The influence of different culture microenvironments on the generation of dendritic cells from non-small-cell lung cancer patients.
BACKGROUND
Monocyte-derived dendritic cells (DCs) are currently under extensive evaluation as cell vaccines for cancer treatment. Many protocols regarding DCs generation in vitro with different protein components, especially autologous proteins, have been described. On the other hand, active tumor-derived factors in patients' serum could impair monocytes, which might result in their abrogated differentiation into DCs in vitro.


METHODS
Autologous DCs from non-small-cell lung cancer (NSCLC)-bearing patients were generated in different culture microenvironments. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence of interleukin-4 and granulocyte-monocyte-stimulating factor with supplementation of 10% autologous serum, 10% allogenic serum, or 2% human albumin. The course of apoptosis, phagocytic ability, and the immunophenotype of the generated DCs were analyzed using flow cytometric methods.


RESULTS
After 48 h of culture, we found a lower percentage of CD1a+/CD14+ and a higher percentage of CD1a+/CD14(-) cells in the culture supplemented with human albumin than in the cultures supplemented with serums. The lowest CD14 antigen expression was found in the human albumin-supplemented 48-h cultures. After 48 h in the cultures carried out with human albumin we found significantly higher percentages of AV+/PI+ cells and AV(-)/PI+ cells than in cultures supplemented with autologous or allogenic serum. We also noted that the expression of FITC-dextran after 4 and 24 h of incubation was significantly higher in the cultures supplemented with both serums than in the HA-SC. The percentage of semi-mature DCs and of CD83 expression was lowest in the culture supplemented with 2% human albumin.


CONCLUSIONS
The kind of culture supplementation had a great impact on the apoptosis of cultured PBMCs. It could also influence the yield of monocyte-derived DCs. It was also confirmed that autologous and allogenic serums provide suitable microenvironments for the generation of autologous DCs from NSCLC patients. The choice of culture supplementation for DC generation is still unsolved and further studies should be undertaken.
Department of Pneumology, Oncology and Allergology, Medical University of Lublin, Jaczewskiego 8, 20-950, Lublin, Poland. pulm.dept@am.lublin.pl
Paweł
P
Krawczyk
Kamila
K
Wojas
Janusz
J
Milanowski
Jacek
J
Roliński
D000368:Aged; D015703:Antigens, CD / Q000276:immunology; D000961:Antilymphocyte Serum; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000276:immunology* / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000276:immunology; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D003713:Dendritic Cells / Q000276:immunology* / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D007167:Immunotherapy; D016753:Interleukin-10 / Q000097:blood / Q000276:immunology; D015847:Interleukin-4 / Q000276:immunology* / Q000378:metabolism; D008175:Lung Neoplasms / Q000097:blood / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000473:pathology; D009367:Neoplasm Staging
D015703:Antigens, CD; D000961:Antilymphocyte Serum; D003470:Culture Media; D016753:Interleukin-10; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor