The first two authors contributed equally to this work.
Evaluating maturation and genetic modification of human dendritic cells in a new polyolefin cell culture bag system
Article first published online: 10 DEC 2009
© 2009 American Association of Blood Banks
Volume 50, Issue 4, pages 843–855, April 2010
How to Cite
Macke, L., Garritsen, H. S.P., Meyring, W., Hannig, H., Pägelow, U., Wörmann, B., Piechaczek, C., Geffers, R., Rohde, M., Lindenmaier, W. and Dittmar, K. E.J. (2010), Evaluating maturation and genetic modification of human dendritic cells in a new polyolefin cell culture bag system. Transfusion, 50: 843–855. doi: 10.1111/j.1537-2995.2009.02520.x
This work was supported by grants from the Federal Ministry of Economics and Technology (Innonet project 16IN170) and the EC (Clinigene-NoE, LSHB-CT-2006-018933).
- Issue published online: 25 MAR 2010
- Article first published online: 10 DEC 2009
- Received for publication July 2, 2009; revision received September 24, 2009, and accepted October 5, 2009.
BACKGROUND: Dendritic cells (DCs) are applied worldwide in several clinical studies of immune therapy of malignancies, autoimmune diseases, and transplantations. Most legislative bodies are demanding high standards for cultivation and transduction of cells. Closed-cell cultivating systems like cell culture bags would simplify and greatly improve the ability to reach these cultivation standards. We investigated if a new polyolefin cell culture bag enables maturation and adenoviral modification of human DCs in a closed system and compare the results with standard polystyrene flasks.
STUDY DESIGN AND METHODS: Mononuclear cells were isolated from HLA-A*0201–positive blood donors by leukapheresis. A commercially available separation system (CliniMACS, Miltenyi Biotec) was used to isolate monocytes by positive selection using CD14-specific immunomagnetic beads. The essentially homogenous starting cell population was cultivated in the presence of granulocyte-macrophage–colony-stimulating factor and interleukin-4 in a closed-bag system in parallel to the standard flask cultivation system. Genetic modification was performed on Day 4. After induction of maturation on Day 5, mature DCs could be harvested and cryopreserved on Day 7. During the cultivation period comparative quality control was performed using flow cytometry, gene expression profiling, and functional assays.
RESULTS: Both flasks and bags generated mature genetically modified DCs in similar yields. Surface membrane markers, expression profiles, and functional testing results were comparable. The use of a closed-bag system facilitated clinical applicability of genetically modified DCs.
CONCLUSIONS: The polyolefin bag–based culture system yields DCs qualitatively and quantitatively comparable to the standard flask preparation. All steps including cryopreservation can be performed in a closed system facilitating standardized, safe, and reproducible preparation of therapeutic cells.