Sobejano de la Merced Carlos, Doveri Lavinia, Muñoz Santoro Tomás, García Javier, Garmendia Junkal, Cortés Domínguez Iván, Díaz Fernández Yuri Antonio, Ortiz de Solórzano Carlos
University Clinic of Navarra Centre for Applied Medical Research, 31008 Pamplona, Spain.
University of Navarra Clinic Cancer Center, 31008 Pamplona, Spain.
ACS Appl Mater Interfaces. 2025 Jan 22;17(3):4529-4542. doi: 10.1021/acsami.4c17706. Epub 2025 Jan 7.
Experimental reproducibility in organ-on-chip (OOC) devices is a challenging issue, mainly caused by cell adhesion problems, as OOC devices are made of bioinert materials not suitable for natural cellularization of their surfaces. To improve cell adhesion, several surface functionalization techniques have been proposed, among which the simple use of an intermediate layer of adsorbed proteins has become the preferred one by OOC users. This way, the cells use surface receptors to adhere to the adsorbed proteins, which are in turn attached to the surface. However, as protein adsorption is based on weak electrostatic bonding between the coating proteins and the substrate, this method produces suboptimal results: as the weak electrostatic bonds break, cells detach, leading to poor, heterogeneous cellularization. To solve this problem, we present a surface functionalization method for polyethylene terephthalate (PETE) membranes, commonly used in multilayer organ-on-chip devices to support cellular layers. This protocol involves hydrolyzation of the membrane, followed by (-dimethylaminopropyl) carbodiimide (EDC) and -hydroxysuccinimide (NHS) activation, resulting in covalent bonding between the membrane and coating proteins, much stronger than the weak electrostatic bonding provided by simple adsorption. As evaluation, we first measured the effect of the functionalization protocol in the morphological and mechanical integrity of the membranes. Next, we confirmed protein coating efficiency using the ζ potential and surface tension of the functionalized membranes coated with collagen type I, polylysine, gelatin, albumin, fetal bovine serum (FBS), and Matrigel. Finally, we showed that our method significantly improves the attachment of epithelial (A549) and endothelial (EA.hy926) cell lines under static conditions, especially in collagen-coated membranes, which were further tested under dynamic conditions, showing statistically significant improvement in cell attachment compared to uncoated or collagen-adsorbed only membranes.
器官芯片（OOC）装置中的实验可重复性是一个具有挑战性的问题，主要是由细胞粘附问题引起的，因为OOC装置由生物惰性材料制成，其表面不适合自然细胞化。为了改善细胞粘附，已经提出了几种表面功能化技术，其中简单地使用吸附蛋白的中间层已成为OOC用户的首选方法。通过这种方式，细胞利用表面受体粘附到吸附的蛋白质上，而这些蛋白质又附着在表面上。然而，由于蛋白质吸附是基于涂层蛋白质与底物之间的弱静电结合，这种方法产生的效果并不理想：随着弱静电键的断裂，细胞会脱落，导致细胞化效果不佳且不均匀。为了解决这个问题，我们提出了一种用于聚对苯二甲酸乙二酯（PETE）膜的表面功能化方法，PETE膜常用于多层器官芯片装置中以支撑细胞层。该方案包括膜的水解，然后用（-二甲基氨基丙基）碳二亚胺（EDC）和-羟基琥珀酰亚胺（NHS）活化，从而在膜和涂层蛋白质之间形成共价键，这比简单吸附提供的弱静电键要强得多。作为评估，我们首先测量了功能化方案对膜的形态和机械完整性的影响。接下来，我们使用涂有I型胶原蛋白、聚赖氨酸、明胶、白蛋白、胎牛血清（FBS）和基质胶的功能化膜的ζ电位和表面张力来确认蛋白质涂层效率。最后，我们表明我们的方法在静态条件下显著改善了上皮细胞系（A549）和内皮细胞系（EA.hy926）的附着，特别是在胶原蛋白涂层的膜中，在动态条件下进一步测试发现，与未涂层或仅吸附胶原蛋白的膜相比，细胞附着有统计学上的显著改善。
