Spheroid cell culture of human skin-derived precursors

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
human skin stem cells


Method keywords
  • tree-dimensional spheroid culture
  • hepatic differentiation
Scientific area keywords
  • spheroids
  • skin stem cells
  • differentiation
Method description

For the generation of the human skin-derived precursors (hSKP) spheroids, ultra-low attachment 96-well plates, cell-repellent (Greiner) are used. hSKP cultured on tissue culture plates are detached with trypple (5 minutes) and collected in a tube. After centrifugation, hSKP are counted at the Burker chamber and seeded at 2,000 cells per well. The 96-well plates are placed on an orbital shaker, shaking at 40 rpm overnight in a humidified incubator. The outer wells are filled with PBS to reduce evaporation of cell culture medium. The day after, hSKP aggregates of approximately 200-250 µm diameter size are formed. Culture medium is refreshed twice in a day every 3 days to allow complete medium replenishment.

Lab equipment
  • Cell culture laboratory;
  • Laminar air flow;
  • Ultra-low attachment 96-well plates;
  • Shaker.
Method status
  • Still in development

Pros, cons & Future potential

  • Emulation of 3D cell organization, simple method, applicable to many cell types.
  • Spheroid formation has been shown to enhance anti-inflammatory effects, augment tissue regenerative and reparative effects, facilitate differentiation potentials of multiple lineages, improve stemness properties of adult stem cells.
  • Spheroid handling and transfer step (e.g. spheroids may be easily lost by aspiration with pipette during refreshment of cell culture medium).
  • Presence of a necrotic core inside the sphere that could lead to poor cell viability in long-term culture.
Future & Other applications

Since spheroid generated from mesenchymal stem cells exert strong anti-inflammatory and immuno-modulatory effects, they could be employed in regenerative medicine and autoimmune diseases.

References, associated documents and other information


Cesarz Z, Tamama K (2016) Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int 2016:. doi: 10.1155/2016/9176357

Tsai A-C, Liu Y, Yuan X, Ma T (2015) Compaction, Fusion, and Functional Activation of Three-Dimensional Human Mesenchymal Stem Cell Aggregate. Tissue Eng Part A 21:1705–1719. doi: 10.1089/ten.tea.2014.0314

Contact person

Alessandra Natale


Vrije Universiteit Brussel (VUB)
Pharmaceutical and Pharmacological Sciences
In Vitro Toxicology and Dermato-Cosmetology
Brussels Region