2d expansion, 2d stimulation, and 3d organoid maturation of adult normal human dermal fibroblasts for tendon research and engineering

用于肌腱研究和工程的三步类器官模型

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure and function. Over the past few years, researchers have strived to better understand the intricate nature of T/L in order to seek effective treatments for T/L disorders3,4,5. T/L are distinguished by a hierarchically organized, extracellular matrix (ECM)-dominated structure, composed primarily of type I collagen fibers and proteoglycans, a feature that is difficult to be replicated in vitro6. Traditional two-dimensional (2D) cell culture models fail to capture the characteristic three-dimensional (3D) organization of T/L tissues, limiting their translational potential as well as hindering the innovative progress in the field of T/L regeneration.
Recently, the development of 3D organoid models has offered new possibilities for advancing basic research and scaffolds-free tissue engineering of various tissue types7,8,9,10,11,12,13. For example, to investigate myotendinous junction, Larkin et al. 2006 developed 3D skeletal muscle constructs together with self-organized tendon segments derived from rat tail tendon10. Moreover, Schiele et al. 2013, by using micromachined fibronectin-coated growth channels, directed the self-assembly of human dermal fibroblasts to form cellular fibers without the assistance of 3D scaffold, an approach that can capture key traits of embryonic tendon development11. In the study by Florida et al. 2016, bone marrow stromal cells were first expanded into bone and ligament lineages, next used to generate self-assembled monolayer cell sheets, which were then implemented to create a multiphasic bone-ligament-bone construct mimicking the native anterior cruciate ligament, a model aiming at improved understanding of ligament regeneration12. To elucidate tendon mechanotransduction processes, Mubyana et al. 2018 utilized a scaffold-free methodology by which single tendon fibers were created and subjected to mechanical loading protocol13. Organoids are self-organized 3D structures that mimic the native architecture, microenvironment, and functionality of tissues. 3D organoid cultures provide a more physiologically relevant model for studying tissue and organ biology as well as pathophysiology. Such models can also be used to induce tissue-specific differentiation of different stem/progenitor cell types14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach9,16. Alternative cell sources can be implemented for the organoid assembly and stimulated toward tenogenic differentiation. One relevant cell type used for demonstration in this study is dermal fibroblasts7,17,18. These cells are easily accessible through a skin biopsy procedure, which is less invasive compared to bone marrow puncture or liposuction and can be multiplied fairly quickly to large numbers due to their good proliferative capacity. In contrast, more specialized cell types, such as T/L-resident fibroblasts, are more challenging to isolate and expand. Therefore, dermal fibroblasts were also used as a starting point for cell reprogramming technologies towards induced pluripotent embryonic stem cells19. Subjecting dermal fibroblasts to specific 3D culture conditions and signaling cues, such as transforming growth factor-beta 3 (TGF&#223;3), which has been reported to act as a key regulator of various cellular processes, including the formation and maintenance of T/L, can potentiate their in vitro tenogenic differentiation leading to the expression of tendon-specific genes and the deposition of T/L-typical ECM20,21.
Here, we describe and demonstrate a previously established and implemented 3-step (2D expansion, 2D stimulation, and 3D maturation) organoid protocol using commercially available normal adult human dermal fibroblasts (NHDFs) as a cell source, offering a valuable model for studying in vitro tenogenesis7. Despite the fact that this model is not equivalent to in vivo T/L tissue, it still provides a more physiologically relevant system that can be used for investigating cellular differentiation mechanisms, mimicking T/L pathophysiology in vitro, and establishing T/L personalized medicine and drug screening platforms. Moreover, in the future, studies can evaluate whether the 3D organoids are suitable for scaffold-free T/L engineering by further optimization as well as utilizable for the development of scaled-up mechanically robust constructs that closely resemble the dimensions and structural and biophysical properties of native T/L tissues.

作者聚焦：使用 3D 类器官模型推进肌腱组织工程

使用人真皮成纤维细胞自组装细胞片培养和手动生成 3D 肌腱/韧带样类器官的演示

We demonstrate a previous established and implement three-step organoid protocol using commercially available, normal human dermal fibroblasts as a cell source. The 3D organoid model holds several advantage for tissue engineering and regenerative medicine application. It mimics closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of tendon tissues.The established 3D tendon-ligament organoid approach can be used as a model for tendon basic and applied research in the following areas. In vitro tenogenesis, disease mechanism, drug testing, and scaffold-free tendon tissue engineering.

用于肌腱研究和工程的成人正常人真皮成纤维细胞的 2D 扩张、2D 刺激和 3D 类器官成熟

首先，离心胰蛋白酶化正常的人真皮成纤维细胞。从离心管中取出上清液。将预热的补充 DMEM 低葡萄糖培养基加入沉淀中，并充分混合以重悬。将正常的人真皮成纤维细胞置于 10 厘米贴壁细胞培养皿中。以横向方式轻轻摇晃盘子。2D 刺激 14 天后，当细胞汇合时，吸出培养基，并使用细胞刮刀轻轻快速地将形成的细胞片从培养皿中分离出来。同时，将细胞片卷成 3D 棒状类器官。将正常的人真皮成纤维细胞类器官转移到 10 厘米宽的非粘附培养皿中。用尺子测量伸长率。然后用镊子握住类器官的一侧，轻轻拉伸类器官，直到其轴向拉长约 10% 使用镊子，手动将金属销穿过类器官的两边压入塑料盘中以固定它。最后，对于 3D 成熟，在孵育前加入 10 毫升预热的补充 DMEM 高糖培养基。在 3D 成熟步骤的第 0 天和第 14 天，3D 类器官的总形态显示出闪闪发光的白色外观。随着时间的流逝，类器官收缩并显得更致密。使用 H E 染色评估类器官的形态。在第一天，类器官显示出不相连的层，主要由被少量基质包围的细胞组成。各层内的细胞表现出圆形和不同大小的细胞核。在第 14 天，观察到伊红信号明显增加，这表明基质沉积和融合层。此外，类器官显示存在包含对齐细胞的区域。大多数细胞核具有相似的大小，偶尔被拉长。

Research

JoVE Journal

Bioengineering

2D Expansion, 2D Stimulation, and 3D Organoid Maturation of Adult Normal Human Dermal Fibroblasts for Tendon Research and Engineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

科研

生物工程