Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures.
BACKGROUND
There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved.


METHODS
The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation.


RESULTS
The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia.


CONCLUSIONS
Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.
Department of Orthopedic Surgery, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Hiromi
H
Kumamaru
Hirokazu
H
Saiwai
Kazu
K
Kobayakawa
Kensuke
K
Kubota
Nico
N
van Rooijen
Kazuhide
K
Inoue
Yukihide
Y
Iwamoto
Seiji
S
Okada
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects*; D050071:Bone Density Conservation Agents / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D004002:Clodronic Acid / Q000494:pharmacology*; D008081:Liposomes / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000187:drug effects*; D061251:Primary Cell Culture / Q000379:methods
D050071:Bone Density Conservation Agents; D008081:Liposomes; D004002:Clodronic Acid