Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

We demonstrate the previous published and implemented three-step organoid protocol using commercially available normal human dermal fibroblasts as a cell source. The 3D organoid model hold several advantages for tissue engineering and for 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 tenogenic, disease mechanism, drug testing, and scaffold-free tendon tissue engineering. To begin, centrifuge trypsinized normal human dermal fibroblasts. Remove the supernatant from the centrifuge tube.

Add prewarmed supplemented DMEM low-glucose medium to the pellet, and mix well to resuspend it. Plate the normal human dermal fibroblasts in a 10-centimeter adherent cell culture dish. Gently rock the dish in a crosswise manner.

After 14 days of 2D stimulation, when the cells have become confluent, aspirate medium, and use a cell scrapper to gently and quickly detach the formed cell sheet from the dish. Simultaneously, roll the cell sheet into a 3D rod-like organoid. Transfer the normal human dermal fibroblast organoids into a 10-centimeter wide, non-adherent Petri dish.

Measure the elongation with a ruler, then hold one side of the organoid with a tweezer, and gently stretch the organoid until it has been elongated axially by approximately 10%Using a tweezer, manually press down the metal pins through both edges of the organoid into the plastic dish to fix it. Finally, for 3D maturation, add 10 milliliters of prewarmed supplemented DMEM high-glucose medium before incubation. The gross morphology of 3D organoids at days zero and 14 of the 3D maturation step showed a glistening white appearance.

Over time, the organoids contracted and appeared denser. The morphology of the organoids was evaluated using H&E staining. On the first day, the organoids showed disconnected layers, primarily composed of cells, surrounded by low amounts of matrix.

The cells within the layers exhibited nuclei with a round shape and varying sizes. On the 14th day, a noticeable increase in the eosin signal was observed, which indicated matrix deposition and fused layers. Moreover, the organoids showed the presence of areas containing aligned cells.

The majority of the nuclei had similar sizes and occasionally were elongated.