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

Demonstrasjon av selvmontert cellearkkultur og manuell generering av en 3D-sene / ligamentlignende organoid ved bruk av humane dermale fibroblaster

2D-utvidelse, 2D-stimulering og 3D-organoid modning av voksne normale humane dermal fibroblaster for seneforskning og engineering

For å begynne, sentrifuge trypsiniserte normale humane dermal fibroblaster. Fjern supernatanten fra det sentrifugerte røret. Tilsett forvarmet tilskudd av DMEM lavt glukosemedium til pelleten, og bland godt for å resuspendere den.Plate de normale humane dermale fibroblastene i en 10 centimeter vedheftende cellekulturfat. Rock retten forsiktig på tvers. Etter 14 dager med 2D-stimulering, når cellene har blitt sammenflytende, aspirerer medium, og bruk en celleskraper for å forsiktig og raskt løsne det dannede cellearket fra parabolen.Samtidig ruller du cellearket til en 3D-stavlignende organoid. Overfør de normale humane dermal fibroblastorganoider til en 10 centimeter bred ikke-adherent petriskål. Mål forlengelsen med en linjal.Hold deretter den ene siden av organoiden med en pinsett, og strekk organoiden forsiktig til den har blitt forlenget aksialt med omtrent 10%Ved hjelp av en pinsett, trykk metallpinnene manuelt gjennom begge kantene av organoiden inn i plastfatet for å fikse det. Til slutt, for 3D-modning, tilsett 10 ml forvarmet supplert DMEM høyt glukosemedium før inkubasjon. Den grove morfologien til 3D-organoider ved dagene null og 14 av 3D-modningstrinnet viste et glitrende, hvitt utseende.Over tid trakk organoidene seg sammen og virket tettere. Morfologien til organoidene ble evaluert ved hjelp av H E-farging. På den første dagen viste organoidene frakoblede lag, hovedsakelig sammensatt av celler omgitt av lave mengder matrise.Cellene i lagene viste kjerner med en rund form og varierende størrelser. På den 14. dagen ble det observert en merkbar økning i eosinsignalet, noe som indikerte matriksavsetning og smeltede lag. Videre viste organoider tilstedeværelsen av områder som inneholder justerte celler.Flertallet av kjernene hadde lignende størrelser, og noen ganger var langstrakte.

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 ...