A simple two-step culture system for the large-scale generation of mature and functional dendritic cells from umbilical cord blood CD34+ cells.
BACKGROUND
In vitro generated dendritic cells (DCs) are widely used as adjuvants in cancer immunotherapy. The major sources for DC generation are monocytes and CD34+ cells. CD34+-derived DCs are less frequently used in clinical applications because it requires complex generation methods. Here a simple method for the large-scale generation of mature functional DCs from umbilical cord blood-derived CD34+ cells is described.


METHODS
CD34+ cells were first expanded with a combination of early acting growth factors in a medium containing autologous plasma. In the second step the DC precursors were further either enriched by plastic adherence or sorted on a cell sorter and differentiated as DCs. DCs generated by both methods were compared for their morphology, phenotype, and different functional variables.


RESULTS
This culture system provided a large-scale expansion of CD34+ cells giving a mean fold increase of 615. The majority of the expanded cells were interstitial DC precursors, that is, CD14+-positive cells. In vitro generated immature DCs could be matured into functional DCs by appropriate maturation stimuli. DCs generated by the plastic adherence method had a better cytokine profile and strong mixed leukocyte reaction compared to those generated by cell sorting.


CONCLUSIONS
A two-step culture system provides a large-scale expansion of CD34+ cells with a preferential lineage commitment toward CD14+ cells. Enrichment of these precursors with a simple plastic adherence technique results in generation of large numbers of mature, functional DCs. This method of in vitro DC generation will have applications in cancer immunotherapy.
Balan
Sreekumar
S
National Centre for Cell Science, Pune University Campus, Pune, India.
Kale
Vaijayanti P
VP
Limaye
Lalita S
LS
D018952:Antigens, CD34 / Q000276:immunology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism
D018952:Antigens, CD34; D016753:Interleukin-10; D018664:Interleukin-12