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Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture
Published on: April 8, 2016
Immunophenotypic analysis of normal human dendritic cells isolated from epidermis and dermis
F Prignano1, G Gerlini, N Pimpinelli
1Department of Human Anatomy and Histology, University of Florence, Italy.
Insights
Skin immune cells, including Langerhans cells (CD1a-positive) and dendritic macrophages (CD36-positive), can be cultured from both epidermis and dermis. Epidermal sheets yield significantly more dendritic cells than dermal sheets in vitro.
Area of Science:
- Dermatology
- Immunology
- Cell Biology
Background:
- The skin immune system involves epidermal Langerhans cells (CD1a+) and dermal dendritic macrophages (CD36+).
- Dendritic cells possess the capability to migrate from skin explants into culture media.
Purpose of the Study:
- To investigate the morphology and immunophenotype of dendritic cells migrating from epidermal and dermal skin layers in vitro.
- To characterize the differences in cell migration and yield between epidermal and dermal sheets.
Main Methods:
- Separation of human skin epidermis and dermis using dispase enzyme.
- Culture of separated tissues for 72 hours to allow cell migration.
- Enrichment, counting, and immunophenotypic labeling (CD1a, CD36, HLADr) of non-adherent migrating cells.
Main Results:
- Migrating cells from both epidermis and dermis exhibit distinct surface projections (thin projections or veils).
- Epidermal sheets produced approximately four times more migrating cells compared to dermal sheets from the same skin sample.
Conclusions:
- Both CD1a-positive Langerhans cells and CD36-positive dendritic macrophages can be successfully isolated from both epidermal and dermal layers using specific separation techniques.
- The number of dendritic cells obtained differs significantly between epidermal and dermal sources, with the epidermis yielding a higher quantity.
Background:
The skin immune system comprises two types of dendritic cells, i.e. CD1a-positive Langerhans cells in the epidermis and CD36-positive dendritic macrophages in the dermis. Dendritic cells can migrate from skin explants into a culture medium.
Methods:
We have examined the morphology and immunophenotype of the dendritic cells migrating from epidermal and dermal sheets in vitro. The epidermis and dermis of keratomes of normal human skin were separated with dispase and cultured for 72 h. At this time, the non-adherent cells in the medium were removed, enriched on a metrizamide or Lymphoprep gradient, counted, prepared by cytospin, and labeled for CD1a, CD36, and HLADr.
Results:
Cells migrating from the epidermis and dermis show many thin projections or a few veils from the cell surface. Approximately four times more cells migrate from epidermal than dermal sheets from the same keratome.
Conclusions:
Using methods to separate the epidermis from the dermis, both CD1a-positive Langerhans cells and CD36-positive dendritic macrophages can be obtained from both tissues, although in different numbers.
