Kauppila M, Mörö A, Valle-Delgado J J, Huhtanen S, Hopia K, Österberg M, Skottman H
Eye Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland.
Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Espoo, 02150 Finland.
Acta Biomater. 2025 Jul 1;201:187-197. doi: 10.1016/j.actbio.2025.05.068. Epub 2025 May 29.
Limbal epithelial stem cells (LSCs) are essential for corneal epithelium regeneration and visual acuity. The limbal niche's physicochemical properties regulate LSC function, but their role is not fully understood. Developing in vitro models that mimic the native niche can enhance our understanding of niche functions, despite the challenges of niche complexity. In this study, we created a 3D bioprinted limbal niche model using a hybrid approach that combines two human pluripotent stem cell-derived LSC (hPSC-LSC) subpopulations (p63+ and ABCG2+ cells) within hyaluronic acid (HA)-based bioinks and a stiff polyacrylamide (PA) gel scaffold produced by conventional gel casting. We analyzed the mechanical properties of the bioinks and assessed cell viability, morphology, and protein expression after one week of culture. Finally, we conducted a proof-of-concept wound healing assay using an alkali burn injury model to assess the functionality of the model for research purposes. The results show that this 3D model effectively replicated the mechanical environment of native tissue, maintains stability for one-week post-printing, and supports LSC viability and normal in vitro phenotype. In addition, the wound healing assay showed a cellular response, indicated by non-simultaneous caspase-3 activation of hPSC-LSC subpopulations for 48 hours post-wounding. This model provides a valuable platform for investigating the limbal niche and advancing cellular therapies applicable to other tissue niches throughout the body. STATEMENT OF SIGNIFICANCE: The corneal limbal niche is crucial for corneal regeneration, creating a high demand for in vitro models. However, current models are not sufficiently replicating the complexity of native tissue and importantly, lack the element of recently demostrated limbal stem cell (LSC) heterogeneity. In this study, we combine three key features of the limbus, including stiffness, architecture and compartmentalization, to create limbal niche-mimicking structures using 3D bioprinting with two human pluripotent stem cell derived LSC (hPSC-LSC) subpopulations. We demonstrate structural stability, native tissue-like mechanical properties, sustained cellular viability, stable hPSC-LSC phenotype post-printing, and a tissue-mimicking response to wounding. This approach offers an innovative strategy to model complex niches and advance the understanding of limbal niche functions.
角膜缘上皮干细胞（LSCs）对角膜上皮再生和视力至关重要。角膜缘生态位的物理化学性质调节LSC功能，但其作用尚未完全明确。尽管生态位复杂存在挑战，但开发模仿天然生态位的体外模型可以增进我们对生态位功能的理解。在本研究中，我们采用一种混合方法创建了一个3D生物打印角膜缘生态位模型，该方法将两种人多能干细胞衍生的LSC（hPSC-LSC）亚群（p63+和ABCG2+细胞）组合在基于透明质酸（HA）的生物墨水以及通过传统凝胶浇铸制备的刚性聚丙烯酰胺（PA）凝胶支架中。我们分析了生物墨水的力学性能，并在培养一周后评估了细胞活力、形态和蛋白质表达。最后，我们使用碱烧伤损伤模型进行了概念验证伤口愈合试验，以评估该模型用于研究目的的功能。结果表明，这个3D模型有效地复制了天然组织的力学环境，在打印后一周内保持稳定，并支持LSC活力和正常体外表型。此外，伤口愈合试验显示出细胞反应，表现为受伤后48小时hPSC-LSC亚群的半胱天冬酶-3激活不同步。该模型为研究角膜缘生态位和推进适用于全身其他组织生态位的细胞治疗提供了一个有价值的平台。重要性声明：角膜缘生态位对角膜再生至关重要，因此对体外模型有很高需求。然而，目前的模型不能充分复制天然组织的复杂性，重要的是，缺乏最近证明的角膜缘干细胞（LSC）异质性元素。在本研究中，我们结合了角膜缘的三个关键特征，包括硬度、结构和分隔，使用3D生物打印和两种人多能干细胞衍生的LSC（hPSC-LSC）亚群创建模仿角膜缘生态位的结构。我们证明了结构稳定性、类似天然组织的力学性能、持续的细胞活力、打印后稳定的hPSC-LSC表型以及对伤口的组织模拟反应。这种方法为模拟复杂生态位和增进对角膜缘生态位功能的理解提供了一种创新策略。
