. When reporting cell culture studies, it is important to fully specify culture surface, surface treatment, and coating protocol since all these factors influence cell attachment, growth, and survival.
神经元细胞的细胞培养对细胞附着和生长的表面有很高要求。天然细胞外基质（ECM）蛋白常被应用于细胞培养表面。当对培养中的细胞施加机械应变时，底物就显得更为重要。这些细胞很容易脱离并死亡，从而无法研究机械因素如何影响这些细胞。例如，机械因素在眼部疾病青光眼的发病过程中很重要，青光眼的特征是视网膜神经节细胞（RGCs）的丧失，视网膜神经节细胞是将视觉信息从视网膜通过视神经传递到大脑的视网膜神经元。高眼压是青光眼的主要危险因素。在此，我们旨在找到一种最佳的包被配方，用于对常用于青光眼体外研究的两种细胞类型进行机械测试：原代大鼠视网膜神经节细胞（RGCs）和神经元PC-12细胞系。玻璃和聚合物盖玻片以及孔板孔用各种底物进行包被：纤连蛋白、胶原蛋白1、RGD肽、聚乙烯亚胺（PEI）、聚-D-赖氨酸（PDL）和层粘连蛋白。我们在500转/分钟和37°C下使用热混合器1分钟，以施加机械应变并在机械刺激过程中以中等通量测试细胞附着。测量细胞密度、形态和细胞死亡情况以评估包被。首先，使用PC-12细胞对各种表面和包被进行筛选，之后用RGCs测试一系列包被策略。对于PC-12细胞，在PBS中使用10μg/mL PDL与2或50μg/mL层粘连蛋白的混合物进行包被（M2）可获得最佳结果。无论有无机械刺激，这都导致了最高的细胞密度。许多其他包被策略未能为PC-12细胞的附着和生长提供有效的底物。包被组成以及包被策略影响细胞附着和存活。与PC-12细胞相反，RGCs在先用10μg/mL PDL然后用2μg/mL层粘连蛋白进行顺序包被（S2）时表现更好。采用此方案时，RGCs显示出最佳的神经突生长和最高的细胞密度。基于PC-12细胞和RGCs之间的这种差异，我们得出结论，最佳包被取决于细胞类型。在报告细胞培养研究时，充分说明培养表面、表面处理和包被方案非常重要，因为所有这些因素都会影响细胞附着、生长和存活。
