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

Organoïdemodel in drie stappen voor onderzoek en engineering van pezen

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.

Auteur in de schijnwerpers: Geavanceerde peesweefselmanipulatie met 3D-organoïde modellen

Demonstratie van zelf-geassembleerde celbladcultuur en handmatige generatie van een 3D-pees/ligament-achtige organoïde met behulp van menselijke dermale 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-expansie, 2D-stimulatie en 3D-organoïderijping van normale menselijke dermale fibroblasten bij volwassenen voor peesonderzoek en -techniek

Centrifugeer om te beginnen getrypsiniseerde normale menselijke dermale fibroblasten. Verwijder het supernatans uit de gecentrifugeerde buis. Voeg voorverwarmd aangevuld DMEM low glucose medium toe aan de pellet en meng goed om het te resuspenderen.Leg de normale menselijke dermale fibroblasten in een 10 centimeter hechtende celkweekschaal. Schud het gerecht voorzichtig kruiselings. Na 14 dagen 2D-stimulatie, wanneer de cellen samenvloeiend zijn geworden, zuigt u het medium op en gebruikt u een celschraper om het gevormde celvel voorzichtig en snel los te maken van de schaal.Rol tegelijkertijd het celvel op tot een 3D-staafachtige organoïde. Breng de normale menselijke dermale fibroblastorganoïden over in een 10 centimeter brede, niet-klevende petrischaal. Meet de rek met een liniaal.Houd vervolgens een kant van de organoïde vast met een pincet en rek de organoïde voorzichtig uit totdat deze axiaal ongeveer 10% is uitgerekt Druk met een pincet handmatig de metalen pinnen door beide randen van de organoïde in de plastic schaal om deze te bevestigen. Voeg ten slotte voor 3D-rijping vóór de incubatie 10 milliliter voorverwarmd aangevuld DMEM hoog glucosemedium toe. De grove morfologie van 3D-organoïden op dag nul en 14 van de 3D-rijpingsstap vertoonde een glinsterend, wit uiterlijk.Na verloop van tijd trokken de organoïden samen en leken ze dichter. De morfologie van de organoïden werd geëvalueerd met behulp van HE-kleuring. Op de eerste dag vertoonden de organoïden losgekoppelde lagen, voornamelijk samengesteld uit cellen omgeven door lage hoeveelheden matrix.De cellen in de lagen vertoonden kernen met een ronde vorm en verschillende groottes. Op de 14e dag werd een merkbare toename van het eosinesignaal waargenomen, wat duidde op matrixafzetting en gefuseerde lagen. Bovendien toonden de organoïden de aanwezigheid van gebieden met uitgelijnde cellen.De meerderheid van de kernen had een vergelijkbare grootte en was soms langwerpig.

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 ...