Evaluating maturation and genetic modification of human dendritic cells in a new polyolefin cell culture bag system.
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.


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.
Macke
Lars
L
Department of Molecular Biotechnology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
Garritsen
Henk S P
HS
Meyring
Wilhelm
W
Hannig
Horst
H
Pägelow
Ute
U
Wörmann
Bernhard
B
Piechaczek
Christoph
C
Geffers
Robert
R
Rohde
Manfred
M
Lindenmaier
Werner
W
Dittmar
Kurt E J
KE
D000328:Adult; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics; D000954:Antigens, Surface / Q000032:analysis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D016922:Cellular Senescence / Q000502:physiology; D002648:Child; D003713:Dendritic Cells / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D018014:Gene Transfer Techniques; D006801:Humans; D007937:Leukapheresis / Q000379:methods; D007962:Leukocytes / Q000166:cytology / Q000502:physiology; D018950:Lipopolysaccharide Receptors / Q000032:analysis / Q000235:genetics; D008297:Male; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis
D015703:Antigens, CD; D000954:Antigens, Surface; D018950:Lipopolysaccharide Receptors