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

Three-Step Organoid Model for Tendon Research and Engineering

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.

Author Spotlight: Advancing Tendon Tissue Engineering with 3D Organoid Models

Dimostrazione della coltura di fogli cellulari autoassemblati e generazione manuale di un organoide 3D simile a tendine/legamento mediante l'utilizzo di fibroblasti dermici umani

Espansione 2D, stimolazione 2D e maturazione 3D di fibroblasti dermici umani normali adulti per la ricerca e l'ingegneria dei tendini

Per iniziare, centrifugare i normali fibroblasti dermici umani tripsinizzati. Rimuovere il surnatante dalla provetta centrifugata. Aggiungere al pellet un mezzo a basso contenuto di glucosio DMEM integrato preriscaldato e mescolare bene per risospenderlo.Placcare i normali fibroblasti dermici umani in un piatto di coltura cellulare aderente di 10 centimetri. Scuotere delicatamente il piatto in modo trasversale. Dopo 14 giorni di stimolazione 2D, quando le cellule sono diventate confluenti, aspirare il terreno e utilizzare un raschietto cellulare per staccare delicatamente e rapidamente il foglio cellulare formato dal piatto.Contemporaneamente, arrotolare il foglio cellulare in un organoide 3D simile a un bastoncino. Trasferire i normali organoidi di fibroblasti dermici umani in una capsula di Petri non aderente larga 10 centimetri. Misurare l'allungamento con un righello.Quindi tenere un lato dell'organoide con una pinzetta e allungare delicatamente l'organoide fino a quando non è stato allungato assialmente di circa il 10%Usando una pinzetta, premere manualmente i perni metallici attraverso entrambi i bordi dell'organoide nel piatto di plastica per fissarlo. Infine, per la maturazione 3D, aggiungere 10 millilitri di terreno ad alto contenuto di glucosio integrato con DMEM preriscaldato prima dell'incubazione. La morfologia grossolana degli organoidi 3D al giorno zero e al giorno 14 della fase di maturazione 3D ha mostrato un aspetto bianco scintillante.Nel tempo, gli organoidi si sono contratti e sono apparsi più densi. La morfologia degli organoidi è stata valutata utilizzando la colorazione HE. Il primo giorno, gli organoidi hanno mostrato strati disconnessi, composti principalmente da cellule circondate da basse quantità di matrice.Le cellule all'interno degli strati mostravano nuclei di forma rotonda e di dimensioni variabili. Il 14° giorno è stato osservato un notevole aumento del segnale eosina, che indicava la deposizione di matrice e strati fusi. Inoltre, gli organoidi hanno mostrato la presenza di aree contenenti cellule allineate.La maggior parte dei nuclei aveva dimensioni simili e occasionalmente erano allungati.

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 ...

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Bioingegneria