Korean J Lab Med.  2009 Aug;29(4):353-360. 10.3343/kjlm.2009.29.4.353.

Analysis of Characteristics of Mononuclear Cells Remaining in the Leukoreduction System Chamber of Trima Accel(R) and Their Differentiation Into Dendritic Cells

• Lee Y
• Kim S
• Lee ST
• Kim HS
• Baek EJ
• Kim HJ
• Lee M
• Kim HO

• KMID: 1096930
• DOI: http://doi.org/10.3343/kjlm.2009.29.4.353

Abstract

BACKGROUND: We investigated the characteristics of the mononuclear cells remaining in the leukoreduction system (LRS) chambers of Trima Accel(R) in comparison with those of standard buffy coat cells, and evaluated their potential for differentiation into dendritic cells.
METHODS
Twenty-six LRS chambers of Trima Accel(R) were collected after platelet pheresis from healthy adults. Flow cytometric analysis for T, B, NK, and CD14+ cells was performed and the number of CD34+ cells was counted. Differentiation and maturation into dendritic cells were induced using CD14+ cells seperated via Magnetic cell sorting (MACS(R)) Seperation (Miltenyi Biotec Inc., USA).
RESULTS
Total white blood cell (WBC) count in LRS chambers was 10.8x108 (range 7.7-18.0x108). The median values (range) of proportions of each cells were CD4+ T cell 29.6% (18.7-37.6), CD8+ T cell 27.7% (19.2-40.0), B cell 5.5% (2.2-12.1), NK cell 15.7% (13.7-19.9), and CD14+ cells 12.4% (8.6-32.3) respectively. Although total WBC count was significantly higher in the buffy coat (whole blood of 400 mL) than the LRS chambers, the numbers of lymphocytes and monocytes were not statistically different. The numbers of B cells and CD4+ cells were significantly higher in the buffy coat than the LRS chambers (P<0.05). The median value (range) of CD34+ cells obtained from the LRS chambers was 0.9x10(6) (0.2-2.6x10(6)). After 7 days of cytokine-supplemented culture, the CD14+ cells were successfully differentiated into dendritic cells.
CONCLUSIONS
The mononuclear cells in LRS chambers of Trima Accel(R) are an excellent alternative source of viable and functional human blood cells, which can be used for research purposes.

Keyword

Trima Accel; Leukoreduction chamber; Dendritic cell

MeSH Terms

Adult
B-Lymphocytes/cytology/immunology
CD4-Positive T-Lymphocytes/cytology/immunology
CD8-Positive T-Lymphocytes/cytology/immunology
Cell Differentiation
Dendritic Cells/*cytology/immunology
Flow Cytometry
Humans
Killer Cells, Natural/cytology/immunology
Plateletpheresis/*instrumentation

Figure

• 

  Fig. 1. Wright-Giemsa stain (×400) of cells isolated from LRS chambers (A), CD14+ cells seperated by MACS® Seperation (B), immature dendritic cells on Day 5 of culture (C), and mature dendritic cells on Day 7 of culture (D).

• 

  Fig. 2. The immunophenotypic characteristics of mature dendritic cells (mDCs) and immature dendritic cells (iDCs) on Day 0, 5 and 7 of culture. Phenotypic analysis was performed with CD14+ cells separated from LRS chamber (A) and buffy coat (D) on Day 0; iDCs generated by culturing CD14+ cells from LRS (B) and buffy coat (E) with GM-CSF and IL-4 for 5 days; and mDCs generated from LRS CD14+ cell-derived iDCs (C) and buffy coat-CD14+cell-derived iDC (F) for additional 48 hr in the presence of TNF- α, IL-1β, IL-6 and PGE2.

• 

  Fig. 3. Mature dendritic cells (mDCs) generated from CD14+ monocytes possess strong allostimulatory capacitites. Allostimulatory capacities of DCs from LRS-derived MNCs (A) and DCs from buffy coat-derived MNCs (B) were analyzed by using identical allogeneic T cells. Allogeneic T cells proliferated more vigorously when they were stimulated with mDCs than stimulated with immature DCs (iDCs) on various DC:T cell ratio. Results shown are mean values (circle)±SD (bar) of triplicate wells.

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