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Immunology JoVE (Open Access) Citable · DOI

A 3D Organotypic Melanoma Spheroid Skin Model

DOI: 10.3791/57500-v
What you'll learn
  • ✓ Generate 3D melanoma spheroids using the hanging drop method
  • ✓ Construct dermal and epidermal layers for organotypic skin models
  • ✓ Culture full-thickness skin reconstructs with integrated tumor spheroids
  • ✓ Validate structural and cellular organization of engineered skin models
Protocol

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Difficulty
advanced
Total time
~5–7 days (spheroid generation 2–3 days; skin construct assembly and maturation 3–5 days)
Model organism
Human primary melanoma cells; human primary dermal fibroblasts; human primary keratinocytes
Biosafety
BSL-1

Steps

1
Generate 3D melanoma spheroids via hanging drop method

Prepare melanoma cell suspensions and deposit them into hanging drops on a non-adherent surface to promote self-aggregation into uniform spheroids. Incubate until spheroids reach the desired size.

▶ 01:20
2
Construct dermal and epidermal skin layers

Seed dermal fibroblasts into collagen-based matrices to form the dermal equivalent, then seed keratinocytes on top to establish the epidermal layer. Allow layers to integrate and differentiate.

▶ 03:16
3
Culture organotypic skin reconstructs at air-liquid interface

Transfer the full-thickness skin reconstructs (with integrated melanoma spheroids) to air-liquid interface cultivation to promote stratification, differentiation, and physiologically relevant tumor-tissue interactions.

▶ 05:08
4
Validate organotypic skin and tumor spheroid architecture

Perform histological, immunofluorescence, and morphological analyses to confirm proper tissue stratification, cellular organization, and successful integration of melanoma spheroids within the engineered skin model.

▶ 06:45
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