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

Dreistufiges Organoid-Modell für die Sehnenforschung und -technik

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.

Autor im Rampenlicht: Weiterentwicklung des Tendon Tissue Engineering mit 3D-Organoidmodellen

Demonstration einer selbstorganisierten Zellblattkultur und manuelle Generierung eines 3D-Sehnen-/Ligamenten-ähnlichen Organoids unter Verwendung humaner dermaler Fibroblasten

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.

2D-Expansion, 2D-Stimulation und 3D-Organoidreifung von adulten normalen menschlichen dermalen Fibroblasten für die Sehnenforschung und -technik

Zentrifugieren Sie zunächst trypsinisierte normale humane dermale Fibroblasten. Entfernen Sie den Überstand aus dem Zentrifugenröhrchen. Geben Sie vorgewärmtes ergänztes DMEM-Medium mit niedrigem Glukosegehalt in das Pellet und mischen Sie es gut, um es wieder zu suspendieren.Die normalen humanen dermalen Fibroblasten werden in eine 10 Zentimeter große adhärente Zellkulturschale gegeben. Schütteln Sie die Form vorsichtig quer. Nach 14 Tagen 2D-Stimulation, wenn die Zellen konfluent geworden sind, aspirieren Sie das Medium und verwenden Sie einen Zellschaber, um das gebildete Zellblatt vorsichtig und schnell von der Schale zu lösen.Rollen Sie gleichzeitig das Zellblatt zu einem 3D-stäbchenartigen Organoid. Übertragen Sie die normalen humanen dermalen Fibroblasten-Organoide in eine 10 Zentimeter breite, nicht adhärente Petrischale. Messen Sie die Dehnung mit einem Lineal.Halten Sie dann eine Seite des Organoids mit einer Pinzette fest und dehnen Sie das Organoid vorsichtig, bis es axial um ca. 10 % verlängert wurde. Drücken Sie mit einer Pinzette die Metallstifte manuell durch beide Kanten des Organoids in die Kunststoffschale, um es zu fixieren. Zum Schluss für die 3D-Reifung vor der Inkubation 10 Milliliter vorgewärmtes, ergänztes DMEM-Medium mit hohem Glukosegehalt hinzufügen. Die grobe Morphologie der 3D-Organoide an den Tagen null und 14 des 3D-Reifungsschritts zeigte ein glitzerndes, weißes Aussehen.Mit der Zeit zogen sich die Organoide zusammen und erschienen dichter. Die Morphologie der Organoide wurde mittels H-E-Färbung untersucht. Am ersten Tag zeigten die Organoide unzusammenhängende Schichten, die hauptsächlich aus Zellen bestanden, die von geringen Mengen an Matrix umgeben waren.Die Zellen innerhalb der Schichten wiesen Kerne mit runder Form und unterschiedlicher Größe auf. Am 14. Tag wurde ein merklicher Anstieg des Eosin-Signals beobachtet, was auf Matrixablagerung und verschmolzene Schichten hindeutete. Darüber hinaus zeigten die Organoide das Vorhandensein von Bereichen, die ausgerichtete Zellen enthielten.Die Mehrzahl der Kerne hatte ähnliche Größen und war gelegentlich länglich.

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 ...

Forschung

Biotechnik