A new approach for the large-scale generation of mature dendritic cells from adherent PBMC using roller bottle technology

Table 2 Yield of mature DC from roller bottle cultures and static flask cultures*

Expt #	Culture system	PBMC seeded per vessel	Average floating cells recovered	Average mature DC (CD83⁺, CD86^hi) recovered	% yield of mature DC
1**	850 cm² roller bottles	900 × 10⁶	106 × 10⁶	85 × 10⁶	9.4%
	T-175 static flasks	180 × 10⁶	20 × 10⁶	19.2 × 10⁶	10.7%
2**	850 cm² roller bottles	900 × 10⁶	100 × 10⁶	82 × 10⁶	9.1%
	T-175 static flasks	180 × 10⁶	16 × 10⁶	10.2 × 10⁶	5.7%
3***	850 cm² roller bottles	800 × 10⁶	84 × 10⁶	23 × 10⁶	2.9%
	T-175 static flasks	200 × 10⁶	29 × 10⁶	6 × 10⁶	3%

*Human peripheral blood leukopheresis products were seeded and monocytes adhered in the two types of culture vessels, as described in the Methods section. After 4 to 5 days of culture with GM-CSF and IL-4, ITIP cocktail was added to induce DC maturation. On average, 2–3 roller bottles or static flasks were set up for each experiment. The floating cells were harvested one day later and viable cellrecovery was determined by Trypan Blue staining with a hemocytometer followed by FACS staining for CD83 and CD86. The number of mature DC was calculated from the percent CD83⁺, CD86^hi cells using the total number of viable floating cells. The results of three separate experiments are shown.
**Experiment was done directly with normal donor non-G-CSF-mobilized leukopheresis products.
***Experiment was done directly with a G-CSF-mobilized normal donor leukopheresis product, accounting for the lower yield of mature DC.