高纯度树突细胞培养方法.pdfVIP

Journal of Immunological Methods 223 1999 77–92 An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow Manfred B. Lutz a, , Nicole Kukutsch a , Alexandra L.J. Ogilvie a , Susanne Roßner a , ¨ Franz Koch b , Nikolaus Romani b , Gerold Schuler a a Department of Dermatology, Uniersity of Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany b Department of Dermatology, Uniersity of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Received 1 March 1998; accepted 20 April 1998 Abstract As dendritic cells DC are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 106 cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1–3 108 immature and mature DC per mouse at 90–95% purity. The major modifications were: i the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, ii a lower plating density of bone marrow cells, iii a prolonged culture period of 10–12 days, iv the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10–12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF- for the last 24