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

Modelo organoide de três etapas para pesquisa e engenharia de tendões

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 em destaque: Avançando na engenharia de tecidos tendinosos com modelos organoides 3D

Demonstração de Cultura de Folha de Células Auto-Montadas e Geração Manual de um Organoide 3D Semelhante a Tendão/Ligamento usando Fibroblastos Dérmicos Humanos

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.

Expansão 2D, estimulação 2D e maturação organoide 3D de fibroblastos dérmicos humanos normais adultos para pesquisa e engenharia de tendões

Para começar, centrifugue fibroblastos dérmicos humanos normais tripsinizados. Remova o sobrenadante do tubo centrifugado. Adicione o meio de baixa glicose DMEM suplementado pré-aquecido ao pellet e misture bem para ressuspendê-lo.Coloque os fibroblastos dérmicos humanos normais em uma placa de cultura de células aderentes de 10 centímetros. Balance suavemente o prato de maneira transversal. Após 14 dias de estimulação 2D, quando as células se tornarem confluentes, aspire o meio e use um raspador de células para separar suave e rapidamente a folha de células formada do prato.Simultaneamente, enrole a folha de célula em um organoide 3D semelhante a uma haste. Transfira os organoides de fibroblastos dérmicos humanos normais para uma placa de Petri não aderente de 10 centímetros de largura. Meça o alongamento com uma régua.Em seguida, segure um lado do organoide com uma pinça e estique suavemente o organoide até que ele seja alongado axialmente em aproximadamente 10%Usando uma pinça, pressione manualmente os pinos de metal através de ambas as bordas do organoide no prato de plástico para fixá-lo. Finalmente, para maturação 3D, adicione 10 mililitros de meio de alta glicose DMEM suplementado pré-aquecido antes da incubação. A morfologia macroscópica dos organoides 3D nos dias zero e 14 da etapa de maturação 3D mostrou uma aparência branca brilhante.Com o tempo, os organoides se contraíram e pareceram mais densos. A morfologia dos organoides foi avaliada por meio da coloração HE. No primeiro dia, os organoides apresentaram camadas desconectadas, compostas principalmente por células circundadas por baixas quantidades de matriz.As células dentro das camadas exibiam núcleos com formato redondo e tamanhos variados. No 14º dia, foi observado um aumento notável no sinal de eosina, o que indicou deposição de matriz e camadas fundidas. Além disso, os organoides apresentaram a presença de áreas contendo células alinhadas.A maioria dos núcleos tinha tamanhos semelhantes e, ocasionalmente, eram alongados.

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 ...

Bioengenharia