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Published on: October 10, 2011
Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays
Isabelle Tchou1, Odile Sabido, Claude Lambert
1Groupe Immunité des Muqueuses et Agents Pathogènes, Equipe d'Accueil, 3064, Faculté de Médecine, Université Jean Monnet de Saint Etienne, 15 rue Ambroise Paré, 42023 Saint-Etienne Cedex, France.
Insights
To study immature Langerhans cells (LCs), researchers compared two purification methods. Ficoll floatation effectively preserved LC immaturity, unlike magnetic bead separation which caused pre-activation.
Area of Science:
- Immunology
- Dermatology
- Cell Biology
Background:
- Langerhans cells (LCs) are immature dendritic cells in the epidermis, crucial for immune responses.
- Accurate assessment of LCs requires maintaining their immature state ex vivo.
- Artefactual maturation during isolation can invalidate experimental results.
Purpose of the Study:
- To compare the efficacy of two methods for purifying immature LCs from human skin epidermis.
- To evaluate the impact of purification techniques on LC phenotype and function.
- To identify a reliable method for obtaining non-activated, immature LCs for research.
Main Methods:
- Isolation of LCs from human skin epidermis.
- Purification using two parallel methods: anti-CD1a magnetic bead separation and a three-step Ficoll floatation process.
- Phenotypic analysis via flow cytometry to assess cell maturation markers.
Main Results:
- Both magnetic bead separation and Ficoll floatation yielded comparable purification efficiency and cell recovery.
- Flow cytometry revealed that anti-CD1a magnetic bead separation induced partial maturation of CD1a(+) LCs.
- Repeated Ficoll floatation successfully maintained the immature phenotype of LCs without pre-activation.
Conclusions:
- Repeated Ficoll floatation is a superior method for purifying immature Langerhans cells ex vivo.
- The use of anti-CD1a magnetic beads can lead to artefactual pre-activation, compromising studies on early LC physiology.
- Researchers should carefully select purification methods to ensure the integrity of immature dendritic cells for experimental validity.
Abstract:
Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.
